Histochemistry and Cytochemistry of Endogenous Animal Lectins

Progr. Histochem. Cytochem. Vol. 33 . No 1(1998) . pp. 1-92

Histochemistry and Cytochemistry of Endogenous Animal Lectins Yoshihiro Akimoto Yasuyuki Imai Jun Hirabayashi Ken-ichi Kasai Hiroshi Hirano With 28 Figures and 5 Tables

GUSTAV FISCHER Jena Stuttgart Lubeck Ulm

YOSHIHIRO AKIMOTO, Ph. D., D. Med. Sci., Associate Professor HIROSHI HIRANO, M. D., (~) Professor and Director Department of Anatomy, Kyorin University School of Medicine Mitaka, Tokyo 181-8611 Oapan) YASUYUKI IMAI, Ph. D., Professor Department of Microbiology of Pharmaceutical Sciences University of Shizuoka Yada, Shizuoka-shi, Shizuoka 422-85260apan) JUN HlRABAYASHl, Ph. D., Assistant Professor KEN-ICHI KASAl, Ph. D., Professor and Director Department of Biological Chemistry Faculty of Pharmaceutical Sciences Teikyo University Sagamiko, Kanagawa 199-0195 Oapan)

Acknowledgement: We wish to express our appreCiatiOn to Mr. M. FUKUDA, Ms. S. MATSUBARA, Ms. C. OKADA, Ms. M. KANAI, and Ms. T. SHIBATA (Laboratory for Electron Microscopy and Department of Anatomy, Kyorin University School of Medicine) for their technical assistances. This work was supported in part by Grants-in Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan, and the Promotion and Mutual Aid Cooperation for Private Schools of Japan.

~ Correspondence to: Prof. HIROSHI HIRANO, Department of Anatomy, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611,Japan, Tel.: 0422-47-5511 (ext. 3416), Fax: 0422-440866, E-mail address:[email protected]

Contents 1 2 2.1 2.1.1 2.1.2 2.1.3 2.1.4 2.1.5 2.1.6 2.2 2.3 3 3.1 3.2 3.3 4 4.1 4.2 4.3 4.4 4.5 5 5.1 5.2 5.2.1 5.2.2 5.2.3 5.3 5.4 5.4.1 5.4.2 5.5 5.6 6 7

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Overview of animallectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Galectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Historical background of galectin . . . . . . . . . . . . . . . . . . . . . . . . . . . Biochemical properties of the galectin family .... . . . . . . . . . . . . . . . Three types of galectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sequence similarity between galectins Controversial tissue and cellular localization of galectins . . . . . . . . . .. Endogenous ligands for galectins C-type lectin I-typelectin Localization of galectins in chick embryonic and adult skin . . . . . . . .. Changes in expression of 14-kDa galectin during epidermal differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. Changes in expression of 16-kDa galectin during epidermal differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. Changes in expression of two (14- and 16-kDa) galectins in the dermis of chick embryonic skin during development . . . . . . . . . . . . . . . . . .. Localization of galectins in other tissues of adult chicken and other vertebrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. Nervous system Striated muscle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. Stomach. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. Intestine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. Lung. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Localization of galectins in human tissues Skin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Skin appendages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. Hair. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sebaceous gland Eccrine sweat gland . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. Placenta. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. Nervous system Central nervous system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. Peripheral nervous system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. Digestive system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. Blood cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. Galectin changes in tumors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. Histochemical localization of the nematode galectin .... . . . . . . . . ..

1 1 4 4 8 9 10 10 11 12 19 20 22 26 30 32 32 34 34 34 34 35 35 39 39 40 40 40 42 42 44 44 47 47 48

VI . Contents

8 8.1 8.1.1 8.1.2

8.1.3 8.1.4 8.1.4.1 8.1.4.2 8.1.4.3 8.1.4.4 8.1.4.5 8.1.4.6 8.1.5 8.1.6 8.2 8.2.1 8.2.2 8.2.3 8.2.4 9 9.1

9.2 10 10.1 10.2 10.2.1 10.2.2 10.2.3 11

Localization of C-type lectins . Mouse macrophage lectin (MMGL) . Background information for MMGL . Production of monoclonal antibodies against MMGL that are useful for immunohistochemical detection . Detection of MMGL in adult mouse tissue extracts . . Immunohistochemical localization of MMGL in adult mouse tissue Lung . Abdominal skin . Muscles . Urinary bladder . Gastrointestinal tract . Thymus . MMGL expression in lung metastatic nodules . Immunohistochemical localization of MMGL in mouse embryos . Immunohistochemical localization of other C-type lectins . Kupffer cell lectin . 180 kDa-mannose specific macrophage lectin . . A cDNA encoding lectin with 8 tandem CRDs Bovine conglutinin . Localization of I-type lectin . Background information for sialoadhesin . Immunohistochemical detection of sialoadhesin . . . . . . . . . . . . . . . . . . Histochemical localization of carbohydrate ligands Relation between endogenous lectin and exogenous lectins as tools for . histochemical detection of sugars . Localization of carbohydrate ligands recognized by L-selectin Background information for glycoprotein ligands for selectins . Reagent to detect glycoprotein ligands for L-selectin . Immunohistochemical detection of glycoprotein ligands for L-selectin . Conclusions .

50 50 50 51 51 51 54

55 55

55 55 58 58 58 62 62 62 63 63 64 64 64 65 65 66 66 66 68

70

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

73

Subject index

91

Abbreviations chicken 14-kDa galectin C-14 C-16 chicken 16-kDa galectin carbohydrate binding protein 35 CBP35 central nervous system CNS carbohydrate recognition domain CRD 4' ,6-diamidine-2' -phenylindole dihydrochloride DAPI dibutyryl cyclic AMP BtlCAMP days post coitus dpc experimental allergic encephalomyelitis EAE extracellular matrix ECM epidermal growth factor EGF fetal calf serum FCS galactose Gal Gal ~1-4 GlcNAc galactose-~ 1-4-N -acetylglucosamine GalNAc N -acetylgalactosamine high endothelial venule HEV horseradish peroxidase HRP heteronuclear ribonucleoprotein hnRNP N -acetyllactosamine LacNAc chimeric protein consisting of L-selectin and immunoglobulin LEC-IgG monoclonal antibody mAb myelin associated glycoprotein MAG mannose binding protein MBP mouse macrophage galactose/N-acetylgalactosamine-specific lectin MMGL natural killer cell NK cell non-obese diabetes NOD O-linked N-acetylglucosamine O-GlcNAc periodic acid Schiff PAS peanut agglutinin PNA Ricinus communis agglutinin RCA SBA soybean agglutinin serine/threonine Ser/Thr Schwann cell myelin protein SMP simian virus 40 large T antigen Tag Vicia villosa agglutinin isolectin B4 VVAB 4

1 Introduction The recognition of carbohydrates by sugar-binding molecules or lectins is involved in many important functions. Many glycobiological investigations have been carried out to elucidate the interactions between cell-surface carbohydrate recognition molecules and carbohydrate ligands expressed on the cell surface or in the extracellular matrix. Recently much attention has been paid to animallectins from the point of view of distinguishing complex cell-surface glycoconjugate structures through carbohydrate recognition (BARONDES 1986, SHARON and LIS 1989). These structures are thought to be implicated in various regulatory phenomena in animals via cell-to-cell or cell-to-extracellular matrix (ECM) recognition, such as cell adhesion, cell migration, morphogenesis, differentiation, neoplastic transformation, metastatic progression and defense against infection by microorganisms (NOWAK et al. 1977; KOBILER et al. 1978; BRILES et al. 1979; MONTELIONE et al. 1981; RAz et al. 1986; HlRABAYASHI and KASAl 1993; BARONDES et al. 1994a). On the other hand, it was recently found that there are many glycoconjugates in the nucleus and cytoplasm (HOLT and HART 1986). A wide variety of nuclear and cytoplasmic proteins, including several structural proteins and transcription factors, are modified by the addition of single N-acetylglucosamine residues glycosidically linked to the hydroxyl side chain of SerfThr residues (O-GlcNAc) (HART et al. 1989). It is suggested that O-GlcNAc is a regulatory modification, being analogous to protein phosphorylation (CHOU et al. 1995; HART 1997). The roles of glycoconjugates in the nucleus and cytoplasm remain to be examined. An increasing amount of data on biochemical and molecular biological aspects of various animal lectins has been accumulated, and has been reviewed by LOTAN (1992), DRICKAMER and TAYLOR (1993), HlRABAYASHI (1993), BARONDES et al. (1994b) and GABIUS (1997). In this review, histochemical and cytochemcial investigations of endogenous animallectins are summarized with the focus on the ~-galactoside-binding lectin galectin and on C-type lectins.

2 Overview of animallectins Since lectins are involved in highly diverse biological phenomena, it is not easy to define them according to their biological roles. Definition based on the phenomenological point of view was once proposed; that is, lectins are proteins having multiple sugarbinding sites and able to cause precipitation of polysaccharides and certain cells by creating cross links and a network between them (GOLDSTEIN et al. 1980). This definition, however, turned out to be inappropirate because a number of monovalent saccharidebinding proteins structurally related to known lectins have been found. At present, we may have to accept the rather vague concept that lectins are sugar-binding proteins other

2 . Y. Akimoto et al.

than sugar-binding antibodies and enzymes acting on sugars. From the viewpoint of the transfer of biological information, lectins are proteins that have a role in deciphering glycocodes; each lectin recognizes a certain sugar structure, and initiates a certain biological phenomenon under a given circumstance. After the discovery of ricin in the castor bean by Stillmark in 1888, many plant lectins have been found. They have been used as very convenient research tools in the biosciences, e. g., for typing ABO blood groups, localization and isolation of glycoconjugates, analysis of the structure of saccharide chains, stimulation of cellular activities, etc., though their phyisological functions have remained ambiguous and assignment of definite roles other than defense has not been easy. However, accumulation of knowledge on animal leetins, which has become more and more extensive over the last two decades, has revealed the involvement of lectins in a variety of important phenomena that take place throughout the life of animals, e. g., fertilization, development, morphogenesis, differentiation, metastasis, apoptosis, etc. A variety of possible roles attributed to lectins are listed in Table 1 and illustrated in Fig. 1. As in the case of plants, animals were also found to have lectins aimed at defense, examples being mammalian serum mannose-binding protein, which recognizes invading bacteria and destroys them by activating the complement system (DRICKAMER et al. 1986); sarcophaga lectin, which is induced in the larva of a fly in response to bodily injury (TAKAHASHI et al. 1985); and macrophage lectin, which serves as a sensor molecule for invading microorganisms (II et al. 1990; SATO et al. 1992). There are many lectins involved mainly in cell-to-cell interaction and cell adhesion: Both sperm and ovum have lectins required for fertilization (WASSARMAN 1987). Homing of lymphocytes and migration of leukocytes are mediated by selectins (LASKY et al. 1989; SIEGELMAN et al. 1989). Lectins are involved in symbiosis between microorganisms and legume roots (Ho et al. 1986); also in bacterial infection to the animal gut (SHARON 1987). Infection of microorganisms by viruses is also mediated by lectins. Mannose 6-phosphate-binding protein is an intracellular carrier molecule for the transfer of lysosomal enzymes from the Golgi apparatus to the lysosome (KORNFELD 1986). Mammalian hepatic lectin (asialoglycoprotein receptor) is the first animal lectin for which a definite biological role was attributed (ASHWELL and HARFORD 1982). Hepatic lectin molecules are located on the surface of liver cells, bind serum glycoproteins that have lost terminal sialic acids of their sugar chains; and internalize serum glycoproteins by endocytosis to remove out dated serum glycoproteins from the blood stream. There are also lectins related to enzymes: i. e., some snake venom proteases have a lectin domain besides the active site (TAKAYA et al. 1992), and thrombomodulin is a regulator of blood clotting enzymes GACKMAN et al. 1987). There are also lectins that seem to be involved in the construction of matrices, e. g., proteoglycan core protein (DOEGE et al. 1986; SAl et al. 1986) and galectins (AKIMOTO et al. 1992, 1995a). Two groups of animallectins have been most extensively studied to date. One is the C-type lectin family (DRICKAMER 1988), and the other the galeetin family (BARONDES

Endogenous animallecrins . 3

Table 1. Possible functions of lectins. Function Defense Against infection

Against herbivora Cell-cell interaction Fertilization Migration Symbiosis Infection Tissue organization Morphogenesis Cell-matrix interaction Tissue organization Adaptor

Example Activator of complement system (e.g., mannose-binding lectin) Sensor for invading organism (e.g., macrophage surface lectin) Immobilization of invading organism (e.g., invertebrate body fluid lectin) Plant toxic lectin (e.g., ricin) Inhibitor of insect amylase (e.g., legume lectin) Sperm lectin, ovum lectin Homing of lymphocyte, migration of leucocyte, metastasis of malignant cell Adhesion of root nodule bacteria to legume root Adhesion of bacteria to animal gut, infection of virus to host cell Condensation of mesenchymal cells (e.g., galectin) Morphogenesis of dermis (e.g., galectin), guidance of axon filament (e.g., galectin) Material for matrix (e.g., proteoglycan core protein) Crosslinker between macromolecules

Regulation Enzyme activity Targetting of enzyme Cellular activity

Thrombomodulin Protease having C-type lectin domain Stimulation of mitosis, induction of apoptosis

Transportation

Carrier of lysosomal enzymes from the Golgi to the lysosome

Clearance

Removal of out-dated serum proteins (e.g., hepatic lectin), scavenging of degraded cells

Weapon

Snake venom lectin

1981, 1984; BARONDES et al. 1994a, b; CARON et al. 1990; DRICKAMER and TAYLOR 1993; HlRABAYASHI 1993, 1994; HlRABAYASHI and KASAl 1993; KASAl 1990; KASAl and HlRABAYASHI 1996). Each family is characterized by a distinct amino acid sequence motif that forms at the sugar-binding site (CRD, standing for carbohydrate-binding domain). The most distinct difference between these two families is the requirement for metal ions. The Ca ion is essential if C-type lectins are to bind to sugar chains, but galeetins do not need any metal ion. Besides the metal ion requirement, there are many other differences between these two families. Other animal lectin families have been proposed, e. g., I-Type lectins form a group of carbohydrate-binding proteins having a structural motif of immunoglobulins.

4 . Y. Akimoto et al.

1) Defense

2) Homing of lymphocytes

3) Fertilization

4) Morphogenesis

Fig. 1. Schematic representation of some possible functions of lectins.

2.1 Galectin 2.1.1 Historical background of galectin

Galectin is a new family name created for a group of animallectins very different from C-type lectins. Since the first report of the presence of a galactoside-specific lectin in electric eel (LEVI and TEICHBERG 1981), animallectins having common properties, that is, specific for p-galactoside and not dependent on metal ion, have been found in various vertebrates (e. g., frog, chicken, mouse, human, etc.). They had been called by different names depending on the researchers (e. g., electrolectin, soluble lectins, galaptins, S-Lac lectins, p-galactoside-binding lectins, S-type lectins, etc.). Since they turned out to have sequences homologous to each other, and the number was growing, the all the time need for a generic family name to avoid inconvenience and confusion became very great. Agreement was reached among concerned researchers to use the new term «galectin» (BARONDES et al. 1994a). It was also agreed to assign a serial number to mammalian galectins in the order of discovery (Table 2).

Endogenous animallectins . 5

In vertebrates, galectins were found in various tissues and cells, e. g., skin, muscle, brain, intestine, liver, kidney, placenta, cultured fibroblasts, established cell lines such as HL60, and tumor cells. A number of observations have indicated their possible involvement in a variety of important phenomena occurring in multicellular animals, such as development, differentiation, morphogenesis, immunity, apoptosis, etc. Metastasis of malignant cells was also reported to be related to the presence of galeetins on the cell surface (RAz and LOTAN 1987). Various cellular activities were found to be triggered by galectins (WELLS and MALLUCI 1991; YAMAOKA et al. 1991). The biological functions of galeetins appear to vary from location to location, and from time to time. From the viewpoint of mechanism, various cellular processes, such as communication, adhesion, transportation, guidance, modulation, signal transduction, etc., seem to be supported by the specific recognition of glycoconjugates by galectins. Galectins found in the early period were non-covalent homodimers of a subunit of about 14 kDa, which comprises the minimum structural element (proto type) of the galectin family. Galectins of this type usually show hemagglutinating activity. Determination of the complete primary structure of chicken 14-kDa galectin (C-14) (HlRABAYASHI et al. 1987b) and cloning of its eDNA (OHYAMA et al. 1986) triggered extensive structural studies on galectins from various sources, and the primary structures of a number of mammalian galeetins were elucidated successively. Various genetically engineered galectins have been produced (ABBOTT and FEIZI 1991; HlRABAYASHI and KASAl 1991, 1994). Alignment of the revealed sequences indicated that the central parts of the galectin molecule are mostly conserved and presumably correspond to the sugarbinding domain (CRD) (Fig. 2). Similarities between mammalian galectins found during this early period of research were significant, and they are now grouped in the galectin-1 category. On the other hand, a larger galactose-binding protein of about 35 kDa (CBP 35) was found in 3T3 cells. Determination of its primary structure revealed that it contained the galectin CRD motif in the C-terminal half region (Fig. 2) alA and WANG 1988). This was the first indication of the existence of the galectin family. The sequences of the Nterminal half, however, exhibited no homology to galectins. This protein, lately named galectin-3, proved to be the same as the proteins that had been called either «IgE-binding protein» of certain leukocytes (ALBRANDT et al. 1987) or «Mac-2» (a macrophage-specific cell-surface antigen) (CHERAYIL et al. 1989). Up to now, discoveries of proteins having the galectin CRD but distinct from known galectins have been continuously reported, especially in the case of mammals (Table 2). The numbered mammalian galeetins so far have reached as many as 9, though it is not yet clear why there should be so many galeetins, and it is not possible to assign a specific role to each member. Though galectins had long only been found in vertebrate species, a galectin of 32 kDa was found in the nematode Caenorhabditis elegans (HlRABAYASHI et al. 1992a, b; ARATA et al. 1996). This was the first discovery not only of a nematode lectin but also of a new

Mammal

Vertebrate

Origin

Proto

Proto Chimera

Tandem-repeat

Proto Tandem-repeat Proto Tandem-repeat

Galectin-2 Galectin-3

Galectin-4

Galectin-5 Galectin-6 Galectin-7 Galectin-8

Structural type

Galectin-l

Designation (common name)

Table 2. Classification of Galectins.

Mouse fibroblast, macrophage, lung Rat leukemia, lung Dog kidney (MOCK) Baby hamster kidney Rat intestine Porcine tongue Rat erythrocytes Rat intestine, colon Human keratinocyte Rat liver

32 29 32 30 36 37 16 34 15 35

14.5 29

Human placenta, skin, brain, lung, liver Rat lung, uterus Mouse fibroblast, muscle Porcine heart Marmoset Human hepatoma Human macrophage

Distribution

14.5

Subunit MW (kDa)

MERKLE et al. 1989 aHSAWA et al. 1990 GITI and BARONDES 1986; GITI et al. 1992 CHERAYIL et al. 1990; RAz et al. 1991; CHERAYIL et al. 1989; Woo et al. 1990; aDA et al. 1991 JIA and WANG 1988; RAz et al. 1989; CHERAYIL et al. 1989; Woo et al. 1990 ALBRANDT et al. 1987; GRITZMACHER et al. 1992 HERRMANN et al. 1993 MEHuL et al. 1994 aDA et al. 1993 CHIU et al. 1994 GITI et al. 1995a GITI et al. 1995b MADSEN et al. 1995; MAGNALDO et al. 1995 HADARI et al. 1995

HlRABAYASHI and KASAl 1988; COURAUD et al. 1989; ABBOTI and FEIZI 1989; HlRABAYASHI et al. 1989; AKIMOTO et al. 1995b; GITI and BARONDES 1991 CLERCH et al. 1988 ABBOTI et al. 1989

References

~

~

8

o

3

>Co

K

a--

Sponge

Nematode

Invertebrate

Eel

Frog

Avian

16K lectin 32K lectin Lectin-l Lectin-2

electrolectin

Proto Tandem-repeat Proto Proto

14

Proto

16 32 15 15

14

16

16

16

Proto

16K lectin

14

Proto Proto Proto

Proto

14K lectin

G. cydonium G. cydonium

C. elegans C. elegans cuticle

Chick liver, skin, embryonic muscle X. laevis skin R, catesbeiana oocyte E. electricus electric organ Conger eel mucus skin

Chick skin, intestine

HlRABAYASHI et al. 1996 HlRABAYASHI et al. 1992a, b; ARATA et al. 1996 PFEIFER et al. 1993 PFEIFER et al. 1993

MURAMOTO and KAMIYA 1992

MARSCHAL et al. 1992 OZEKI et al. 1991 PAROUTAUD et al. 1987

ODA et al. 1983; OHYAMA et al. 1986; HlRABAYASHI et al. 1987b; OHYAMA and KASAl 1988; AKIMOTO et al. 1992 SAKAKURA et al. 1990; AKIMOTO et al. 1993

tTl

::>

"

~.

~

"

e.

3'

::>

.,

o e In

::>

(1)

a
o

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8 . Y. Akimoto et al.

type of galectin family. The molecular architecture of the 32-kDa molecule was unique because two CRD motifs were found to be tandemly repeated in a single polypeptide, suggesting that it is a divalent monomer (Fig. 2). The existence of a mammalian galectin having a similar protein architecture was also reported lately (named galectin-4) (aDA et a1. 1993). In addition, a proto type C. elegans galectin having a molecular weight of approximately 16 kDa was found (HlRABAYASHI et a1. 1996). The nematode is therefore likely to be equiped with a set of galectins similar to those in vertebrates, though a chimera-type galectin has not yet been found. Soon after the discovery of the galectin in C. elegans, the existence of galectins in the marine sponge Geodia cydonium was reported (PFEIFER et a1. 1993). This finding has a significant meaning in galectin research because the phylogenically oldest metazoan phylum (Porifera) already had galectins. Galectins therefore must have appeared on earth with the rise of multicellular animals to carry out certain fundamental tasks; thus they became distributed widely throughout the animal kingdom. Very recently, it was reported that galectins were found in a mushroom (COOPER et a1. 1997).

2.1.2 Biochemical properties of the galectin family

Galectins are water-soluble proteins. For the initial process of purification, however, it is necessary to add some hapten sugars such as lactose to the extraction buffer so as to dissociate them from insoluble substances in the starting tissues. This does not mean they are integrated into lipid membrane, but they appear to bind to sugar chains of insoluble glycoconjugates such as glycoproteins, glycolipids, and proteoglycans. Since the sugar-binding activity of galectins is often lost in the absence of a reducing agent such as ~-mercaptoethanol, the presence of a free SH group(s) was once thought to be essential (LEVI and TEICHBERG 1981). However, various experiments including chemical modification (WHITNEY et a1. 1986) and site-directed mutagenesis (HlRABAYASHI and KASAl 1991) showed that SH groups were not essential for sugar binding. The residues proven to be indispensable for the sugar-binding activity by site-directed mutagenesis are shown by block capital letters in Fig. 2. These residues were found by X-ray crystallography to form hydrogen bonds with principal hydroxyl groups in lactose. The N-termini of all galectins studied so far are found to be blocked by an acetyl group (HlRABAYASHI et a1. 1987b; HlRABAYASHI and KASAl 1988). Although galectins occasionally contain cysteine residues, no disulfide bond is formed and all SH groups are in a free state. Cloning of cDNAs revealed that galectins are synthesized without a signal sequence (OHYAMA et a1. 1986). These characteristics strongly suggest that galectins are designed as intracellular proteins. However, galectins were very often found outside cells. Hence, they must be externalized by an unknown mechanism that does not depend on the signal peptide (COOPER and BARONDES 1990).

44

.

46

.jlJ

HF pRF vc

Endogenous animallectins . 9

.

3

61

K

weER FPF e

R

0 C

C

I

t

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hnRNP-like domain

l-

---ll.--..L.J.!I.-

CRD-1

Chimera

.:....-._ _- - I - L - L J J l - - - - - - - J

Tandem-repeat

CRD-2

Fig.2. Schematic representation of the three types of galectins (proto-, chimera- and tandemrepeat). The proto-type is composed of only a single lectin domain. The chimera-type is composed of two parts; a C-terminal half containing the galectin CRD and an N-terminal half of unknown function. The tandem-repeat type is composed of two homologous CRD domains. Above the proto-type structure, conservative residues are shown in single letters. Large capital letters denote the most important residues for sugar binding. Asterisks denote residues mostly conserved in all galectins studied so far. C indicates cysteine residues, the oxidation of which results in loss of sugar-binding activity. Residue numbers are those of human galectin-l (HlRABAYASHI and KASAl 1988). Arrowheads indicate the positions in which introns are inserted. X is the modified residue at the N-terminal.

2.1.3 Three types of galectins Galectins can be classified into three types according to their molecular architecture, that is, proto, chimera and tandem-repeat types (Fig. 2) (HlRABAYASHI et al. 1992). The proto-type galectin comprises small proteins of about 15,000 daltons containing only one CRD domain, with the most common one being mammalian galectin-1. In addition, mammalian galectins-2 (GITT et al. 1992), -5 (GITT et al. 1995) and -7 (MADSEN et al. 1995; MAGNALDO et al. 1995) have also been found to be proto types. Of these, galectins1, -2, and -7 exist as non-covalent homodimers under physiological conditions, whereas galectin-5 has been reported to exist as a monomer. In chickens two proto-type galectins (C-14 and C-16, named according to the apparent molecular weights deduced from SDS-PAGE), have been found (HlRABAYASHI et al. 1987a; SAKAKURA et al. 1990). The chimera type, which has been found only in mammals alA and WANG 1988) and chick (NURMINSKAYA and LINSENMAYER 1996), has a molecular weight about twice that of the proto type (30-35Kdas), and is composed of two different domains. The C-terminal half is the galectin domain containing one CRD. The N-terminal half, exhibiting no homology to galectins, is related to components of the heteronuclear ribonucleoprotein com-

10 . Y. Akimoto et al.

plex (hnRNP). C. elegans 32-kDa galectin as well as mammalian galectins-4 (ODA et al. 1993), -6 (GITT et al. 1995b) and -8 (HADARI et al. 1995) belong to the tandem-repeat type. They contain two CRD domains in a single polypeptide chain. From a functional viewpoint, these three types must behave differently. Proto-type galeetins from divalent homodimers and, consequently, cross-link two glycoconjugates of a very similar nature. Tandem-repeat types are divalent, but the specificity and binding strength of the two binding sites are not necessarily the same (ODA et al. 1993; ARATA et al. 1997). They can therefore form bridges between glycoconjugates of different types. Chimera types have one sugar-binding site and other unknown regions, which might interact with biomolecules other than sugars. They can therefore serve as cross-linkers between glycoconjugates and other biomolecules - in other words, as adaptor molecules between different kinds of biomolecules or else as heterobifunctional cross-linkers. 2.1.4 Sequence similarity between galectins

Galectin-1 is the most common galeetin in mammalian species, and its respective primary structures are very similar to each other. About 90% of the amino acid residues are identical. As for galectins of non-mammalian species, it is not easy to correlate anyone of them to anyone of the mammalian galectins. For example, though two proto-type galectins (C-14 and C-16) have been found in chickens, neither is similar to anyone of the mammalian galectins (SAKAKURA et al. 1990). Both chicken galectins differ from mammalian galectin-1 to approximately the same extent, i. e., the degree of amino acid substitution between C-16 and mammalian galectin-1 is 59-63 per 135, and that between C-14 and mammalian galectin-1 is 58-60 per 135. On the other hand, the degree of substitution between the two chicken galectins is 71 per 134. This suggests that divergence of the two chicken proto-type galectins occurred a very long time ago, presumably close to the time of divergence of birds and mammals. Divergence of galectins seems to have occurred independently in birds and mammals. 2.1.5 Controversial tissue and cellular localization of galectins

It is difficult to generalize about the location of galectins in the animal body because it depends on such various factors as time, tissue, and circumstance. In vertebrates, galectins appear in a variety of tissues and cells at programmed times. Galectins are usually found not in a free state but bound noncovalently to galactoside-containing sugar chains of water-insoluble substances of tissues or cells. Lines of immunohistochemical evidence have indicated a mysterious feature about the loclization of galectins (ODA et al. 1989; AKIMOTO et al. 1992, 1993, 1995). They have been found at a variety of sites, both inside and outside cells. In the former case, both the cytoplasm and nucleus are sites of localization, though nothing is known about endogenous intracellular ligands. In the latter case, they are either attached to the cell surface or localized in the intercellular spaces

Endogenous animallectins . 11

between closely packed cells. They are also found in the extracellular matrix in the case of connective tissues. Since almost all known glycoconjugates are localized extracellularly, it is not easy to understand why proteins supposed to interact with glycoconjugates should be found intracellularly. However, from the viewpoint of protein structure, there are lines of evidence showing that galectins are designed as proteins to play roles inside cells, e. g., acetylated N-terminus (HlRABAYASHI et al. 1987; HlRABAYASHI and KAsAl 1988), cysteine residues in fully free states (HlRABAYASHI et al. 1987; HlRABAYASHI and KASAl 1988), lack of signal sequences (OHYAMA et al. 1986), and biosynthesis ocurring on free ribosomes (WILSON et al. 1989), etc. This suggests that their roles at time of first appearance must have been within cells, and that their extracellular functions were acquired in the course of development of multicellular metazoan systems. Hence, elucidation of their intracellular functions is very important. However, little research has been done from such a viewpoint except for that on the possible function of galectin-3 as one of the components of the splisosomes in the nucleus, where galectin-3 carries out splicing of precursors of mRNA (DAGHER et al. 1995). Extracellular galectins do not seem mere cellular waste. They seem to be secreted on purpose. Though the process of externalization of galectin-1 from muscle cells was studied extensively (COOPER et al. 1990), the detailed molecular mechanism without signal sequences, remains to be solved. Another problem is that galeetins are generally inactivated if certain SH groups are oxidized. If they remain within cells, such a risk is not high. However, when they are externalized, the risk of oxidative inactivation becomes high. Do galectins abundantly localized extracellularly retain sugar-binding activity? If they are already inactivated, what is the significance of their presence? A report has appeared suggesting that inactivated galectin-1 acquired TGF activity (YAMAOKA et al. 1991). Vertebrate galectins might have dual potential functions depending on the oxidative state, switching from one to the other in different situations. 2.1.6 Endogenous ligands for galectins

There are many candidates of endogenous ligands for galeetins, such as glycoproteins, glycolipids, and proteoglycans. This makes the situation rather complicated and confirmation of true ligands difficult. On the other hand, information on intracellular glycoconjugates is poor except for proteins having O-linked N-acetylglucosamine (HART et al. 1989). Since no cytoplasmic or nuclear glycoconjugate having galactoside has been identified, nothing is known about intracellular ligands for galectins. Though galeetins bind galactose in vitro, their endogenous ligands are not free monosaccharides but sugar chains attached to glycoconjugates. The key structure recognized by galectins is the disaccharide unit, N-acetyllactosamine (Gal ~ 1-4GlcNAc, LacNAc) (ABBOTT et al. 1988; LEFFLER and BARONDES 1986; LEE et al. 1990). The LacNAc structure is included in a variety of glycolipids, and serves as a backbone for various glycosig-

12 . Y. Akimoto et al.

nals such as blood group determinants and Lewis antigens. Polymerized LacNAc structures, i. e., polylactosamine chains, are also often found in such glycoconjugates as fibronectin, laminin, lysosome-associated membrane protein (LAMP), etc. Thus, the LacNAc unit seems one of the important keywords in glycobiology and galectins are in charge of deciphering this keyword. It is well known that the arrangement of the LacNAc unit changes in the course of mammalian development. For example, poly LacNAc chains on the fetal erythrocyte surface (i antigen) are linear, while those on the adult eryhtrocyte surface (I antigen) are branched. The biological meaning of such a change remains unexplained, but it undoubtedly alters the nature of the interaction between galectins and the LacNAc unit. Though the affinities of galectins for various galactoside-containing oligosaccharides slightly differ in each case, their fundamental specificity for LacNAc seems to have been preserved since the appearance of the ancestor galectin. This suggests that the keyword, LacNAc, was also created at the very beginning of life on earth, and that the deciphering or decoding of this glycocode by the galectin family has continuously been one of the most important processes in living organisms.

2.2 C-type lectins C-type lectins are a family of animallectins that require calcium for their binding to carbohydrate ligands. C-type lectins have a common type of carbohydrate recognition domain (CRD), termed C-type CRD (DRICKAMER 1988). Crystallographic studies have revealed that calcium plays critical roles in the formation of a network of coordinated hydrogen bonds that stabilize the ternary complex of protein, calcium, and carbohydrate ligands (WEIS et al. 1992). C-type CRDs are present in a variety of context of molecular organization such as transmembrane glycoprotein, soluble proteins and extracellular matrix proteins. This characteristic suggests that C-type lectins perform diverse biological functions in which carbohydrate recognition is a key feature. These include clearance of molecules from blood circulation (hepatocyte asialoglycoprotein receptors), internalization of foreign and self derived materials (alvoelar macrophage lectin), a role in humoral self defense mechanism (collectins), cell-cell adhesion (selectins), and transmembrane signaling to cells (natural killer cell receptors). Structural features and typical examples of C-type lectins are summarized in Fig. 3 and Table 3. C-type lectins can be categorized on the basis of molecular organization. The structure reflects the biological function performed by these molecules: The first category of C-type lectin comprises proteoglycan core peptides. Proteoglycans are constituents of extracellular matrix. One type of proteoglycans is a subfamily containing a C-type carbohydrate recognition domain as a part of core peptide. This category includes versican, aggrecan, neurocan, and brevican. The name «lectican» has been recently proposed for these proteoglycans (RUOSLAHTI 1996). They contain two

Endogenous animallectins . 13

1) Lectican (a proteoglycan ore) H

2) type

3)

n receptor

ollectins

H

4) electin

H2

C)() (n

=2,6

o 9)

5) Tran membr ne lectin with tandem

H2~""'"

.t'

H RD

H

(n = 8 10

Y -rich domain

o

-type Ig-like domain

RD

O dom -like in link proteinlike dom in

complement regulatory domain fibronectin type If domain

Fig. 3. Schematic representation of the structural features of C-type lectins.

tr n membrane domain

2-2) Macrophage type II lectin peritoneal macrophages MMGL Mouse M-ASGP-BP Rat peritoneal macrophages HML Human monocytes cultured with IL-2 2-3) Kupffer cell lectin liver Kupffer cell Rat receptor

Hep G2 hepatoma Human MHL-1 Mouse liver liver CHL Chicken 2-1-2) Minor receptors (HL-2/3) RHL-2/3 liver Rat H2 Human Hep G2 hepatoma MHL-2 Mouse liver

HI

SATO et al. 1992 II et al. 1990 SUZUKI et al. 1996 HOYLE and HILL 1988

Fuc, GaINAc, Gal 88 kDa

HALBERG et al. 1987 SPIESS and LODISH 1985 SANFORD and DOYLE 1990 Gal, GalNAc Gal, GalNAc Gal, GalNAc

Gal, GalNAc Gal, GalNAc Gal, GalNAc

49 kDa, 54 kDa 46 kDa 45 kDa, 51 kDa

DRICKAMER et al. 1984; HOLLAND et al. 1984 SPIESS et al. 1985 TAKEZAWA et al. 1993 MELLOW et al. 1988

DOEGE et al. 1987 DOEGE et al. 1991 ZIMMERMANN and RUOSLAHTI 1989 SHINOMURA et al. 1993 ITo et al. 1995 RAUCH et al. 1992 YAMADA et al. 1994

References

42 kDa 42 kDa 38 kDa

Gal,GaiNAc Gal, GalNAc GlcNAc

Gal, GalNAc

Haptenic sugars (ligand 2 )

46kDa 42 kDa 26 kDa

42 kDa

Molecular mass of monomers

2) Type II receptors 2-1) Hepatocyte asialoglycoprotein receptor 2-1-1) Major receptor (HL-l) RHL-l Rat liver

cDNA source!

221 kDa core 220 kDa core 265 kDa core 388 kDa core 300-550 kDa core 136 kDa core 145 kDa core

Species

1) Proteoglycan core peptides chondrosarcoma aggrecan Rat costal chondrocytes Human verSlcan Human fibroblasts Chick limb buds Mouse aortic endothelial cells brain Rat neurocan brevican Bovine brain

Molecules

Table 3. C-type lectin family.

~

~

8

0

3

~

>-

K

. j>.

-

cloned human NK cells IL-2 activated NK cells

Human Human

Human

2-5) CD69 CD69

lung

lung

Rat

Human

SP-D

liver serum liver lung fetal lung lung

Dog Rabbit Human

Rat Bovine

SP-A

3) Collectins MBP conglutinin

43 kDa

29-36 kDa 29-36 kDa 32kDa 35 kDa 43 kDa

29kDa 48kDa

20kDa

2-7) mast cell receptor MAFA Rat

mast cell line (RBL-2H3)

25kDa

27-33 kDa

2-6) low affinity immunoglobulin E receptor CD23 Human B lymphoblast

PMA-activated lymphocytes

38-40 kDa

IL-2 activated NK cells

Mouse

NKR-P1C (NKl.l) NKG2 CD94 43 kDa

30kDa 44 kDa 45-50 kDa

2-4) Natural killer (NK) cell receptors NKR-P1A Rat IL-2-activated NK cells Ly49A Mouse EL-4 T lymphoma MBL-2 T lymphoma

GUTHMANN et al. 1995

KIKUTANI et al. 1986, IKUTA et al. 1987

LOPEZ-CABRERA et al. 1993

HOUCHINS et al. 1991 CHANG et al. 1995

RYAN et al. 1992

GIORDA et al. 1990 YOKOYAMA et al. 1989 CHAN and TAKEI 1989

DRICKAMER et al. 1986 Man, GlcNAc Man, GlcNAc, iC3b LEE et al. 1991 Lu et al. 1993 BENSON et al. 1985 BOGGARAM et al. 1988 WHITE et al. 1985 FLOROS et al. 1986 Man, Fuc, Glc, SHIMIZU et al. 1992 Maltose Man, Fuc, Glc, Lu et al. 1992 Maltose

(IgE)

sulfated polysaccharides, (Class I MHC)

t .n

-

~.

<">

ib

S" a.

b

~

'"o::l

CIQ

o

trl

::l 0-

Human

P-selectin

IL-1-stimulated endothelial cells umbilical vein endothelial cells

5) Transmembrane protein with tandem CRDs placenta macrophage Human cultured monocytes mannose receptor thymus, dendritic cells DEC-205 Mouse PLA2-I Bovine MDBK (kidney cell line) Rabbit skeletal muscle cells

Human

lymphocytes

Human

E-selectin

spleen

Mouse

4) Selectins L-selectin

cDNA source!

Species

Molecules

Table 3. Continued.

TEDDER et al. 1989; CAMERINI et al. 1989 BEVILAcQua et al. 1989

LASKY et al. 1989; SIEGELMAN et al. 1989

References

Man, GlcNAc, Fuc TAYLOR et al. 1990 EZEKOWITZ et al. 1990 JIANG et al. 1995 (phospholipase A 2) ISHIZAKI et al. 1994 (phospholipase A 2) LAMBEAU et al. 1994

205 kDa 190 kDa 180 kDa

sialyl-LeX, PSGL-1 JOHNSTON et al. 1989

sialyl-LeX, ESL-1

sulfated-sialyl-LeX, GlyCAM-1, CD34, Sgp200, MAdCAM-1

Haptenic sugars (ligand 2)

180 kDa

140 kDa

115 kDa

90kDa

90kDa

Molecular mass of monomers

...-

~

~

8

o

S

a-

:>

K

'"

2

1

32kDa 123 kDa 18 kDa 15 kDa 13 kDa 22kDa

venom

whole body

coelomic fluid coelomic fluid

100 kDa

umbilical vein endothelial cells fat body of larvae amebocytes

Gal

Gal

Gal

GIGA et al. 1987 MURAMOTO and KAMIYA 1990

SUZUKI et al. 1990

TAKEYA et al. 1992

TAKAHASHI et al. 1985 MUTA et al. 1991

WEN et al. 1987

Underlined material indicates a source of purified protein not of eDNA Ligands in the parenthesis are recognized by a carbohydrate independent manner. Haptenic sugars: Fuc, fucose; Gal, galactose; GaINAc, N-acetylgalactosamine; Glc, glucose; GlcNAc, N-acetylglucosamine; Man, mannose.

6) Miscellaneous thromboHuman modulin Flesh fly SPL Limulus Horseshoe factor C Crab RVV-X Snake (subunit LCt) Polyandroca Sea squirt rpa lectin Echinoidin Sea urchin BRA-2 Acorn barnacle

0>

-"

",.,S·

e..

§.

'"o::> :;; .,

(1Q

o

tTl ::>

0-

18 . Y. Akimow et al.

epidermal growth factor (EGF)-like domains, one C-type lectin domain, and one complement regulatory domain at the carboxyl terminus. The second category comprises type II receptors. These molecules are called type II receptors because of their molecular features (BEZOUSKA et al. 1991). They are transmembrane glycoproteins with a single transmembrane domain and with a C-type CRD at extracellular C-terminus (type II transmembrane orientation). This group includes asialoglycoprotein receptor of hepatocytes (HOLLAND et al. 1984; HALBERG et al. 1987), macrophage galactoselN-acetylgalactosamine specific lectin from mouse (SATO et al. 1992), rat (II et al. 1990) and human (SUZUKI et al. 1996), Kupffer cell lectin (HOYLE and HILL 1988, 1991), natural killer (NK) cell receptors (YOKOYAMA and SEAMAN 1993; GIORDA et al. 1990; CHANG et al. 1995), CD69 (LOPEZ-CABRERA et al. 1993), low affinity IgE Fe receptor (CD23) (KIKUTANI et al. 1986; IKUTA et al. 1987), and a mast cell receptor (GUTHMANN et al. 1995). Most of the C-type lectins belonging to this group contain consensus cytoplasmic tyrosine motifs related to rapid endocytosis OADOT et al. 1992). A major function of asialoglycoprotein receptors on hepatocytes is currently believed to be clearance of desialylated serum glycoproteins from blood circulation by internalization (ASHWELL and HARFORD 1982; ISHIBASHI et al. 1994). In contrast, some such as NK cell receptors appear to be involved in transmembrane signaling events (GIORDA et al. 1990; KARLHOFER et al. 1992). In the case of natural killer cell receptors, cytoplasmic tyrosine motifs are reported to be responsible for transmembrane signal transduction (OLCESE et al. 1996). The third group comprises soluble molecules containing a C-type CRD at the Cterminus with a N-terminal collagen-like domain. These molecules are called collectins (MALHOTRA et al. 1994). This group includes mannose binding protein (MBP), conglutinin, lung surfactants SP-A and SP-D. MBP is known to activate the classical pathway of complement, and conglutinin is known to bind to a complement fragment iC3b. Lung surfactants are believed to be involved in natural defense mechanisms through binding to certain infectious organisms (MALHOTRA et al. 1994). The fourth group comprises selectins. Selectins contain N-terminal single C-type CRD followed by an EGF-like domain and multiple complement regulatory domains as extracellular domains. They have single transmembrane domain and C-terminal short cytoplasmic tail (type I transmembrane orientation) (LASKY et al. 1989; SIEGELMAN et al. 1989; BEVILACQUA et al. 1989; JOHNSTON et al. 1989). Selectins are adhesion molecules generally involved in leukocyte-endothelial cell adhesive interaction through recognition of corresponding carbohydrate ligands. Another important class of adhesion molecules on leukocytes are integrins. Unlike selectins, integrins bind to respective protein ligands through protein-protein interaction. Typical examples in which selectins are involved include leukocyte extravasation during inflammation and lymphocyte homing to secondary lymphoid organs. L-selectin is a molecule on leukocytes while E-selectin and P-selectin are present on activated endothelial cells. P-selectin is also present on activated platelets. Interaction between selectins and their carbohydrate ligands is invol-

Endogenous animallectins . 19

ved as an initial step of leukocyte extravasation (called as leukocyte rolling or tethering) (LAWRENCE and SPRINGER 1991; ROSEN et al. 1992; LASKY 1992; SPRINGER 1994). Leukocytes in blood flow slow down as a result of this adhesive interaction at the luminal surface of venules where appropriate ligands (for L-selectin) or appropriate selectins (Eselectin or P-selectin) are expressed. Subsequently, signaling events to leukocyte mediated by soluble mediators such as chemokines induce the activation of adhesive activity of integrins on leukocytes (SPRINGER 1994). Firm adhesion mediated by leukocyte integrins and molecules belonging to the immunoglobulin superfamily on endothelial cells takes place. These processes are considered a prerequisite for diapedesis (leukocyte extravasation). The fifth group comprises type I transmembrane glycoprotein with multiple C-type CRD. A mannose specific lectin of 175-180 kDa, originally described as rat alveolar macrophage lectin (HALTIWANGER and HILL 1986a, b), has been cloned from the cDNA library of human placenta (TAYLOR et al. 1990), cultured human monocytes (EZEKOWITZ et al. 1990), and mouse peritoneal macrophages (HARRIS et al. 1992). This transmembrane lectin has an N-terminal cystein-rich domain followed by a fibronectin type II repeat and 8 tandem C-type CRDs as extracellular domains. It has been reported to play a role in endocytosis of mannose-rich glycoconjugate as well as in phagocytosis of yeasts (EZEKOWITZ et al. 1990). Recently, several molecules with similar domain organization have been reported. These include phospholipase A 2 receptor (ISHIZAKI et al. 1994; LAMBEAU et al. 1994), DEC-205 molecule (a mouse dendritic cell antigen defined by mAb NLDC-145) GIANG et al. 1995), and mRNA encoding a molecule with unknown function (Wu et al. 1996). These molecules have 8 tandem CRDs with the exception of DEC-205, which contains 10 tandem CRDs. It has been proposed that C-type CRD could be categorized into two groups in terms of amino acid motifs which are critical for interaction with carbohydrate ligands and calcium ions (DRICKAMER 1992; WEIS et al. 1992). One of the CRD motifs is the QPD (Gln-Pro-Asp) motif that relates galactose specificity; the other is the EPN (Glu-ProAsn) motif that relates mannose specificity. Aggrecan, versican, hepatic asialoglycoprotein receptors, macrophage galactose/N-acetylgalactosamine specific lectin, Kupffer cell lectin belong to the former group. In contrast, MBP, conglutinin, avian hepatic lectin, SP-D, 180 kDa-macrophage lectin, selectins belong to the latter group.

2.3 I-type lectin

There are several type I transmembrane glycoproteins with immunoglobulin-like domains that exhibit carbohydrate binding activity. Since these molecules contain extracellular immunoglobulin domains, the name «I-type lectin» has been proposed (POWELL and VARKI 1995). This group includes sialoadhesin on macrophages, CD22 on B lymphocytes, myelin associated glycoprotein (MAG) on myelinating Schwann cells and

20 . Y. Akimoto et al.

oligodendrocytes, and CD33 on myelomonocytic cells (CROCKER et al. 1994; POWELL and VARKI 1995). In addition, domain structure and amino acid sequence homology revealed that another cell surface molecule, Schwann cell myelin protein (SMP) on myelinated and non-myelinated Schwann cells and oligodendrocytes, belongs to this group (CROCKER et al. 1994). In the case of sialoadhesin, study of macrophage adhesion molecules that recognize carbohydrate ligands containing sialic acid revealed its molecular structure as composed of 17 immunoglobulin-like domains (CROCKER et al. 1994). In contrast, molecules such as CD22, CD33 and MAG had been characterized as immunoglobulin superfamily proteins before their carbohydrate binding activity was established. These different approaches merged into a common notion of an I-type lectin. Another collective feature of this group is its binding specificity to carbohydrate determinants containing terminal sialic acids. Although there are differences in the fine specificity of lectins, involvement of sialic acid has been demonstrated for sialoadhesin (CROCKER et al. 1991, 1994), for CD22 (ENGEL et al. 1993; POWELL and VARKI 1994), for MAG (YANG et al. 1996) and for CD33 (FREEMAN et al. 1995).

3 Localization of galectins in chick embryonic and adult skin In vertebrates, galectins have been isolated from several tissues of calf and chick (DE WAARD et al. 1976; DEN and MALINZAK 1977; NOWAK et al. 1977; KOBILER et al. 1978; BRILES et al. 1979; MONTELIONE et al. 1981). Chick embryos have at least two different kinds of endogenous galectins, the 16- and 14-kDa lectins (aDA and KAsAl 1983), which are identical or very similar to chicken lactose-lectin I and II (CLL-I and II), respectively (BEYER et al. 1980; BARONDES and HAYWOOD-REID 1981). In the chick embryo, 16-kDa galectin has been detected in the liver, pancreas, and skeletal muscle (DEN and MALINZAK 1977); whereas the 14-kDa type has been found in many tissues including the small intestines, thymus, and skin (aDA and KAsAl 1983; CERRA et al. 1984). The latter, in particular, is very abundant in the tarsometatarsal and dorsal skin of chick embryos (aDA and KASAl 1983). 14-kDa galectin increases in amount in vivo during the course of differentation (aDA and KASAl 1983). Both cDNA and genomic DNA for 14-kDa galectin were cloned, and the nucleotide sequence as well as the complete amino acid sequence was determined (OHYAMA et al. 1986; OHYAMA and KASAl 1988; HlRABAYASHI et al. 1987b). These studies revealed that the 14-kDa galectin contained no signal sequence but did have some regions homologous to carbohydrate binding protein 35 (CPB 35). Why does chick have two different isotypes of galectins? Where and when are these two galectins expressed? Biochemical and histochemical studies on their distribution and fate during differentiation should give a clue to their roles. Chick embryonic skin is a good experimental model system for such objectives. In vivo, the chick embryonic tarsometatarsal skin remains undifferentiated until 13 days of incubation; thereafter the keratinization of the epidermis starts, and is completed on day 17 (Fig. 4). Organ cul-

Endogenous animallectins . 21

Fig. 4. Electron micrographs of 13- (a) and 17- (b) day-old chick embryonic epidermis from the tarsometatarsal region. Osmium-fixed and Epon-embedded section. D: dermis, P: peridermal cells. a: Epidermis consists of the superficial (S), intermediate (I), and basal (B) cell layers. Keratinization has not yet occurred. b: Several layers of the superficial cells are keratinized. Peridermal cells (P) are above the keratinized layer (K). - a: x 1,900, b: x 1,800.

tures from explants of chick embryonic skin not only enable normal differentiation (keratinization of the epidermis) in vitro (TAKATA et al. 1981; TAKATA and HIRANO 1983), but also allow deviant differentiation, such as mucous metaplasia, in which the epidermis is converted to a mucus-secreting tissue by pretreatment with retional (HIRANO et al. 1985; OBINATA et al. 1991a, b). Taking advantage of this experimental model, we were able to obtain some information on the distribution and fate of the 14-kDa (Section 3.1) and 16-kDa (Section 3.2) galectins plus their gene expression in the epidermis with special reference to the keratinization process (AKIMOTO et al. 1992, 1993). On the other hand, the dermis has an important role in the morphogenesis and cytodifferentiation of the epidermis (DHOUAILLY et al. 1978; OBINATA et al. 1987). We therefore also examined the location and fate of the two galectins in the dermis of the skin (AKIMOTO et al. 1995a) (Section 3.3).

22 . Y. Akimoto et al.

Table 4. Changes in expression of 14-kDa and 16-kDa galectins in the epidermis of chick embryonic skin. in vitro

tn VlVO

13 days

17 days

HC':'

± ±

+ +

+ +

± ±

+

+

+

+

Retinol

14-kDa galectin immunostaining in situ hybridization 16-kDa galectin immunostaining in situ hybridization Keratinization Mucous metaplasia

+

+

+

+

+

':. HC: hydrocortisone.

3.1 Changes in expression of 14-kDa galectin during epidermal differentiation Changes in the expression pattern of the gene for 14-kDa galectin of the chick embryo were examined immunohistochemically during epidermal differentiation in vivo and in vitro with special reference to the detailed localization of 14-kDa galectin (HIRANO et al. 1988; aDA et al. 1989; AKIMOTO et al. 1992), as summarized in Tables 4 and 5, and Fig. 5. Gene expression was visualized by the HRP-staining method following in situ hybridization, with sulfonated cDNA being employed as a probe. 14-kDa galectin gene expression (mRNA) was detected mainly in the intermediate layer of the epidermis; faint

Fig. 5. The schema of the changes in expression of 14-kDa and 16-kDa galectins in the chick embryonic skin. The localization of the galectins is described by dots. 14- and 16-kDa galectins exist in the tarsometatarsal skin of chick embryos. Their expression and localization are closely correlated with keratinization and retinol-induced mucous metaplasia of the skin. The stainings of 14- and 16-kDa galectins are faint in the epidermis of 13-day-old embryos, gradually increase in intensity during epidermal keratinization, and become intensely positive in the intermediate layer of the differentiating epidermis (17-day-old embryonic skin in ovo or skin cultured in the presence of hydrocortisone in vitro). The expression of the 14-kDa galectin is suppressed by retinol, while the 16-kDa galectin expression increases in the epidermis by retinol, especially in the superficial cells of the epidermis. On the other hand, in the dermis, 14kDa galectin expression increases after 13 days, while 16-kDa galectin expression decreases after 13 days. 14- and 16-kDa galectins are abundant in the extracellular matrix of dermis, especially in the basement membrane, and dermal fibroblasts.

Endogenous animal leetins . 23

14

a galectin

@.'~.'~ '
@

•

l

00

~

ee>

1 da ''';'01

6 ) '.Gi;)

C§>

17 da

ni one

CQ)

ucou metapla ia

16 kDa galectin

0 0 ,

G::>

~

ee>'

17 da

~s~:r!j;~··

ucou metapla ia

<).

•

Gi;)

.~

0 0

'~'."@'.

t
24 . Y. Akimoto et al.

in 13-day-old embryos, it gradually increased in intensity during epidermal differentiation to become intensely positive in the 17-day-old embryo. The expression of the gene in the epidermis of skin explants was suppressed by retinol, which induces mucous metaplasia of the epidermis in vitro (Fig. 6). Anti-14-kDa galectin reaction was mainly positive in the intermediate layer of the differentiating epidermis, coinciding chronologically with expression of the gene at the

c

o

o d

..

Endogenous animallectins . 25

light microscopic level (Fig. 7). Immuno-electron microscopy revealed that the positive reaction was primarily localized in desmosomes, in tonofilament bundles anchored to the desmosomes, along the outer surface of the plasma membrane, and in the intercellular space (Fig. 8). Essentially the same staining pattern was observed in differentiating epidermis in vitro. The positive reaction was markedly reduced in the epidermis, where differentiation had been suppressed in vitro by the addition of retinol. In view of the localization and timing of 14-kDa galectin expression in the course of skin differentiation, this galectin seems to playa role in epidermal differentiation by binding to complementary sugars, probably polylactosaminoglycans (ODA and KASAl 1984), located on epidermal cell surfaces.

Table S. Changes in expression of 14-kDa and 16-kDa galectin mRNA in the epidermis and dermis of chick skin during development. Day 8

10

13

17

20

Adult

In the epidermis 14-kDa galectin 16-kDa galectin

++

++

± ±

+ +

++ ++

++ ++

In the dermis 14-kDa galectin 16-kDa galectin

++ +

++

++

+ ++

++

++

+

+

+

Keratinization

Fig. 6. In situ localization of 14-kDa (a, b) and 16-kDa (c, d) galectin mRNA in sections of 13day-old chick embryonic skin cultured in vitro. Arrowheads indicate the basal surface of the epidermis. D: dermis, E: epidermis. a: Chick embryonic skin cultured in the presence of hydrocortisone; i. e. keratinization has occurred in the epidermis. The hybridization signals are localized mainly in the intermediate layer. The basal cell layer is slightly stained. b: Chick embryonic skin cultured in the presence of retinol. Differentiation has been suppressed by retinol. The epidermis is only slightly stained, while the dermis is moderately stained. c: Chick embryonic skin cultured in the presence of hydrocortisone. The hybridization signals are localized mainly in the intermediate layer. The basal region of the basal cell is also stained. d: Chick embryonic skin cultured in the presence of retinol. Differentiation has been suppressed as in (b). All layers of the epidermis are intensely stained. - a and c: X 1,200, band d: X 1,000.

26 . Y. Akimoto et al.

3.2 Changes in expression of 16-kDa galectin during epidermal differentiation In early studies, 16-kDa chicken galectin was shown to occur predominantly in embryonic muscle and adult liver, whereas 14-kDa chicken galectin was found to be abundant in embryonic skin and adult intestine. 16-kDa galectin was also reported to contribute to cartilage differentiation and dermal condensation in the feather region of chick embryonic dorsal skin (KITAMURA 1981; MATSUTANI and YAMAGATA 1982). However, our biochemical studies revealed that, like 14-kDa galectin, 16-kDa galectin also exists in embryonic skin in significant amount. Thus, immunohistochemical localization of both isolectins is meaningful from a comparative viewpoint. Immunostaining was weak in undifferentiated epidermis from 13-day-old embryos, whereas it became intense in keratinized epidermis, particularly in the intermediate cells from 17-day-old embryos (AKIMOTO et al. 1993) (Figs. 5 and 7, Tables 4 and S). Essentially the same staining pattern was observed in cultured skin, in which keratinization had been induced in vitro by the addition of hydrocortisone (20 nM). When skin was cultured in the presence of retinol (29Itg/ml), mucous metaplasia of the epidermis was induced (Fig. Sa and b) while, at the same time, changes in localization of 16-kDa galectin occurred. That is, galectin expression was increased in the epidermis, especially in the superficial cells of the epidermis in which mucous metaplasia had been induced (Fig. 9). Immunoelectron microscopic observation revealed that 16-kDa galectin was located along the plasma membrane, in the intercellular space, and in the desmosomes of the keratinized epidermis, and also detected in the mucous granules and on the microvilli of the superficial cells in the mucous metaplastic epidermis (Fig. 10). 16-kDa galectin as well as 14-kDa galectin may be involved in epidermal differentiation. Retinol induces the secretion of 16-kDa galectin from the epidermis, 16-kDa galectin could cross-link glycoproteins in the glycocalyces of the microvilli. A complex glycoconjugate mass may thus coat the epidermal surface and so protect the epidermis from irritation and/or infection. There have been several reports that the galectins were secreted even in the absence of a typical signal sequence (BEYER et al. 1979; BARONDES and HAYWOOD-REID 1981;

Fig. 7. Immunohistochemical localization by light microscopy of 14-kDa (a, c) and 16-kDa (b, d) lectins in the skin from 8-day-old (a, b), and 17-day-old (c, d) chick embryos. Stained with rabbit anti-lectin antiserum and visualized by immunofluorescence with rhodamine-conjugated goat anti-rabbit IgG (red). DNA was stained with DAPI (blue). Positive staining of the 14-kDa lectin is evident at a very low level in 8-day-old chick embryonic dermis (a). In 17-day-old chick embryonic dermis, intense staining was observed, and some nuclei of fibroblasts showed white fluorescence due to the relatively high concentration of the dyes (c). Intense staining of 16-kDa galectin was observed in 8-day-old dermis (b). Positive staining in 17-day-old dermis was weaker than in earlier specimens (d). Arrowheads indicate basal surface of epidermis. E: epidermis, D: dermis. - a, b: X 650, c: X 980, d: X 990.

Endogenous arumallectins . 27

28 . Y. Akimoto et al.

:,

.t

\.

)- '.

.. \

~

\

Fig. 8. Immunohistochemical localization by electron microscopy of the 14-kDa galectin in the epidermis from the tarsometatarsal region of a 17-day-old chick embryo. Formaldehyde-fixed and Lowicryl K4M-embedded sections were reacted with rabbit anti-lectin serum and then reacted with colloidal gold-conjugated goat anti-rabbit IgG. In the intermediate layer, desmosomes (arrows), both tonofilament bundles (arrowheads) and the cytoplasm in the immediate vicinity of the bundles, and the intercellular space are intensely labeled. K, alpha-keratin. - X 57,000.

et al. 1984; BOLS et al. 1986; WASANO and YAMAMOTO 1989; COOPER and 1990; HARRISON and WILSON 1992). Though the secretory mechanism of 16-kDa chicken galectin remains unknown, a novel mechanism has been demonstrated for mouse galectin-1: the mouse galectin becomes concentrated in evaginations of the plasma membrane of cultured muscle cells, which then become pinched off to form a labile lectin-rich extracellular matrix (COOPER and BARONDES 1990; HARRISON and WILSON 1992). CERRA

BARONDES

Endogenous animallectins . 29

Fig. 9. Light micrographs of cultured 13-day-old chick embryonic skin. Skin explants were incubated for 1 day in the medium BG]b containing 5% delipidized FCS and hydrocortisone (20 nM) with (b, d) or without (a, c) retinol (20 mM) and then for 6 days in BG]b in the presence of Bt2cAMP (2 mM). (a, b) Sections were stained with PAS and slightly counterstained with hematoxylin. a: The PAS reaction is negative in all layers of the keratinized epidermis. b: PAS-positive metaplastic cells (arrow) are seen in the superficial and in the intermediate layers of the epidermis, which is not so thick as in (a). c, d: Immunohistochemical localization by light microscopy of the 16-kDa galectin. Stained with rabbit anti-16-kDa lectin antiserum and with HRP-conjugated goat anti-rabbit IgG. c: In hydrocortisone-treated chick embryonic epidermis, intense staining (arrows) is observed mainly in the intermediate and keratinized layers. d: Intense staining is found in the superficial cells of the epidermis, and mucous granules (arrows) are also stained, in the cultures treated with retinol. Arrowheads indicate basal surface of epidermis. - X 990.

30 . Y. Akimoto et al.

Fig. 10. Immuno-electron microscopical localization of the 16-kDa galectin in the epidermis from the tarsometatarsal region of a chick embryonic epidermis cultured in the presence of hydrocortisone and retinol for 1 day and then Bt2cAMP for 8 days. Formaldehyde-fixed and frozen section was reacted with rabbit anti-lectin antiserum and then reacted with HRP-conjugated goat antirabbit IgG F(ab'h. Desmosomes (arrowheads) are stained very weakly in the epidermis. Positive staining is detected in the mucous granules (MG) and on the microvilli (arrows) of the superficial cells of the epidermis in which mucous metaplasia had been induced. - X 18,000.

3.3 Changes in expression of two (14- and 16-kDa) galectins in the dermis of chick embryonic skin during development These 14- and 16-kDa galectins were immunohistochemically located at different stages of development (AKIMOTO et al. 1995a) (Table 5, and Fig. 5). Light microscopic observation showed that whereas positive staining for 14-kDa galectin was negative at days 8 and 10, it became intense after day 13 (Fig. 7). In contrast, staining for 16-kDa galectin was intense at days 8, 10, and 13, but became weak after day 17 when keratiniza-

Endogenous animallectins . 31

tion of the epidermis was completed (Fig. 7). Such a different feature in the expression of closely related isolectins reminds us of two other developmentally regulated isolectins, discoindins-l and 2 from cellular slime mold (BARONDES and HAYWOOD-REID 1979). Immunoelectron microscopic observation revealed that both 14- and 16-kDa galectins were located on the basement membrane, in the extracellular matrix, and in both the cytoplasm and the nucleus of dermal fibroblasts (Fig. 11). Distribution of the two isolectins was also examined in the dermis of cultured skin explants in vitro. The results were almost the same as those obtained in ovo when the skin explant was keratinized in the presence of hydrocortisone. However, in the skin explant where keratinization was prevented and mucous metaplasia was induced by addition of retinol, the distribution of 14-kDa galectin in the dermis was not significantly affected, whereas this was the case in the epidermis. These results indicate that (1) expression of the two isolectins is differently regulated in both dermis and epidermis, (2) 16-kDa galectin is involved in the early stage of formation of the dermis and the base-

Fig. II. Immunohistochemical localization by electron microscopy of the 14-kDa galectin in the epidermis from the tarsometatarsal region of a 17-day-old chick embryo. Formaldehyde-fixed and Lowicryl K4M-embedded section was reacted with rabbit anti-lectin serum and then reacted with colloidal gold-conjugated goat anti-rabbit IgG. At the boundary between the epidermis and the dermis, the basement membrane and immediate environs (arrowheads) are labeled. Gold particles are also seen in the extracellular matrix of the dermis. Be: basal cell, D: dermis. - X 60,000.

32 . Y. Akimoto et al.

ment membrane and is replaced by 14-kDa galeetin as keratinization of the epidermis occurs, and (3) the expression of the two isolectins in the dermis is not significantly affected by the induction of mucous metaplasia in contrast to the drastic change in 14-kDa galeetin in the epidermis.

4 Localization of galectins in other tissues of adult chicken and other vertebrates 4.1 Nervous system

Chick 14-kDa galectin was localized in the capillaries of the adult chicken brain (Fig. 12a). On the other hand, in the rat brain the expression of galectin-1 and complementary glycoconjugates can be correlated with significant events in brain development (LI et al. 1992). Galectins have been detected in embryonic brain and spinal cord (KOBILER and BARONDES 1977; KOBILER et al. 1978; EISENBARTH et al. 1978). Galectins represent ligands that might mediate interactions between lactoseries structures on dorsal root ganglion neurons and spinal cord cells. Rat 14-kDa galectin-l and 29-kDa galectin-3 have been localized in a subset of dorsal root ganglion neurons and in their central terminals in the superficial dorsal horn (REGAN et al. 1986). Both galectins are synthesized in the population of dorsal root ganglion neurons that expresses cell-surface lactoseries glycoconjugates (REGAN et al. 1986; HYNES et al. 1988). Dorsal horn neurons that express lactoseries structures do not appear to synthesize these galectins. Galectin-l is first detectable in dorsal root ganglion neurons and sensory axons in the dorsal root entry zone at embryonic day (E)13-14, whereas galectin-3 cannot be detected before E15. From E16 both galectins are present in sensory afferent fibers as they enter the spinal cord. At present, a role for these molecules in sensory neuron development has not been established. However, several possibilities may be envisaged. For example, the transient expression of lactoseries structures on embryonic dorsal horn neurons, combined with the onset of galeetin synthesis, could provide a signaling mechanism that both initiates the ingrowth and restricts the dorsoventral projection of sensory axons that express lactoseries oligosaccharides. Fig. 12. Immunofluorescence localization of 14-kDa galectin in adult chicken tissues. Semithin frozen sections stained with antiserum against 14-kDa galeetin and rhodamine-labeled goat-anti rabbit IgG. Nuclei were simultaneously stained with DAPI. a: Cerebrum. 14-kDa galectin is seen in the blood vessels. (arrows). b: Pectoral muscle. 14-kDa galectin is seen in the endomysium (small arrows) and blood vessel (large arrow). c: Stomach. The baso-lateral plasma membrane of epithelial cells is stained. d: Small intestine. 14-kDa galectin is abundant in the lamina propria (L). Arrows indicate the goblet cells. E: epithelium. - x 500.

Endogenous animal leetins . 33

34 . Y. Akimoto et al.

Galectin-1 (14-kDa) is expressed by non-neuronal cells in the rat olfactory nerve (MAHANTHAPPA 1994). The galeetin binds and co-localizes with two ligands (a ~­ lactosamine-containing glycolipid and laminin) in the rat olfactory system. The galectin promotes primary olfactory neuron adhesion to two laminin family members as well as intercellular adhesion. Thus, the galectin in vivo could promote olfactory axon fasciculation by crosslinking the neurons to the extracellular matrix.

4.2 Striated muscle Chick 14-kDa galeetin was localized in the connective tissues surrounding striated muscle cells and in the capillaries (Fig. 12b). In mouse myoblast cultures, galectin-1 is abundant on the cell surface, intracellularly, and concentrated at ruffled edges of migrating cells (HARRISON and WILSON 1992). After fusion to form multinucleate myotubes, intracellular galectin is less abundant and concentrated at the periphery of myotubes, from where galeetin appears to be released in vesicles packed with galectin that bud off from the myotube surface (HARRISON and WILSON 1992).

4.3 Stomach Chick 14-kDa galectin was localized in the lamina propria and on the baso-lateral plasma membrane of the epithelial cells in the fundic gland (Fig. 12c). Also, thin discontinuous fascicles or small aggregates of immunoreactive cells were detectable in the lamina propria of the stomach of adult rats (WASANO et al. 1990).

4.4 Intestine 14-kDa galectin was localized in the lamina propria of adult chicken small intestine (Fig. 12d), but not found in the goblet cells. CLL-II was localized in the secretory granules of the goblet cell of the intestinal mucosa and in the mucin lining the intestinal mucosa of adult chicken intestinal villi (BARONDES and HEIWOOD-REID 1981; CERRA et al. 1984).

4.5 Lung In bovine lung, galectin-1 was found in squamous alveolar epithelial (type I) cells and is concentrated extracellularly in elastic fibers of pulmonary parenchyma (CERRA et al. 1984). Rat lung 29-kDa galectin-3 is secreted by bronchiolar Clara cells into the airways

Endogenous animallectins . 35

(WASANO and YAMAMOTO 1989). The extracellular localization of the galeetin suggests that it functions by interaction with extracellular glycoconjugates.

5 Localization of galectin in human tissues 5.1 Skin

It was reported that the keratinocytes of human skin produce a galectin-3 (33-kDa IgE-binding protein) that binds to Langerhans cells, where it modulates their capacity to bind glycoform IgE (WOLLENBERG et al. 1993). In parallel, galectin-1 has been found in human tissues. Indeed, we isolated a galectin-1 from human placenta (HlRABAYASHI and KASAl 1984), and showed, based on immunological (HlRABAYASHI et al. 1987a) and sequence analysis (HlRABAYASHI et al. 1987b, 1989; HlRABAYASHI and KASAl 1988), that human galectin-1 is quite similar to chick 14- and 16-kDa galectins. From analysis of both complete amino acid and nucleotide sequences of a full-length eDNA for the human galectin-1 (HlRABAYASHI and KASAl 1988; HlRABAYASHI et al. 1989), this galectin was found to be composed of 134 amino acid residues, 56% of which are identical with chick 14-kDa galectin. The human galectin-1 has some regions homologous to the Cterminal half of CBP35 and 33-kDa galectin (ALBRANDT et al. 1987, LAING et al. 1989). Moreover, the human galectin-1 shows about 90% similarity with rat galectin. No signal sequence exists in the initiator region of the eDNA. Light microscopic localization of a galectin-1 has been reported in various normal and malignant tissues including cutaneous tissue (ALLEN et al. 1991; SKRINCOSKY et al. 1993). According to ALLEN et al. (1991), in cutaneous tissue the extracellular matrix contains abundant galectins. Immunostaining of galectin-1 was observed in the cell membrane of cells in the basal and spinous layers of the epidermis (AKIMOTO et al. 1995b) (Fig. 13a). In the dermis, immunostaining for galectin-1 was positive in the extracellular matrix and in fibroblasts (Fig. 13a). At the electron microscopic level, the galectin was located primarily along the plasma membrane of keratinocytes and in both the cytoplasm and nucleus of Langerhans cells in the epidermis; whereas in the dermis it was detected in the extracellular matrix and in both the nucleus and cytoplasm of fibroblasts (Fig. 14). The gene expression of the galectin-1 was visualized by the HRP-staining method following in situ hybridization. Expression was detected in the cytoplasm of cells in the basal and spinous layers of the epidermis; whereas in the dermis it was detected in the cytoplasm of fibroblasts (AKIMOTO et al. 1995b). These results suggest that galectins are important for cell-cell contact and/or adhesion in the epidermis and for cell-extracellular matrix interaction in the dermis. Our light microscopic results are consistent with those reported by ALLEN et al. (1991), who reported that galectin-1 exists in a small amount in the epidermis but abundantly in the dermis. The location of galectin-1 is different from that of galectin-

36 . Y. Akimoto et al.

Endogenous animallectins . 37

3 (IgE-binding protein, WOLLENBERG et al. 1993), for the former is primarily located on the cell surface, whereas the latter galectin exists mainly in the cytoplasm of keratinocytes. Changes in glycoconjugate structures have important roles in development and differentiation (KAWAI et al. 1979; BARNES 1988). These changes are observed in both developing chick embryonic epidermis (TAKATA and HIRANO 1983) and normal human skin (OOKUSA et al. 1983). In the epidermis of the 13-day-old chick embryo, glycoconjugates containing B-galactose, N-acetylglucosamine and N-acetylgalactosamine residues are not present (TAKATA and HIRANO 1983; AKIMOTO et al. 1995c). As the epidermis develops toward keratinization, however, these glycoconjugates accumulate on the cell surface of the intermediate cells. Such changes chronologically correspond to expression of galectins during epidermal differentiation (AKIMOTO et al. 1992, 1993). This strongly suggests that galectins recognize these glycoconjugates on the cell surface and are involved in cell-to-cell interaction. The site of galectin-1 in normal human skin is consistent with the binding pattern of RCA, which binds to glycoconjugates containing Bgalactose and N -acetyllactosamine (OOKUSA et al. 1983). Our results indicate that glycoconjugates containing galactose and lactosamine (B-galactose 1-4 N-acetylglucosamine), for which the galectin-1 has affinity, accumulate on the cell surface as the keratinocyte migrates and differentiates from the basal layer to the upper layers of the epidermis and that the 14-kDa galectin recognizes these glycoconjugates. Galectin-1 is abundantly present in the extracellular matrix of the dermis. Fibronectin, a major component of the extracellular matrix, has polylactosaminoglycan in its structure (ZHU et al. 1984), the latter being a possible substance recognized and bound by the galectins (ODA and KASAl 1984; MERKLE and CUMMINGS 1988; SOLOMON et al. 1991; ZHOU and CUMMINGS 1993; OZEKI et al. 1995). Galectins are also known to recognize laminin (Woo et al. 1990; COOPER et al. 1991; CASTRONOVO et al. 1992; SATO and HUGHES 1992). There are also various glycoproteins along the plasma membrane, in the intercellular spaces of the epidermis, and in the extracellular matrix of the dermis (HOLTON et al. 1990; KApPRELL et al. 1985; KOCH et al. 1990; KREIS and VALE

Fig. 13. Immunohistochemical localization by light microscopy of galectin-1 in human integumentary system. Sections were stained with antibody against galectin-1 and HRP-labeled goat anti-rabbit IgG. a: Thick skin of finger tip. The plasma membrane of the epithelial cells in the prickle cell layer (P) and basal layer (B) is stained. The fibroblasts and extracellular matrix of the dermis (D) are stained. H: horny layer, C: clear layer, G: granular layer. Arrowheads indicate basal surface of epidermis. b: Hair follicle (HF). The basal cells (arrows) in the external root sheath of hair follicle are stained preferentially. c: Sebaceous glands (SG). The basal cells (arrows) of the sebaceous glands are stained. d: Eccrine sweat glands (E). Secretory cells and myoepithelial cells of eccrine sweat glands are stained (arrowheads). - a: X 400, b: X 200, c: X 160, d: X 200.

38 . Y. Akimoto et al.

Fig. 14. Ultrastructural localization of galectin-1 by immunogold labeling method in which an ultrathin cryosection of human skin was reacted with antibody against galectin-1 and then with colloidal gold-conjugated goat anti-rabbit IgG. Boundary between epidermis and dermis. Basal lamina (BL) is seen in the lower right. Arrowheads indicate colloidal gold particles localized along the plasma membrane and in the intercellular space around a basal cell (Be) of the epidermis. Colloidal gold particles are also localized in the matrix of the cytoplasm of a Langerhans cell (Le). Reticular layer of basement membrane and extracellular matrix in the dermis (D) are densely labeled by colloidal gold particles as well. - X 26,000.

Endogenous animallectins . 39

1993). Studies are now in progress to identify these glycoproteins recognized by galectin-1. Double staining with anti-galectin-1 and anti-CDla revealed that galcetin-1 is located not only in keratinocytes but also in Langerhans cells (AKIMOTO et al. 1995b). This galectin-1 has been shown to have an extensive sequence similarity with IgEbinding protein (epsilon BP, 33-kDa galectin) of human skin, which is localized on the surface of Langerhans cells and modulates their binding capacity for IgE (WOLLENBERG et al. 1993). However, galectin-1 seems to be produced by both keratinocytes and Langerhans cells, whereas the 33-kDa galectin is produced by kerationocytes only and subsequently binds to the surface of Langerhans cells (WOLLENBERG et al. 1993). From these results, we propose that galectin-1 as well as 33-kDa galectin (IgE-binding protein) may function in modulating the binding capacity of Langerhans cells for glycoconjugates, which cells are known to be involved in the immune response of the skin (HosOI et al. 1993). Several other galectins have been isolated from human tissues and cells (SPARROW et al. 1987; CHERAYIL et al. 1989; LAING et al. 1989; GIrr et al. 1992; BARONDES et al. 1994b). However, the physiological functions of these galectins, aside from the function of 33-kDa IgE-binding galectin, are unclear. In view of the location of galectin-1 and 14-kDa chick galectin expression in human skin and chick skin, it seems reasonable to suggest that this galectin may playa role in cell-to-cell and cell-to-extracellular matrix interaction by binding to complementary sugars located on the cell surface and extracellular matrix. Recently it was reported that keratinocyte expresses galectin-7, a monomeric ~-ga­ lactoside-binding protein (MADSEN et al. 1995; MAGNALDO et al. 1995). Immunofluorescent study of human tissues with a specific galectin-7 antibody revealed that galectin-7 is localized mainly in the stratified squamous epithelium of skin, i. e., in basal and suprabasal layers of the epidermis. Its expression is moderately suppressed by retinoic acid (MAGNALDO et al. 1995) as is that of 14-kDa lectin by retinol in chick embryonic epidermis (ODA et al. 1989; AKIMOTO et al. 1992). Galectin-7 is thought to playa role in the cell-to-cell and/or cell-to-extracellular matrix interaction necessary for normal growth control. 5.2 Skin appendages 5.2.1 Hair

Hair follicles are tubular invaginations of the epidermis that can produce hairs. The walls of hair follicles are made up of the external root sheath, which is a tubular invagination of the epidermis, and also of the internal root sheath, which is a sleeve-like lining made of soft keratin. The basal cells of the external root sheet of hair follicles contain an abundance of galectin-1 (ALLEN et al. 1991) (Fig. 13b).

40 . Y. Akimoto et al.

5.2.2 Sebaceous gland

Sebaceous glands are simple alveolar glands that open into the upper part of the hair follicle and produce a complex oily material called sebum. Cells resulting from mitosis in the basal layer synthesize and accumulate lipids as they become displaced toward the interior of the gland. The basal cells of the sebaceous gland contain abundant galectin-l (Fig. 13c). 5.2.3 Eccrine sweat gland

This is a simple tubular gland with an irregularly coiled secretory portion and a slightly helical duct that takes sweat to the skin surface. Most the secretory cells are low columnar in profile and have rather pale-staining cytoplasm. Myoepithelial cells, specialized for contraction, are arranged helically around the periphery of the secretory portion. Some secretory cells and the myoepithelial cells are rich in galectin-l (Fig. 13d).

5.3 Placenta Galectin-l was found to be abundant in stroma of placental villi and decidual cells (Fig. 15). In placental villi, fibroblasts showed positive staining for the lectin, whereas the syncytiotrophoblast and cytotrophoblast showed no positive staining. In the decidua, some decidual cells and extracellular matrix showed positive staining. Decidua consists of maternal cells and extravillous trophoblast. Extravillous trophoblast invading the maternal tissues did not contain galectin-l; however, maternal cells did express it (BEVAN et al. 1994). Electron-microscopic examination revealed that in the stroma of placental villi galectin-l exists both in the cytoplasm and in the nucleus of fibroblasts, whereas it exists both in cytoplasm of decidual cells and in the extracellular matrix (Figs. 16, 17). These results suggest that galectin-l may play an important role as a component of extracellular matrix and fibroblast in stroma of villi and decidual cell. BEVAN et al. (1994) reported immunohistochemical localization of a galectin-l at the human maternofetal interface. Strong reactivity was found in decidual stromal cells

Fig. IS. The localization of galectin-I in villi (a, b) and decidua (c) of human placenta at term. a: Semithin frozen section was stained with antibody against galectin-I and rhodamine-labeled goat-anti-rabbit IgG. Nuclei were simultaneously stained with DAPI. Galectin-I is seen in the fibroblasts (arrowheads) in the stroma of the villus. S: syncytiotrophoblast, C: cytotrophoblast. b: Nomarski differential interference-contrast image of the same specimen. c: This section was stained with anti-galectin-I and HRP-labeled goat anti-rabbit IgG. Nuclei were counterstained with hematoxylin. Some of the decidual cells are stained. - a and b: X 750, c:

x 420.

Endogenous animallectins . 41

c

42 . Y. Akimoto et al.

Fig. 16. Survey view of a human placental villus by electron microscopy. Formaldehyde-fixed and embedded in LR white resin. Rectangle indicates the area shown enlarged in Fig. 17. S: syncytiotrophoblast, C: cytotrophoblast, B: blood vessel. - x 1,700.

throughout gestation, and endometrial stromal cells were also positive. This galectin was detected in neither the villous syncytiotrophoblast nor the underlying cytotrophoblast in first-trimester tissue. Nor was it detectable in villous or extravillous trophoblast populations at term. They concluded that this galectin is not a component of the immunosuppressive factors associated with syncytiotrophoblast membranes, but may have a role in either the decidual control of trophoblast migration or some functions unrelated to pregnancy or both.

5.4 Nervous system 5.4.1 Central Nervous System (CNS)

The cerebellar cortex consists of three layers, that is, the molecular layer (outermost), the layer of Purkinje cells (middle), and the granular layer (innermost). Galectin-l is found in the neurons that are present between the molecular and granular layers (Fig. 18a).

Endogenous animallectins . 43

Fig. 17. Enlargement of the rectangle in Fig. 16 for the ultrastructural localization of galectin-l. Ultrathin section was labeled with antibody against galectin-l, then with affinity-purified goat anti-rabbit IgG-colloidal gold (10 nm in diameter) conjugate. Colloidal gold particles representing galectin-l are seen in the cytoplasm and nucleus of the fibroblast present in the stroma of a placental villus. N: nucleus. - x 35,000.

44 . Y. Akimoto et al.

5.4.2 Peripheral nervous system (PNS)

Galectin-l exists in parasympathetic ganglions. It is also seen in the ganglion cells of the myenteric plexus, which lies between the inner circular and outer longitudinal layers of smooth muscle in the muscularis externa of the digestive tract, and in those of the submucosal plexus, which lies in the submucosa of the digestive tract (Fig. 18b). In the pancreas, galectin-l is localized in the ganglion cells that form one of the intramural ganglia (Fig. 18c). In the peripheral nerve fiber bundles, the galectin exists in both axon and Schwann cell (Fig. 18d).

5.5 Digestive system In the tongue, galectin-l was localized in the lamina propria and in the stratified squamous epithelium. Galectin exists predominantly in the basal cells and on the plasma membrane of the intermediate cells. In the esophagus, galeetin was localized in the lamina propria and in the stratified squamous epithelium (Fig. 19a). In the epithelium, the cell membrane of the intermediate cell layer was strongly positive. The localization of galectin-l is consistent with the binding patterns of PNA (peanut agglutinin) and SBA (soybean agglutinin) (YAMAGUCHI et al. 1985), which are known to bind specifically to D-galactose and N-acetyl-Dgalactosamine, respectively. In the duodenum and ileum, galectin-l was detected in the lamina propria and submucosa (Fig. 19b, c). It was not seen in the epithelium of villi and crypts. In the colon, galectin-l exists prominently in the basement membrane and in the lamina propria (ALLEN et al. 1991). Collagen fibrils and macrophages were densely immunostained (Fig. 19d). The cytoplasm of epithelial cells stained lightly for galectin1, whereas the galectin-l was not present in the mucous granules or secreted mucin. Galectin-l was also present in capillary walls.

Fig. 18. Immunohistochemical localization by light microscopy of the galectin-l in human nervous tissues. Sections were stained with anti-galectin-l and HRP-Iabeled goat anti-rabbit IgG. a: Cerebellum. Neurons (small arrows) interspersed between Purkinje cells are stained. M: molecular layer, G: granular layer. Large arrows indicate the Purkinje cells. b: Ileum. Meissner's plexus (MP) are stained. C: crypt, L: lamina propria, MM: muscularis mucosa, S: submucosa. c: Pancreas. Ganglion (arrow) in the lobule of pancreas is stained intensely. d: Omohyoid muscle. Perineuriun (arrow) and axon (arrowheads) in the nerve fiber bundle are intensely stained. SM: striated muscle, B: blood vessels. - a: x 430, b: x 400, c, d: x 320.

Endogenous animal leetins . 45

46 . Y. Akimoto et al.

Endogenous animallectins . 47

5.6 Blood cells Galectin-1 was isolated from human peripheral leukocytes (ALLEN et al. 1991). The galectin was distributed throughout the cytoplasm of B lymphoblastoid cells rather than being localized on the cell surface. A galectin-3 (hL-31, Mac-2) is expressed in human peripheral blood monocytes and macrophages. Galectin-3 is expressed on the surface of human monocytes. Cell surface galectin-3 increased in level progressively as monocytes differentiated into macrophages (LIU et al. 1995). Galectin-3 is also found in the nucleus and/or cytoplasm of human peripheral blood basophils, where the cytoplasmic labeling is concentrated over secretory granules (CRAIG et al. 1995). Thus, basophils may release galectin-3 when activated to degranulate (CRAIG et al. 1995). The processes leading to increased galectin-3 expression in HL-60 cells, a human promyelocytic leukemia cell line, may be specific to differentiation along the monocyte/macrophage pathway (NANGIA-MAKKER et al. 1993).

6 Galectin changes in tumors Galectins have been used as marker proteins for studying formation and metastasis of tumors (RAz et al. 1988; GABIUS et al. 1986a, b; LOTAN et al. 1989; ALLEN et al. 1991). In cutaneous tissues, although the extracellular matrix and hair follicle cells contain abundant galectin-1 (ALLEN et al. 1991), this galectin is absent in basal cell carcinoma and associated stroma (ALLEN et al. 1991). Basal cell carcinomas also express little or no 30-kDa galeetin, whereas nonmalignant basaloid cells, squamous cell carcinomas, melanoma, and nevi contain an abundance of this lectin (SANTA-LuCIA et al. 1991). Some malignant tissues may therefore be characterized by a deficiency of galectin (ALLEN et al. 1991). Thus, the immunodetection of galectin may aid the dermatopathologist in difficult histologic diagnosis.

Fig. 19. Immunohistochemical localization by light microscopy of galectin-1 in the human digestive system. Sections were stained with anti-galectin-1 and HRP-Iabeled goat anti-rabbit IgG. a: Esophagus. The plasma membrane of the cells located in the middle and superficial layers of the epithelium is stained preferentially. The fibroblasts and the extracellular matrix in the lamina propria are also positive for galectin-l. E: epithelium, L: lamina propria. Arrowheads indicate basal surface of epithelium. b: Duodenum. Lamina propria and submucosa are stained. C: crypt, B: Brunner's gland. c: Ileum. Lamina propria is stained preferentially. E: epithelium, L: lamina propria. Arrows indicate the goblet cells. d: Colon. Lamina propria is stained. C: crypt. Arrows indicate the goblet cells. - a: X 400, b: X 160, c: X 400, d: X 200.

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Galectin-l (MW 14.5 kDa) and galectin-3 (MW 31 kDa) have been found in a variety of normal and malignant cells and are considered to be involved in the regulation of cell growth, cell adhesion, and metastasis. The human colon carcinoma cell line KM12 expresses only galectin-3. Sodium butyrate, which is a differentiation-inducing agent for colon carcinoma cells, induces an increase in the expression of galectin-l as well as its glycoconjugate ligands (carcinoembryonic antigen, lamp-I, and lamp-2) (OHANNESIAN et al. 1994). By immunoprecipitation from radioiodinated cell-surface proteins as well as by indirect immunofluorescence labeling, butyrate-induced galectin-l can be detected on the cell surface. Galectin-l expression may be associated with the differentiation of human colon carcinoma cells (KMI2). Thus, tumor cell differentiation is accompanied by the regulation of lectin expression. Several glycoproteins (carcinoembyronic antigen, lamp-I, and lamp-2) function as its endogenous ligands in the process of colon carcinoma adhesion and metastasis.

7 Histochemical localization of the nematode galectin Caenorhabiditis elegans 32-kDa galectin is the first invertebrate galectin to be found so far (HlRABAYASHI et al. 1992a, b). It was also the first demonstration of the existence of the tandem repeat type galectin. C. elegans is a very useful experimental animal for the study of the molecular events underlying differentiation and development, since it is composed of a small number of cells (about 1,000), is easily maintained, has short life cycle of 3 days, and can be observed with the aid of a microscope. C. elegans hatches and develops through four larval stages separated by moults, prior to becoming fertile. Extensive genetic analyses have been performed and a variety of mutants obtained. Figure 20 shows the immunohistochemical localization of 32-kDa galectin in a section through almost the entire body length (a small part of the tail is missing) of an adult (ARATA et al. 1996). The galectin was visualized with rhodamine-conjugated secondary antibody and DNA was counterstained with DAPL Clusters of blue spots seen around the adult, which indicate the position of cell nuclei, are mainly cross-section of larval animals. The most intense red fluorescence was found in the cuticle of the adult (C in Fig. 20). However, labeling of the cuticle was only seen in adult animals carrying eggs, while larval animals (L in Fig. 20), which can be identified by DAPI fluorescence or by Nomarski differential interference-contrast images, were not labeled. Intense labeling was also found in the terminal bulb of the adult pharynx (T in Fig. 20), a structure that ingests, concentrates, and processes food before pumping it into the gut. The cuticle of C. elegans is a multilayered, extracellular structure protecting the animal from its environment under a variety of conditions. The cuticles of the L1, dauer, L4, and adult stages are reported to differ in both structure and protein composition. 32-kDa

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Fig. 20. Immunohistochemical localization of 32-kDa galectin in a cryostat section of C. elegans. Galectin was detected by the indirect immunofluorescence method using rhodamine-conjugated secondary antibody. The section was counterstained with DAPI to show DNA. a: Galectin is localized in the adult cuticle (C) and in the terminal bulb of the pharynx (T). Larval animals (L) are not stained. V: vulva, E: eggs, G: germ cell nuclei. b: Nomarski differential interference-contrast image. - x 150.

galectin may be an essential part of the adult cuticular matrix. It is interesting that both vertebrate and invertebrate galectins have a closely related role and the final stage of development, that is, the construction of the outer barrier of the body, although the structures of the cuticle of C. elegans and the skin of vertebrates are obviously quite different. As to the endogenous ligands, the unc-6 gene was found to encode a laminin-related protein that is required for the guidance of pioneer axons and migration of cells along the body wall in C. elegans (ISHII et al. 1992). In mammals, laminin is a candidate for the endogenous receptor of galectins. Hence, the unc-6 gene product might be an endogenous ligand for 32-kDa galectin.

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8 Localization of C-type lectins 8.1 Mouse macrophage lectin (MMGL) 8.1.1 Background information for MMGL MMGL was originally identified form Streptococcus pyogenes (OK-432)-induced peritoneal exudate macrophages showing tumoricidal activity (ODA et al. 1988, 1989; SATO et al. 1992). It is a Mr 42,000 type II transmembrane glycoprotein with single C-terminal C-type CRD (SATO et al. 1992) containing QPD motif. Galactose (Gal) and N-acetylgalactosamine (GaINAc) specificity of this lectin has been further established using soluble recombinant lectin consisting of extracellular domains produced by a bacterial expression system (SATO et al. 1992; IMAI and IRIMURA 1994; YAMAMOTO et al. 1994). A recently developed ELISA-based carbohydrate binding assay demonstrated that galactose is a 3-fold potent inhibitor as compared with GalNAc (IMAI and IRIMURA 1994). Using oligosaccharides and an immobilized recombinant form of MMGL, this lectin had significant affinity to triantennally and tetra-antennally complex type sugar chains with terminal galactose (YAMAMOTO et al. 1994). Using N -terminal glycopeptides from human erythrocyte glycophorin A, it has also been demonstrated that MMGL has affinity to glycopeptides containing Gal~I-3GalNac-SeriThr (Tf-antigen) as well as those containing three consecutive GalNAc-SeriThr (Tn-antigen) (YAMAMOTO et al. 1994). This is consistent with an observation to the effect that truncation of O-linked sugar chains using benzyl-GalNAc treatment of mouse P815 mastocytoma cells resulted in an increased accessibility of the treated cells to MMGL and macrophages (SAKAMAKI et al. 1995). In fact, the benzyl-GaINAc-treated P815 cells had increased binding sites for peanut agglutinin (PNA) reactive to Tf antigen and for Vicia villosa agglutinin B4 (VVAB 4 ) reactive to Tn antigen (SAKAMAKI et al. 1995). It has been reported that MMGL is involved in the binding of macrophages to tumor cells (ODA et al. 1989; SAKAMAKI et al. 1995) and in in vitro tumoricidal activity on the part of activated macrophages (ODA et al. 1989; KAWAKAMI et al. 1994). It is also reported that MMGL and its rat homologue (M-ASGP-BP) (II et al. 1990) are involved in internalization of asialoglycoprotein (OZAKI et al. 1992,1993; KAWAKAMI et al. 1994). The presence of cytoplasmic tyrosin motifs (YENL for MMGLand YENF for rat M-ASGPBP) is consistent with their involvement in a rapid endocytosis pathway (JADOT et al. 1992). Under pathological conditions, mRNA of the rat M-ASGP-BP has been reported to be selectively expressed in rat cardiac allograft with arteriosclerosis during the process of chronic rejection using a differential mRNA display method (UTANS et al. 1994; RUSSELL et al. 1994). By exploiting the cross reactivity of a polyclonal anti-rat hepatic asialoglycoprotein receptor antibody for rat M-ASGP-BP, expression of the lectin has been demonstrated in cells localized in perivascular regions of the cardiac allografts (RUSSELL et al. 1994).

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8.1.2 Production of monoclonal antibodies against MMGL that are useful for immunohistochemical detection

We developed rat monoclonal antibodies (mAb) against MMGL by immunizing rat with natural MMGL affinity purified from RAW264.7 mouse macrophage cell line. Screening of mAbs was carried out (KIMURA et al. 1995) using the conventional ELISAbased antibody assay binding to the natural and the recombinant forms of MMGL as well as by inhibition of carbohydrate binding using the ELISA-based carbohydrate binding assay (lMAI and IRIMURA 1994). The reactivity of mAbs was further characterized by flow cytometry and immunoprecipitation analyses using various mouse macrophage cell lines (KIMURA et al. 1995). A non-blocking mAb LOM-14 turned out to produce best results after immunohistochemcial staining of frozen sections of mouse tissue. Blocking mAbs, which interfere with lectin activity on the part of MMGL, also produced positive signals for MMGL, however, the signal intensity was relatively weak as compared with mAb LOM-14. We therefore generally used mAb LOM-14 as an immunohistochemical reagent. We carefully evaluated the cross reactivity of mAb LOM-14 to mouse hepatic lectins, because there is a significant homology in the amino acid sequence (SATO et al. 1992; TAKEZAWA et al. 1993; SANFORD and DOYLE 1990). We affinity purified MMGL and mouse hepatic lectins from mouse macrophage cell line J774A.l and from mouse liver, respectively. We proved that mAb LOM-14 does not cross react to mouse hepatic lectins (MIZUOCHI et al. 1997). 8.1.3 Detection of MMGL in adult mouse tissue extracts

By a combination of affinity chromatography using galactose-Sepharose column and immunoblotting analyses using mAb LOM-14, we detected a 42 kDa component representing MMGL in tissue detergent extract from various mouse tissues (MIZUOCHI et al. 1997). The use of affinity chromatography has two advantages. First, proteins that exhibit calcium-dependent binding to galactose can be concentrated from protein mixture in the tissue lysates. Second, only active lectins that retain carbohydrate binding activity can be analyzed. MMGL was widely distributed throughout mouse tissue with the exception of brain, peripheral blood, liver, kidney, and small intestine. The tissue that had the greatest amount of MMGL relative to the amount of protein in the extract was skin. Significant amounts of MMGL were detected in the extracts from heart, lung, stomach, large intestine, cecum/appendix, urinary bladder, testis, thymus, spleen, lymph nodes, bones (including bone marrow) and skeletal muscle. 8.1.4 Immunohistochemical localization of MMGL in adult mouse tissue

We examined the localization of cells expressing MMGL by immunohistochemical methods using frozen sections. Immunohistochemical staining using mAb LOM-14 was

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Fig.22. Immunohistochemical localization of MMGL in mouse lung. Frozen sections of lung from normal CD-l (ICR) mice were incubated with mAb LOM-14. The bound antibodies were visualized (red) using biotinylated anti-rat kJA plus alkaline phosphatase-streptavidin. Nuclei were stained with hematoxylin. EP: bronchiolar epithelium, P: lung parenchyma. Arrow points to a representative cell with positive reaction. A chain of MMGL-positive cells is observed beneath the bronchiolar epithelium. MMGL-positive cells are absent from lung parenchyma. - X 940.

Fig.21. Immunohistochemical localization of MMGL in mouse lung. Frozen sections of lung from normal CD-l (ICR) mice (a) or from B6C3Fl mice with metastatic nodules of OV2944HM-l ovarian tumor cells (b) were incubated with rat anti-MMGL mAb LOM-14. The bound antibodies were visualized (red) using biotinylated anti-rat k/A plus either Texas Red-streptavidin (a) or Texas Red avidin D (b). DNA was stained with DAPI. BY: blood vessels, P: lung parenchyma, MN: metastatic nodules. Arrows point to representative cells with positive reaction. Arrowheads in (b) indicate border between nodules and lung tissue. Cells with positive staining for MMGL were observed in the connective tissue surrounding blood vessels, but were absent from the parenchyma consisting of lung wall and alveoli. MMGL-positive cells were also observed within the lung metastatic nodules while they were absent from lung parenchyma. - X 460.

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carried out on tissue that produced a significant signal for MMGL by immunoblot analyses. A common feature of cellular localization was that expression of MMG L is restricted to macrophages in the connective tissue (IMAI et al. 1995; MIZUOCHI et al. 1997).

8.1.4.1 Lung Light microscopic detection revealed that expression of MMGL was restricted to stellate-shaped cells in the connective tissue surrounding blood vessels and respiratory epithelium (IMAI et al. 1995) (Figs.21 and 22). Cells in the alveolar regions, including alveolar macrophages, did not express MMGL. Cells within the respiratory epithelium or on the luminal side of the airway, where alveolar macrophages and dendritic cells were present, did not express MMGL (Fig. 22). The pattern of expression was different from those of known cell markers for macrophage subsets including CDII b integrins (Mac-l antigen) (IMAI et al. 1995; BREEL et al. 1988; BILYKI and HOLT 1991). Electron microscopic detection using colloidal gold also support the idea that MMGL is present in macrophages localized in the connective tissue surrounding bronchioles (Fig. 23). On the other hand, alveolar macrophages with phagocytic inclusions found on the luminal surface (the other side of the connective tissue) of bronchiolar epithelium did not express MMGL. Electron microscopic results also demonstrated the presence of MMGL in intracellular vesicles of the connective tissue macrophages (IMAI et al. 1995)

Fig. 23. Immunohistochemical localization of MMGL in normal mouse lung by electron microscopy. MMGL was detected by mAb LOM-14 and colloidal gold-conjugated goat anti-rat IgG. a: Connective tissue area surrounding blood vessels and bronchioles. A cell with arrow b (in the connective tissue) is shown at higher magnifications in (b). b: Colloidal gold particles representing MMGL are indicated by arrows. Cells in the connective tissue contain intracellular vesicles with MMGL in the luminal side. A: arteriole, B: bronchiole, EP: epithelium, I: interstitium, M: mitochondrion, N: nucleus. - a: X 940, b: X 22,000.

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(Fig. 23). The presence of a consensus sequence (YXXL) related to rapid endocytosis GADOT et al. 1992) in the cytoplasmic domain of MMGL (SATO et al. 1992) plus an ability to internalize asialoglycoprotein into MMGL-positive peritoneal macrophages (KAWAKAMI et al. 1994) suggested that MMGL may be present in intracellular vesicular compartments including endosomes. The immunohistochemical results are the first evidence to show the presence of MMGL within the intracellular vesicles. A remaining question is whether these vesicles are endosomes. In other words, the question is now whether these vesicles are coated and, if so, what kind of coat is associated with the vesicles. Another interesting point for clarification is whether MMGL-positive connective tissue macrophages express class II major histocompativility complex (MHC) molecules, and if so, whether class II MHC molecules could colocalize with MMGL within the vesicles. On the latter point it is particularly important to determine whether MMGL is involved in antigen internalization and the processing of prior antigen presentation on class II MHC molecules. 8.1.4.2 Abdominal skin MMGL-positive cells are abundant in the dermis and subcutaneous tissue, whereas they are absent from the epidermis (Fig.24a, b). These cells are stellate-shaped, as revealed by a higher magnification. A similar distribution for MMGL-positive cells was seen in skin from other sites, including the pinna and soles of paws. These results suggest the presence of MMGL in dermal macrophages and its absence from Langerhans cells in the epidermis. 8.1.4.3 Muscles Chains of MMGL-positive cells were detected in the interstitium along myocardiac fibers (Fig. 24c). Cardiac muscle cells were negative for MMGL. Perivascular connective tissue contained MMGL-positive cells. Endothelial cells of blood vessels were negative. Connective tissue in skeletal muscle (e. g., of the femur), typically along the endomysium and perimysium and surrounding blood vessels, contained MMGL-positive cells (Fig. 24d). Endothelial cells of blood vessels and skeletal muscle cells were negative. 8.1.4.4 Urinary bladder The lamina propria, submucosa and smooth muscle layers contained scattered cells positive for MMGL (Fig.2Sa). Transitional epithelia did not contain MMGL-positive cells. 8.1.4.5 Gastrointestinal tract The lamina propria and submucosa of the large intestine contained MMGL-positive cells (Fig.2Sb, c, and d). Periphery of lymphoid aggregates in the submucosa also contained MMGL-positive cells. A similar pattern of distribution for MMGL-positive cells was seen in the stomach and cecum!appendix.

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Fig. 24. Immunohistochemical localization of MMGL in the skin (a, b), heart (c), and skeletal muscle (d) by light microscopy. A boxed area in (a) is shown at a higher magnification in (b). In the skin, MMGL-positive cells are observed in dermis and subcutaneous layer, whereas those are absent from epidermis. MMGL positive cells have stellate-shape with elongated pseudopodia (b). In heart, MMGL-positive cells are observed between myocardial fibers (c) and connective tissue surrounding the cardiac blood vessels (not shown). In skeletal muscle (d), MMGL-positive cells are observed in the connective tissue surrounding blood vessels and are also present along endomysium and perimysium. Arrows indicate MMGL-positive cells. Bv: blood vessels, D: dermis, E: epidermis, Em: endomysium, H: hair follicles, M: myocardial fibers, Mf: muscle fibers, Pm: perimysium, S: subcutaneous layer. - a: X 130, b: x 750, c: x 300, d: x 400.

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Fig. 25. Immunohistochemical localization of MMGL in urinary bladder (a), and large intestine (b-d) by light microscopy. A boxed area in (b) is shown at a higher magnification in (c). In urinary bladder (a), MMGL-positive cells are observed in lamina propria, submucosa and muscularis mucosa but are absent from transitional epithelia. In large intestine, MMGL-positive cells are observed in lamina propria and submucosa. They are also observed on the periphery of the lymphoid aggregate (d). Arrows indicate MMG L-positive cells. E: epithelium, L: crypts of Lieberkiihn, La: lymphoid aggregate, Lp: lamina propria, Sm: submucosa, Te: transitional epithelium. - a: X 270, b: x 200, c: X 800, d: X 100.

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8.1.4.6 Thymus MMGL-positive cells were present along the capsule and septa in a high density (Fig. 26). The cortex was essentially devoid of MMGL-positive cells and a sparse distribution of MMGL-positive cells was seen in the medulla. 8.1.5 MMGL expression in lung metastatic nodules

We examined whether tumor-infiltrating macrophages could express MMGL in vivo. To this purpose, we used an experimental lung metastasis system in which highly metastatic mouse ovarian tumor OV2944-HM-1 cells (HM-1) (HASHIMOTO et al. 1989) were intravenousely injected into a syngenic host, and frozen sections from lung with established metastatic nodules were immunohistochemically examined. We discovered that macrophages expressing MMGL are frequently present within the lung metastatic nodules (Fig. 21 b). On the other hand, the distribution of MMGL-positive macrophages outside the nodules was restricted to the connective tissue, and the alveolar regions were essentially devoid of MMGL-positive macrophages as seen for normal mouse lung. A remaining question is how MMGL-positive connective tissue macrophages could infiltrate or coexist within the metastatic nodules. One of the interesting possibilities is that MMGL is involved in the cellular trafficking of macrophages. To address this question, we recently carried out cell trafficking experiments using a mouse cytotoxic T cell clone that had been transfected with an expression vector containing MMGL eDNA. We found that the MMGL-transfected cells displayed a preferential localization in the lung metastatic nodules, while vector-transfected cells did not (ICHI! et al. 1997). This result suggests the possibility that tumor-infiltrating macrophages may utilize MMGL to accumulate in the tumor site. Another question is whether these tumor-infiltrating macrophages could contribute to host defense mechanisms or facilitate in situ tumor growth in the metastatic nodules. These questions are currently under investigation. 8.1.6 Immunohistochemical localization of MMGL in mouse embryos

Another interesting issue is when and where MMGL production starts during embryonic development. It is also important to understand the relationship with the development of macrophages in embryos. Thus we performed biochemical and immunohistochemical studies on MMGL expression during mouse embryogenesis (MIZUOCHI et al. 1998). A 42 kDa band representing the signal for MMGL was first detected in detergent extract as early as 11 days post coitus (dpc) using affinity chromatography and immunoblot analyses. The signal intensity was increased during the stages of embryogenesis, and major increases in the signal intensity were seen at 12 dpc and 14 dpc. Immunohistochemical localization of MMGL in mouse embryos was analyzed using mAb LOM-14. A representative sagittal section of 14 dpc embryos is shown in Fig. 27. MMGL is expressed in mesenchymal cells, including mesenchyme of midbrain region

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Fig. 26. Immunohistochemical localization of MMG L in thymus by light microscopy. A region in (a) indicated by an asterisk is shown at a higher magnification in (b). MMGL-positive cells (arrows) are observed along capsule and .septa (b) as well as in medulla (c). Ca: capsule, C: cortex, s: septa, M: medulla. - a: X 60, b: X 700, c: X 430.

(Fig. 27d) and beneath embryonic epidermis (not shown). The expression of MMGL in mesenchymal cells localized beneath embryonic skin was observed during the embryonic stages examined (from 12 dpc to 18 dpc). Fetal liver, which performs hematopoiesis and produces monocyte/macrophages during these stages, did not produce positive signals. In addition, a conspicuous signal for MMGL was observed in an intermediate cartilage tissue. Chondroblasts in the transient cartilage during endochondral ossification process produced intense signals representing MMGL at 14 dpc embryos (Fig.27b and c). Positive reaction representing mAb LOM-14 binding to MMGL was found along the rough endoplasmic reticulum (Fig.28). The signal intensity was decreased as calcification proceeded in the intermediate cartilage. In 16 dpc embryos, the expression of

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c

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Fig.28. Ultrastructural localization of MMGL in chondrocytes within cartilage of 14 dpc mouse embryo by reaction with mAb LOM-14 as revealed by the HRP method. Arrows indicate positive reaction representing MMGL among rough endoplasmic reticulum. N: nucleus, Mt: mitochondrion. - X 7,100.

Fig. 27. Immunohistochemical localization of MMGL in 14 dpc mouse embryos by light microscopy. a: A sagittal section of 14 dpc embryo. b: Higher magnification of the area indicated by small letter b (vertebral body) in the panel a. All chondroblasts in the vertebral bodies are stained with mAb LOM-14. c: Higher magnification of the area indicated by small letter c in the panel b. Positive reaction was detected in cytoplasm of chondroblasts (arrows) in the intermediate cartilage tissue of vertebral body. d: Higher magnification of the area indicated by small letter d (midbrain region) in the panel a. Arrows indicate MMGL-positive cells. H: heart, I: intestine, K: kidney, Li: liver, Lu: lung, C: vertebral bodies. a: X 7, b: X 100, c: X 590, d: X 590.

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MMGL in the chondroblast declined. In 18 dpc embryos and in newborn mice, chondroblasts were scarcely MMGL-positive. Furthermore, the clacified area within the intermediate cartilage was devoid of MMGL-positive cells. In adult mice, chondrocytes localized in the xiphoid process and in the cartilage of auricle were devoid of MMGLpositive cells. These results suggest that MMGL in the intermediate cartilage may be involved in the process of mesenchymal cell condensation as cell to cell or cell to extracellular matrix (ECM) adhesion molecules. An alternative possibility is that MMGL may be involved in the remodeling of ECM proteins that chondroblasts perform by capture and endocytosis of precursor proteins present in the intermediate cartilage tissue. The latter possibility suggests that MMGL plays a role in the remodeling of intermediate cartilage tissue in mouse embryos.

8.2 Immunohistochemical localization of other C-type leetins 8.2.1 Kupffer cell lectin

A type II receptor (Mr = 88,000) specific for fucose, galactose and N-acetylgalactosamine was characterized from rat liver (HALTIWANGER et at. 1986; HOYLE and HILL 1988, 1991). This lectin contains the QPD motif in CRD. Light microscopic study on frozen sections using rabbit polyclonal antibody revealed that this lectin was localized in Kupffer cells in rat liver (HALTIWANGER et at. 1986). The staining pattern was different from that produced by rabbit polyclonal anti-galactose lectin (asialoglycoprotein receptor) antibody, which stained hepatocytes. When rat liver was examined after intravenous injection of colloidal iron, cells containing iron particles were specifically stained with the antiserum. This result further supports the Kuppfer cell specificity of this lectin. It is also reported that lung, spleen, peritoneal macrophages, and blood monocytes did not express this lectin (HALTIWANGER et at. 1986). 8.2.2 180 kDa-mannose specific macrophage lectin

A type I transmembrane lectin (Mr = 180,000) specific for mannose, fucose, and Nacetylglucosamine were originally characterized from rabbit and rat alveolar macrophages, respectively (WILEMAN et at. 1986; HALTIWANGER and HILL 1986a). Subsequently, cDNA was obtained from human placenta (TAYLOR et at. 1990), cultured human monocytes (EZEKOWITZ et al. 1990), and mouse peritoneal macrophages (HARRIS et al. 1992). The genomic organization of the human (KIM et at. 1992) and mouse (HARRIS et al. 1994) receptors was published. In the case of human receptor, it was reported that CRD #4, #5, and #7 were required for high affinity binding and endocytosis of multivalent glycoconjugates (TAYLOR et at. 1992). The CRD #4 and #5 contain the EPN motif. A rabbit polyclonal antibody against rat alveolar macrophage lectin showing no cross reactivity to the rat Kupffer cell lectin was produced and used to examine tissue localiza-

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tion in frozen sections of rat liver (HALTIWANGER and HILL 1986). The antibody did not stain hepatocytes but it did nonparenchymal cells. When colloidal iron was injected prior to immunohistochemical determinations, the antibody stains cells containing iron particles (Kupffer cells) and cells without particles (endothelial cells). Further results were obtained by a double staining experiment. It was observed that there are two types of nonparenchymal cells: cells with double positive for the alveolar macrophage lectin and Kupffer cell lectin, and cells positive only for the alveolar macrophage lectin. This was interpreted to mean that in the liver the former are Kupffer cells and the latter endothelial cells. By immunoblot analyses using the polyclonal antibody, they detected significant signals for the lectin in the detergent extracts from isolated alveolar macrophages, lung, liver, spleen, and leg muscle. They also reported that much weaker signals were detected in heart muscle, brain, small intestine and kidney whereas no signals were detected from erythrocyte, buffy coat of blood, and plasma. 8.2.3 A cDNA encoding lectin with 8 tandem eROs

A mouse eDNA encoding a type I transmembrane protein with 8 tandem CRDs has been cloned from mouse heart eDNA library using homology with E-selectin CRD (Wu et al. 1996). An interesting feature of this molecule is that CRD # 1 contains Q PD motif whereas CRD #2 contains EPN motif. The presence of CRDs probably with distinct specificity within the molecule may suggest a promiscuous nature for the specificity of this molecule. Although molecular characterization on the protein level has not been reported, in situ hybridization study revealed the expression of mRNA in mouse tissue including lung, kidney glomeruli, and choroid plexus. In addition, the mRNA was seen in mouse embryos as early as 7 dpc, and their expression was also observed in chondrocytes in cartilaginous regions of mouse embryos (Wu et al. 1996). 8.2.4 Bovine conglutinin

Bovine conglutinin is a collectin found in bovine serum. It has N-terminal collagenous domain and C-terminal single C-type CRD (MALHOTRA et al. 1994). Through the collagenous domain each monomer forms a trimer. Then the trimer forms a tetramer by disulphide linkage, so that the assembled conglutinin contains 12 CRDs. Conglutinin has been reported to bind a complement fragment iC3b through carbohydrate moiety on the a-chain of iC3b, and the binding was calcium dependent and inhibitable by monosaccharides, N-acetylglucosamine, and mannose (HIRANI et al. 1985). Conglutinin binds to circulating immune complex and iC3b-coated bacteria and enhance their clearance. Binding of conglutinin to yeast mannan has also been reported (LIM and HOLMSKOV 1996). Although conglutinin is a serum protein, its tissue distribution was studied using rabbit polyclonal antibody against bovine conglutinin (HOLMSKOV et al. 1992). Light microscopic examination of frozen sections from bovine tissue was carried out. Positive

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sites were hepatocytes, endothelial cells, follicular dendritic cells, and macrophages. The cytoplasm of hepatocytes was intensely stained, suggesting that the major site of biosynthesis was hepatocytes. Connective tissue, biliary ducts and portal tracts were negative. Follicular dendritic cells in the germinal center of lymph nodes were stained, as were also cells with dendritic morphology in the germinal center of the spleen and tonsils. Alveolar macrophages in lung and microglia cells in the brain were stained. Endothelial cells, especially glomerulus of the kidney and the capillary sinusoids of the adrenal cortex were intensely stained. Endothelial cells in other sites such as those of blood vessels in the liver, brain, thymus, lymph nodes, and spleen were stained. Lymph node high endothelial cells (HEV) were stained.

9 Localization of I-type lectin 9.1 Background information for sialoadhesin Sialoadhesin was originally identified as a lectin-like molecule of resident macrophages from mouse bone marrow. The definition of sialoadhesin activity was based on a rosette formation between bone marrow macrophages and sheep erythrocytes (CROCKER and GORDON 1986). The binding of sheep erythrocytes to macrophages was inhibited by sialidase treatment of sheep erythrocytes and the binding was inhibited by oligosaccharides and glycolipids containing sialic acid, suggesting that sialoadhesin recognizes glycoconjugates containing sialic acid on sheep erythrocytes (CROCKER and GORDON 1986). Subsequently, a rat blocking antibody (SER-4) that interferes with the binding of macrophages to sheep erythrocytes was developed (CROCKER and GORDON 1989). Purification using mAb SER-4 affinity chromatography was performed, and the activity of the purified sialoadhesin (Mr = 185,000) in binding to sheep eryhtrocytes was directly established (CROCKER et al. 1991). The carbohydrate specificity was characterized as Neu5Aca2-3Gal~1-3GaINAc (CROCKER et al. 1991). Molecular cloning of sialoadhesin revealed a type I transmembrane glycoprotein containing 17 immunoglobulin-like extracellular domains (CROCKER et al. 1994).

9.2 Immunohistochemical detection of sialoadhesin Immunohistochemical studies have been performed using mAb SER-4 and frozen tissue sections. In bone marrow, macrophages forming a network of stromal cells were reported to produce positive signals (CROCKER and GORDON 1989). On the other hand, bone marrow monocytes were negative with respect to SER-4 staining. The expression of sialoadhesin in the bone marrow stromal macrophages was implicated in cellular interactions of stromal macrophages with developing myeloid cells (CROCKER et al.

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1995). In lymph nodes, stromal macrophages in the subcapsular sinus and medullary cords were stained. A striking pattern of localization was observed in the spleen. Stellate cells within the inner region of the marginal zone at periphery of white pulp produced a strongly positive reaction, while weaker staining was observed on macrophages in the marginal zone and red pulp. On the other hand, the thymus did not contain cells showing detectable staining with SER-4. Kupffer cells in the liver were weakly positive. Epidermal Langerhans cells and microglia cells in the brain were negative. Several other tissue macrophages - including lung interstitial macrophages, a subpopulation of bronchoalveolar macrophages, dermal histiocytes, macrophages in the lamina propria of the small intestine - were also reported to be positive. In a study trying to elucidate the biological functions of sialoadhesin, a role in lymphocyte adhesion to macrophages in lymphoid tissue has been proposed (VAN DEN BERG et al. 1992). In an in vitro frozen section assay in which adhesion of lymphocytes to tissue sections was observed, sialoadhesin dependent adhesive interaction of lymphoma cells to the marginal zone of spleen and to the subcapsular sinus and medulla of lymph nodes was demonstrated. The adhesive interaction was inhibited by mAb SER-4 and was sialic acid dependent (VAN DEN BERG et al. 1992). Another study demonstrated that sialoadhesin preferentially binds to granulocytes rather than to lymphocytes (CROCKER et al. 1995). Also demonstrated was the preferential binding of sialoadhesin to neutrophils using a native form of sialoadhesin, sialoadhesin-immunoglobulin Fc chimera, and COS-1 cells transfected with sialoadhesin cDNA (CROCKER et al. 1995). Another interesting feature of sialoadhesin is that expression appears to be regulated by an unknown serum factor. Expression of sialoadhesin on thioglycolate-elicited peritoneal macrophages was increased during culture in the presence of mouse serum (CROCKER and GORDON 1989). In contrast to the absence of sialoadhesin on microglia cells, its presence on macrophages outside the blood brain barrier (choroid plexus, pituitary gland, subfornical organ, and leptomeninges) was reported (CROCKER and GORDON 1989; GORDON et al. 1992). Furthermore, when CNS was injured and the blood-brain barrier damaged, sialoadhesin was induced on a proportion of microglia (PERRY et al. 1992). Expression of sialoadhesin was reported to occur in an area wher plasma extravasation took place upon CNS injury.

10 Histochemical localization of carbohydrate ligands 10.1 Relation between endogenous lectins and exogenous lectins as tools for

histochemical detection of sugars Endogenous lectins have been introduced for use in glycohistochemical analysis. Their application in histochemistry is valuable when it comes to demonstrating the presence of the actual ligand structures in situ.

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Plant lectins have been popularly used in histochemistry, as they are commercially more readily available than animallectins. However, plant lectin may not always exhibit identical binding properties. Exogenous plant lectins are foreign substances for the cell and tissues, and thus their binding does not constitute true physiological sugar binding. So, to detect the sugar-binding site in situ, the endogenous animal lectin is more useful. Neoglycoconjugate, a synthetic probe that is chemically glycosylated, has recently been used histochemically (GABIUS and BARDOSI 1991; DANGUY et al. 1995). The use of labeled neoglycoconjugates in histology and pathology is called reverse lectin histochemistry. Neoglycoconjugates are used for the detection and localization of the endogenous lectin in situ. The application of both neoglycoconjugates and endogenous lectins is useful for the study of interaction between ligand and lectin. 10.2 Localization of carbohydrate ligands recognized by L-selectin. As an example of experiments in which a recombinant form of endogenous lectin was successfully utilized as an immunohsitochemical probe to detect carbohydrate ligands, we describe results on L-selectin ligands. Lymphocyte homing to lymph nodes is initiated by specific adhesive interaction between L-selectin on lymphocytes and its carbohydrate ligands displayed on high endothelial venules (HEV) of lymph nodes (ROSEN et al. 1992; LASKY 1992). To detect L-selectin ligands on lymph node HEY; on blood vessels in inflamed tissue and on other tissue sites, a recombinant form of L-selectin was utilized as a probe. 10.2.1 Background information for glycoprotein ligands for selectins

Several glycoproteins have been identified as glycoprotein ligands which have molecular features suitable for optimal presentation of carbohydrate ligands to CRD of selectins. Since L-selectin is a molecule on leukocytes, ligands for L-selectin were found on endothelial cells such as high endothelial cells (HEC) in lymph nodes. On the other hand, E-selectin and P-selectin expression is induced on the endothelial side, and glycoprotein ligands for these selectins are found on corresponding leukocytes. As HEV-associated glycoprotein ligands for L-selectin, GlyCAM-1, CD34, Sgp200, and MAdCAM-1 have been reported (IMAI et al. 1991; LASKY et al. 1992; BAUMHUETER et al. 1993; HEMMERICH et al. 1994b; BRISKIN et al. 1993; BERG et al. 1993). Glycoprotein ligands for P-selectin and E-selectin have been identified from granulocytes as PSGL-1 and ESL-1, respectively (SAKO et al. 1993; STEEGMAIER et al. 1995). 10.2.2 Reagent to detect glycoprotein ligands for L-selectin

A construct encoding a chimeric protein consisting of extracellular domains of Lselectin and Fc portion (hinge region, CH 2 and CH 3 domains) of human IgG has been

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expressed in human kidney 293 cells, and the chimeric protein has been produced. (WATSON et al. 1990). This reagent, termed LEC-IgG, has been extensively characterized in terms of carbohydrate binding specificity as compared with native L-selectin purified from mouse spleen (IMAI et al. 1990; WATSON et al. 1990). The use of chimeric protein has a couple of advantages. First, the chimeric protein can be purified by a simple step using immobilized Protein A. Second, conventional immunochemical reagents such as protein A and anti-IgG antibody, are readily applicable. Using the chimeric protein, glycoprotein ligands on lymph node HEV have been identified. GlyCAM-1 is a Mr = 50,000 mucin-like glyoprotein originally characterized as sulfated, fucosylated and sialylated glycoprotein (termed Sgp50) (IMAI et al. 1991). Subsequent cDNA cloning revealed that two regions containing clusters of serine and threonine that are potential sites for O-glycosylation (LASKY et al. 1992). There is no transmembrane domain in GlyCAM-1 sequence, suggesting that GlyCAM-1 is a soluble glycoprotein secreted from HEC cells. Another glycoprotein ligand is CD34 (Mr = 90,000) originally characterized as another species of sulfated, fucosylated, and sialylated glycoprotein (termed Sgp90) (IMAI et al. 1991). Subsequently, Sgp90 was shown to be identical with CD34 (BAUMHUETER et al. 1993). CD34 is a transmembrane glycoprotein with potential sites for clustered O-linked glycosylation. The third species of glycoprotein ligand detected using LEC-IgG chimeric protein is Sgp200 (Mr = 200,000), though peptide core has not been characterized (HEMMERICH et al. 1994b). These glycoprotein ligands have been shown to satisfy every feature known for HEV ligands for L-selectin. For example, involvement of sialic acid on HEV ligands has been demonstrated in earlier studies (ROSEN et al. 1985). Removal of sialic acid from Sgp50 (GlyCAM-1), Sgp90 (CD34), and Sgp200 terminated their ability to bind to L-selectin (IMAI et al. 1991; HEMMERICH et al. 1994b). The interaction between these ligands to L-selectin was inhibited by a mAb MEL-14 (IMAI et al. 1991), an original blocking antibody against mouse L-selectin that interferes with the adhesion of lymphocytes to the lymph node HEV (GALLATIN et al. 1983). It was also demonstrated (IMAI et al. 1991; HEMMERICH et al. 1994b) that these ligands have epitopes recognized by mAb MECA 79, which was developed against molecules (termed peripheral lymph node vascular addressin) on lymph node HEV and which blocks lymphocyte adhesion to the lymph node HEV (STREETER et al. 1988). Another important feature of L-selectin ligands is sulfation on carbohydrate moiety (IMAI et al. 1991; IMAI and ROSEN 1993). Functional requirement of sulfation has been demonstrated for GlyCAM-1 (IMAI et al. 1993), and for CD34 and Sgp200 (HEMMERICH et al. 1994b). Recent studies partially identified carbohydrate structures from GlyCAM1 as sialylated, fucosylated, and sulfated O-linked oligosaccharides that may represent a carbohydrate ligand structure for L-selectin (HEMMERICH et al. 1994a, 1995; HEMMERICH and ROSEN 1994). The carbohydrate structures contain a sialyl-Le structure, which has been shown to be recognized by all three selectins (PHILLIPS et al. 1990; POLLEY et al. 1991; FOXALL et al. 1992; IMAI et al. 1992); it undergoes modification by

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sulfation either on the galactose residue or on the N-acetylglucosamine residue. However, more complex carbohydrate structures from GlyCAM-1 remain to be investigated, especially with multiple sulfation (IMAI and ROSEN 1993). In addition, MECA 79 antibody is now believed to recognize a modification of carbohydrate moiety by sulfate displayed on HEV carbohydrate ligands (HEMMERICH et al. 1994b). 10.2.3 Immunohistochemical detection of glycoprotein ligands for L-selectin

There are two ways to detect glycoprotein ligands for L-selectin. First, a chimeric protein consisting of L-selectin and immunoglobulin (LEC-IgG) can be used in the presence of calcium as an immunohistochemical reagent. To detect binding of the chimeric protein, anti-human IgG can be used. Second, polyclonal or monoclonal antibody against core protein of the ligands, or antibody raised against a synthetic peptide predicted from cDNA sequence of the ligands could be used. However, there is an important difference between these procedures. The chimeric protein can only detect biologically active ligands with appropriate post-translational modification including glycosylation and sulfation, whereas the antibody detects not only active ones but their precursors. On the other hand, the antibody can detect an individual glycoprotein ligand, while detection by chimeric protein reflects the total activity of the biological ligands. In an earlier study, the activity of the chimeric protein as an immunohistochemical reagent was extensively characterized (WATSON et al. 1990). Frozen sections of mOU5e lymph nodes and Peyer's patches were incubated with LEC-IgG in the presence of calcium. LEC-IgG stained lymph node HEV but did not stain Peyer's patch HEY. This specificity is consistent with the specificity of L-selectin as a lymphocyte adhesion molecule (GALLATIN et al. 1983; LASKEY et al. 1989; SIEGELMAN et al. 1989). Calcium dependent binding of LEC-IgG satisfies the nature of C-type lectin. Furthermore, the binding of LEC-IgG was inhibited by the presence of mAb MEL-14 (a blocking antibody to Lselectin) (GALLATIN et al. 1983) and by fucoidin, a polysaccharide that inhibit lymphocyte adhesin to lymph node HEV (STOOLMAN et al. 1984; GEOFFROY and ROSEN 1989). Molecular cloning of GlyCAM-1 and molecular identification of Sgp90 as CD34 first made the use of antibody feasible. Polyclonal antibody against predicted peptides for GlyCAM-1 stained lymph node HEV (LASKY et al. 1992), while polyclonal antibody against a recombinant mouse CD34 stained lymph node HEV (BAUMHUETER et al. 1993). As extensions of these studies, comparative studies using LEC-IgG and anti-peptide antibodies against GlyCAM-1 have been carried out. Colloidal gold conjugates of LECIgG were used in a study (KIKUTA and ROSEN 1994). Light microscopic and electron microscopic observations using LEC-IgG mapped the localization of L-selectin ligands on the luminal surface of lymph node HEC as well as on the trans-Golgi network (TGN) and peripheral vesicles in the cytoplasm of HEC, while cis- and medial-Golgi cisterna were not stained. On the other hand, anti-GlyCAM-1 antibody bound to intra-

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cellular organelles responsible for biosynthesis (endoplasmic reticulum, Golgi apparatus, TGN, electron-lucent cytoplasmic vesicles, and some electron-dense lysosomal vesicles) but failed to bind to the luminal surface of HEC. The presence of active ligands detected by LEC-IgG on the luminal surface of HEC revealed a functional role for the ligands as cell-cell adhesion molecules. In contrast, the absence of GlyCAM-1 on the luminal surface coupled with its presence in the cytoplasmic vesicles suggests that GlyCAM-1 is not an adhesion molecule on the surface of HEV but predominantly a secreted molecule. If this is the case, CD34 and Sgp200 would account for the staining of LEC-IgG on the surface of HEC. However, the possibility that GlyCAM-1 has a peripheral association with plasma membranes with a weak affinity in an in vivo condition is not formally ruled out. A recent study demonstrated that GlyCAM-1 could work as a signaling molecule for lymphocytes, resulting in integrin activation on lymphocytes (HWANG et al. 1996; GIBLIN et al. 1997). This result may suggest a possibility that GlyCAM-1 mainly works as secreted soluble molecule from HEC. Intracellular distribution of ligands showing that GlyCAM-1 acquires ligand activity in TGN is consistent with the findings of a study of biosynthesis, in which criticalsulfation on GlyCAM-1 was shown to take place in the TGN as the last step of post-translational modification (CROMMIE and ROSEN 1995). The comparison of reactivity to LEC-IgG and to antibody against peptide core has also provided further information on the nature of ligands. These studies indicated that ligand glycoproteins fail to express ligand activity unless appropriate post-translational modifications take place. One example is GlyCAM-1 (DOWBENKO et al. 1993). Immunohistochemical observation using anti-GlyCAM-1 antibody revealed the presence of GlyCAM-1 in epithelial cells of lactating mouse mammary gland and in murine milk. However, these molecules are not sulfated and failed to bind to LEC-IgG. Another example is CD34 (FINA et al. 1990; BAUMHUETER et al. 1994). CD34 was generally detected on endothelial cells of blood vessels using polyclonal antibody against recombinant mouse CD34, whereas the binding of LEC-IgG was restricted to lymph node HEV (BAUMHUETER et al. 1994). GlyCAM-1 in mammary gland and CD34 on both endothelial cells other than HEV and hematopoietc progenitor cells (SIMMONS et al. 1992) clearly demonstrates the fact of their different types of glycoforms that lack ligand activity for L-selectin. Immunohistochemical studies on L-selectin ligands have also been carried out to investigate a process of leukocyte infiltration during pathological conditions. As a possible cause for lymphocyte and macrophage trafficking during the process of demyelinating diseases of the CNS, such as multiple screlosis in humans and experimental allergic encephalomyelitis (EAE) in animals, antigen independent mechanisms of leukocyte adhesion to myelin have been proposed in addition to the myelin basic protein-specific T cell response (HUANG et al. 1991). L-selectin dependent lymphocyte adhesion to cerebellar white matter has been described. In parallel with this, it has also been shown that LEC-IgG exhibits calcium-dependent binding to the white matter of mouse cerebellum.

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In the inflamed pancreas of non-obese diabetic (NOD) mice, some HEV-like vessels were shown to be positive for CD34 and GlyCAM-1, and were also shown to bind to LEC-IgG (BAUMHUETER et al. 1994). In an independent study, mAb MECA 79 (reactive to peripheral lymph node vascular addressin) stained several HEV-like vessels in an inflamed islet at later stages of insulitis (HANNINEN et al. 1993). Also reported was another experimental model for inflammation of pancreas developed by autoimmune processes. Transgenic mice expressing the simian virus 40 large T antigen (Tag) under the control of the rat insulin gene regulatory region were examined (ONRUST et al. 1996). In RIP1-TagS mice with delayed onset of Tag expression, an immune response against Tag was provoked, and intense infiltration of lymphocytes and macrophages into islets occurred. In RIP1-TagS pancreas, binding of LEC-IgG was demonstrated on the infiltrated islet vascular endothelium. Furthermore, binding of mAb MECA 79, polyclonal anti-mouse recombinant CD34 and polyclonal anti-GlyCAM-1 were also demonstrated. Some of the vessels exhibited HEV-like morphology. Interestingly, the presence of GlyCAM-1 was restricted to endothelial cells with an HEC-like morphology. The upregulation of binding sites for mAb MECA 367, which defines MAdCAM-1, was also demonstrated. These results suggest that interaction between L-selectin and its ligands (GlyCAM-1, CD34, MAdCAM-1) on endothelial cells is involved during the process of lymphocyte infiltration in pancreatic inflammation. The upregulation of MAdCAM-l also indicates that the adhesive interaction between a4~7 integrins on lymphocytes and MAdCAM-l on endothelial cells may also contribute to lymphocyte infiltration. These results clearly demonstrate that the use of endogenous lectin molecules (or genetically engineered recombinant molecules such as LEC-IgG) in combination with antibodies against core peptides of the corresponding ligand molecules (carbohydrate presenting molecules) is extremely useful for investigating ligands by immunohistochemical analysis. It is also important to check whether the binding specificity of endogenous lectins or recombinant ones is consistent with the binding specificity of the lectins naturally displayed on the cell surface or expressed in in vivo environments before using them as immunohistochemical probes. Sialidase sensitivity and calcium dependence for LEC-IgG binding to lymph node HEV are typical examples of such test.

11 Conclusions Although most work is presently being done with mammalian galectins, nonmammalian galectins are attracting increasing attention. Whereas it is easy to recognize the same lectin in different mammalian species because of the great conservation of amino acid sequence, it is difficult to infer the relationship particular mammalian lectins have to those found in lower vertebrates and invertebrates. The first invertebrate galectin to be discovered was the 32-kDa one in the nematode C. elegans. More recently, a prototype 16-kDa galectin from the same animal was also sequenced (HIRABAYASHI et al.

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1996). Other galectins were also found in sponges (PFEIFER et al. 1993). The detailed localization and function of 32-kDa galeetin in C. elegans are now under study. The nematode could be an excellent model animal for elucidating the role of galectin. The discovery of galeetins in invertebrates implies the fundamental roles of galectins in the animal body. As shown in this review, histochemistry and cytochemistry have revealed the dual localization of galectins, both inside and outside the cell, in various tissues at various developmental stages. The localization of the galeetins on plasma membrane, desmosome and extracellular matrix indicates their involvement in cell-cell interactions and in cell-extracellular matrix interactions. In the process of development, growth, differentiation, and morphogenesis, the galectins seem to play important roles in various tissues by recognizing the complementary glycoconjugates. Furthermore, in adult tissues galectins are involved in the maintenance of tissue structure and function. The existence of galectin in both nucleus and cytoplasm indicates other functions, such as cell growth regulation (CHIAROITTI et al. 1991; WELLS and MALLUCCI 1991), regulation of RNA transcription (MOUTSATSOS et al. 1987; JIA and WANG 1988; DAGHER et al. 1995), and nuclear import-export of glycoconjugates (NIGG et al. 1991; DUVERGER et al. 1993). While galeetins function both inside and outside the cells, most C-type lectins appear to work extracellularly as cell surface molecules, as secreted soluble molecules, or as components of extracellular matrices. Recent advances in the study of leukocyte endothelial adhesive interaction have provided an excellent example of specific carbohydrate recognition, in which cell surface selectins recognize defined carbohydrate motifs displayed on carbohydrate presenting molecules. Immunohistochemical studies using antibodies against selectins as well as those against their ligands have significantly contributed to our understanding of the physiological and pathological roles selectins and their ligands play. It should be noted that a recombinant soluble form of selectins (selectin-immunoglobulin chimera) was useful for detecting biologically active glycoform of selectin ligands. On the other hand, antibodies against the protein core of ligands could be useful for detecting an individual type of carbohydrate-presenting molecules (for example, GlyCAM-1), while the positive signals do not necessarily mean the presence of the biologically active glycoform. Immunohistochemical studies using antibodies against cell surface C-type lectins other than selectins have also provided basic information in elucidating biological roles of the endogenous lectins. Such studies have revealed the presence of a macrophage lectin on a subset of tissue macrophages and on tumorinfiltrating macrophages, and even on developing chondroblasts during embryogenesis. To further investigate the roles of the cell surface endogenous lectins, application of blocking antibodies (capable of interfering with lectin-carbohydrate ligand interaction), of the recombinant soluble form of the lectins, and of cells transfected with an expression vector encoding the endogenous lectin eDNA would all be useful (in addition to immunohistochemical analysis).

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Compared with the nucleic acids and proteins, the idiosyneracies of each specific function of many carbohydrates still remain to be unraveled. The interaction between the galectins and glycoconjugates should be one of the most basic mechanisms in biological regulations. Study of the animallectins may lead to decipherment of the glycocode. The application of histo- and cytochemistry together with biochemical and molecular biological techniques is very useful for elucidating of the role of animallectins.

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References ABBOlT, W. M., FEIZI, T.: Evidence that the 14 kDa soluble ~-galactoside-bindinglectin in man is encoded by a single gene. - Biochem. J. 259, 291-294 (1989). -: Soluble 14-kDa ~-galactoside-specificbovine lectin: evidence from mutagenesis and proteolysis that almost the complete polypeptide chain is necessary for integrity of the carbohydrate recognition domain. - ]. BioI. Chern. 266,5552-5557 (1991). ABBOlT, W. M., HOUNSELL, E. E, FEIZI, T.: Further studies of oligosaccharide recognition by the soluble 13 kDa lectin of bovine heart muscle. - Biochem. J. 252, 283-287 (1988). ABBOlT, W. M., MELLOR, A., EDWARDS, Y., FEIZI, T.: Soluble bovine galactose-binding lectin. cDNA cloning reveals the complete amino acid sequence and an antigenic relationship with the major encephalitogenic domain of myelin basic protein. - Biochem. J. 259, 283-290 (1989). AKIMOTO, Y., HlRABAYASHI,]., KASAl, K., HIRANO, H.: Expression of the endogenous 14-kDa ~-galactoside-binding lectin galectin in normal human skin. - Cell Tissue Res. 280, 1-10 (1995b). AKIMOTO, Y., KAWAKAMI, H., ODA, Y., OBINATA, A., ENDO, H., KASAl, K., HIRANO, H.: Changes in expression of the endogenous ~-galactoside-binding14 kDa lectin of chick embryonic skin during epidermal differentiation. - Exp. Cell Res. 199, 297-304 (1992). AKIMOTO, Y., OBINATA, A., ENDO, H., FURUKAWA, K., AOKI, D., NOZAWA, S., HIRANO, H.: Immunocytochemical localization of the protein reactive to human ~-1, 4-galactosyltransferase antibodies during chick embryonic skin differentiation. - Anat. Res. 243, 109-119 (1995c). AKIMOTO, Y., OBINATA, A., HlRABAYASHI,J., SAKAKURA, Y., ENDO, H., KASAl, K., HIRANO, H.: Secretion of endogenous 16-kDa ~-galactoside-bindinglectin from vitamin A-pretreated chick embryonic cultured skin. - Exp. Cell Res. 205, 251-260 (1993). -: Changes in expression of two endogenous ~-galactoside-bindingisolectins in the dermis of chick embryonic skin during development in vivo and in vitro. - Cell Tissue Res. 279, 3-12 (1995a). ALBRANDT, K., ORIDA, N. K., LIU, E- T.: An IgE-binding protein with a distinctive repetitive sequence and homology an IgG receptor. - Proc. Natl. Acad. Sci. USA 84, 6859-6863 (1987). ALLEN, H.]., SUCATO,D., GOlTSTINE,S., KISAILUS,E., NAVA,H., PETRELLI,N., CASTILLO,N., WILSON, D.: Localization of endogenous ~-galactoside-bindinglectin in human cells and tissues. - Tumor BioI. 12, 52-60 (1991). ARATA, Y., AKIMOTO, Y., HlRABAYASHI,J., KASAl, K., HIRANO, H.: An immunohistochemical study of the 32-kDa galectin (~-galactoside-bindinglectin) in the nematode Caenorhabditis elegans. - Histochemical J. 28, 201-207 (1996). ARATA, Y., HlRABAYASHI,]., KASAl, K.: The two lectin domains of the tandem-repeat 32-kDa galectin of the nematode Caenorhabditis elegans have different binding properties. Studies with recombinant protein. -]. Biochem. (Tokyo) 121,1002-1009 (1997). ASHWELL, G., HARFORD,J.: Carbohydrate-specific receptors of the liver. - Ann. Rev. Biochem. 51, 531-554 (1982). BARNES, D. M.: Orchestrating the sperm-egg summit. - Science 239, 1091-1092 (1988). BARONDES, S. H.: Lectins: Their multiple endogenous cellular functions. - Ann. Rev. Biochem. 50, 207-231 (1981). -: Soluble lectins: A new class of extracellular proteins. - Science 223, 1259-1264 (1984). -: Vertebrate Lectins: Properties and functions. - In: The Lectins (eds. I. E. LIENER, N. SHARON, and I.J. GOLDSTEIN), pp. 437-466 - Academic Press, Orlando, FL 1986. BARONDES, S. H., CASTRONOVO, v., COOPER, D. N. CUMMINGS, R. D., DRICKAMER, K., FEIZI, T., GIlT, M. A., HlRABAYASHI, J., HUGHES, C., KASAl, K., LEFFLER, H., LIU, E-T., LOTAN, R., MERCURIO, A. M., MONSIGNY, M., PILLAI, S., POIRER, E, RAz, A., RIGBY, P. W. I., RINI, J. M.,

w.,

74 . Y. Akimoto et al. WANG, ]. L.: Galectins: A family of animal ~-galactoside-binding lectins. - Cell 76, 597-598 (1994a). BARONDES, S. H., COOPER, D. N. W., GIIT, M. A., LEFFLER, H.: Galectins: Structure and function of a large family of animallectins. J. BioI. Chern. 269, 20807-20810 (1994b). BARONDES, S. H., HAYWOOD-REID, P. L.: Comparison of developmentally regulated lectins from three species of cellular slime mold. - Biochim. Biophys. Acta 550, 297-308 (1979). -: Externalization of an endogenous chicken muscle lectin with in vivo development. - ]. Cell BioI. 91,568-572 (1981). BAUMHUETER, S., DYBDAL, N., KYLE, c., LASKY, L. A.: Global vascular expression of murine CD34, a sialomucin-like endothelial ligand for L-selectin. - Blood 84, 2554-2565 (1994). BAUMHUETER, S., SINGER, M. S., HENZEL, W., HEMMERICH, S., RENZ, M., ROSEN, S. D., LASKY, L. A.: Binding of L-selectin to the vascular sialomucin CD34. - Science 262,436-438 (1993). BENSON, B., HAWGOOD, S., SCHILLING,l, CLEMENTS,]., DAMM, D., CORDELL, B., WHITE, R. T.: Structure of canine pulmonary surfactant apoprotein: cDNA and complete amino acid sequence. - Proc. Nat!. Acad. Sci. USA 82,6379-6383 (1985). BERG, E. L., MCEVOY, L. M., BERLIN, c., BARGATZE, R. F., BUTCHER, E. c.: L-selectin-mediated lymphocyte rolling on MAdCAM-l. - Nature (Lond.) 366,695-698 (1993). BEVAN, B. H., KILPATRICK, D. c., LISTON, W. A., HIRABAYASHI,l, KASAl, K.: Immunohistochemicallocalization of a ~-D-galactoside-binding lectin at the human maternofetal interface. - Histochem.J. 26,582-586 (1994). BEVILACQUA, M. P., STENGELIN, S., GIMBRONE, M. A.]., SEED, B.: Endothelial leukocyte adhesion molecule 1: an inducible receptor for neutrophils related to complement regulatory proteins and lectins. - Science (Wash.DC) 243, 1160-1165 (1989). BEYER, E. c., TOKUYASU, K. T., BARONDES, S. H.: Localization of an endogenous lectin in chicken liver, intestine, and pancreas. - J. Cell BioI. 82, 565-571 (1979). BEYER, E. c., ZWEIG, S. E., BARONDES, S. H.: Two lactose binding lectins from chicken tissues. - ]. BioI. Chern. 255, 4236-4239 (1980). BEZOUSKA, K., CRICHLOW, G. V., ROSE,]. M., TAYLOR, M. E. DRICKAMER, K.: Evolutionary conservation of intron position in a subfamily of genes encoding carbohydrate-recognition domains. J. BioI. Chern. 266, 11604-11609 (1991). BILYK, N., HOLT, P. G.: The surface phenotypic characterization of lung macrophages in C3H/HeJ mice. - Immunology 74,645-651 (1991). BOGGARAM, v., QING, K., MENDELSON, C. R.: The major apoprotein of rabbit pulmonary surfactant. Elucidation of primary sequence and cyclic AMP and developmental regulation. - ]. BioI. Chern. 263, 2939-2947 (1988). BOLS, N. c., ROBERSON, M. M., HAYWOOD-REID, P. L., CERRA, R. F., BARONDES, S. H.: Secretion of a cytoplasmic lectin from Xenopus laevis skin. - ]. Cell. BioI. 102, 492-499 (1986). BREEL, M., VAN DER ENDE, M., SMINIA, T., KRAAL, G.: Subpopulations of lymphoid and nonlymphoid cells in bronchus-associated lymphoid tissue (BALT) of the mouse. - Immunology 63,657-662 (1988). BRILES, E. B., GREGORY, W., FLETCHER, P., KORNFELD, S.: Vertebrate lectins. Comparison of properties of ~-galactoside-bindinglectins from tissues of calf and chicken. - J. Cell BioI. 81, 528537 (1979). BRISKIN, M.l, MCEVOY, L. M., BUTCHER, E. c.: MAdCAM-1 has homology to immunoglobulin and mucin-like adhesion receptors and to IgAl. - Nature (Lond.) 363,461-464 (1993). CAMERINI, D., JAMES, S. P., STAMENKOVIC, 1., SEED, B.: Leu-8/TQl is the human equivalent of the Mel-14 lymph node homing receptor. - Nature (Lond.) 342,78-82 (1989). CARON, M., BLADIER, D., JOUBERT, R.: Soluble galactoside-binding vertebrate lectins: a protein family with common properties. - Int. J. Biochem. 22, 1379-1385 (1990).

Endogenous animal lectins . 75

CASTRONOVO, V., LUYTEN, E, VAN DEN BRULE, E, SOBEL, M. E.: Identification of a 14-kDa laminin binding protein (HLBP14) in human melanoma cells that is identical to the 14-kDa galactoside binding lectin. - Arch. Biochem. Biophys. 297, 132-138 (1992). CERRA, R.E, HAYWOOD-REID, P.L., BARONDES, S.H.: Endogenous mammalian lectin localized extracellularly in lung elastic fibers. - ]. Cell BioI. 98, 1580-1589 (1984). CHAN, P.-Y., TAKEI,E: Molecular cloning and characterization of a novel murine T cell surface antigen, YE1/48 1. - ] . Immunol.142, 1727-1736 (1989). CHANG, C., RODRIGUEZ, A., CARRETERO, M., LOPEZ-BoTET, M., PHILLIPS,]. H., LANIER, L. L.: Molecular characterization of human CD94: a type II membrane glycoprotein related to the C-type lectin superfamily. - Eur. J. Immunol. 25, 2433-2437 (1995). CHERAYIL, B.J, CHAITOVITZ, S., WONG, C, PILLAI, S.: Molecular cloning of a human macrophage lectin specific for galactose. - Proc. Natl. Acad. Sci. USA 87, 7324-7328 (1990). CHERAYIL, B. J., WEINER, S. J., PILLAI, S.: The Mac-2 antigen is a galactose-specific lectin that binds IgE. - J. Exp. Med. 170, 1959-1972 (1989). CHIARIOTTI, L., WELLS, v., BRUNI, C B., MALLUCCI, L.: Structure and expression of the negative growth factor mouse p-galactoside binding protein gene. - Biochim. Biophys. Acta (Arnst.) 1089,54-60 (1991). CHIU, M. L., PARRY, D. A. D., FELDMAN, S. R., KLAPPER, D. G., O'KEEFE, E.J.: An adherens junction protein is a member of the family of lactose-binding lectins. - ]. BioI. Chern. 269, 3177031776 (1994). CHOU, T-Y., DANG, C v., HART, G. W.: Glycosylation of the c-Myc transactivation domain. Proc. Natl. Acad. Sci. USA 92,4417-4421 (1995). CLERCH, L. B., WHITNEY, P., HASS, M., BREW, K., MILLER, T, WERNER, R., MASSARO, D.: Sequence of a full-length cDNA for rat lung p-galactoside-binding protein: Primary and secondary structure of the lectin. - Biochemistry 27, 692--699 (1988). COOPER, D. N. W., BARONDES, S. H.: Evidence for export of a muscle lectin from cytosol to extracellular matrix and for a novel secretory mechanism. - J. Cell. BioI. 110, 1681-1691 (1990). COOPER, D. N. W., MASSA, S. M., BARONDES, S. H.: Endogenous muscle lectin inhibits myoblast adhesion to laminin. - J. Cell BioI. 115, 1437-1448 (1991). COOPER, D. N. W., BOULIANNE, R. P., CHARLTON, S., FARRELL, E. M., SUCHER, A., Lu, B. C.: Fungal galectins, sequence and specificity of two isolectins from Coprinus cinereus. - ]. BioI. Chern. 272,1514-1521 (1997). COURAUD, P.-O., CASENTINI-BOROCZ, D., BRINGMAN, TS., GRIFFITH,]., MCGROGAN,M., NEDWIN, G. E.: Molecular cloning, characterization, and expression of a human 14-kDa lectin. - ]. BioI. Chern. 264,1310-1316 (1989). CRAIG, S. S., KRISHNASWAMY, P, IRANI, A-M. A., KEPLEY, C L., LIU, E-T, SCHWARTZ, L. B.: Immunoelectron microscopic localization of galectin-3, an IgE binding protein, in human mast cells and basophils. - Anat. Rec. 242, 211-219 (1995). CROCKER, P. R., FREEMAN, S., GORDON, S., KELM, S.: Sialoadhesin binds preferentially to cells of the granulocyte lineage. - J. Clin. Invest. 95,635-643 (1995). CROCKER, P. R., GORDON, S.: Properties and distribution of a lectin-like hemagglutinin differentially expressed by murine stromal tissue macrophages. - ]. Exp. Med. 164, 1862-1875 (1986). -: Mouse macrophage hemagglutinin (sheep erythrocyte receptor) with specificity for sialylated glycoconjugates characterized by a monoclonal antibody. - ]. Exp. Med.169, 1333-1346 (1989). CROCKER, P. R., KELM, S., DUBOIS, C, MARTIN, B., MCWILLIAM, A. S., SHorrON, D. M., PAULSON, ]. C, GORDON, S.: Purification and properties of sialoadhesin, a sialic acid-binding receptor of murine tissue macrophages. - EMBO J.lO, 1661-1669 (1991). CROCKER, P. R., MUCKLOW, S., BOUCKSON, v., MCWILLIAM, A., WILLIS, A. C., GORDON, S., MILON, G., KELM, S., BRADFIELD, P.: Sialoadhesin, a macrophage sialic acid binding receptor

76 . Y. Akimoto et al.

for haemopoietic cells with 17 immunoglobulin-like domains. - EMBO J. 13, 4490-4503 (1994). CROMMIE, D., ROSEN, S.D.: Biosynthesis of GlyCAM-1, a mucin-like ligand for L-selectin. - J. BioI. Chern. 270, 22614-22624 (1995). DAGHER, S. E, WANG, J. L., PATI'ERSON, R.J.: Identification of galectin-3 as a factor in pre-mRNA splicing. - Proc. Natl. Acad. Sci. USA 92,1213-1217 (1995). DANGUY, A., KAYSER, K., BOVIN, N. v., GABIUS, H.-J.: The relevance of neoglycoconjugates for histology and pathology. - Trends in Glycosci. Glycotech. 7, 261-275 (1995). DEN, H., MALINZAK, D. A.: Isolation and properties of ~-D-galactoside-specificlectin from chick embryo thigh muscle. - J. BioI. Chern. 252,5444-5448 (1977). DE WAARD, A., HICKMAN, S., KORNFELD, S.: Isolation and properties of ~-galactosidebinding lectins of calf heart and lung. - J. BioI. Chern. 251, 7581-7587 (1976). DHOUAILLY, D., ROGERS, G. E., SENGEL, P.: The specification of feather and scale protein synthesis in epidermal-dermal recombinations. - Dev. BioI. 65, 58-68 (1978). DOEGE, K., FERNANDEZ, P., HASSELL, J. R., SASAKI, M., YAMADA, Y.: Partial cDNA sequence encoding a globular domain at the C-terminus of the rat cartilage proteoglycan. - J. BioI. Chern. 261, 8108-8111 (1986). DOEGE,K., SASAKI,M., HORIGAN, E., HASSELL,J.R., YAMADA, Y: Complete primary structure of the rat cartilage proteoglycan core protein deduced from cDNA clones. - J. BioI. Chern. 262, 17757-17767 (1987). DOEGE, K.J., SASAKI,M., KIMURA, T., YAMADA, Y.: Complete coding sequence and deduced primary structure of the human cartilage large aggregating proteoglycan, aggrecan: Humanspecific repeats, and additional alternatively spliced forms. - J. BioI. Chern. 266, 894-902 (1991). DOWBENKO, D., KIKUTA, A., FENNIE, c., GILLETI', N., LASKY, L. A.: Glycosylation-dependent cell adhesion molecule 1 (GlyCAM 1) mucin is expressed by lactating mammary gland epithelial cells and is present in milk. - J. Clin. Invest. 92, 952-960 (1993). DRICKAMER, K.: Two distinct classes of carbohydrate-recognition domains in animallectins. - J. BioI. Chern. 263, 9557-9560 (1988). -: Engineering galactose-binding activity into a C-type mannose-binding protein. - Nature (Lond.)360, 183-186 (1992). DRICKAMER, K., TAYLOR, M. E.: Biology of animallectins. - Annu. Rev. Cell BioI. 9, 237-264 (1993). DRICKAMER, K., DORDAL, M. S., REYNOLDS, L.: Mannose-binding proteins isolated from rat liver contain carbohydrate-recognition domains linked to collagenous tails. Complete primary structures and homology with pulmonary surfactant apoprotein. - J. BioI. Chern. 261, 68786887 (1986). DRICKAMER, K., MAMON,J. E, BINNS, G., LEUNG,J. 0.: Primary structure of the rat liver asialoglycoprotein receptor. Structural evidence for multiple polypeptide species. - J. BioI. Chern. 259, 770-778 (1984). DUVERGER, E., CARPENTIER, V., ROCHE, A.-C., MONSIGNY, M.: Sugar-dependent nuclear import of glycoconjugates from the cytosol. - Exp. Cell Res. 207, 197-201 (1993). EISENBARTH, G. S., RUFFOLO, R. R., WALHSH, E S., NIERENBERG, M.: Lactose sensitive lectin of chick retina and spinal cord. - Biochem. Biophys. Res. Commun. 83, 1246-1252 (1978). ENGEL, P., NOJIMA, Y, ROTHSTEIN, D., ZHOU, L.-J., WILSON, G. L., KEHRL, J. H., TEDDER, T. E: The same epitope on CD22 of B lymphocytes mediates the adhesion of erythrocytes, T and B lymphocytes, neutrophils, and monocytes. - J. Immunol. 150, 4719-4732 (1993). EZEKOWITZ, R. A. B., SASTRY, K., BAILLY, P. WARNER, A.: Molecular characterization of the human macrophage mannose receptor: demonstration of multiple carbohydrate recognition-like domains and phagocytosis of yeasts in Cos-1 cells. - J. Exp. Med. 172, 1785-1794 (1990).

Endogenous animal leetins . 77

FINA, L., MOLGAARD, H. V., ROBERTSON, D., BRADLEY, N. J., MONAGHAN, P., DELIA, D., SUTHERLAND, D. R., BAKER, M. A., GREAVES, M. E: Expression of the CD34 gene in vascular endothelial cells. - Blood 75,2417-2426 (1990). FLOROS,J., STEINBRINK, R., JACOBS, K., PHELPS, D., KRIZ, R., RECNY, M., SULTZMAN, L., JONES, S., TAEUSCH, H. W., FRANK, H. A., FRITSCH, E. E: Isolation and characterization of eDNA clones for the 35-kDa pulmonary surfactant-associated protein. - J. BioI. Chern. 261, 9029-9033 (1986). FOXALL, C., WATSON, S.R., DOWBENKO, D., FENNIE, C., LASKY, L.A., KISO, M., HASEGAWA, A., ASA, D., BRANDLEY, B. K.: The three members of the selectin receptor family recognize a common carbohydrate epitope, the sialyl Lewis' oligosaccharide. - J. Cell. BioI. 117, 895-902 (1992). FREEMAN, S. D., KELM, S., BARBER, E. K., CROCKER, P. R.: Characterization of CD33 as a new member of the sialoadhesin family of cellular interaction molecules. - Blood 85, 2005-2012 (1995). GABIUS, H.-J.: Animallectins. - Europ. J. Biochem. 243, 543-576 (1997). GABIUS, H.-J., BARDOSI,A.: Neoglycoproteins as tools in glycohistochemistry. - Prog. Histochern. Cytochem. 22,1-16, (1991). GABIUS, H.-J., BREHLER, R., SCHAUER, A., CRAMER, E: Localization of endogenous lectins in normal human breast, benign breast lesions and mammary carcinomas. - Virchows Arch. [B] 52, 107-115 (1986a). GABIUS, H.-J., ENGELHARDT, R., REHM, S., BARONDES, S. H., CRAMER, E: Presence and relative distribution of the endogenous ~-galactoside-specificlectins in different types of rat tumors. Cancer J. 1, 19-21 (1986b). GALLATIN, W. M., WEISSMAN, 1. L., BUTCHER, E. c.: A cell-surface molecule involved in organspecific homing of lymphocytes. - Nature (Lond.) 304,30-34 (1983). GEOFFROY, J. S., ROSEN, S. D.: Demonstration that a lectin-like receptor (gp90 MEL) directly mediates adhesion of lymphocytes to high endothelial venules of lymph nodes. - J. Cell BioI. 109, 2463-2469 (1989). GIBLIN, P. A., HWANG, S. T, KATSUMOTO, T R., ROSEN, S. D.: Ligation of L-selectin on T lymphocytes activates ~ 1 integrins and promotes adhesion to fibronectin. - J. Immunol. 159, 3498-3507 (1997). GIGA, Y., IKAI, A., TAKAHASHI, K.: The complete amino acid sequence of Echinoidin, a lectin from the coelomic fluid of the sea urchin Anthocidaris crassispina. Homologies with mammalian and insect lectins. - J. BioI. Chern. 262, 6197-6203 (1987). GIORDA, R., RUDERT, W. A., VAVASSORI, C., CHAMBERS, W. H., HISERODT, J. C., TRUCCO, M.: NKR- PI, a signal transduction molecule on natural killer cells. - Science 249, 1298-1300 (1990). GIll, M. A., BARONDES, S. H.: Evidence that a human soluble ~-galactoside-binding lectin is encoded by a family of genes. - Proc. Natl. Acad. Sci. USA 83, 7603-7607 (1986). -: Genomic sequence and organization of two members of a human lectin gene family. - Biochemistry 30,82-89 (1991). GITT, M. A., COLNOT, C., XIA, Y. R., ATCHISTON, R. E., LusIs, A.J., PIORIER, E, BARONDES, S. H., LEFFLER, H.: Galectin-6: a new mammalian galectin. - Glycoconjugate J. 12 (Abstracts of WIIIth International Symposium on Glycoconjugates), 548 (1995b). GIll, M. A., MASSA, S. M., LEFFLER, H., BARONDES, S. H.: Isolation and expression of a gene encoding L-14-II, a new human soluble lactose-binding lectin. -]. BioI. Chern. 267, 1060110606 (1992). GIll, M.A., WISER, M.E, LEFFLER, H., HERRMANN,J., XIA, Y.-R., MASSA, S.M., COOPER, D. N. W., LUSIs, A.J., BARONDES, S. H.: Sequence and mapping of galectin-5, a ~-galactoside­ binding lectin, found in rat erythrocytes. - ]. BioI. Chern. 270, 5032-5038 (1995a). GOLDSTEIN, 1.]., HUGHES, R. c., MONSIGNY, M., OSAWA, T, SHARON, N.: What should be called a lectin? - Nature (Lond.), 285, 66 (1980).

78 . Y. Akimoto et al.

GORDON, S., LAWSON, L., RABINOWITZ, S., CROCKER, P. R., MORRIS, L., PERRY, V. H.: Antigen markers of macrophage differentiation in murine tissues. - Curro Top. Microbiol. Immunol. 181,1-37(1992). GRITZMACHER, C. A., MEHL, V. S., LIU, E-T.: Genomic cloning of the gene for an IgE-binding lectin reveals unusual utilization of 5' untranslated regions. - Biochemistry 31,9533-9538 (1992). GUTHMANN, M. D., TAL, M., PECHT, I.: A secretion inhibitory signal transduction molecule on mast cells is another C-type lectin. - Proc. Natl. Acad. Sci. USA 92,9397-9401 (1995). HADARI, Y. R., PAZ, K., DEKEL, R., MESTROVIC, T., ACCILI, D., ZICK, Y.: Galectin-8. A new rat lectin, related to galectin-4. - J. BioI. Chern. 270, 3447-3453 (1995). HALBERG, D.E, WAGER, R.E., FARRELL, D.C., HILDRETH, J.IV., QUESENBERRY, M.S., LOEB, J. A., HOLLAND, E. c., DRICKAMER, K.: Major and minor forms of the rat liver asialoglycoprotein receptor are independent galactose-binding proteins. Primary structure and glycosylation heterogeneity ofminor receptor forms. - J. BioI. Chern. 262,9828-9838 (1987). HALTIWANGER, R. S., HILL, R. L.: The isolation of a rat alveolar macrophage lectin. - J. BioI. Chern. 261,7440-7444 (1986a). -: The ligand binding specificity and tissue localization of a rat alveolar macrophage lectin. - J. BioI. Chern. 261,15696-15702 (1986b). HALTIWANGER, R. S., LEHRMAN, M. A., ECKHARDT, A. E., HILL, R. L.: The distribution and localization of the fucose-binding lectin in rat tissues and the identification of a high affinity form of the mannose/N-acetylglucosamine-binding lectin in rat liver. - J. BioI. Chern. 261, 7433-7439 (1986). HARRIS, N., PETERS, L. L., EICHER, E. M., RITS, M., RASPBERRY, D., EICHBAUM, Q. G., SUPER, M., EZEKOWITZ, A. B.: The exon-intron structure and chromosomal localization of the mouse macrophage mannose receptor gene Mrel: Identification of a Ricin-like domain at the N-terminus of the receptor. - Biochem. Biophys. Res. Commun. 198, 682-692 (1994). HARRIS, N., SUPER, M., RITS, M., CHANG, G., EZEKOWITZ, A. B.: Characterization of the murine macrophage mannose receptor: demonstration that the downregulation of receptor expression mediated by interferon-y occurs at the level of transcription. - Blood 80, 2363-2373 (1992). HARRISON, E L., WILSON, T.J. G.: The 14 kDa I)-galactoside binding lectin in myoblast and myotube cultures: localization by confocal microscopy. - J. Cell. Sci. 101, 635-646 (1992). HART, G. W.: Dynamic O-linked glycosylation of nuclear and sytoskeletal proteins. - Annu. Rev. Biochem. 66, 315-335 (1997). HART, G. W., HALTIWANGER, R. S., HOLT, G. D., KELLY, W. G.: Glycosylation in the nucleus and cytoplasm. - Ann. Rev. Biochem. 58, 841-874 (1989). HASHIMOTO, M., NIWA, 0., NITTA, Y., TAKEICHI, M., YOKORO, K.: Unstable expression ofE-cadherin adhesion molecules in metastatic ovarian tumor cells. - Jpn. J. Cancer Res. 80, 459-463 (1989). HEMMERICH, S., BERTOZZI, C. R., LEFFLER, H., ROSEN, S. D.: Identification of the sulfated monosaccharides of GlyCAM-1, an endothelial-derived ligand for L-selectin. - Biochemistry 33, 4820-4829 (1994a). HEMMERICH, S., BUTCHER, E. c., ROSEN, S. D.: Sulfation-dependent recognition of high endothelial venules (HEV)-ligands by L-selectin and MECA 79, an adhesion-blocking monoclonal antibody. - J. Exp. Med. 180, 2219-2226 (1994b). HEMMERICH, S., LEFFLER, H., ROSEN, S.D.: Structure of the O-glycans in GlyCAM-1, an endothelial-derived ligand for L-selectin. - J. BioI. Chern. 270, 12035-12047 (1995). HEMMERICH, S., ROSEN, S. D.: 6' -sulfated sialyl Lewis x is a major capping group of GlyCAM-l. - Biochemistry 33,4830-4835 (1994). HERRMANN, J., TURCK, C. W., ATCHISON, R. E., HUFLEJT, M. E., POULTER, L., GITT, M. A., BURLINGAME, A. L., BARONDES, S. H., LEFFLER, H.: Primary structure of the soluble lactose binding lectin L-29 from rat and dog and interaction of its non-collagenous proline-, glycine-, tyrosinrich sequence with bacterial and tissue collagenase. - J. BioI. Chern. 268, 26704-26711 (1993).

Endogenous animal leetins . 79

HlRABAYASHI,].: A general comparison of two major families of animallectins. - Trends in Glycosci. Glycotech. 5, 251-270 (1993). -: Two distince families of animallectins: speculations on their raisons d'etre. - In: Lectins, Biology, Biochemistry, Clinical Biochemistry (VAN DRIESSCHE, E., FISCHER,J., BEECKMAN, S., BOGHANSEN, T C, eds), vol. 10, pp. 205-219 - Textop, Hellerup, Denmark 1994. HlRABAYASHI,J., AYAKI, H., SOMA, G., KASAl, K.: Cloning and nucleotide sequence of a full-length cDNA for human 14 kDa ~-galactoside-bindinglectin. - Biochim. Biophys. Acta 1008, 85-91 (1989). HIRABAYASHI,J., KASAl, K.: Human placenta ~-galactopside-bindinglectin. Purification and some properties. - Biochem. Biophys. Res. Commun. 122, 938-944 (1984). -: Complete amino acid sequence of a ~-galactoside-binding lectin from human placenta. - ]. Biochern. 104, 1-4 (1988). -: Effect of amino acid sustitution by site-directed mutagenesis on the carbohydrate recognition and stability of human 14 kDa ~-galactoside-bindinglectin. - ]. BioI. Chern. 266, 23648-23653 (1991 ). -: The family of metazoan metal-independent ~-galactoside-bindinglectins: structure, function and molecular evolution. - Glycobiology 3,297-304 (1993). -: Further evidence by site-directed mutagenesis that conserved hydrophilic residues form a carbohydrate-binding sites of human galectin-1. - Glycoconjugate J. 11, 437-442 (1994). HIRABAYASHI,J., KAWASAKI, H., SUZUKI, K., KASAl, K.: Complete amino acid sequence of 14-kDa ~-galactoside-bindinglectin of chick embryo. - ]. Biochem.101, 775-787 (1987b). HIRABAYASHI, J., ODA, Y., OOHARA, T, YAMAGATA, T, KASAl, K.: Immunological cross-reactivity between ~-galactoside-binding lectins of vertebrates. Possible relationship of antigenicity to oligomeric structure. - Biochim. Biophys. Acta (Arnst.) 916,321-327 (1987a). HIRABAYASHI, ]., SATOH, M., KASAl, K.: Evidence that Caenorhabditis elegans 32-kDa ~-galac­ toside-binding protein is homologous to vertebrate ~-galactoside-bindinglectins: cDNA cloning and deduced amino acid sequence. -]. BioI. Chern. 267,15485-15490 (1992b). HIRABAYASHI,]., SATOH, M., OHYAMA, Y., KASAl, K.: Purification and characterization of ~-galacto­ side-binding proteins from Caenorhabditis elegans. - ]. Biochem. (Tokyo) 111, 553-555 (1992a). HIRABAYASHI,J., UBUKATA, T., KASAl, K.: Purification and molecular characterization of a novel 16kDa galcetin from the nematode Caenorhabditis elegans. - J. BioI. Chern. 271, 2497-2505 (1996). HIRANI, S., LAMBRIS,]. D., MULLER-EBERHARD, H.].: Localization of the conglutinin binding site on the third component of human complement. -]. Immunol.134, 1105-1109 (1985). HIRANO, H., AKIMOTO, Y., KAWAKAMI, H., ODA, Y., KASAl, K.: Localization of endogenous ~-ga­ lactoside-binding lectin in embryonic chick epidermis. - In: Glycoconjugates in Medicine (OHYAMA, M., MURAMATsu, T, eds.), pp. 8-13. - Professional Postgraduate Services, Tokyo 1988. HIRANO, H., OISHI, N., OBINATA, A., ENDO, H.: Metaplastic changes in chick embryonic skin induced by vitamin A. - In: Basement Membranes (SHIBATA, S., ed), pp. 395-406. - Elsevier Science Publishers B.Y., Amsterdam 1985. Ho, S.-C, MALEK-HEDAYAT, S., WANG,]. L., SCINDLER, M.: Endogenous lectins from cultured soybean cells: isolation of a protein immunologically cross-reactive with seed soybean agglutinin and analysis of its role in binding of Rhizobium japonicum. - J. Cell. BioI. 103, 1043-1054 (1986). HOLLAND, E. c, LEUNG,J. 0., DRICKAMER, K.: Rat liver asialoglycoprotein receptor lacks a cleavable NHz-terminal signal sequence. - Proc. Nat!. Acad. Sci. USA 81,7338-7342 (1984). HOLMSKOV, U., TEISNER, B., PEDERSEN, N. T, LAURSEN, S. B., RASMUSSEN, H. B., JENSENIUS,]. C: Tissue localization of conglutinin, a bovine C-type lectin. - Immunology 76,169-173 (1992). HOLT, G. D., HART, G. W.: The subcellular distribution of terminal N-acetylglucosamine moieties. Localization of a novel protein-saccharide linkage, O-linked GIcNAc. - J. BioI. Chern. 261,8049-8057 (1986).

80 . Y. Akimoto et al.

HOLTON, l L., KENNY, T. P., LEGAN, P. K., COLLINS, l E., KEEN, IN., SHARMA, R., GARROD, D. R.: Desmosomal glycoproteins 2 and 3 (desmocollins) show N-terminal similarity to calcium-dependent cell-cell adhesion molecules. - l Cell Sci. 97, 239-246 (1990). HosoI, l, MURPHY, G. E, EGAN, C. L., LERNER, E. A., GRABBE, S., ASAHINA, A., GRANSTEIN, R. D.: Regulation of Langerhans cell function by nerves containing calcitonin gene-related peptide. - Nature (Lond.), 363, 159-163 (1993). HOUCHINS, l P., YABE, T., MCSHERRY, c., BACH, E H.: DNA sequence analysis of NKG2, a family of related cDNA clones encoding type II integral membrane proteins on human natural killer cells. - l Exp. Med. 173, 1017-1020 (1991). Hoyle, G. W., Hill, R. L.: Molecular cloning and sequencing of a cDNA for a carbohydrate binding receptor unique to rat Kupffer cells. - l BioI. Chern. 263, 7487-7492 (1988). -: Structure of the gene for a carbohydrate-binding receptor unique to rat Kupffer cells. - l BioI. Chern. 266, 1850-1857 (1991). HUANG, K., GEOFFROY, J. S., SINGER, M. S., ROSEN, S. D.: A lymphocyte homing receptor (Lselectin) mediates the in vitro attachment of lymphocytes to myelinated tracts of the central nervous system. - l Clin. Invest. 88, 1778-1783 (1991). HWANG, S. T., SINGER, M. S., GIBLIN, P. A., YEDNOCK, T. A., BACON, K. B., SIMON, S. I., ROSEN, S. D.: GlyCAM-l, a physiologic ligand for L-selectin, activates ~2 integrins on naive peripheral lymphocytes. - l Exp. Med. 184, 1343-1348 (1996). HYNES, M. A., BUCK, L. B., CASANO, E I., HUANG, K. K., BARONDES, S. H., JESSELL, T. M.: Cloning and expression of a soluble 14-kDa ~-galactoside binding lectin in rat nervous system. - Soc. Neurosci.14, 71 (1988). HANNINEN, A., TAYLOR, C., STREETER, P. R., STARK, L. S., SARTE, l M., SHIZURU, l A., SIMELL, 0., MICHIE, S. A.: Vascular addressins are induced on islet vessels during insulitis in nonobese diabetic mice and are involved in lymphoid cell binding to islet endothelium. - J. Clin. Invest. 92, 2509-2515 (1993). !cHII, S., IMAI, Y., IRIMURA, T.: Tumor site-selective localization of an adoptively transferred T cell line expressing a macrophage lectin. - J. Leukoc. BioI. 62,761-770 (1997). II, M., KURATA, H., !TOH, N., YAMASHINA, I., KAWASAKI, T.: Molecular cloning and sequence analysis of cDNA encoding the macrophage lectin specific for galactose and N-acetylgalactosamine. - J. BioI. Chern. 265, 11295-11298 (1990). IKUTA, K., TAKAMI, M., KIM, C. W., HONIO, T., MIYOSHI, T., TAGAYA, Y., KAWABE, T., YODOI, l: Human lymphocyte Fc receptor for IgE: Sequence homology of its cloned cDNA with animal lectins. - Proc. Nat!. Acad. Sci. USA 84, 819-823 (1987). IMAI, Y., AKIMOTO, Y., MIZUOCHI, S., KIMURA, T., HIRANO, H., IRIMURA, T.: Restricted expression of galactoseIN-acetylgalactosamine-specific macrophage C-type lectin to connective tissue and to metastatic lesions in mouse lung. - Immunology 86,591-598 (1995). IMAI, Y., IRIMURA, T.: Quantitative measurement of carbohydrate binding activity of mouse macrophage lectin. - l Immunol. Meth.I71, 23-31 (1994). IMAI, Y., LASKY, L. A., ROSEN, S. D.: Further characterization of the interaction between L-selectin and its endothelial ligands. - Glycobiology 2,373-381 (1992). -: Sulphation requirement for GlyCAM-l, an endothelial ligand for L-selectin. - Nature (Lond.) 36~ 555-557(1993~

IMAI, Y., ROSEN, S. D.: Direct demonstration of heterogenous, sulfated O-linked carbohydrate chains on an endothelial ligand for L-selectin. - Glycoconjugate l 10, 34-39 (1993). IMAI, Y., SINGER, M. S., FENNIE, c., LASKY, L. A., ROSEN, S. D.: Identification of a carbohydratebased endothelial ligand for a lymphocyte homing receptor. - J. Cell BioI. 113, 1213-1221 (1991). IMAI, Y., TRUE, D. D., SINGER, M. S., ROSEN, S. D.: Direct demonstration of the lectin activity of gp90MEL, a lymphocyte homing receptor. - l Cell BioI. 111, 1225-1232 (1990).

Endogenous animallectins . 81

ISHIBASHI, S., HAMMER, R. E., HERZ, J.: Asialoglycoprotein receptor deficiency in mice lacking the minor receptor subunit. - J. BioI. Chern. 269, 27803-27806 (1994). ISHII, N., WADSWORTH, W.G., STERN, B.D., CULOTTI, J.G., HEDGECOCK, E.M.: UNC-6, a laminin-related protein, guides cell and pioneer axon migrations in C. elegans. - Neuron 9, 873881 (1992). ISHIZAKI,J., HANASAKI, K., HIGASHINO, K., KISHINO,]., KIKUCHI, N., OHARA,O., ARITA, H.: Molecular cloning of pancreatic group I phospholipase A 2 receptor. - J. BioI. Chern. 269, 58975904 (1994). !To, K., SHINOMURA, T., ZAKO, M., UIITA, M., KIMATA, K.: Multiple forms of mouse PG-M, a large chondroitin sulfate proteoglycan generated by alternative splicing. - J. BioI. Chern. 270, 958965 (1995). JACKMAN, R. W., BEELER, D. L., FRITZE, L., SOFF, G., ROSENBERG, R. D.: Human thrombomodulin gene is intron depleted: Nucleic acid sequences of the cDNA and gene predict protein structure and suggest sites of regulatory control. - Proc. Nat!. Acad. Sci. USA 84, 6425-6429 (1987). JADOT, M., CANFIELD, W. M., GREGORY, W., KORNFELD, S.: Characterization of the signal for rapid internalization of the bovine mannose 6-phosphate/insulin-like growth factor-II receptor. - J. BioI. Chern. 267,11069-11077 (1992). JIA, S., WANG, J. L.: Carbohydrate binding protein 35: complementary DNA sequence reveals homology with proteins of the heterogeneous nuclear RNP. - J. BioI. Chern. 263, 6009-6011 (1988). JIANG, W., SWIGGARD, W.J., HEUFLER, C., PENG, M., MIRZA, A., STEINMAN, R. M., NUSSENZWEIG, M. c.: The receptor DEC-205 expressed by dendritic cells and thymic epithelial cells is involved in antigen processing. - Nature (Lond.) 375, 151-155 (1995). JOHNSTON, G. 1., COOK, R. G., McEVER, R.P.: Cloning of GMP-140, a granule membrane protein of platelets and endothelium: sequence similarity to proteins involved in cell adhesion and inflammation. - Cell 56, 1033-1044 (1989). KAPPRELL, H.-P., COWIN, P., FRANKE, W. W., PONSTING, H.: Biochemical characterization of desmosomal proteins isolated from bovine muzzle epidermis: amino acid and carbohydrate composition. - Europ. J. Cell BioI. 36,217-229 (1985). KARLHOFER, EM., RIBAUDO, R. K., YOKOYAMA, W. E.: MHC class I alloantigen specificity of Ly49+ IL-2-activated natural killer cells. - Nature (Lond.) 358,66-70 (1992). KASAl, K.: Biochemical properties of vertebrate 14K I)-galactoside-binding lectins. - In: Advances in Lectin Research (FRANZ, H., KASAl, K., KOCOUREK,]., OLSNES, S., SHANNON, L. M., eds.), Vol. 3, pp 10-35 - VEB Verlag Volk und Gesundheit, Berlin 1990. KASAl, K., HlRABAYASHI,J.: Galectins: a family of animallectins that decipher glycocodes. - J. Biochern. 119, 1-8 (1996). KAWAKAMI, K., YAMAMOTO, K., TOYOSHIMA, S., OSAWA, T., IRIMURA, T.: Dual function of macrophage galactose/N-acetylgalactosamine-specific lectins: Glycoprotein uptake and tumoricidal cellular recognition. - Jpn. J. Cancer Res. 85,744-749 (1994). KAWAI, N., NISHIYAMA, E, HIRANO, H.: Changes of lectin-binding on the embryonic muscle cell surface in the developing ascidian. Halocynthia aurantium. - Exp. Cell Res. 122, 293-304 (1979). KIKUTA, A., ROSEN, S. D.: Localization of ligands for L-selectin in mouse peripheral lymph node high endothelial cells by colloidal gold conjugates. - Blood 84, 3766-3775 (1994). KIKUTANI, H., INUI, S., SATO, R., BARSUMIAN, E. L., OWAKI, H., YAMASAKI, K., KAISHO, T., UCHIBAYASHI, N., HARDY, R. R., HIRANO, T., TSUNASAWA, S., SAKIYAMA, E, SUEMURA, M., KISHIMOTO, T.: Molecular structure of human lymphocyte receptor for immunoglobulin E. - Cell 47, 657-665 (1986).

82 . Y. Akimoto et al.

KIM, S.}., RUIZ, N., BEZOUSKA, K., DRICKAMER, K.: Organization of the gene encoding the human macrophage mannose receptor (MRC1). - Genomics 14,721-727 (1992). KIMURA, T, IMAI, Y, IRIMURA, T: Calcium-dependent conformation of a mouse macrophage calcium-type lectin. Carbohydrate binding activity is stabilized by an antibody specific for a calcium-dependent epitope. - J. BioI. Chern. 270, 16056-16062 (1995). KITAMURA, K.: Distribution of endogenous l3-galactoside-specific lectin, fibronectin and type I and III collagens during dermal condensation in chick embryos. - J. Embryol. expo Morph. 65, 41-56 (1981). KLION, A. D., DONELSON, J. E.: OvGaIBP, a filarial antigen with homology to vertebrate galactoside-binding proteins. - Mo. Biochem. Parasitol. 65, 305-315 (1994). KOBILER, D., BARONDES, S. H.: Lectin activity from embryonic chick brain, heart and liver: Changes with development. - Dev. BioI. 60, 326-330 (1977). KOBILER, D., BEYER, E. c., BARONDES, S. H.: Developmentally regulated lectins from chick muscle, brain, and liver have similar chemical and immunological properties. - Dev. BioI. 64, 265272 (1978). KOCH, P.J., WALSH, M.J., SCHMELZ, M., GOLDSCHMIDT, M. D., ZIMBELMANN, R., FRANKE, W. W.: Identification of desmoglein, a constitutive desmosomal glycoprotein, as a member of the cadherin family of cell adhesion molecules. - Eur. J. Cell BioI. 53, 1-12 (1990). KORNFELD, S. : Trafficking of lysosomal enzymes in normal and disease states. - J. Clin. Invest. 77, 1-6 (1986). KREIS, T, VALE, R.: Guidebook to the Extracellular Matrix and Adhesion Proteins. - Oxford University Press, Oxford 1993. LAING, J. G., ROBERTSON, M. W., GRITZMACHER, C. A., WANG, J. L., LIU, E- T: Biochemical and immunological comparisons of carbohydrate-binding protein 35 and an IgE-binding protein. - J. BioI. Chern. 264, 1907-1910 (1989). LAMBEAU,G., ANCIAN,P., BARHANIN,J., LAZDUNSKI,M.: Cloning and expression of a membrane receptor for secretory phospholipases A z. - J. BioI. Chern. 269, 1575-1578 (1994). LASKY, L. A.: Selectins: interpreters of cell-specific carbohydrate information during inflammation. - Science 258, 964-969 (1992). LASKY, L. A., SINGER, M. S., DOWBENKO, D., IMAI, Y, HENZEL, W.J., GRIMLEY, C., FENNIE, C., GILLETT, N., WATSON, S. R., ROSEN, S. D.: An endothelial ligand for L-selectin is a novel mucin-like molecule. - Cell 69, 927-938 (1992). LASKY, L. A., SINGER, M. S., YEDNOCK, T A., DOWBENKO, D., FENNIE, C., RODRIGUEZ, H., NGUYEN, T, STACHEL, S., ROSEN, S. D.: Cloning of a lymphocyte homing receptor reveals a lectin domain. - Cell 56, 1045-1055 (1989). LAWRENCE, M. B., SPRINGER, T. A.: Leukocytes roll on a selectin at physiologic flow rates: distinction from and prerequisite for adhesion through integrins. - Cell 65, 859-873 (1991). LEE, R. T, IcHIKAWA, Y, ALLEN, H.J., LEE, C. Y.: Binding characteristics of galactoside-binding lectin (galaptin) from human spleen. - J. BioI. Chern. 265, 7864-7871 (1990). LEE, Y-M., LEIBY, K. R., ALLAR, J., PARIS, K., LERCH, B., OKARMA, T B.: Primary structure of bovine conglutinin, a member of the C-type animal lectin family. - J. BioI. Chern. 266, 27152723 (1991). LEFFLER, H., BARONDES, S. H.: Specificity of binding of three soluble rat lung lectins to substituted and unsubstituded mammalian l3-galactosides. - J. BioI. Chern. 261,10119-10126 (1986). LEFFLER, H., MASIARZ, E R., BARONDES, S. H.: Soluble lactose-binding vertebrate lectins: a growing family. - Biochemistry 28, 9222-9229 (1989). LEVI, G., TEICHBERG, V. I.: Isolation and physicochemical characterization of electrolectin, a I3-Dgalactoside binding lectin from the electric organ of Electrophorus electricus. - J. BioI. Chern. 256,5735-5740 (1981).

Endogenous animallectins . 83

LI, W. X., JOUBERT-CARON, R., OUMAMI, H. E. L., BLADIER, D., CARON, M., BAUMANN, N.: Regulation of a ~-galactoside-binding lectin and potential ligands during postnatal maturation of rat brain. - Dev. Neurosci. 14, 290-295 (1992). LIM, B.-L., HOLMSKOV, D.: Expression of the carbohydrate recognition domain of bovine conglutinin and demonstration of its binding to iC3b and yeast mannan. - Biochem. Biophys. Res. Commun. 218, 260-266 (1996). LIU, F.- T., Hsu, D. K., ZUBERI, R. 1., KUWABARA, 1., CHI, E. Y., HENDERSON, W. R. JR.: Expression and function of galectin-3, a ~-galactoside-binding lectin, in human monocytes and macrophages. - Amer. J. Pathol. 147, 1016-1028 (1995). LOPEZ-CABRERA, M., SANTIS, A. G., FERNANDEZ-RUIZ, E., BLACHER, R., ESCH, F., SANCHEZMATEOS, P., SANCHEZ-MADRID, F.: Molecular cloning, expression, and chromosomallocalization of the human earliest lymphocyte activation antigen AIM/CD69, a new member of the Ctype animal lectin superfamily of signal-transmitting receptors. - J. Exp. Med. 178, 537-547 (1993). LOTAN, R.: ~-galactoside-bindingvertebrate lectins: synthesis, molecular biology, function. - In: Glycoconjugates (ALLEN, H. J., KISAILUS, E. c., eds.), pp 635-671- Marcel Dekker, New York (1992). LOTAN, R., LOTAN, D., CARRALERO, D.: Modulation of galactoside-binding lectins in tumor cells by differentiation-inducing agents. - Cancer Lett. 48, 115-122 (1989). Lu, J., LAURSEN, S. B., THIEL, S., JENSEN IUS, J. c., REID, K. B.: The cDNA cloning of conglutinin and identification of liver as a primary site of synthesis of conglutinin in members of the bovidae. - Biochem. J. 292, 157-162 (1993). Lu, J., WILLIS, A. c., REID, K. B.: Purification, characterization and cDNA cloning of human lung surfactant protein D. - Biochem. J. 284, 795-802 (1992). MADSEN, P., RASMUSSEN, H. H., FLINT, T., GROMOV, P., KRUSE, T. A., HONORE, B., VORUM, H., CELIS, J. E.: Cloning, expression, and chromosome mapping of human galectin-7. - J. BioI. Chern. 270,5823-5829 (1995). MAGNALDO, T., BERNERD, F., DARMON, M.: Galectin-7, a human 14-kDa S-lectin, specifically expressed in keratinocytes and sensitive to retinoic acd. - Dev. BioI. 168, 259-271 (1995). MAHANTHAPPA, N. K., COOPER, D. N. W., BARONDES, S. H., SCHWARTING, G. A.: Rat olfactory neurons can utilize the endogenous lectin, L-14, in a novel adhesion mechanism. - Development 120,1373-1384 (1994). MALHOTRA, R., Lu, J., HOLMSKOV, D., SIM, R. B.: Collectins, collectin receptors and the lectin pathway of complement activation. - Clin. Exp. Immunol. 97, (Suppl. 2), 4-9 (1994). MARSCHAL, P., HERRMANN, J., LEFFLER, H., BARONDES, S. H., COOPER, D. N. W.: Sequence and specificity of a soluble lactose-binding lectin from Xenopus laevis skin. - J. BioI. Chern. 267, 12942-12949 (1992). MATSUTANI,E., YAMAGATA, T.: Chick endogenous lectin enhances chondrogenesis of cultured chick limb bud cells. - Dev. BioI. 92, 544-548 (1982). MEHUL,B., BAWUMIA,S., MARTIN, S. R., HUGHES, R. c.: Structure of baby hamster kidney carbohydrate-binding protein CBP-30, an S-type animal lectin. - J. BioI. Chern. 269, 1825018258 (1994). MELLOW, T. E., HALBERG, D., DRICKAMER, K.: Endocytosis of N-acetylglucosamine-containing glycoproteins by rat fibroblasts expressing a single species of chicken liver glycoprotein receptor. - J. BioI. Chern. 263, 5468-5473 (1988). MERKLE, R. K., CUMMINGS, R. D.: Asparagine-linked oligosaccharides containing poly-Nacetyllactosamine chains are preferentially bound by immobilized calf heart agglutinin. - J. BioI. Chern. 263, 16143-16149 (1988). MERKLE, R. K., ZHOU, Q., SCHULTZ, T. K., HARPER, W. B., CUMMINGS, R. D.: Characterization of an S-type lectin purified from porcine heart. - Arch. Biochem. Biophys. 274, 404-416 (1989).

84 . Y. Akimoto et al.

MIZUOCHI, S., AKIMOTO, Y., IMAI, Y., HIRANO, H., IRIMURA, T: Unique tissue distribution of a mouse macrophage C-type lectin. - Glycobiology 7, 137-146 (1997). -: Immunohistochemical study on a macrophage calcium-type lectin in mouse embryos: transient expression in chondroblasts during endochondral ossification. - Glycoconjugate J. 15, 397-404 (1998). MONTELIONE, G. T., CALLAHAN, S., PODLESKI, T R.: Physical and chemical characterization of the major lactose-blockable lectin activity from fetal calf skeletal muscle. - Biochim. Biophys. Acta (Arnst.) 670,110-123 (1981). MOUTSATSOS, 1. K., WADE, M., SCHINDLER, M., WANG, J. L.: Endogenous lectins from cultured cells: Nuclear localization of carbohydrate-binding protein 35 in proliferating 3T3 fibroblasts. - Proc. Nat!. Acad. Sci. USA 84, 6452-6456 (1987). MURAMOTO, K., KAMIYA, H.: The amino-acid sequence of a lectin of the acorn barnacle Megabalanus rosa. - Biochim. Biophys. Acta (Arnst.) 874, 285-295 (1986). -: The amino-acid sequence of multiple lectins of the acorn barnacle Megabalanus rosa and its homology with animal1ectins. - Biochim. biophys. Acta (Arnst.) 1039, 42-51 (1990). -: The amino-acid sequence of a lectin from conger eel, Conger myriaster, skin mucus. - Biochim. Biophys. Acta 1116, 129-136 (1992). MUTA, T, MIYATA, T, MISUMI, Y., TOKUNAGA, E, NAKAMURA, T, TOH, Y., IKEHARA, Y., IWANAGA, S.: Limulus factor C. An endotoxin-sensitive serine protease zymogen with a mosaic structure of complement-like, epidermal growth factor-like, and lectin-like domains. - J. BioI. Chern. 266,6554-6561 (1991). NANGIA-MAKKER, P., OCHIENG, J., CHRISTMAN, J. K., RAz, A.: Regulation of the expression of galactoside-binding lectin during human monocytic differentiation. - Cancer Res. 53, 50335037 (1993). NIGG, E. A., BAEUERLE, P. A., LUHRMANN, R.: Nuclear import-export: in search of signals and mechanisms. - Cell 66, 15-22 (1991). NOWAK, T P., KOBILER, D., ROEL, L. E., BARONDES, S. H.: Developmentally regulated lectin from embryonic chick pectoral muscle. Purification by affinity chromatography. - J. bioI. Chern. 252, 6026-6030 (1977). NURMINSKAYA, M., LINSENMAYER, T E: Identification and characterization of up-regulated genes during chondrocyte hypertrophy. - Devei. Dynamics 206,260-271 (1996). OBINATA, A., AKIMOTO, Y., HIRANO, H., ENDO, H.: Stimulation by Bt2cAMP of epidermal mucous metaplasia in retinol-pretreated chick embryonic cultured skin, and its inhibition by herbimycin A, an inhibitor for protein-tyrosine kinase. - Exp. Cell Res. 193, 36-44 (1991a). -: Short term retinol treatment in vitro induces stable transdifferentiation of chick epidermal cells into mucus-secreting cells. - Roux's Arch. Dev. BioI. 200, 289-295 (1991 b). OBINATA,A., KAwADA,M., ENDO, H.: Induction of epidermal mucous metaplasia by culture of recombinants of undifferentiated epidermis and retinol-treated dermis in a chemically defined medium. - Dev. BioI. 123, 59-62 (1987). ODA, S., SATO, M., TOYOSHIMA, S., OSAWA, T: Purification and characterization of a lectin-like molecule specific for galactose/N-acetyl-galctosamine from tumoricidal macrophages. - J. Biochern. 104, 600-605 (1988). -: Binding of activated macrophages to tumor cells through a macrophage lectin and its role in macrophage tumoricidal activity. - J. Biochem.105, 1040-1043 (1989). ODA, Y., HERRMANN, J., GITT, M. A., TURCK, C. W., BURLINGAME, A. L., BARONDES, S. H., LEFFLER, H.: Soluble lactose-binding lectin from rat intestine with two different carbohydratebinding domains in the same peptide chain. - J. BioI. Chern. 268, 5929-5939 (1993). ODA, Y., KASAl, K.: Purification and characterization of ~-galactoside-bindinglectin from chick embryonic skin. - Biochim. Biophys. Acta (Arnst.) 761, 237-245 (1983).

Endogenous animal leetins . 85

-: Photochemical cross-linking of ~-galactoside-bindinglectin to polylactosamino-proteoglycan of chick embryonic skin. - Biochem. Biophys. Res. Commun. 123, 1215-1220 (1984). ODA, Y., LEFFLER, H., SAKAKURA, Y., KASAl, K., BARONDES, S. H.: Human breast carcinoma cDNA encoding a galactoside-binding lectin homologous to mouse Mac-2 antigen. - Gene (Amst.) 99,279-283 (1991). ODA, Y., OHYAMA, Y., OBINATA, A., ENDO, H., KASAl, K.: Endogenous ~-galactoside-bindinglectin expression is suppressed in retinol-induced mucous metaplasia of chick embryonic epidermis. - Exp. Cell Res. 182, 33-43 (1989). OHANNESIAN, D. W., LOTAN, D., LOTAN, R.: Concomitant increases in galectin-l and its glycoconjugate ligands (carcinoembryonic antigen, lamp-I, and lamp-2) in cultured human colon carcinoma cells by sodium butyrate. - Cancer Res. 54, 5992-6000 (1994). OHSAWA, E, HIRANO, E, NATORI, S.: Purification and properties of a ~-galactoside-binding lectin from neonatal marmoset. - J. Biochem.107, 431-434 (1990). OHYAMA, Y., HlRABAYASHI,J., ODA, Y., OHNO, S., KAWASAKI, H., SUZUKI, K., KASAl, K.: Nucleotide sequence of chick 14K ~-galactoside-binding lectin mRNA. - Biochem. Biophys. Res. Commun. 134, 51-56 (1986). OHYAMA, Y., KASAl, K.: Isolation and characterization of the chick 14K ~-galactoside-binding lectin gene. - J. Biochem. 104, 173-177 (1988). OLCESE, L., LANG, P., VELY, E, CAMBIAGGI, A., MARGUET, D., BLERY, M., HIPPEN, K. L., BIASSONI, R., MORETTA, A., MORETTA, L., CAMBIER, J. c., VIVIER, E.: Human and mouse killer-cell inhibitory receptors recruit PTPIC and PTPID protein tyrosine phosphatases. - J. Immuno!. 156, 4531-4534 (1996). ONRUST, S. V, HARTL, P. M., ROSEN, S. D., HANAHAN, D.: Modulation of L-selectin ligand expression during an immune response accompanying tumorigenesis in transgenic mice. - J. Clin. Invest. 97, 54-64 (1996). OOKUSA, Y., TAKATA, K., NAGASHIMA, M., HIRANO, H.: Distribution of glycoconjugates in normal human skin using biotinyllectins and avidin-horseradish peroxidase. - Histochemistry 79,1-7 (1983). OZAKI, K., II, M., ITOH, N., KAWASAKI, T.: Expression of a functional asialoglycoprotein receptor through transfection of a cloned cDNA that encodes a macrophage lectin. - J. Bio!. Chem. 267, 9229-9235 (1992). OZAKI, K., ITOH, M., KAWASAKI, T: Role of tyrosine-5 in the cytoplasmic tail of the macrophage asialoglycoprotein receptor in the rapid internalization of ligands. - J. Biochem. 113, 271-276 (1993). OZEKI, Y., MATSUI, T, NITTA, K., KAWAUCHI, H., TAKAYANAGI, Y., TITANI, K.: Purification and characterization of ~-galactoside binding lectin from frog (Rana catesbeiana) eggs. - Biochem. Biophys. Res. Commun.178, 407-413 (1991). OZEKI, Y., MATSUI, T, YAMAMOTO, Y., FUNAHASHI, M., HAMAKO,J., TITANI, K.: Tissue fibronectin is an endogenous ligand for galectin-l. - Glycobiology 5,255-261 (1995). PAROUTAUD, P., LEVI, G., TEICHBERG, V I., STROSBERG, A. D.: Extensive amino acid sequence homologies between animallectins. - Proc. Nat!' Acad. Sci. USA 84, 6345-6348 (1987). PERRY, V H., CROCKER, P. R., GORDON, S.: The blood-brain barrier regulates the expression of a macrophage sialic acid-binding receptor on microglia. - J. Cell Sci. 101, 201-207 (1992). PFEIFER, K., HAASEMANN, M., GAMULIN, V, BRETTING, H., FAHRENHOLZ, E, MULLER, W. E.: Stype lectins occur also in invertebrates: high conservation of the carbohydrate recognition domain in the lectin genes from the marine sponge Geodia Cydonium. - Glycobiology 3, 179184 (1993). PHILLIPS, M. L., NUDELMAN, E., GAETA, E C. A., PEREZ, M., SINGHAL, A. K., HAKOMORI, S., PAULSON, J. c.: ELAM-1 mediates cell adhesion by recognition of a carbohydrate ligand, sialylLeX. - Science 250, 1130-1132 (1990).

86 . Y. Akimoto et al. POLLEY, M.l, PHILLIPS, M. L., WAYNER, E., NUDELMAN, E., SINGHAL, A. K., HAKOMORI, S., PAULSON, J.C: CD62 and endothelial cell-leukocyte adhesion molecule 1 (ELAM-l) recognize the same carbohydrate ligand, sialyl-Lewis x. - Proc. Natl. Acad. Sci. USA 88,6224-6228 (1991 ). POWELL, L. D., VARKI, A.: The oligosaccharide binding specificities of CD22~, a sialic acid-specific lectin of B cells. - J. BioI. Chern. 269, 10628-10636 (1994). -: I-type lectins. - J. BioI. Chern. 270,14243-14246 (1995). RAUCH, U., KARTHIKEYAN, L., MAUREL,P., MARGOLIS, R. U., MARGOLIS, R. K.: Cloning and primary structure of neurocan, a developmentally regulated, aggregating chondroitin sulfate proteoglycan of brain. - J. BioI. Chern. 267,19536-19547 (1992). RAz, A., CARMI, P., PAZERINI, G.: Expression of two different endogenous galactoside-binding lectins sharing sequence homology. - Cancer Res. 48, 645-649 (1988). RAz, A., CARMI, P., RAz, T., HOGAN, v., MOHAMED, A., WOLMAN, S. R.: Molecular cloning and chromosomal mapping of a human galactoside-binding protein. - Cancer Res. 51, 2173-2178 (1991 ). RAz, A., LOTAN, R.: Endogenous galactoside-binding lectins: a new class of functional tumor cell surface molecules related to metastasis. - Cancer Metast. Rev. 6, 433-452 (1987). RAz, A., MEROMSKY, L., LOTAN, R.: Differential expression of endogenous lectins on the surface of nontumorigenic, tumorigenic, and metastatic cells. - Cancer Res. 46, 3667-3672 (1986). RAz, A., PAZERINI, G., CARMI, P.: Identification of the metastasis-associated, galactoside-binding lectin as a chimeric gene product with homology to an IgE-binding protein. - Cancer Res. 49, 3489-3493 (1989). REGAN, L.l, DODD,J., BARONDES, S. H., JESSELL, T. M.: Selective expression of endogenous lactose-binding lectins and lactoseries glycoconjugates in subsets of rat sensory neurons. Proc. Natl. Acad. Sci. USA 83, 2248-2252 (1986). ROBERTSON, M. ALBRANDT, K., KELLER, D., LIU, E- T.: Human IgE-binding protein: A soluble lectin exhibiting a highly conserved interspecies sequence and differential recognition of IgE glycoforms. - Biochemistry 29, 8093-8100 (1990). ROSEN, S. D., IMAI, Y, SINGER, M. S., HUANG, K.: L-selectin; a lectin-like leukocyte adhesion protein. - Trends in Glycosci. Glycotech. 4,1-13 (1992). ROSEN, S. D., SINGER, M. S., YEDNOCK, T. A., STOOLMAN, L. M.: Involvement of sialic acid on endothelial cells in organ-specific lymphocyte recirculation. - Science 228, 1005-1007 (1985). RUOSLAHTI, E.: Brain extracellular matrix. - Glycobiology 6,489-492 (1996). RUSSELL, M. E., UTANS, U., WALLACE, A. E, LIANG, P., ARCECI, R. J., KARNOVSKY, M.J., WYNER, L. R., YAMASHITA, Y, TARN, C: Identification and upregulation of galactoseIN-acetylgalactosamine macrophage lectin in rat cardiac allografts with arteriosclerosis. - J. Clin. Invest. 94, 722730 (1994). RYAN, J. c, TURCK,J., NIEMI, E. C, YOKOYAMA, W. M., SEAMAN, W. E.: Molecular cloning of the NKl.l antigen, a member of the NKR-Pl family of natural killer cell activation molecules. - J. Immunol.149, 1631-1635 (1992). SAl, S., TANAKA, T., KOSHER, R. A., TANZER, M. L.: Cloning and sequence analysis of a partial eDNA for chicken cartilage proteoglycan core protein. - Proc. Natl. Acad. Sci. USA 83,50815085 (1986). SAKAKURA, Y, HlRABAYASHI,l, ODA, Y, OHYAMA, Y, KASAl, K.: Structure of chicken 16-kDa ~­ galactoside-binding lectin. - J. BioI. Chern. 265, 21573-21579 (1990). SAKAMAKI, T., IMAI, Y., IRIMURA, T.: Enhancement in accessibility to macrophages by modification of mucin-type carbohydrate chains on a tumor cell line: role of a C-type lectin of macrophages. - J. Leukoc. BioI. 57, 407-414 (1995).

w.,

Endogenous animallectins . 87

SAKO, D., CHANG, X.-J., BARONE, K. M., VACHINO, G., WHITE, H. M., SHAW, G., VELDMAN, G. M., BEAN, K. M., AHERN, T.]., FURIE, B., CUMMING, D. A., LARSEN, G. R.: Expression cloning of a functional glycoprotein ligand for P-selectin. - Cell 75, 1179-1186 (1993). SANFORD, J. P., DOYLE, D.: Mouse asialoglycoprotein receptor eDNA sequence: conservation of receptor genes during mammalian evolution. - Biochim. Biophys. Acta (Arnst.) 1087, 259-261 (1990). SANTA-LuCIA, P., WILLSON, B. D., ALLEN, H.J.: Localization of endogenous ~-galactoside­ binding lectin as a means to distinguish malignant from benign skin tissue. - J. Dermatol. Surg. Oncol.17, 653-655 (1991). SATO, M., KAWAKAMI, K., aSAWA, T., TOYOSHIMA, 5.: Molecular cloning and expression of eDNA encoding a galactose/N-acetylgalactosamine-specific lectin on mouse tumoricidal macrophages. - J. Biochem.ll1, 331-336 (1992). SATO,S., HUGHES, R. c.: Binding specificity of a baby hamster kidney lectin for H type I and II chains, polylactosamine glycans, and appropriately glycosylated forms of laminin and fibronectin. - J. BioI. Chern. 267, 6983-6990 (1992). SHARON, N.: Bacteriallectins, cell-cell recognition and infectious disease. - FEBS Lett. 217, 145157 (1987). SHARON, N., LIS, H.: Lectins as cell recognition molecules. - Science 246,227-234 (1989). SHIMIZU, H., FISHER,J. H., PAPST, P., BENSON, B., LAU, K., MASON, R.]., VOELKER, D. R.: Primary structure of rat pulmonary surfactant protein D. eDNA and deduced amino acid sequence. - J. BioI. Chern. 267,1853-1857 (1992). SHINOMURA, T., NISHIDA, Y., ITo, K., KIMATA, K.: eDNA cloning of PG-M, a large chondroitin sulfate proteoglycan expressed during chondrogenesis in chick limb buds. Alternative spliced multiforms of PG-M and their relationships to versican. - J. BioI. Chern. 268, 14461-14469 (1993). SIEGELMAN, M. H., VAN DE RIJN, M., WEISSMAN, 1. L.: Mouse lymph node homing receptor eDNA clone encodes a glycoprotein revealing tandem interaction domains. - Science 243, 1165-1172 (1989). SPIESS, M., LODISH, H. E: Sequence of a second human asialoglycoprotein receptor: Conservation of two receptor genes during evolution. - Proc. Nat!. Acad. Sci. USA 82: 6465-6469 (1985). SPIESS, M., SCHWARTZ, A. L., LODISH, H. E: Sequence of human asialoglycoprotein receptor eDNA. An internal signal sequence for membrane insertion. - J. BioI. Chern. 260, 1979-1982 (1985). SIMMONS, D. L., SATTERTHWAITE, A. B., TENEN, D. G., SEED, B.: Molecular cloning of a eDNA encoding CD34, a sialomucin of human hematopoietic stem cells. - J. Immunol. 148, 267-271 (1992). SKRINCOSKY, D. M., ALLEN, H.]., BERNACKI, R.J.: Galaptin-mediated adhesion of human ovarian carcinoma A 121 cells and detection of cellular galaptin-binding glycoproteins. - Cancer Res. 53,2667-2675 (1993). SOLOMON, J. c., STOLL, M.S., PENFOLD, P., ABBOTT, W. M., CHILDS, R. A., HANFLAND, P., FEIZI, T.: Studies of the binding specificity of the soluble 14,000-dalton bovine heart muscle lectin using immobilised glycolipids and neoglycolipids. - Carbohydr. Res. 213, 293-307 (1991). SPARROW, C. P., LEFFLER, H., BARONDES, S. H.: Multiple soluble ~-galactoside-binding lectins from human lung. - J. BioI. Chern. 262, 7383-7390 (1987). SPRINGER, T. A.: Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. - Cell. 76, 301-314 (1994). STEEGMAIER, M., LEVINOVITZ, A., ISENMANN, 5., BORGES, E., LENTER, M., KOCHER, H. P., KLEUSER, B., VESTWEBER, D.: The E-selectin-ligand ESL-l is a variant for a receptor for fibroblast growth factor. - Nature (Lond.) 373,615-620 (1995).

88 . Y. Akimoto et al.

STOOLMAN, L. M., TENFORDE, T. S., ROSEN, S. D.: Phosphomannosyl receptors may participate in the adhesive interaction between lymphocytes and high endothelial venules. - J. Cell BioI. 99, 1535-1540 (1984). STREETER, P. R., ROUSE, B. T. N., BUTCHER, E. C: Immunohistologic and functional characterization of a vascular addressin involved in lymphocyte homing into peripheral lymph nodes. - J. Cell BioI. 107, 1853-1862 (1988). SUZUKI, N., YAMAMOTO, K., TOYOSHIMA, S., OSAWA, T., IRIMURA, T.: Molecular cloning and expression of cDNA encoding human macrophage C-type lectin. Its unique carbohydrate binding specificity for Tn antigen. - J. Immunoi. 156, 128-135 (1996). SUZUKI, T., TAKAGI, T., FURUKOHRI, T., KAWAMURA, K., NAKAUCHI, M.: A calcium-dependent galactose-binding lectin from the Tunicate Polyandrocarpa misakiensis. Isolation, characterization, and amino acid sequence. - J. BioI. Chern. 265,1274-1281 (1990). TAKAHASHI, H., KOMANO, H., KAWAGUCHI, N., KITAMURA, N., NAKANISHI, S., NATORI, S.: Cloning and sequencing of cDNA of Sarcophaga peregrina humoral lectin induced on injury of the body wall. - J. BioI. Chern. 260, 12228-12233 (1985). TAKATA, K., HIRANO, H.: Changes in soybean agglutinin (SBA) and peanut agglutinin (PNA) binding pattern in the epidermis of the developing chick embryo. - Develop. Growth Differ. 25, 299-305 (1983). TAKATA,K., OBINATA, A., ENDO, H., HIRANO, H.: Induction of the a-type keratinization by hydrocortisone in embryonic chick skins grown in a chemically defined medium. An electron microscopic study. - Dev. BioI. 85, 370-379 (1981). TAKEYA, H., NISHIDA, S., MIYATA, T., KAWADA, S., SAISAKA, Y., MORITA, T., IWANAGA, S.: Coagulation factor X activating enzyme from Russell's viper venom (RVV-X). A nobel metalloproteinase with disintegrin (platelet aggregation inhibitor)-like and C-type lectin-like domains. - J. BioI. Chern. 267, 14109-14117 (1992). TAKEZAWA, R., SHINZAWA, K., WATANABE, Y., AKAIKE, T.: Determination of mouse major asialoglycoprotein receptor cDNA sequence. - Biochim. Biophys. Acta (Arnst.) 1172, 220-222 (1993). TAYLOR, M. E., BEZOUSKA, K., DRICKAMER, K.: Contribution to ligand binding by multiple carbohydrate-recognition domains in the macrophage mannose receptor. - J. BioI. Chern. 267, 1719-1726 (1992). TAYLOR, M. E., CONARY, J. T., LENNARTZ, M. R., STAHL, P. D., DRICKAMER, K.: Primary structure of the mannose receptor contains multiple motifs resembling carbohydrate-recognition domains. - J. BioI. Chern. 265, 12156-12162 (1990). TEDDER, T.E, ISAACS, CM., ERNST, T.J., DEMETRI, G.D., ADLER, D.A., DISTECHE, CM.: Isolation and chromosomal localization of cDNAs encoding a novel human lymphocyte cell surface molecule, LAM-I. Homology with the mouse lymphocyte homing receptor and other human adhesion proteins. - J. Exp. Med.170, 123-133 (1989). UTANS, U., LIANG, P., WYNER, L. R., KARNOVSKY, M.]., RUSSELL, M. E.: Chronic cardiac rejection: Identification of five upregulated genes in transplanted hearts by differential mRNA display. Proc. Nat!. Acad. Sci. USA 91,6463-6467 (1994). VAN DEN BERG, T. K., BREVE, J.J. P., DAMOISEAUX, J. G. M. C, Dopp, E. A., KELM, S., CROCKER, P. R., DIJKSTRA, C D., KRAAL, G.: Sialoadhesin on macrophages: Its identification as a lymphocyte adhesion molecule. - J. Exp. Med.176, 647-655 (1992). WASANO, K., HIRAKAWA, Y., YAMAMOTO, T.: Immunohistochemical localization of 14 kDa Bgalactoside-binding lectin in various organs of rat. - Cell Tissue Res. 259, 43-49 (1990). WASANO, K., YAMAMOTO, T.: Rat lung 29 kD B-galactoside-binding lectin is secreted by bronchiolar Clara cells into airways. - Histochemistry 90,447-451, (1989). WASSARMAN, P. M.: The biology and chemistry of fertilization. - Science 235,553-560 (1987).

Endogenous animallectins . 89

WATSON, S. R., IMAI, Y., FENNIE, c., GEOFFROY, J. S., ROSEN, S. D., LASKY, L. A.: A homing receptor-IgG chimera as a probe for adhesive ligands of lymph node high endothelial venules. J. Cell BioI. 110, 2221-2229 (1990). WEIS, W. I., DRICKAMER, K., HENDRICKSON, W. A.: Structure of a C-type man nose-binding protein complexed with an oligosaccharide. - Nature (Lond.) 360,127-134 (1992). WELLS, V., MALLUCCI, L.: Identification of an autocrine negative growth factor: mouse ~-galacto­ side-binding protein is a cytostatic factor and cell growth regulator. - Cell 64, 91-97 (1991). WEN, D., DITTMAN, W. A., YE, R. D., DEAVEN, L. L., MAJERUS, P. W., SADLER,J. E.: Human thrombomodulin: complete eDNA sequence and chromosome localization of the gene. - Biochemistry 26,4350-4357 (1987). WHITE, T, DAMM, D., MILLER, J., SPRATT, K., SCHILLING, J., HAWGOOD, S., BENSON, B., CORDELL, B.: Isolation and characterization of the human pulmonary surfactant apoprotein gene. Nature (Lond.) 317, 361-363 (1985). WHITNEY, P. L., POWELL,J. T, SANFORD, G. L.: Oxidation and chemical modification of lung ~­ galactoside-specific lectin. - Biochem. J. 238, 683-689 (1986) WILEMAN, T E., LENNARTZ, M. R., STAHL, P. D.: Identification of the macrophage mannose receptor as a 175-kDa membrane protein. - Proc. Natl. Acad. Sci. USA 83,2501-2505 (1986). WILSON, TJ. G., FIRTH, M. N., POWELL,J. T, HARRISON, E L.: The sequence of the mouse 14 kDa ~-galactoside-bindinglectin and evidence for its synthesis on free cytoplasmic ribosomes. Biochem. J. 261, 847-852 (1989). WOLLENBERG, A., DE LA SALLE, H., HANAU, D., LIU, E-T, BIEBER, T: Human keratinocytes release the endogenous ~-galactoside-bindingsoluble lectin immunoglobulin E (IgE-binding protein) which binds to Langerhans cells where it modulates their binding capacity for IgE glycoforms. - J. Exp. Med.178, 777-785 (1993). Woo, H.-J., SHAW, L. M., MESSIER, J. M., MERCURIO, A. M.: The major non-integrin laminin binding protein of macrophages is identical to carbohydrate binding protein 35 (Mac-2). - J. BioI. Chern. 265, 7097-7099 (1990). Wu, K., YUAN, J., LASKY, L. A.: Characterization of a novel member of the macrophage mannose receptor type C lectin family. - J. BioI. Chern. 271, 21323-21330 (1996). YAMADA, H., WATANABE, K., SHIMONAKA, M., YAMAGUCHI, Y.: Molecular cloning of brevican, a novel brain proteoglycan of the aggrecan/versican family. - J. BioI. Chern. 269, 10119-10126 (1994). YAMAGUCHI, Y., ITo, M., SAITO, S., AOYAGI, T, TAKATA, K., HIRANO, H.: Light and electron microscopic study of lectin binding sites in human esophageal epithelium. - Acta Anat. Niponica 60,104-108 (1985). YAMAMOTO, K., ISHIDA, C., SHINOHARA, Y., HASEGAWA, Y., KONAMI, Y., OSAWA, T, IRIMURA, T: Interaction of immobilized recombinant mouse C-type macrophage lectin with glycopeptides and oligosaccharides. - Biochemistry 33, 8159-8166 (1994). YAMAOKA,K., OHNO,S., KAWASAKI, H., SUZUKI,K.: Overexpression of a ~-galactoside binding protein causes transformation of BALB3T3 fibroblast cells. - Biochem. Biophys. Res. Commun.179, 272-279 (1991). YANG, L.J.-S., ZELLER, C.B., SHAPER, N.L., Krso,M., HASEGAWA, A., SHAPIRO, R.E., SCHNAAR, R. L.: Gangliosides are neuronal ligands for myelin-associated glycoprotein. - Proc. Nat!. Acad. Sci. USA 93,814-818 (1996). YOKOYAMA, W. M., JACOBS, L. B., KANAGAWA, 0., SHEVACH, E. M., COHEN, D. I.: A murine T lymphocyte antigen belongs to a supergene family of type II integral membrane proteins. - J. Immunol.143, 1379-1386 (1989). YOKOYAMA, W. M., SEAMAN, W. E.: The Ly-49 and NKR-P1 gene families encoding lectin-like receptors on natural killer cells: the NK gene complex. - Annu. Rev. Immunol.11, 613-635 (1993).

90 . Y. Akimoto et al.

ZHOU, Q., CUMMINGS, R. D.: L-14 lectin recognition of laminin and its promotion of in vitro cell adhesion. - Arch. Biochem. Biophys. 300,6-17 (1993). ZHU, B. C.-R., FISHER, S. F., PANDE, H., CALAYCAY,J., SHIVELY, J. E., LAINE, R. A.: Human placental (fetal) fibronectin: increased glycosylation and higher protease resistance than plasma fibronectin. - J. BioI. Chern. 259, 3962-3970 (1984). ZIMMERMANN, D. R., RUOSLAHTI, E.: Multiple domains of the large fibroblast proteoglycan, versican. - EMBO J. 8, 2975-2981 (1989).