Carbohydrate recognition systems: functional triads in cell—cell interactions

679

Carbohydrate recognition systems: functional triads in cell-cell interactions Paul R Crocker* and Ten Feizit Considerable progress is being made in our understanding of the molecular basis for mammalian carbohydrate recognition systems. Selectins, related proteins and sialoadhesins are carbohydrate-binding proteins which serve as receptors in the orchestration of innate and acquired immune responses, inflammation and other forms of cell-cell communication. Protein structural studies and gain-of-function and loss-of-function mutations are providing clues to ways in which the receptors interact with monosaccharide elements of the oligosaccharide ligands. Binding experiments using oligosaccharides on lipid or protein carriers indicate that modes of presentation such as the clustered state and the manner of display on proteins are crucial factors determining whether a functional triad of receptor and ligand + carrier (counter-receptor) is formed.

Addresses *ICRF Molecular Haemopoiesis Laboratory, Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Headington, Oxford, OX3 9DU, UK; e-mail: [email protected] +The Glycosciences Laboratory, Imperial College School of Medicine, Northwick Park Hospital, Watford Road, Harrow, Middlesex HA1 3UJ, UK; e-mail: [email protected] Current Opinion in Structural Biology 1996, 6:679-691

© Current Biology Ltd ISSN 0959-440X Abbreviations CAM cell adhesion molecule CHO Chinese hamster ovary OR consensusrepeat EGF epidermal growth factor Fuc fucose GalNAc N-acetyl galactosamine Gal galactose GIc glucose GIcNAc N-acetyl glucosamine MAG myelinassociated glycoprotein MBP mannose-binding protein NeuAc N-acetylneuraminic acid NeuGc N-glycolyl neuraminic acid

Introduction Much has happened in the past decade to change radically the general perception that oligosaccharides of mammalian cells are post-translational modifications of glycoproteins often of uncertain biological significance. T h e field of glycobiology has gained remarkable momentum with the delineation of oligosaccharides as key players in recognition systems [1,2]. Unlike the usual situation of receptor-ligand pairs [3], however, a picture is emerging of recognition systems that operate as triads: receptors, ligands and carriers. Here the receptors are lectins [4], the ligands are oligosaccharides, and when they are optimally assembled and presented on carriers (proteins or lipids),

functional counter-receptors are formed, as depicted in Figure 1. Such glycorecognition systems are now established as being among essential components of the cell-cell interaction cascades, particularly in the orchestration of the innate immune responses and inflammation, and they almost certainly play a role in communications in the nervous system. It has also emerged that carbohydrate recognition systems are important intracellularly during protein folding [5-9], in addition to their well documented role in the routing of newly synthesized lysosomal enzymes [10]. T h e ligands that are involved in these various functions are by no means unique. On the one hand, for protein folding, they are intermediates in the conserved biosynthetic pathways of N-glycosylation. On the other hand, for effector functions in inflammation, the ligands can be, for example, blood group related oligosaccharides (carbohydrate differentiation antigens) [11[ that are expressed selectively on various cell types of the body. Here we focus primarily on recent progress in the delineation of functional triads (receptors, ligands and carriers) involved in certain cell-cell interactions, and mainly on the selectin and sialoadhesin systems (Fig. 2). First, we discuss the principles that are emerging on ways in which receptors and monosaccharides are ligated. Second, we address ways in which macromolecular carriers of oligosaccharide ligands can exert a profound influence on availability or accessibility of the ligands for receptor binding.

Receptors Mammalian lectins have been classified into discrete families based on similarities in primary amino acid sequence [4]. Among these are Ca2+-dependent (C-type) lectins, galectins, mannose-6-phosphate-binding lectins (P-type) and immunoglobulin-like lectins (I-type) [12]. Progress is being made towards understanding the molecular basis for their recognition of carbohydrates through a combination of techniques such as X-ray crystallography, molecular modelling, site-directed mutagenesis, N M R analysis and various ligand binding experiments.

Selectins and related C-type lectins as receptors To date, a limited number of X-ray crystal structures have been determined for C-type lectins complexed with ligands. These include the structures of one of the serum collectins, mannose-binding protein (MBP-A, also known as the mannan-binding protein) of the rat complexed with oligomannose chains [13], a mutant form of this receptor complexed with [3-methyl galactose and N-acetyl galactosamine [14"] and the rat liver mannose-binding

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Carbohydrates and glycoconjugates

Figure 1

(a)

Functional Triad

c

(b)

Non-functional Triad

Schematic representation of receptor-ligand assemblies. (a) Functional. (b) Non-functional. The key ligating elements (ligands) are particular oligosaccharides that must be effectively presented on protein or lipid carriers, which together serve as functional counter-receptors. The receptor protein is the product of a single or a relatively small number of genes; by contrast, the counter-receptor is typically the product of many genes, the majority of which are involved in post-translational and post-biosynthetic modifications. There are so many factors involved in determining whether a glycoprotein (or a glycolipid) does indeed function as a counter-receptor that in such glycorecognition systems it seems appropriate to reserve the term ligand to the oligosaccharide and the term counter-receptor to the functional saccharide-carrier assembly.

protein (MBP-C) complexed with methyl glycosides of mannose, N-acetyl glucosamine and fucose [15"]. In all cases, binding of sugars is mediated principally by the formation of direct coordination bonds between a Ca 2+ ion bound to the lectin domain and two appropriately positioned hydroxyls in the sugar ring; these hydroxyls also form hydrogen bonds with amino acid side chains that ligate the Ca2+. Three of the five amino acids that coordinate the Ca 2+ are conserved in C-type lectins, and alterations of the other two can change the specificity of C-type lectins in a defined manner [16]. T h e crystal structure of the carbohydrate recognition domain and adjoining

EGF-like domain of E-selectin without complexed ligand has revealed a very similar overall fold to that of MBPs, but has not given a clear insight into how the oligosaccharide ligand, 3'-sialyl-Le x (S-Lex; Fig. 3), is bound [17]. Extensive mutagenesis studies on E- and P-selectins have been performed, and two distinct schools of thought have arisen as to how S-Le x is recognized by these lectins. It is generally considered likely that the fucose residue of S-Lex is coordinated to a Ca 2+ ion via the equatorial C2-OH and C3-OH groups, as recently revealed in the crystal structure of MBP-C complexed with fucose [15"']. T h e difference in opinion relates to the positioning of the other sugar components of S-Le x, especially sialic acid. For both E- and P-selectin, conservative substitutions of certain basic residues, especially Lysll3 in P- and E-selectin and Arg97 in E-selectin, abrogates binding to HL60 cells and S-Le x [17-21]. These observations have led to suggestions that Lysll3 or Arg97 is involved in a charge-paired electrostatic interaction with the carboxylate group of sialic acid. Arg97 is not conserved in P-selectin, however, and subsequent mutagenesis experiments have shown that in E-selectin aspartate or serine can be substituted for it without affecting binding to HL60 cells [22"]. T h e importance of L y s l l l and Lysll3 in charge interactions has also been addressed by mutation to cysteine followed by chemical modification [23]. These studies have shown that the charge, but not the length of the side chain at position 111, is important for P-selectin binding. T h e failure to restore binding of the chemically modified Lysll3Cys mutation could be interpreted as showing the importance of both length and charge of this residue, as suggested in other studies [21]. More extensive mutagenesis experiments have questioned the importance of Lysll3 in charge interactions, however, as for both P- and E-selectins, mutation of Lysll3 to glutamine or glutamic acid has no effect on binding to HL60 cells and S-Le x [22°,24"]. As discussed below, P-selectin resembles L- but not E-selectin in that it can also bind sulphatide. T h e results of mutagenesis studies are not in complete accord, one suggesting that the binding sites for S-Le x and sulphatide may overlap [21], and another that they may be distinct [22"]. One of the caveats of site-directed mutagenesis studies that are used to examine loss of binding to ligands is that changes distant from the binding site may alter binding indirectly by affecting the orientation of amino acids that are involved directly in ligating sugar. For C-type lectins, clear examples of this problem have been demonstrated in elegant studies with mutant forms of MBP-A [25"']. An alternative approach to probing the carbohydrate binding site of selectins is to introduce novel binding properties that mimic those of a naturally occurring lectin. Such 'gain-of-function' mutations may give more insight into binding mechanisms than 'loss-of-function' mutations. This strategy has been attempted for both E- and P-selectins in which a single amino acid substitution

Carbohydrate recognition systems Crocker and Feizi

681

Figure 2 Schematic representation of selectin type receptors, their ligands and their carriers as triads in cell-cell recognition. Selectins (left) play a key role in leucocyte migration in inflammation and in lymphocyte recirculation. In inflammation the induced expression of P- and E-selectins on the surface of endothelial cells leads to the rolling of neutrophils and their subsequent margination and diapedesis into tissues. In lymphocyte homing, L-selectin on leucocytes recognizes counter-receptors ('addressins') on high endothelial venules, leading to leucocyte entry into the lymph node parenchyma. The sialoadhesins (right) are expressed in a cell-type restricted manner and are involved in diverse biological functions ranging from myeloid cell production (sialoadhesin and CD33), activation of B lymphocytes (CD22) and regulation of neuronal cell growth and myelination in the nervous system (MAG). PSGL-1, P-selectin glycoprotein ligand; ESL-1, E-selectin ligand; GlyCAM-1, glycosylation dependent cell adhesion molecule; MadCAM-1, mucosal addressin cell adhesion molecule; EGF, epidermal growth factor. Further information on the biology of selectins and sialoadhesins can be found in [96,97].

Sialoadhesin Triads (Proposed)

Selectin Triads

and sialoadhesin

Endothefium Platelets

Macrophages

Leucocytes

I P-selecli~

I i;~i

,% Endothelium

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Leucocytes

/

~1 E-selectin

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Ii'"

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CD22

Oligodendrocytes Schwanncells

t t

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~

GlyCAM-1 @

.....

Myeloidcells

Myelold

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MadCAM tReceptor domains

(;~ Lectin Cqype <'* EGF-like o Complementqike

o

Lectin, Ig like, V-set Ig-likeC2-sel

['

3'-sialylN-glycans

Ligands ) O-glycans (sialo/sulpho/tuco}

3'-s~alylO-glycans

N-gl ycans (sialo,'fuco! I~ Li piddinked-glycans (sialo/fuco)

6'-sialyl N-glycans

~. Sulpho-tyroslne

Carriers Mucin like Cysteinerich • Ig-hke < Lipid

(Ala77Lys) results in loss of binding to S-Le x but gain of binding to oligomannose, thus mimicking the specificity of MBP-A [22°,24"]. T h e authors of these studies argue that a c o m m o n region of the lectin domain is involved in binding both S-Lex and oligomannose, and this interpretation is supported by modelling studies in which the N M R - d e t e r m i n e d solution conformation of S-Le x bound to E-selectin [26] was docked onto the X-ray crystal structure of E-selecdn [17]. In a separate gain-of-function study [27"], however, it was found that the specificity of MBP-A could be altered to include S-Le x as well as mannose binding, by substituting a stretch of three positively charged residues ( L y s l l l - L y s l l 3 ) which had been proposed on the basis of other studies as being critical in forming a charge interaction with the sialic acid of S-Le x or with sulphates attached to related oligosaccharides [21,23]. This approach [27 °]

~1=~: Proteins:)r lipids tc be defined

provides a useful model for binding of selectins to their oligosaccharide ligands. Although, the degree to which it truly mimicks E- and P-selectin binding specificity remains to be determined. T h e X-ray crystal structure of a C-type lectin domain with bound S-Lex is awaited with interest. S i a l o a d h e s i n s as r e c e p t o r s T h e sialoadhesins form a distinct subgroup within the Ig superfamily. Each contains a single N-terminal V-set Ig-like domain and varying numbers of C2-set domains (Fig. 2). T h e Ig fold consists of a sandwich of two J] sheets, each consisting of antiparallel J3 strands of five-ten amino acids. T h e core of the fold is made up of three [3 strands, labelled ABE and GFC. V-set domains contain two extra ]3 strands in the G F C [3 sheet, ginving rise to a GFCC'C"[3 sheet. For sialoadhesin,

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Carbohydratesand glycoconjugates

the N-terminal V-set Ig-like domain has been shown to be both necessary and sufficient to mediate sialic acid dependent binding of the correct specificity [28"]; however, for CD22 the V-set and adjacent C2-set Ig-like domain appear to constitute a 'minimal unit' for sialic acid dependent binding [28°-30"], although the precise role of the C2-set domain remains to be determined. Further insight into the molecular basis for sialic acid dependent binding by members of the sialoadhesin family has come from site-directed mutagenesis studies. For both sialoadhesin and CD22, nonconservative substitutions of predicted surface residues on the V-set domains led to the identification of a subset of amino acids potentially involved in binding [31",32"]. In both proteins, the great majority of these amino acids were positioned on the G, F and C [3 strands of the GFCC'C"I3 sheet, circumscribing a conserved arginine on the F strand. When this arginine was further mutated conservatively to alanine or lysine, sialic acid dependent binding of both sialoadhesin and CD22 was disrupted, arguing that this residue plays a key role in sialic acid dependent binding. Interestingly, the GFC [3 sheet of the Ig domain is thought to be used by other members of the Ig superfamily such as CD2 and the vascular and intercellular cell adhesion molecules (VCAMs and ICAMs, respectively), all of which are involved in protein-protein interactions. As indicated by the X-ray crystal structures of CD2 [33] and VCAM-1 [34], this face of the lg domain may be more favourably exposed to ligands than the ABED [~ sheet. Once again, the X-ray crystal structures of lectin domains complexed with carbohydrate ligands are awaited with interest.

Receptor topography It is important to consider the molecular topography of carbohydrate-binding receptors at the cell surface. Relevant parameters include the length of the molecules and their accessibility to ligands on apposing cells, the dimensions and composition of neighbouring macromolecules, the ability of the receptors to cluster in the membrane, and, in some cases, their localization within subdomains such as microvilli. T h e differing lengths of the selectins is determined by the number of consensus repeats (CRs), ranging from two in L-selectin to nine in human P-selectin. Stable cell lines of Chinese hamster ovary (CHO) cells have been derived that express truncated forms of P-selectin containing varying numbers of CRs [35]. Unde~ static assay conditions, all the constructs bound equal numbers of neutrophils, whereas under physiological flow conditions, five or more CRs within one construct were required to achieve wild-type binding levels. These results suggest that the lectin domain of P-selectin must be appropriately extended at the cell surface to mediate efficient attachment of moving neutrophils. Likewise, for sialoadhesin, it has been proposed that the unusual length of its extracellular region (17 Ig domains) is required to project its lectin domain in order to minimize potentially

inhibitory cis interactions with oligosaccharides on neighbouring glycoproteins [36]. T h e occurrence of such cis interactions has been demonstrated experimentally using CD22 (seven Ig domains) and CD33 (two Ig domains). T h e lectin functions of both proteins are inhibited when they are expressed on cells that bear the appropriate sialyl linkage (Fig. 2) [37,38"]. The low affinities of many endogenous lectins for their oligosaccharide ligands means that to obtain high avidity binding, both receptor and ligand need to be clustered. Clustering of receptors can occur constitutively in the plasma membrane, as shown for CD22 [39*], or by passive diffusion on encountering a counter-receptor. T h e localization of L-selectin and the counter-receptor for P-selectin (PSGL-1) to microvilli of neutrophils [40-42] is also likely to be important in promoting cell-cell interactions under shear flow. For L-selectin, it is interesting that this localization can occur even if the cytoplasmic tail is truncated to prevent interactions with the actin cytoskcleton [41].

Ligands and counter-receptors: lessons from antibodies It is emerging that several principles established previously with carbohydrate antigen-antibody systems apply equally to the interactions between endogenous carbohydrate-binding proteins and their ligands. It is therefore appropriate to briefly highlight some pertinent aspects. First, because of their low binding affinities, antibodies to the major blood group antigens (A, B, H, I,e a and Le b) and related carbohydrate differentiation antigens absolutely require their oligosaccharide epitopes to be in the clustered state (for example on epithelial mt, cins or on glycolipids) for binding to be observed in conventional binding assays; however, the free epitopebearing oligosaccharides effectively inhibit the binding when present in high concentrations [11,43,44]. As with the selectin ligands (see [45"1 and references therein), the binding determinants here are relatively rigid entities located at the nonreducing ends of oligosaccharide chains of varying backbone length. With antibodies to this family of oligosaccharides, the part played by the protein and lipid is no more than to present the oligosaccharide antigen. Second, there are nontrivial issues of cry'pticity at the surface of cells, particularly crypticity of carbohydrate antigens on glycolipids such that the antigens may not be detectable immunochemicallx, although chemically they are shown to be present in substantial amounts 146]. Thus, it is not a surprising phenomenon, and it is not difficult to envisage that a carbohydrate ligand may not be detectable within the complex microenvironment of the cell membrane because of limitations of oligosaccharide chain length and flexibility, as well as density of tim ligand in the presence of a diverse array of neighbouring molecules that may dwarf, crowd out and even interact with the ligand. T h e orientation of the ligands on carrier proteins and lipids are additional factors that may

Carbohydrate recognition systems Crocker and Feizi

683

Figure 3 Sequences and abbreviations for some of the oligosaccharides referred to in this review. With the exception of the globo-Lex, the two O-glycans of GlyCAM-1 (with and without 6-O-sulphation at GIcNAc, indicated by +HSO3), and the trisaccharide designated 3-0, these sequences occur as the peripheral domains of (poly)lactosamine backbones (type I, GalI31-3GIcNAc; type 2, GalI~I-4GIcNAc) of varying length.*For additional references to selectin ligands see [47]. tAbbreviations: E, E-selectin; L, L-selectin; P, P-selectin; Sn, sialoadhesin; CD, cluster of differentiation; MAG, myelin-associated glycoprotein; + indicates an interaction; - indicates no interaction; nt, not tested, ttMost likely ligands for L-selectin. SCD22, which has been investigated in more detail than the other sialoadhesins, has been shown to interact also with NeuAcc~2-6GlcNAc and NeuAcct2-6GalNAc sequences.

Ligands for selectins*

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+

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GalBI-3GIcNAc 13 [ctlA HSO~ Fuc

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+

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GalB 1-4GIcNAcB I-3GalB1-4GIcNAcB 1-3GalB I-

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GalBI-4GIcNABI.~6 GalNAcB l - 3 G a l a I-3GaIBI 4 G l c 3 Gall~| /

HNK 1

GIcABI-3GalB1-4GIcNAcBI-3GalBI-

13 HSOa GIyCAM- I

HSO~ Fuc r6 Ira.3 GalBI~GIcNAcBI\ 1~2,3 16 6 GalNAc NeuAc + H S O 3 3 GaIBI /

1~2,3

NeuAc Ligands for sialoadhesins 3-O

Galfil - 3GalNAc

3-N

GalBI-3/4GIcNAc ] ot2,3 NeuAc

6-N

GalBI4GIcNAc [ ct2,6 NeuAc

+

la2.3

NeuAc

determine whether a given epitope- or ligand-bearing glycoconjugate can be recognized. Simply for the purposes of inhibiting carbohydrate binding, for use as potential anti-inflammatory compounds, knowledge of the ligands would suffice; however, for a complete understanding of the biological effects, it is essential to identify the counter-receptors.

Ligands for E-selectin Among peripheral blood cells in the human, Le x antigen is a distinctive marker of granulocytes. S-Le x and the internally fucosylated analogue VIM-2 (Fig. 3) are antigens shared among granulocytes and monocytes [47]. Thus, when lectin-like domains were discovered on the endothelial adhesion molecules (E- and P-selectins) that

+S

bind to these blood cells, intensive research activities were directed to the above carbohydrate antigens as the candidate ligands [1,48]. T h u s far there has been general agreement that S-Le x and its isomer, S-Le a (the latter is an oligosaccharide occurring on epithelial rather than blood cells in the human), arc bound by the selectins. T h e 3'-O-sulphated-Le x and -Le a (Su-Le x and Su-Le a) are also bound by E- and L-selectins [49,50,51 °] as well as by P-selectin ([52]; RA Childs, T Feizi, unpublished data). Initial binding experiments with glycolipids extracted from leucocytes raised the possibility that among the heterogeneous long oligosaccharide chain sialoglycolipids, those with the VIM-2 sequence may be bound by E-selectin [53]. T h e r e has been no direct support for this so far. Rather, the inhibition of the E-selectin-myeloid cell

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Carbohydrates and glycoconjugates

interactions by monoclonal antibody CSLEX-1 (directed at S-Lex), but not by anti-VIM-2 [54], has indicated that for high avidity E-selectin binding, fucosylation of the subterminal rather than the internal N-acetyl glucosamine of the sialo-oligosaccharide sequence is a requirement (see Fig. 3). Four recent papers [55°-58 "] have rekindled interest in long-chain glycolipid ligands for E-selectin. Both in the extracts of human [55°-57"], and murinc [58 °] myeloid cells, and in murine kidney [58"], examined by chromatogram binding assays, E-selectin has been shown to bind only to long-chain, slowly migrating glycolipids. These have Rf values substantially less than those of S-Le x hexasaccharide based on the tetrasaccharide backbone (GalI]I--+4GlcNAcI31--+3GalI31-4GIc) [59] and of the VIM2 octasaccharide-based on the hexasaccharide backbone [60]. All are highly heterogeneous, and extremely minor components among the glycolipid populations. By using advanced mass spectrometry and IH-NMR in an unusually large scale study (starting with 1.2 litres of packed HL60 cells), sequence information was obtained on mononosialogangliosides based on backbones of disaccharides to dodecasaccharides [55",56°]. Monofucosyl S-Le x sequences could not be detected, however. Rather, many sequences with fucose at one or more of the inner N-acetyl glucosamines were identified. Fucose situated at penultimate N-acetyl glucosamine was detected only in the presence of one or two inner fucoses. E-selectin binding was observed in the regions of chromatograms containing these internally fucosylated glycolipids. Stroud et al. [56"] cite similar binding patterns with monosialogangliosides from normal human leucocytes, and suggest that E-selectin binds to the internally fucosylated monosialogangliosides. They also state that the CSLEX-1 antibody may not after all be monospecific for the S-Lex sequence and suggest that the antibody cross-reacts, not only with internally fucosylated sialo-oligosaccharide sequences such as the VIM-2 sequence, but also with the nonfucosylated sialo-oligosaccharide backbones. We concur with the authors that further investigation is needed of the CSLEX-1 antibody specificity. The question raised by these data is whether the binding observed was to relatively abundant components, or to minor components that could not be detected by the physico-chemical techniques used. A more reassuring picture regarding antibody specificity and the hitherto accepted sialyl ligands for E-selectin emerges from another study [57 °] in which there was excellent resolution and fractionation of glycolipids from human granulocytes. By a combination of immunochemical and mass spectrometric experiments Mtithing et al. identified unambiguously a S-Le x glycolipid based on a hexasaccharide backbone which was bound by CSLEX-1 but not VIM-2 antibody. Two other monofucosyl gan-

gliosides, based on the octasaccharide backbone with fucose on the inner (second) N-acetyl glucosamine and differing only in their ceramide moieties, were unreactive with CSLEX-1, but, as expected, were bound by the VIM-2 antibody. Several other fucogangliosides based on the octasaccharide or decasaccharide backbone, not fully sequenced, were bound by one or other antibody but not both. According to these results, the specificity of CSLEX-1 is unchallenged; the S-Lex sequence based on the hexasaccharide backbone is to be found on human granulocytes. In agreement with [55°,56 °] there is, in addition, an array of monosialyl-gangliosides based on octasaccharide to decasaccharide backbones with various internal fucosylation patterns. As E-selectin can bind, albeit with lower avidity, to clustered asialo-Lex sequence [1], the internal fucosylation motifs are indeed potential ligands for this adhesion molecule.

Murine leucocytes, in sharp contrast with those of the human, do not express immunochemically detectable Le x and S-Lex antigens [61] nor the Le a and S-Lea antigens [62]. Two approaches have been made to identifying E-selectin ligands in mice by exploiting the neoglycolipid technology [58°]. Having found evidence for the presence of (poly)lactosamine-type sequences among the E-selectin-binding glycolipid population extracted from the neutrophilic cell line 32Dc13, Osanai et al. [58 °] used endo-lB-galactosidase to release susceptible oligosaccharides of this series from the surface of these cells. Oligosaccharide fractions were converted into neoglycolipids, and the main E-selectin-binding component was identified by thin layer chromatography liquid secondary ion mass spectrometry (TLC-LSIMS), as a sialo-fuco-oligosaccharide of S-Lea/Le x type. This neoglycolipid was bound by monoclonal anti-S-Le a (2D3) but not by CSLEX-1. Several questions are raised by this finding. First, is S-Le a the main E-selectin-binding ligand on these cells, and is it also present on circulating myeloid cells of the mouse? Second, are there additional ligands based on sequences that are resistant to endo-13galactosidase, and which would have been missed by this approach? Third, why is the S-Le a antigen not detectable immunochemically either on the murine leucocytes or on the cell line 32Dc13 investigated? A possible answer to this question is that the display of the saccharide epitope on these cells is suboptimal for antibody binding, perhaps because of crowding by other selectin ligands that are not recognized by the antibody. Fourth, can the S-Le a on the murine cells be generated by fucosyltransferase VII, Fuc-T VII, an established oc 1-3 fucosyltransferase [63] whose products appear to be critical ligands for E-selectin at the murine leucocyte surface [64°°]? The findings [58°] raise the possibility either that Fuc-T VII may also act as a 4-fucosyltransferase or that an additional enzyme (a 4-fucosyltransferase) may act in concert with Fuc-T VII to generate ligand densities above the threshold needed for selectin-mediated adhesion.

Carbohydrate recognition systems Crocker and Feizi

A second class of glycolipid bound by E-selectin has been characterized in the kidney extracts of BALB/c mice [58°]. By chromatogram binding experiments and in situ LSIMS using neoglycolipids derived from oligosaccharides released with endoglycoceramidase, in conjunction with compositional and linkage analyses, the oligosaccharide moiety was identified as the Lex-active extended, branched globo sequence [65] (Fig. 3). Evidence was found for the presence of novel sialyl analogues of the branched globo sequence, raising the possibility that fucosyl (S-Le x) analogues may also be present although not detected. It will be interesting to determine the cellular distribution of the globo-Lex-type sequence, especially among epithelia and malignant cells derived from them, as its presence in the kidney is genetically determined [65], and it may therefore contribute to the metastatic potential of epithelial tumours in various inbred strains of mice.

Ligands for L-selectin Sulphate is an important component of the ligands for L-selectin. This has been clearly shown by metabolic studies of the endothelial glycoprotein counter-receptors [2] and by binding experiments with structurally defined oligosaccharides of the Le a, Le x and sulphoglucuronyl series (HNK-1) (Fig. 3) and sulphated nonfucosylated glycolipids [1]. From binding experiments with the recombinant soluble protein, it is clear that there is an overall preference for the sulphated Le a and Lc x sequences over the sialyl analogues, to the extent that the nonfucosylated sulphated backbones are bound (albeit with lower avidities), but not the sialyl analogues [51"]. L-selectin interacts with blood group or ganglio series sequences that have a terminal galactose that is variously sulphated, for example, 3-0, 4-0, 6-0, 3,4-O-disulphated or 3,6-O-disulphated [1,66]. There is increasing evidence that L-selectin also binds to glycosaminoglycans. When presented as neoglycolipids, glycosaminoglycan disaccharides of keratan sulphate, heparin and chondroitin sulphate types are bound [51°], leading to the conclusion that clustered oligosaccharides with 6-0 sulphation of N-acetyl galactosamine, N-acetyl glucosamine or glucosamine, 4-0 sulphation of N-acetyl galactosamine, 2-O-sulphation of uronic acid, N-sulphation of glucosamine, and (to a lesser extent) the nonsulphated disaccharides containing uronic acid can all support L-selectin binding. Endothelial cells contain heparan sulphate glycosaminoglycans with various sulphation patterns, including nonsulphated glucosamine, to which immobilized L-selectin binds with high avidity [67°]. All this suggests that endothelial glycosaminoglycans may be among the natural ligands for L-selectin. Such interactions of L-selectin have been proposed [51 °] to provide a link between the selectin-mediated and the integrin-mediated adhesion systems in leucocyte extravazation cascades, as glycosaminoglycans serve as reservoirs for inflammato~- chemokines (short-range stimulators of lymphocyte migration which trigger integrin activation [68]).

685

Progress has been made in characterizing oligosaccharides of the endothelial counter-receptor for L-selectin, GIyCAM-1, in the mouse. A profiling study was performed on the full-length oligosaccharides released by ]3-elimination from the purified glycoprotein [69°]. The oligosaccharides, although heterogeneous, appear rather less so than those typically found in mixtures of mucin glycoproteins produced by epithelial cells, particularly with respect to their cores and backbone chain lengths. Two heptasaccharides were sequenced (Fig. 3) and found to contain S-Lex capping groups with 6-O-sulphation of the terminal galactose, with or without 6-O-sulphation of the penultimate N-acetyl glucosamine (Fig. 3). Could these represent the preferred oligosaccharide ligands for L-selectin? In a different approach [70], mucin preparations and their O-sialoglycoprotease-treated forms from epithelial cells were separated into fractions which were bound or not bound by immobilized L-selectin. Oligosaccharides then released from the mucins were examined for size and charge, but no distinctive features were observed for the bound and nonbound fractions. The low affinity of immobilized L-selectin for the free oligosaccharides released from the mucins precluded the enrichment of fractions with any minor oligosaccharide components specifically bound by L-selectin.

Are oligosaccharides alone sufficient for selectin binding? This is a recurring question. Certainly, under static assay conditions, oligosaccharide ligands presented in a clustered state, linked to either lipid or protein, are avidly bound by E-, L- and P-selectins [1,48,51°]. Also, glycosaminoglycans are able to support L-selectin binding both as neoglycolipids [51 °] and as oligosaccharides released from proteoglycans [67"]. In vitro experiments, under conditions of physiological flow, now clearly show that lipid-linked oligosaccharides, such as S-Le x glycolipids and S-Lc a neoglycolipids based on tetrasaccharide backbones, can mediate E- and L-selectin-dependent leucocyte tethering and rolling [71°]. The important influence of the modes of immobilization/presentation of the oligosaccharides on selectin recognition have been highlighted by some of the data from in vitro assay systems. For example, a trisulphated heparin disaccharide in the form of neoglycolipid is bound by L-selectin when presented on plastic microwells, but not on chromatograms [51°]. It has been suggested that such differences in selectin recognition in vitro may translate into differential L-sectin binding to a given saccharide ligand in different microenvironments in vivo.

Ligands for sialoadhesins Human CD22 has been investigated for its interactions with a range of structurally defined oligosaccharides [39°]. The conclusion is that the minimum structure recognized by CD22 is a commonly occurring sequence,

686

Carbohydratesand glycoconjugates

N-acetyl neuraminic acid 6-1inked to Gal, or GlcNAc, or GalNAc (Fig. 2). T h e carbohydrate-binding specificities of the four members of the sialoadhesin family have been compared by performing binding experiments with human erythrocytes that have been desialylated and resialylated using 3- and 6-sialyhransferases that have well defined specificities towards galactose-terminating iV- and O-glycans (Fig. 2) [38",72]. Aside from linkage specificity, sialoadhesins exhibit differential recognition of various forms of sialic acid which could be important in the fine-tuning of interactions at the cell surface [73,74]. In addition to binding to oligosaccharides carried on glycoproteins, sialoadhesin and myelin-associated glycoprotein (MAG) have been shown to bind to gangliosidcs in chromatogram binding assays [75,76]. In accord with binding specificities observed for derivatized erythrocytes [72], the binding preference of sialoadhesin and MAG is towards gangliosides with 3-sialyl termini.

C o u n t e r - r e c e p t o r s for selectins Determining the physiological counter-receptors on intact cell membranes for endogenous lectins such as the selectins is a particularly challenging task, given that they can interact with multiple oligosaccharide sequences, and P- and L-selectins can, in addition, bind to simple motifs such as sulphated galactose (sulphatide). A common approach to identifying glycoproteins that may serve as counter-receptors for selectins is to perform binding or enrichment experiments using lysates of the target cells. A disadvantage of this approach is that macromolecules that depend on ordered clustering (on the membrane) for binding activity may be missed [1]. Conversely, false positive reactions may result if macromolecules are artificially aggregated under the assay conditions. A further risk is the possible isolation of intracellular or otherwise inaccessible glycoproteins which, although possessing high avidity for the lectin, may not be available at the cell surface to mediate cell-cell interactions. This possibility has been raised [70] for a putative counter-receptor for E-selectin that has been designated 'E-selectin ligand-l' (ESL-1) [77"], which has been isolated independently from rat cerebellum as a marker of the Golgi apparatus. Apparently, it is not expressed on the surface of neuronal cells [78], but it has been detected at the surface of mouse neutrophils (D Vestweber, personal communication). The soluble recombinant P-selectin (selectin-IgG chimera) binds with high avidity to immobilized S-Lex sequence in a clustered display [48]. Although multiple glycoproteins carry the S-Lex sequence on myeloid cells, attempts to identify the counter-receptors have so far revealed a single glycoprotein, 'P-selectin glycoprotein ligand-l' (PSGL-1), the assembly of which has been the subject of several investigations reviewed here. Similarly, for L- and E-selectins, discrete glycoproteins can be revealed by affinity purification from target cell lysates [2]; PSGL-1 features

among these in the case of E-selectin [79,80,81",82"] (Fig. 2). Human PSGL-1 is a 402 residue disulphide-linked homodimeric type 1 membrane glycoprotein with an cxtracellular domain that contains multiple O-linked glycans and three potential sites for addition of N-glycans [79,83]. On human myeloid cells this glycoprotein has features of a physiological counter-receptor for P-selectin. A specific monoclonal antibody, PL1, directed to the N terminus of PSGL-1 is able to block rolling of neutrophils on P-selectin both in vitro [42] and in vivo [84]. Furthermore, PSGL-1 has been shown to be localized by immunoelectron microscopy to the tips of neutrophil microvilli [42], and by rotary shadowing and electron microscopy it can be visualized as an extended molecule of approximately 50 nm [85]. Taken together, these findings indicate that PSGL-1 is optimally orientated at the leucocyte cell surface to mediate effective interactions with P-sclectin expressed on endothelial cells. Using monoclonal antibodies that recognize protein determinants of PSGL-1 [42,86"], it has been shown that the protein is expressed on all blood leucocytes, but not necessarily in a form that binds P-selectin. For example, T lymphocytes were shown to express high levels of PSGL-1 constitutively, but only when these cells had been activated were they able to bind to P-selectin in a P S G L - l - d e p e n d e n t manner [86"]. Where examined, activities of enzymes involved in biosynthesis of S-Le x have been demonstrated as essential in cells for the production of a functional PSGL-I. Lymphocyte activation was accompanied by increased activity of 131-6 GIcNAc transferase (known as the core 2 enzyme) and c~1-3 fucosyltransferase, but no apparent increase in the levels of the PSGL-1 protein [86"]. Using stably transfected C H O cells, which do not express ~1-3 fucosyltransferase and lack O-linked glycans containing core 2, it was shown that the generation of high-avidity forms of PSGL-1 depended on the coexpression of the core 2 enzyme and an c~1-3 fucosyhransferase [82"]. T h e core 2 enzyme converts the GalI31-3GalNAc (core 1) to GaI131-3(GIcNAcI31-6)GalNAc (core 2) which is apparently a good acceptor for c~1-3 fucosyhransferase. Also, human cancer cells that do not express PSGL-1, but that do express the appropriate glycosyhransferases for synthesis of S-Lex, bind poorly to P-selectin [87"]. By transfecting these cells with PSGL-1 cDNA, it was possible to induce high levels of binding to P-selectin. Correct glycosylation of O-linked rather than N-linked glycans of PSGL-1 is a requirement for high-avidity binding [42,79,81"], In a construct where the O-glycosylation sites were provided wholly by CD43, however, the binding of P-selectin was not lost 188"']. Therefore, a unique protein moiety of PSGL-1 does not appear to be critical for recognition by P-sclectin. At the N-terminal region of the mature PSGL-1 protein there are three clustered tyrosine residues which are

Carbohydrate recognition systems Crockerand Feizi

potential sites of sulphation. Three research groups have demonstrated independently that sulphation of at least one of those tyrosines is an additional requirement for high-avidity binding by P-selectin [81",88°',89"]. This contrasts with observations on the L-selectin counter-receptor GlyCAM-1 [2], where sulphation is predominantly on oligosaccharidc chains. By generating deletion mutants it was shown that constructs expressing only the first 19 or 70 amino acids of mature PSGL-1, which contain the three tyrosines and two potential O-glycosylation sites, supported binding of P- but not E-selctin, although the intensity of P-selectin binding was considerably less than to the full-length construct [81",88"*]. When the three tyrosine residues were replaced by tryptophan, there was a marked reduction in avidity of P-selectin binding such that binding could only be detected in a muhivalent cell binding assay [81"}. Treatment with sialidase, or excluding the presence of a fucosyltransferase resulted in complete loss of P-selectin binding. Moreover, the binding to the sulphated tyrosine-containing 19 amino acid fragment was abolished when Thrl6, a potential O-glycosylation site, was replaced with alanine. Thus, O-glycosylation of the polypeptide with S-Le x (or a related sequence) is essential to generate sufficient avidity for detection of binding. Collectively these data suggest that for P-selectin, in contrast with E-selectin, there is cooperativity between oligosaccharides and sulphotyrosines. A major challenge for the future is to establish whether a given P-selectin molecule binds simultaneously or alternatively to oligosaccharide and sulphotyrosine.

Counter-receptors for CD22 Identification of counter-receptors for members of the sialoadhesin family is so far limited to CD22. Similarly to selectins, this protein is able to bind with a high degree of specificity only to selected glycoproteins. An early investigation suggested that the leucocyte common antigen, CD45, could function as a counter-receptor for CD22 [90] but doubts about this were raised when it was shown that the type of antibody preparation (ascites) that was used to block the binding to CD45 contains glycoproteins that are potent inhibitors of the lectin activity of CD22 [91]. Attempts to precipitate counter-receptors on T and B lymphocytes for CD22 using recombinant Fc-chimaeras have, nevertheless, confirmed that CD45 is recognized by CD22, along with other glycoproteins including CD22 itself (see [29"] and references therein). In a recent study based on plasmon resonance spectroscopy, it was shown that a recombinant form of routine CD22 bound avidly to purified, native rat CD45 {31"]. In contrast, a recombinant form of rat CD45 expressed in C H O cells that carry exclusively 3'-sialyl oligosaccharides was not recognized by CD22 unless it was desialylated, and then resialylated enzymatically to carry NeuGcc~2-6Gal~l-4GlcNAc, one of the ligands for murine CD22 [73]. T h e selective binding of CD22 to subsets of glycoproteins has also been demonstrated by affinity chromatography

687

of glycoproteins in human plasma [92]. Although many plasma glycoproteins car D" the 6'-sialyl-oligosaccharide structures recognized by CD22, only two glycoproteins, haptoglobin and IgM, were bound. Thus, CD22 binds differentially to its oligosaccharidc ligands when presented on different proteins.

The collectins and c o m p l e m e n t C3 glycoprotein Remarkable effects (permissive or nonpermissive) can be exerted by a carrier protein in the presentation of an oligosaccharide ligand. This has been highlighted by results of binding experiments in a model system using the collectins, conglutinin, MBP-A and collectino43 (CL-43) as the receptors, and the complement glycoprotein C3 and derived glycopeptides as potential counter-receptors [93,94"]. C3 contains an oligomannose chain (Man8 or Man9) at Asn917 of the alpha chain. T h e free oligosaccharides, Man8 and Man9, are ligands for all three collectins but are cryptic (inaccessible) on the unmodified protein. T h e alpha chain of (;3 becomes sequentially clipped proteolytically in the complement cascade to yield the glycopeptides C3b, iC3b, and C3c, but without loss of oligosaccharide. It was found that MBP-A did not bind to any of the C3 forms produced in the complement cascade. In contrast, the sequential proteolytic cleavages were found to reveal or render cryptic the oligosaccharide ligand, but differentially for conglutinin and CL3, such that conglutinin bound in a carbohydrate-dependent manner to C3b and iC3b but not to C3c [94",95], whereas CL-43 bound only to C3c [93]. Conclusion Now that several of the natural ligands for lectin-type receptors have been defined, attention is being focused on factors that determine and regulate assembly of the ligands into effective counter-receptors. It is clear that for E- and L-selectins, the presence of clustered oligosaccharide ligands on lipids can suffice, not only for binding under static conditions, but also for binding under conditions of physiological flow. P-selectin also binds well to such clustered oligosaccharides in the absence of protein. Both P- and L-selectins also bind to simple sulphated motifs on monosacchatides, when these are ch, stered. When oligosacchaxide ligands are located on glycoproteins, however, there are variable effects: either high-avidity binding may result from clustered displa.~; oi the carrier protein may exert considerable constraints on ligand availability for binding. Thus, P-selectin binds with only low avidity to the mucin-like S-Lex-bearing region of the glycoprotein PSGL-1; the binding avidity is increased when there are also sulphated tyrosines at the N-terminal region of the glycoprotein. T h e molecular basis of the mutual dependence of the two types of ligand on this glycoprotein remains to be determined. Is it that there is suboptimal clustering or orientation of oligosaccharides on PSGL-1, and this is compensated for by the presence of the alternative ligands, the sulphated tyrosines? Or is

688

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there a need for dual a t t a c h m e n t of a given P-selectin molecule to the two different ligands? Or is there more to it than that? As for the sialoadhesins and the collectins, what are the factors that d e t e r m i n e w h e t h e r they bind to their oligosaccharide ligands borne on various proteins?

Note added in

proof

Since submitting this review a paper has been published [98"] which describes the construction of a series of soluble domain swap chimaeras b e t w e e n E- and L-selectins.

References and recommended

into E-selectin/ligand interaction from the crystal structure and mutagenesis of the lec/EGF domains. Nature 1994, 367:532-538, 18.

Erbe DV, Wolitzky BA, Presta LG, Norton CR, Ramos RJ, Burns DK, Rumberger JM, Rao BN, Foxall C, Brandley BK, Lasky LA: Identification of an E-selectin region critical for carbohydrate recognition and cell adhesion. J Ceil Biol 1992, 119:215-227. [Published erratum appears in J Cell Biol 1993, 120:1071].

19.

Erbe DV, Watson SR, Presta LG, Wolitzky BA, Fexall C, Brandley BK, Lasky LA: P- and E-selectin use common sites for carbohydrate ligand recognition and cell adhesion. J Cell Biol 1993, 120:1227-1235.

20.

Hollenbaugh D, Bajorath J, Stenkamp R, Aruffo A: Interaction of P-selectin (CD62) and its cellular ligand: analysis of critical residues. Biochemistry 1993, 32:2960-2966.

21.

Bajorath J, Hollenbaugh D, King G, Harte W Jr, Eustice DC, Darveau RP, Aruffo A: CD62/P-selectin binding sites for myeloid cells and sulfatides are overlapping. Biochemistry 1994, 33:1332-1339.

reading

Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest • = of outstanding interest 1.

Feizi T: Oligosaccharides that mediate mammalian cell-cell adhesion. Curt Opin Struct Biol 1993, 3:701-710.

2.

Rosen SD, Bertozzi CR: Leukocyte adhesion: two selectins converge on sulphate. Curr Bio/1996, 6:261-264.

22. *

Revelle BM, Scott D, Kogan TP, Zheng J, Beck PJ: Structurefunction analysis of P-selectin-sialyl LewisX-binding interactions. J Biol Chem 1996, 271:4289-4297. See annotation [27"]. 23.

Hollenbaugh D, Aruffo A, Senter PD: Effects of chemical modification on the binding activities of P-selectin mutants. Biochemistry 1995, 34:56?8-5684.

3.

Liebecq C: International Union of Biochemistry and Molecular Biology. Biochemical Nomenclature and Related Documents. London: Portland Press; 1992.

4.

Drickamer K: Increasing diversity of animal lectin structures. Curt Opin Struct Bio11995, 5:612-616.

5.

Hebert DN, Foellmer B, Helenius A: Glucose trimming and reglucosylation determine glycoprotein association with calnexin in the endoplasmic reticulum. Cell 1995, 81:425-433.

25. •.

6.

Ware FE, Vassilakos A, Peterson PA, Jackson MR, Lehrman MA, Williams DB: T h e molecular chaperone calnexin binds GlclMangGIcNAc2 oligosaccharide as an initial step in recognizing unfolded glycoproteins. J Biol Chem 1995, 270:4697-4704.

Three elegant studies [14",15"',25"] combine X-ray crystallography with site-directed mutagenesis to explore the structural basis of monosaccharide recognition by C-type lectins.

7.

Sousa M, Parodi AJ: The molecular basis for the recognition of misfolded glycoproteins by the UDP-GIc:glycoprotein glucosyltransferase. EMBO J 1995, 14:4196-4203.

8.

Peterson JR, era A, Van PN, Helenius A: Transient, lectin-like association of calreticulin with folding intermediates of cellular and viral glycoproteins. Mol Biol Cell 1995, 6:1173-1184.

9.

Spire RG, Zhu Q, Bhoyroo V, S61ing H: Definition of the lectinlike properties of the molecular chaperone, calreticulin, and demonstration of its copurification with endomannosidase from rat liver golgi. J Bio/Chem 1996, 271:11586-11594.

10.

Komfeld S: Structure and function of the mannose 6phosphate/insulin-like growth factor-II receptors. Annu Rev Bioohem 1992, 61:307-330.

11.

Feizi T: Demonstration by monoclonal antibodies that carbohydrate structures of glycoproteins and glycolipids are once-developmental antigens. Nature 1985, 314:53-57

12.

Powell LD, Varki A: I-type lectins. J Bio/Chem 1995, 270:14243-14246.

13.

Weis WI, Drickamer K, Hendrickson WA: Structure of a C-type mannose-binding protein complexed with an oligosaccharide. Nature 1992, 360:127-134.

14. See

Kolatkar AR, Weis WI: Structural basis of galactose recognition by C-type animal lectins. J Bio/Chem 1996, 271:6679-6665. annotation [25"'].

15. •.

Ng KK, Drickamer K, Weis WI: Structural analysis of monosaccharide recognition by rat liver mannose-binding protein. J Biol Chern 1996, 271:663-674. See annotation [26"].

24.

Kogan TP, Revelle BM, Tapp S, Scott D, Beck PJ: A single amino acid residue can determine the ligand specificity of E-selectin. J Biol Chem 1995, 270:14047-14055. See annotation [27"]. •

Iobst ST, Drickamer K: Selective sugar binding to the carbohydrate recognition domains of the rat hepatic and macrophage asialoglycoprotein receptors. J Biol Chern 1996, 271:6686-6693.

26.

Cooke RM, Hale RS, Lister SG, Shah G, Weir MP: The conformation of the sialyl Lewis X ligand changes upon binding to E-selectin. Biochemistry 1994, 33:10591-10596.

27.

Blanck O, Iobst ST, Gabel C, Drickamer K: Introduction of selectin-like binding specificity into a homologous mannosebinding protein. J Biol Chem 1996, 271:7289-7292. In three studies [22",24",27"], site-directed mutagenesis of E-selectin [24"], P-selectin [22"[ and mannose-binding protein [27"] resulted in changes in carbohydrate-binding specificities. •

28.

Nath D, Van der Merwe PA, Kelm S, Bradfield P, Crocker PR: The amino-terminal immunoglobulin-like domain of sialoadhesin contains the sialic acid binding site. J Bio/Chern 1995, 270:26184-26191. See annotation [30"]. •

29. •

Law CL, Aruffo A, Chandran KA, Doty RT, Clark EA: Ig domains 1 and 2 of murine CD22 constitute the ligand-binding domain and bind multiple sialylated ligands expressed on B and T cells. J Immune/1995, 155:3368-3376. See annotation [30"]. 30.

Engel P, Wagner N, Miller AS, Tedder TF: Identification of

•

t h e ligand-binding domains of CD22, a member of the

immunoglobulin superfamily that uniquely binds a sialic aciddependent ligand, J Exp Med 1995, 181:1581-1566. By creating deletion mutants, three papers [28"-30"] identified the sialic acid binding region of CD22 as being within the N-terminal two Ig-like domains. In addition, Nath et al. [28"] showed that the N-terminal V-set domain of sialoadhesin is both necessary and sufficient for sialic acid dependent binding. 31. •

16.

Drickamer K: Engineering galactose-binding activity into a Ctype mannose-binding protein. Nature 1992, 360:183-186.

Van der Merwe PA, Crocker PR, Vinson M, Barclay AN, Schauer R, Kelm S: Localization of the putative sialic acid-binding site on the immunoglobulin superfamily cell-surface molecule CD22. J Bio/Chem 1996, 271:9273-9280. See annotation [32"].

17.

Graves BJ, Crowther RL, Chandran C, Rumberger JM, Li S, Huang KS, Presky DH, Familletti PC, Wolitzky BA, Burns DK: Insight

32. •

Vinson M, Van der Merwe PA, Kelm S, May A, Jones EY, Crocker PR: Characterisation of the sialic acid binding site in

Carbohydrate recognition systems Crocker and Feizi

sialoadhesin by site-directed mutagenesis. J B/el Chem 1996, 271:9267-9272. In two studies [31",33°], the results of site-directed mutagenesis experiments indicated that the sialic acid binding sites of sialeadhesin and CD22 are located within the GFCCC sheet of the N-terminal Ig-like domains. 33.

Jones EY, Davis ~J, Williams AF, Harlos K, Stuart DI: Crystal structure at 2.8A resolution of a soluble form of the cell adhesion molecule CD2. Nature 1992,360:232-239.

34.

Jones EY, Harlos K, Bottomley MJ, Robinson RC, Driscoll PC, Edwards RM, Clements JM, Dudgeon TJ, Stuart Dh Crystal structure of an integrin-bindjng fragment of vascular cell adhesion molecule-1 at 1.8 A resolution. Nature 1995, 373:539-544.

35.

Patel KD, Nollert MU, McEver RP: P-selectin must extend a sufficient length from the plasma membrane to mediate rolling of neutrophils. J Ceil Biol 1995, 131:1893-1902.

36.

Crocker PR, Mucklow S, Bouckson V, McWilliam A, Willis AC, Gordon S, Milon G, Kelm S, Bradfield P: Sialoadhesin, a macrophage sialic acid binding receptor for haemopoietic cells with 17 immunoglobulin-like domains. EMBO J 1994, 13:4490-4503.

37.

Braesch-Andersen S, Stamenkovic I: SialylatJon of the B lymphocyte molecule CD22 by alpha 2,6-sialyltransferase is implicated in the regulation of CD22-mediated adhesion. J Bio/ Chem 1994, 269:11783-11786.

Freeman SD, Kelrn S, Barber EK, Crocker PR: Characterization of CD33 as a new member of the sialoadhesin family of cellular interaction molecules. Blood 1995, 85:2005-2012. This is the first demonstration that CD33 can function as a sialic acid binding protein.

48.

Bevilacqua MP, Nelson RM: Selectins. J Clin Invest 1993, 91:379-387.

49.

Y u e nC-T, Lawson AM, Chai W, Larkin M, Stoll MS, Stuart AC, Sullivan FX, Ahem TJ, Feizi T: Novel sulfated ligands for the cell adhesion molecule E-selectin revealed by the neoglycolipid technology among O-linked oligosaccharides on an ovarian cystadenoma glycoprotein. Biochemistry 1992, 31:9126-9131.

50.

Green PJ, Tamatani T, Watanabe T, Miyasaka M, Hasegawa A, Kiso M, Yuen C-T, Stoll MS, Feizi T: High affinity binding of the leucocyte adhesion molecule L-selectin to 3'-sulphated-Lea and -Lex oligosaccharides and the predominance of sulphate in this interaction demonstrated by binding studies with a series of lipid-linked oligosaccharides. Biochem Biophys Res Commun 1992, 188:244-251.

51. •

Green PJ, Yuen C-T, Childs RA, Chai W, Miyasaka M, Lemoine R, Lubineau A, Smith B, Ueno H, Nicolaou KC, Feizi T: Further studies of the binding specificity of the leukocyte adhesion molecule, L-selectin, towards sulphated oligosaccharides - suggestion of a link between the selectin- and the integrinmediated lymphocyte adhesion systems. Glycobio/ogy 1995, 5:29-38. The binding of recombinant soluble rat L-selectin was demonstrated to a range of sulphated oligosaccharides of the blood group Lea and Lex and glycosaminoglycan series. 52.

Brandley BK, Kiso M, Abbas S, Nikrad P, Srivasatava O, Foxall C, Oda Y, Hasegawa A: Structure-function studies on selectin carbohydrate ligands. Modifications to fucose, sialic acid and sulphate as a sialic acid replacemenL Glycobiology 1993, 3:633-641.

53.

Tiemeyer M, Swiedler SJ, Ishihara M, Moreland M, Schweingruber H, Hirtzer P, Brandley BK: Carbohydrate ligands for endothelium leukocyte adhesion molecule-1. Proc Nat/Acad Sci USA 1991,

38. •

Powell LD, Jain RK, Matta KL, Sabesan S, Varki A: Characterization of sialyloligosaccharide binding by recombinant soluble and native cell-associated CD22. Evidence for a minimal structural recognition motif and the potential importance of multisite binding. J Bio/Chem 1995, 270:7523-7532. This is a thorough study of the reactivities of human CD22 with various 6'sialyl oligosaccharides.

39. •

40.

Picker U, Warnock RA, Burns AR, Doerschuk CM, Berg EL, Butcher EC: The neutrophil selectin LECAM-1 presents carbohydrate ligands to the vascular selectins ELAM-1 and GMP-140 Ceil 1991, 66:921-933. [Published erratum appears in Co//1991, 67:1267].

41.

Pavalko FM, Walker DM, Graham L, Goheen M, Doerschuk CM, Kansas GS: The cytoplasmic domain of L-selectin interacts with cytoskeletal proteins via alpha-actinin: receptor positioning in microvilli does not require interaction with alphaactinin. J Ceil Bio/1995, 129:1155-1164.

42.

Moore KL, Patel KD, Bruehl RE, Li F, Johnson DA, Lichenstein HS, Cummings RD, Bainton DE McEver RP: P-selectin glycoprotein ligand-1 mediates rolling of human neutrophils on P-selectin. J Ceil Biol 1995, 128:661-671.

43.

Watkins WM: Biochemistry and genetics of the ABO, Lewis and P blood group systems. Adv Hum Genet 1980, 10:1-136, 379-385.

44.

Kabat EA: Contributions of quantitative immunochemistry to knowledge of blood group A, B, H, Le, I and i antigens. Am J C/in Pathol 1982, 78:281-292.

45. •

Kogelberg H, Frenkiel TA, Homans SW, Lubineau A, Feizi T: Conformational studies on the selectin and natural killer cell receptor ligands sulfo- and sialyl-lacto-N-fucopentaoses (SuLNFPII and SLNFPll) using NMR spectroscopy and molecular dynamics simulations. Comparisons with the nonacidic parent molecule LNFPII. Biochemistry 1996, 35:1954-1964. In the sulphated Lea pentasaccharide (as with th3 sialyl analogue), two rigid domains, the Lea trisaccharide at the nonreducing end, and the disacchafide lactose sequence at the reducing end, were shown to be joined by a flexible linkage. 46.

Hakomori S: Tumor-associated carbohydrate antigens. Annu Roy Immunol 1984, 2:103-126.

47

Feizi T: Cell-cell adhesion and membrane glycosylation. Curt Opin Struct Bio11991, 1:766-770.

689

88:1138-1142.

54.

Lowe JB, Steolman LM, Nair RP, Larsen RD, Berhend TL, Marks RM: ELAM-l-dependent cell adhesion to vascular endothelium determined by a transfected human fucosyltransferase cDNA. Ceil 1990, 63:475-484.

55. •

Stroud MR, Handa K, Salyan MEK, Ito K, Levery SB, Hakomori S, Reinhold BB, Reinhold VN: Monosialogangliosides of human myelogenous leukemia HL60 cells and normal human leukocytes. 1. Separation of E-selectin binding from nonbinding gangliosides, and absence of sialosyI-Lex having tetraosyl to octaosyl core. Biochemistry 1996, 35:758-769. See annotation [57"]. 56. •

Stroud MR, Handa K, Salyan MEK, Ito K, Levery SB, Hakomori S, Reinhold BB, Reinhold VN: Monosialogangliosides of human myelogenous leukemia HL60 cells and normal human leukocytes. 2. Characterization of E-selectin binding fractions, and structural requirements for physiological binding to Eselectin. Biochemistry 1996, 35:770-778. See annotation [57"]. 57. •

M(JthingJ, Spanbroek R, Peter-Katalinic J, Hanisch FG, Hanski C, Hasegawa A, Unland F, Lehmann J, Tschesche FI, Egge H: Isolation and structural characterization of fucosylated gangliosides with linear poly-N-acetyllactosaminyl chains from human granulocytes. Glycobiology 1996, 6:147-156. These are investigations [55"-57 °] of the sequences of the highly heterogeneous, long-chain monosialogangliosides isolated from human myeloid cells, together with their interactions with some monoclonal antibodies [56°,57 "] and E-selectin [56"]. 58. •

Osanai T, Feizi T, Chai W, Lawson AM, Gustavsson ML, Sudo K, Araki M, Araki K, Yuen C-T: Two families of murine carbohydrate ligands for E-selectin. Biochem Biophys Res Commun 1996, 218:610-615. Neoglycolipid technology was used to characterize E-selectin-binding oligosaccharide sequences at the surface of a murine myeloid cell line and on glycolipids isolated from kidneys of BALB/c mice. 59.

Fukushima K, Hirota M, Terasaki PI, Wakisaka A, Togashi H, Chia D, Suyama N, Fukushi Y, Nudelman E, Hakomori S: Characterisation of sialosylated Lewis x as a new tumorassociated antigen. Cancer Res 1984, 44:5279-5285.

60.

Macher BA, Buehler J, Scudder P, Knapp W, Feizi T: A novel carbohydrate differentiation antigen on fucogangliosides of human myeloid cells recognized by monoclonal antibody VIM2. J Biol Chem 1988, 263:10186-10191.

690

Carbohydrates and glycoconjugates

61.

Thorpe S J, Feizi T: Species differences in the expression of carbohydrate differentiation antigens on mammalian blood cells revealed by immunofluorescence with monoclonal antibodies. Biosei Rap 1984, 4:6?3-685.

62.

Ito K, Handa K, Hakomori S: Species-specific expression of sialosyI-Le(x) on polymorphonuclear leukocytes (PMN), in relation to selectin-dependent PMN responses. G/ycoconjugate J 1994, 11:232-237.

63.

Smith PL, Gersten KM, Petryniak B, Kelly RJ, Rogers C, Natsuka Y, AIford JA III, Scheidegger EP, Natsuka S, Lowe JB: Expression of the (x(1-3)fucosyltransferase Fuc-TVII in lymphoid aggregate high endothelial venules correlates with expression of Lselectin ligands. J Bio/Chem 1996, 271:8250-8259.

Maly P, Thall AD, Petryniak B, Rogers CE, Smith PL, Marks RM, Kelly RJ, Gersten KM, Cheng G, Saunders TL et al.: The ~(1,3)fucosyltransferase Fuc-TVII controls leukocyte trafficking through an essential role in L-, E-, and P-selectin ligand biosynthesis. Ceil 1996, 86:643-653. A dominant role for fucosyltransferase VII in generating the oligosaccharide ligands for E- and L-selectins was shown in mice deficient in this enzyme.

77. •

Steegmaler M, Levinovitz A, Isenmann S, Borges E, Lenter M, Kocher HP, Kleuser B, Vestweber D: The E-selectin-ligand ESL-1 is a variant of a receptor for fibroblast growth factor. Nature 1995, 373:615-620. ESL-1, with two N-glycosylation sites, cotransfected with fucosyltransferase Ill is shown to be bound by E- but not L-selectin when expressed in cells. 78.

Gonatas JO, Mourelatos Z, Stieber A, Lane WS, Brosius J, Gonatas NK: MG-160, a membrane sialoglycoprotein of the medial cisternae of the rat Golgi apparatus, binds basic fibroblast growth factor and exhibits a high level of sequence identity to a chicken fibroblast growth factor receptor. J Ceil Sci 1995, 108:457-467.

79.

Sako D, Chang XJ, Barone KM, Vachino G, White HM, Shaw G, Veldman GM, Bean KM, Ahem TJ, Furie B e t a/.: Expression cloning of a functional glycoprotein ligand for P-selectin. Ceil 1993, 75:1179-1186.

80.

Asa D, Raycroft L, Ma L, Aeed PA, Kaytes PS, Elhammer AP, Gang J: The P-selectin glycoprotein ligand functions as a common human leukocyte ligand for P- and E-selectins. J Biol Chem 1995, 270:11662-11670.

64. •.

65.

Sekine M, Nakamura K, Suzuki M, Inagaki F, Yamakawa T, Suzuki A: A single autosomal gene controlling the expression of the extended globoglycolipid carrying SSEA-1 determinant is responsible for the expression of two extended globogangliosides. J Biochem (Tokyo) 1988, 103:722-729.

66.

Bertozzi CR, Fukuda S, Rosen SD: Sulfated disaccharide inhibitors of L-selectin: deriving structural leads from a physiological selectin ligand. Biochemistry 1995, 34:14271-14278.

67. •

Norgard-Sumnicht K, Varki A: Endothelial heparan sulfate proteoglycans that bind to L-selectin have glucosamine residues with unsubstituted amino groups. J Bio/Chem 1995, 270:12012-12024. Heparan sulphate glycosaminoglycans released from endothelial proteogiycans were shown to be bound by L-selectin. 68.

Tanaka Y, Adams DH, Shaw S: Proteoglycans on endothelial cells present adhesion-inducing cytokines to leukocytes. Immuno/Today 1993, 14:111-115.

Hemmedch S, Leffler H, Rosen SD: Structure of the O-glycans in GlyCAM-1, an endothelial-derived ligand for L-selectin. J Biol Chem 1995, 270:12035-12047. Oligosaccharide sequences were determined for two O-glycans isolated from GlyCAM-1, which is a counter-receptor for the murine L-selectin.

81. •o

Sako D, Comess KM, Barone KM, Camphausen RT, Cumming DA, Shaw GD: A sulfated peptide segment at the amino terminus of PSGL-1 is critical for P-selectin binding. Ceil 1995, 83:323-331. See annotation [89"]. 82.

Li F, Wilkins PP, Crawley S, Weinstein J, Cummings RD, McEver RP: Post-translational modifications of recombinant P-selectin glycoprotein ligand-1 required for binding to P- and E-selectin. J Bio/Chem 1996, 271:3255-3264. See annotation [87°]. •

83.

McEver RP: Selectins. Curr Opin Immuno/1994, 6:75-84.

84.

Norman KE, Moore KL, McEver RP, Lay K: Leukocyte rolling in vivo is mediated by P-selectin glycoprotein ligand-1. B/ood 1 9 9 5 , 86:4417-4421.

85.

Li F, Erickson HP, James JA, Moore KL, Cummings RD, McEver RP: Visualization of P-selectin glycoprotein ligand-1 as a highly extended molecule and mapping of protein epitopes for monoclonal antibodies. J Bio/Chem 1996, 271:6342-6348.

69.

•

70.

Kim YJ, Varki A: Subsets of sialylated, sulfated mucins of diverse origins are recognized by L-selectin. Lack of evidence for unique oligosaccharide sequences mediating binding. G/ycobio/ogy 1996, 6:191-208. C r o t t e t P,

71. •

AIon R, Feizi T, Yuen C-T, Fuhlbrigge RC, Springer TA: Glycolipid ligands for selectins support leukocyte tethering and rolling under physiologic flow conditions. J/mmuno/1995, 154:5356-5366. Lipid-linked oligosaccharide ligands for E- and L-selectins were shown to support cell tethering and rolling under conditions of physiologic flow.

86. •

Vachino G, Chang XJ, Veldman GM, Kumar R, Sako D, Fouser LA, Berndt MC, Cumming DA: P-selectin glycoprotein ligand-1 is the major counter-receptor for P-selectin on stimulated T cells and is widely distributed in nonfunctional form on many lymphocytic cells. J Biol Chem 1995, 270:21966-219?4. See annotation [87°]. 87.

Handa K, White T, Ito K, Fang H, Wang S, Hakomod S: P-selectin-dependent adhesion of human cancer cells: requirement for co-expression of a 'PSGL-l-like' core protein and the glycosylation process for sialosyI-Lex or sialosyI-Lea. Int J Oncol 1995, 6:773-781. Three papers [82",86",87 "] focus on PSGL-1 expressed in different cell types. Each paper demonstrates the importance of co-expression of appropriate glycosytransferases for high avidity binding of PSGL-1 by P-selectin. •

72.

K e l mS, Pelz A, Schauer R, Filbin MT, Tang S, De Bellard ME, Schnaar RL, Mahoney JA, Hartnell A, Bradfietd P, Crocker PR: Sialoadhesin, myelin-associated glycoprotein and CD22 define a new family of sialic acid-dependent adhesion molecules of the immunoglobulin superfamily. Curt Biol 1994, 4:965-972.

88. •.

73.

K e l mS, Schauer R, Manuguerra JC, Gross HJ, Crocker PR: Modifications of cell surface sialic acids modulate cell adhesion mediated by sialoadhesin and CD22. Glycoconjuage J 1994, 11:576-585.

89. •-

74.

Sjoberg ER, Powell LD, Klein A, Varki A: Natural ligands of the B cell adhesion molecule CD22 beta can be masked by 9-0acetylation of sialic acids. J Ceil Biol 1994, 126:549-562.

75.

Crocker PR, Kelm S, Dubois C, Martin B, McWilliam AS, Shotton DM, Paulson JC, Gordon S: Purification and properties of sialoadhesin, a sialic acid-binding receptor of murine tissue macrophages. EMBO J 1991, 10:1661-1669.

90.

Stamenkovic I, Sgroi D, Aruffo A, Sy MS, Anderson T: The B lymphocyte adhesion molecule CD22 interacts with leukocyte common antigen CD45RO on T cells and alpha 2 - 6 sialyltransferase, CD75, on B cells. Ceil 1991,66:1133-1144.

76.

Yang U, Zeller CB, Shaper NL, Kiso M, Hasegawa A, Shapiro RE, Schnaar RL: Gangliosides are neuronal ligands for myelinassociated glycoprotein. Proc Nat/Acad Sci USA 1996, 93:814-818.

91.

Engel P, Nojima Y, Rothstein D, Zhou U, Wilson GL, Kehrl JH, Tedder TF: The same epitope on CD22 of B lymphocytes mediates the adhesion of erythrocytes, T and B lymphocytes, neutrophils, and monocytes. J/mmuno/1993, 150:4719-4732.

Pouyani T, Seed B: PSGL-1 recognition of P-selectin is controlled by a tyrosine sulfation consensus at the PSGL-1 amino terminus. Ceil 1995, 83:333-343. See annotation [89°°]. Wilkins PP, Moore KL, McEver RP, Cummings RD: Tyrosine sulfation of P-selectin glycoprotein ligand-1 is required for high affinity binding to P-selectin. J Bio/Chem 1995, 270:22677-22680. Using different experimental approaches, three papers [81"°,88"*,89°°], demonstrate the presence of sulphotyrosines on PSGL-1 and their cooperativity with O-glycosidic ligands to generate high-avidity counter-receptors for P-selectin.

Carbohydrate recognition systems Crocker and Feizi

92.

Hanasaki K, Powell LD, Varki A: Binding of human plasma sialoglycoproteins by the B cell-specific lectin CD22. Selective recognition of immunoglobulin M and haptoglobin. J Bio/Chem 1995, 270:7543-7550.

93.

Soils D, Feizi T, Yuen CT, Lawson AM, Harrison RA, Loveless RW: Differential recognition by conglutinin and mannanbinding protein of N-glycans presented on neoglycolipids and glycoproteins with special reference to complement glycoprotein C3 and ribonuclease B. J Biol Chem 1994, 269:11555-11562.

94. •

Loveless RW, Holmskov U, Feizi T: Collectin-43 is a serum lectin with a distinct pattern of carbohydrate recognition. Immunology 1995, 85:651-659. In this and in an earlier study [93] the complement glycoprotein C3 and its glycopeptides derived in the complement cascade were used as model counter-receptors to extend knowledge on the influence of carrier protein in recognition of oligosaccharide ligands by the collectins. 95.

Laursen SB, Thiel S, Teisner B, Holmskov U, Wang Y, Sim RB, Jensenius JC: Bovine conglutinin binds to an oligosaccharide

691

determinant presented by iC3b but not by C3, C3b or C3c. Immunology 1994, 81:648-654. 96.

McEver RP, Moore KL, Cummings RD: Leukocyte trafficking mediated by selectin-carbohydrate interactions. J Biol Chem 1995, 270:11025-11028.

97.

Crocker PR, Kelm S, Hartnell A, Freeman S, Nath D, Vinson M, Mucklow S: Sialoadhesin and related cellular recognition molecules of the immunoglobulin superfamily. Biochem Soc 7-tans 1996, 24:150-156.

98. •.

Kolbinger F, Patton JT, Geisenhoff G, Aenis A, Li X, Katopodis AG: The carbohydrate-recognition domain of E-selectin is sufficient for ligand binding under both static and flow conditions. Biochemistry 1996, 35:6385-6392. A series of soluble domain swap chimaeras between E- and L- selectins have been constructed that show that the selective binding of the lectin domain of E-selectin to a preparation of S-Lea trisaccharide, and of L-selectin to sulphatide, are uninfluenced by the origins of either the EGF or CR domains to which they are attached.