Lectins

Lectins C. BROWN

JAY

Department of Microbiology, University of Virginia School of Medicine, Charlottesville, Virginia AND

RICHARDC.HUNT Department of Biochemistry, Oxford University, Oxford, England I. Introduction

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11. Lectin Biochemistry

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A. Blood Croup Specificity . B. Lectin Purification . . . . C. Lectin Structure . . . . Lectin-Induced Lymphocyte Mitogenesis A. Nature o f t h e Mitogenic Response . B. Mechanism of Lectin-Induced Mitogenesis Selective Agglutination of Transformed Cells A. Correlation of Lectin Agglutinability with . . . . . Transformation . B. Lectin Agglutination and Tumorigenicity C. Transient Agglutinability of Untransformed D. Mechanism of Agglutination . . . Interaction of Lectins with Cells Infected by NononcogenicViruses . . . . . Interaction of Lectins with Developing Cells A. Eggs and Embryos .- . . . . B. Male Germinal Cells . . . . Biochemistry of Cell Surface Lectin Receptors Lectin Toxicity . . . . . . The Biological Role of Lectins . . . References . . . . . . .

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I. Introduction Lectins are proteins that can bind noncovalently to specific carbohydrate groups without modifying them chemically. Binding is reversible, and all lectins have more than one specific carbohydratecombining site. No enzymic activity has as yet been associated with any purified lectin molecule. The presence of more than one carbohydrate-combining site allows individual lectin molecules to serve as cross-linking agents, and in fact lectins were first identified in extracts of plant seeds found to contain soluble factors that agglutinate red 277

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JAY C. BROWN AND RICHARD C. HUNT

blood cells. The factors responsible for hemagglutination were called phytohemagglutinins (from the Greek phyton, meaning “plant”), and one still encounters this term. The word “lectin” was first proposed by Boyd (1954) to take account of the fact that similar hemagglutinating factors can also be isolated from animal sources. Since individual plant or animal species ordinarily contain only one type of lectin, lectins are named with reference to their species of origin. For example, one speaks of wheat germ agglutinin (WGA) or soybean lectin, and we adhere to this system of nomenclature in this article. Cell biologists have made the most conspicuous use of lectins as probes in studies of cell surface structure and function. Lectins are bound specifically to carbohydrate-containing groups on the cell surface, and biochemical or microscopic methods are employed to examine the consequences of such binding. Striking results are often observed. For instance, some lectins induce mitosis in resting lymphocytes (see Section 111),some agglutinate neoplastic, transformed cells but not their normal, noncancerous counterparts (see Section IV), and many have been employed to demonstrate significant changes in cell surface architecture following virus infection (see Section V) and during development (see Section VI). One lectin, concanavalin A (Con A), has even been employed as the basis of a method for physically isolating the plasma membranes of Neurospora crassa (Scarborough, 1975). In this article we describe the ways lectins have been used to study animal cell surfaces, and we attempt to evaluate the results obtained. Special attention is devoted to studies published since this subject was last reviewed in the Znternational Review of CyOther recent reviews which have covered tology (Nicolson, 1974~). some of the same material include those by Toms and Western (1971), Sharon and Lis (1972), Lis and Sharon (1973b), Burger (1973), Rapin and Burger (1974), Nicolson (1976a,b), and Sharon (1977). Collections of papers edited by Cohen (1974) and by Chowdhury and Weiss (1975) are also relevant. Even brief attention to this literature will convince the reader that the potential value of lectins as structural and functional membrane probes is only just beginning to be realized. 11. Lectin Biochemistry

A. BLOOD GROUPSPECIFICITY The hemagglutinating activity of plant extracts was first observed by Stillmark (see Boyd, 1963), who studied Ricinus communis (castor bean) agglutinin (RCA). Much later, Landsteiner (see Boyd, 1963) noted that crude lectin preparations did not agglutinate all red cells to

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279

the same extent; agglutination was found to depend on the species and blood type of the donor. Similarly, Renkonen (1948)observed that extracts of Vicia cracca agglutinated human blood type-A cells more strongly than cells of groups B and 0. These original observations were quickly and thoroughly extended in an effort to identify lectins suitable for use in blood typing. For example, Boyd and Reguera (1949) extracted 262 types of seeds from 63 different plant families and tested the extracts for hemagglutinating activity; 191 did not agglutinate, 46 showed agglutination that was not specific to any blood group, and 25 agglutinated cells of only a particular blood group. An even more extensive study was carried out by Allen and Brilliantine (1969).Material from 2663 plants was prepared and tested for hemagglutination; 1635 extracts were inactive, 711 were nonspecific agglutinins, 227 lysed red cells of all types, and 90 caused specific agglutination. Studies on the blood group specificity of lectins and their use in blood typing has been thoroughly reviewed (Makela, 1957; Bird, 1959a; Boyd, 1963).Table I lists some of the lectins found to be blood group-specific. The differential agglutination of red cells based on blood type clearly indicates that different lectins must bind to different chemical groups on the red cell surface. The identity of the group recognized by a given lectin is ordinarily determined by measuring the ability of simple monosaccharides or oligosaccharides to inhibit lectin-induced hemagglutination. Inhibition is usually found to be quite specific for a particular monosaccharide. For example, Morgan and Watkins (1959) showed that agglutination by lectins specific for blood group A was inhibited b y N-acetyl-Dglucosamine, while those specific for blood group 0 were inhibited by a-methyl-L-fucose. Similar studies have been carried out to determine the saccharide-binding specificity of most lectin preparations, and some of the results are given in Table 11. Although the most commonly studied lectins have been extracted from plants, particularly from the seeds of Leguminoseae, many other sources have been found to contain lectins. These include plant roots (Saint-Paul, 1961; Allen and Neuberger, 1973),fungi (Elo et al., 1951; Tktry et al., 1954; Saint-Paul, 1961; Sage and Connett, 1969), and the hemolymph of a variety of invertebrates including the horseshoe crab (Cohen et al., 1965, 1974; Marchalonis and Edelman, 1968), sea hare (Pauley et al., 1971), snail (Cohen et al., 1965; Prokop et al., 1968), lobster (Cornick and Stewart, 1973), earthworm (Cooper et al., 1974), and oyster (Tripp, 1966, 1974). Other sources include the ova of fish (Prokop et al., 1967,1968;Pardoe and Uhlenbruck, 1970),slime molds (Rosen et al., 1973, 1974, 1975; Simpson et al., 1974),the electric eel (Teichberg et at., 1975), toad eggs (Wyrick et al., 1974), rabbit liver

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JAY C. BROWN AND RICHARD C. HUNT

TABLE I BLOOD GROUP-SPECIFIC LECTINS Blood grow A

A+B

B

Lectin source Clitocybe nebularis Crotalaria aegyptiaca Dolichos bijorus (horse gram) Hyptis suaveolens Lathyrius sylvestris Phaseolus lunatus (lima bean) Vicia cracca Bandeiraea simplicifolia Calpurina aurea Coronilla varia Crotalaria mucronata Crotalaria striata Sophora japonica Euonymus spp. Marasmius oreades Polyporus fomentarius

0

M N

Vitis aestivalis Cytisus sessilifolius Laburnum alpinum Lotus tetragonolobus Ononsis spinosa Ulex europeus (I and 11) Xylaria polymorpha Iberia amava Vicia graminia

Reference TBtry et al. (1954) Bachrach et a!. (1957) Bird (1951, 1952) Bird (1959b, 1960) Cazal and Lalaurie (1952) Boyd and Reguera (1949) Koulumies (1949) Makela and Makela (1956) Makela (1957) Kriipe and Braun (1952) Ottensooser and Sat0 (1963) Makela and Makela (1956) Kriipe and Braun (1952) Potapov (1968, 1970); Ottensooser et al. (1968) Elo et al. (1951) Saint-Paul (1961); Gillespie and Gold (1960) Boyd and Reguera (1949) Renkonen (1948) Renkonen (1948) Renkonen (1948) Saint-Paul (1961); Herzog (1959) Cazal and Lalaurie (1952) Saint-Paul (1961) Boyd (1963) Saint-Paul (1961)

(Lunney and Ashwell, 1976), eel serum (Bezkorovainy et al., 1971; Desai and Springer, 1973),and the albumin gland of mollusks (Prokop et al., 1965; Kriipe and Pieper, 1966; Ishiyama et al., 1974; Hammarstrom, 1974).

B. LECTINPURIFICATION Many of the crude extracts found to contain hemagglutinins have now been fractionated to yield purified lectin preparations. In some cases purification has been accomplished by traditional, methods of protein chemistry, although quite often more modern techniques of affinity chromatography have been employed. The latter procedures exploit the binding of lectins to specific saccharide groups, and they

TABLE I1 BIOCHEMICALPROPERTIES OF SOME IMPORTANT LECXINS

Source Plant lectins Abrus pecatorius

Molecular weight

Number of subunits

60,000

2

Nontoxic

126,000

4

Peanut lectin

110,000

Lectin

Glycoprotein

Inhibitor

References'

Abrin Yes

DGalactose

1

Yes

DGalactose

2

4

30,& (A) and 35,000 (B) 2 x 33,800 and 2 x 32,200 27,500

No

Dgalactose

3

114,000

4

28,500

Yes

Dgalactose

4

Con A

102,000

4

25,500

No

5

Sunn hemp lectin Horse gram lectin Soybean lectin

120,000

-

Yes

DGlucose, Dmannose DGalactose

111,000

4

110,000

Toxic

Arachis hypogaea Bandeiraea simplicifolia Canaualia ensiformis Crotolaria juncea Dolichos bifirus Glycine mar

Subunit molecular weight

-

6

Yes

DgalNAc

7

4

26,000 and 26,500 30,000

Yes

DgalNAc, Dgalactose DMannose, Dglucose

8

Lens cvlinaris

LCA-A

48,000

2

24,500

Yes

LCA-B A B C

48,000 120,000 58,000 117,000

2 4 2 4

24500

Yes

Lotus tetragonolobus

27,000

-

-

L-Fucose L-Fucose L-Fucose

9

10 (Continued)

TABLE I1 (Continued)

Source Phaseolus lunatus

Lectin Lima bean lectin 1 2

Phaseolus uulgaris

Phytolacca americana Pisum satioum

Ricinus communis

PHA 1 2 Pokeweed niitogen Pea lectin

Castor bean lectin RCAI RCAn

Solanum tuberosum Suphora japonica

Potato lectin

Subunit molecular weight

Molecular weight

Number of subunits

180,000 or 269,000" 90,000or 138,000"

-

31,000

Yes

DgalNAc

11

128,000 128,OOO 32,oocI

4 4

29,000 33,000

Yes Yes Yes

DgalNAc DgalNAc

12

4 9 , W or

4 4

8000 and x 15,000 or 10,OOO and x 18,000

Yes

55,000

2x 2 2x 2

DMannose, Dglucose DMannose, Dglucose

120,000

4

Yes

60,000

2

120,000

4

2 x 29,500 and 2 x 37,000 1 x 29,500 and 1 x 34,000 46,000

132,000135,000

4

33,000

Yes

-

-

Glycoprotein

Yes

Yes Yes

Inhibitor

-

References'

13

14

DGalactose, DgalNAc DGalactose, DgalNAc DglcNAc

15

Dgalactose, DgalNAc

17

16

Triticum uulgaris

WGA

34,000

2

17,000

Yes or no"

DglcNAc, NANAb

18

L-Fucose Di-N-acetylchitobiose DgalNAc Di-N-acetylchitobiose

19 20

Ulex europaeus

Gorse lectin

I

11

170,000 170,000

Wistaria floribunda

WFH WFM

136,000 67,000

4 2

35,000 32,000

Yes Yes

Eel antihuman blood group protein Snail hemagglutinin Horseshoe crab agglutinin

123,000

3

40.000

Yes

L-Fucose

22

79,000

6

13,000

Yes

23

-

22,500

DgalNAc, DglcNAc -

Animal lectins Anguilla rostrata Helix pomatia Limulus pol yphemus

400,000

Yes Yes

21

Disagreement between reports. N-Acetylneuraminic acid. References: (1)McPherson and Rich, 1973; Olsnes et al., 1974a, 1975 (2) Olsnes et al., 1974a; Wei et al., 1975 (3)Lotan et al., 1975b (4) Hayes and Goldstein, 1974,1975 (5)Goldstein et al., 1965; Goldstein and So, 1965;Agrawal and Goldstein, 1968a,h; Ahe et al., 1971; Entlicher et al., 1971; McKenzie et al., 1972 (6)Ersson et al., 1973 (7) Etzler and Kabat, 1970; Carter and Etzler, 1975a,b (8)Lis et al., 1966a, 1970; Lotan et al., 1974,1975a (9) Entlicheret al., 1969; Howardet al., 1971; Stein et al., 1971(10) Springer and Williamson, 1962; Yarivet al., 1973; Pereira and Kabat, 1974 (11)Could and Scheinberg, 1970; Galbraith and Goldstein, 1970 (12) Allen et al., 1969; Kornfeld and Kornfeld, 1970; Allan and Crumpton, 1971 (13)Reisfeld et al., 1967 (14) Entlicher et al., 1969, 1970; Trowbridge, 1974; Marik et al., 1974; Van Wauwe et al., 1975 (15) Nicolson and Blaustein, 1972; Nicolson et al., 1974; Olsnes et al., 1974a, 1975 (16) Allen and Neuberger, 1973 (17) Poreiz, 1973; Poretz et al., 1974 (18)LeVine et al., 1972; Nagata and Burger, 1972, 1974; Allen et al., 1973; Greenaway and LeVine, 1973 (19) Matsumoto and Osawa, 1969,1970 (20) Toyoshima et al., 1971; Toyoshima and Osawa, 1975 (21) Marchalonis and Edelman, 1968 (22) Springer et al., 1965 (23) Hammarstrom and Kabat, 1969; Hammarstrom, 1973.

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JAY C. BROWN AND RICHARD C. HUNT

may result in a one-step procedure yielding a homogeneous protein. For example, Con A can be purified in this way by taking advantage of its specific binding to Sephadex, a glucose polymer. The lectin can be eluted from Sephadex with glucose (Agrawal and Goldstein, 1967) or by lowering the pH (Olson and Liener, 1967). Similarly, galactosebinding lectins (such as those from R. communis, Momordia charantia, Abrus pecatorius, and Crotalaria juncea) can be purified by affinity chromatography on agarose gels (Nicolson and Blaustein, 1972; Tomita et al., 1972b; Ersson et al., 1973). Affinity columns for other lectins require attachment of a specific receptor to an insoluble matrix. This has been achieved by cyanogen bromide activation of Sepharose (Cuatrecasas, 1970), or by copolymerization of the receptor with the N-carboxy anhydride of L-leucine (Tsuyuki et al., 1956).The former method was employed, for example, to insolubilize ovomucoid for the purification of WGA (LeVine et al., 1972), and the latter to insolubilize hog A + H blood group substance for the preparation of Dolichos bijlorus lectin (Kaplan and Kabat, 1966; Etzler and Kabat, 1970; Etzler, 1973).Table I11 lists some of the lectins that have been purified and indicates the method of purification employed. Difficulties encountered in lectin purification have been connected with the fact that lectins can occur in different polymeric states. For example, Con A exists as a dimer in solution below pH 5.6 and as a tetramer above pH 5.6 (Kalb and Lustig, 1968; Abe et al., 1971; McKenzie et al., 1972) Lectin properties can also differ as a result of the presence of ions (Agrawal and Goldstein, 1968a; Uchida and Matsumoto, 1972) or sugars (Agrawal and Goldstein, 196813).Also, lectin purification has occasionally been complicated by the presence in extracts of more than one form of the same lectin. Such isolectins have identical saccharide-binding specificities and similar, but not identical, structures. Isolectins have now been identified in the case of D. biflorms lectin (Carter and Etzler, 1975a), lentil lectin (Tichi et al., 1970; Howard et al., 1971), lima bean lectin (Gould and Scheinberg, 1970),red kidney bean agglutinin (Yachnin and Svenson, 1972; Miller et al., 1973; Pusztai and Watt, 1974),soybean lectin (Lis et al., 196613; Catsimpoolas and Meyer, 1969), and WGA (Allen and Neuberger, 1973; Rice and Etzler, 1975).Much less frequently a single biological source may be found to yield multiple lectins having different structures and saccharide-binding specificities. The most prominent example of this situation is the gorse (Ulex europeus) lectins UEA, and UEA,; UEAl binds fucose, while UEAn binds di-N-acetyldichitobiose (D-glcNAc), (Matsumoto and Osawa, 1969, 1970). I

TABLE 111 LECTIN PURIFICATION Lectin and source

Method of purification

Abrin (Abrus pecotorius)

(NH,),SO, precipitation; affinity chromatography on Sepharose; elute with galactose Extract jack bean meal with NaCI; (NH,),SO, precipitation; affinity chromatography on Sephadex G-50; elute lectin with glucose or lower pH Affinity chromatography on Sepharose; ellite with lactose Alcohol precipitation Affinity chromatography on polyleucyl-hog blood group A + H substance; elute with N-acetylglucosamine Extract with NaCI; (NH,),SO, precipitation UEA: CM-cellulose, Sephadex G-200, Biogel P-200. UEA 11: DEAE cellulose; electrophoresis; biogel P-200 Affinity chromatography. Lectin I on fucose coupled to starch gel; elute with glycine-HC1. Lectin I1 on tri-N-acetylchitotriose-starch gel; elute with glycine-HC1 buffer Extract in NaCI; DEAE-cellulose chromatography Affinity chromatography on Sephadex; elute with glucose

Con A (Conouolia enisformis, jack bean)

Crotolnriu junceo lectin Dolichos bijlorus lectin (horse gram)

Gorse lectins (Ulex europeus): UEA I binds fucose; UEA I1 binds (DglcNAc),

Lentil lectin (Lens culinuris)

Lima bean lectin (Phaseolus lunutus)

Extract with NaCI; (NH,),SO, precipitation; affinity chromatography on polyleucyl type-A blood group substance; elute with N-acetylglucosamine

Reference Tomita et oZ. (1972b)

Agrawal and Goldstein (1965, 1967, 1973); Olson and Liener (1967) Ersson et al. (1973) Bird (1959a) Kaplan and Kabat (1966); Etzler and Kabat (1970); Etzler (1973) Matsumoto and Osawa (1969, 1970) Osawa and Matsumoto (1973)

Matsumoto and Osawa (1972)

Howard and Sage (1969); Sage and Green (1973) Entlicher et ul. (1970); Tichi et al. (1970); Toyoshima et al. (1970); Howard et al. ( 1971) Galbraith and Goldstein (1970, 1973)

(Continued )

286

JAY C. BROWN AND RICHARD C. HUNT

TABLE 111 (Continued) Lectin and source

Lotus tetragonolobus lectin Mormordia charantia lectin

Mushroom lectin (Agaricus bisporus) Pea lectin (Pisum sativum) Potato lectin (Solanum tuberosum)

PHA (Phaseolus uulgaris)

RCA

Robin (Robinia pseudoacacia, block locust)

Method of purification Extract in phosphate buffer; (NH,),SO, precipitation; gel filtration on Biogel A0.5 or Biogel P-300 Extract with phosphate-buffered saline; affinity chromatography on Sepharosefucose; elute with fucose (NH4),S04precipitation; affinity chromatography on Sepharose 4B; elute with galactose Extract in NaCl solution; DEAE-cellulose, Sephadex G-100, phosphocellulose chromatography (NH,),SO, precipitation; affinity chromatography on Sephadex G-150; elute with glycine-HC1 (NH,),SO, precipitation; DEAE-cellulose, CM-cellulose, Sephadex G-100, SP-Sephadex chromatography Ethanol precipitation; (NH,),SO, precipitation SE-Sephadex chromatography; Sephadex-G150 filtration; phosphocellulose chromatography Affinity chromatography on Sepharose-th yroglobulin; elute with glycine-HC1 (NH,),SO, precipitation; affinity chromatography on agarose; elute with galactose or lactose. Separates into two fractions on Sephadex G-200, called RCA, and RCA,, Extract in phosphate-buffered saline; (NH4)*S04 precipitation; DEAE-cellulose and CM-cellulose chromatography

Reference Could and Scheinberg (1970) Blumberg et al. (1972); Yariv et al. (1973) Tomita et al. (1972b)

Sage and Connett (1969); Presant and Komfeld (1972) Entlicher et al. (1970)

Allen and Neuberger (1973)

Rigas and Osgood (1955) Kornfeld et al. (1973)

Matsumoto and Osawa (1972) Nicolson and Blaustein (1972); Tomita et al. (1972b)

Bourrillon and Font (1968)

287

LECTINS TABLE 111 (Continued) Lectin and source

Method of purification

Sophora japonica lectin

Extract in phosphate-buffered saline; ethanol precipitation; affinity chromatography on polyleucylhog gastric mucin; elute with galactose Extract in water; (NH,),SO, precipitation; dialyze against ethanol; chromatography on calcium phosphate and DEAE-cellulose Extract in NaCl solution; (NH4),S04precipitation; affinity chromatography on Sepharose-galactose; elute with galactose Affinity chromatography on Sepharose -bl ood group-A substance; elute with acetate buffer Extract wheat germ with NaCI; (NH,),SO, precipitation; affinity chromatography on ovomucoid-Sepharose; elute with 0.1 N acetic acid Affinity chromatography on chitin column; elute with 0.05 N HCI Affinity chromatography on N-acetylglucosamine-Sepharose; elute with N-acetylglucosamine or 0.1 N acetic acid Extract in NaCI; (NH,),SO, precipitation; SE-Sephadex chromatography; Sepharose-6B filtration

Soybean lectin (Clycine max)

Vicia cracca lectin

WGA (Triticum uulgaris)

Wistaria floribunda lectin

Reference Poretz (1972); Poretz et al. (1974)

Liener and Pallansch (1952); Liener (1953); Wada et al. (1958);Lis et al. (1966a); Lis and Sharon (1973a) Gordon et al. (1972, 1973)

Sundberg et QZ. (1970)

LeVine et al. (1972); Marchesi (1973)

Bloch and Burger (1974) Lotan et al. (1973a); Shaper et al. (1973)

Toyoshima et aZ. (1971); Osawa and Toyoshima (1972)

288

JAY C. BROWN AND RICHARD C. HUNT

C. LECTINSTRUCTURE

1. Concanavalin A Although many lectins have been chemically purified, relatively few have been subjected to detailed structural analysis. By far the best studied lectin structure is that of Con A. Chemical studies of Con A have shown that it is a tetramer of identical protomers, each of which has a molecular weight of 25,500. The protomer polypeptide chain consists of 237 amino acid residues whose sequence has been determined (Wang et al., 1975a; Cunningham et al., 1975);Con A contains no covalently bound carbohydrate. Individual Con-A protomers contain binding sites for two metal ions, one for Mn2+and one for Ca2+, plus one saccharide-binding site. Mn2+must be bound before Ca2+, and both metal ions are required for optimal saccharide binding (Yariv et al., 1968). In solution the Con-A tetramer dissociates into two identical dimers below pH 5.6 (Becker et al., 1971; Hardman and Ainsworth, 1972a), and it self-associates in a time-dependent fashion to form high-molecular-weight aggregates above neutral pH (McKenzie et al., 1972). Studies on the specificity of the Con-A saccharide-binding site have revealed that Con A binds specifically to mannosyl and also to glucosyl groups at the nonreducing ends of oligosaccharide chains (Goldstein et al., 1965, 1974; Poretz and Goldstein, 1970). Certain internal mannosyl groups can also be accommodated. Con A can be induced to form crystals suitable for structural studies using x-ray diffraction techniques, and two laboratories have now completed this analysis (Hardman and Ainsworth, 1972b; Becker et al., 1975; Reeke et al., 1975). Their results are in good overall agreement. The Con-A monomer (protomer) that forms the crystallographic asymmetric unit has been found to be a compact, dome-shaped structure having a height of 43 A and a cross section of 39 x 40 A. Two such protomers are joined at their bases to form a dimer having roughly the shape of a prolate ellipsoid of revolution (84 x 40 x 39 A) in which the protomers are related by a twofold axis of rotation. Tetrameric Con A is formed by the association of two dimers to create a roughly tetrahedral structure in which the protomers are related by three twofold crystallographic axes; the tetramer therefore has D z symmetry, as shown in Fig. 1. The dimers referred to above, which exist in solution at low pH, are formed between subunits I and I1 or I11 and IV but not, for example, between I and I11 or I and IV (Reeke et al., 1975), as shown in Fig. 1. The most conspicuous feature of the Con-A polypeptide is the large

LECTINS

289

FIG. 1. Schematic representation of the Con-A tetramer. The manganese and calcium sites are indicated by Mn and Ca, respectively. The saccharide-binding site is indicated by S and the major cleft by I. Adapted from Reeke et al. (1975).

amount of p structure found in this molecule; almost 55% of the amino acid residues are involved in two regions of an antiparallel pleated sheet. One of these regions, the “back” pleated sheet, consists of 64 amino acid residues arranged in six antiparallel chains found in the back of the molecule, as iIlustrated in Fig. 2. Except for a slight curl at the top, this region is relatively flat. It forms the back of the major cleft to the lower right in Fig. 2 and contains most of the amino acid residues involved in intersubunit interactions. Details of the intersubunit contacts are given by Reeke et al. (1975).The second major region of /3 structure, the “front” pleated sheet, consists of 57 amino acid residues arranged in seven antiparallel chains which lie on top of the back pleated sheet with their long axes oriented at approximately a 45” angle to those of the back sheet. The front pleated sheet is twisted through an angle of about go”, and it forms the upper portion of the major cleft which lies roughly between the back and front pleated sheets and opens at the lower right in Fig. 2. The amino- and carboxyterminal amino acids are found to the right of the front pleated sheet, and the metal ion-binding sites are just above it. The Mn2+ and Ca2+ ions are found 4.6 A apart in the upper portion of the molecule, and

290

JAY C. BROWN AND RICHARD C. HUNT

FIG. 2. Stereo view of the Con-A protomer with the back region of the p structure darkened. The back B structure forms a vertical plane with a curl at the top left and a small bent portion at the far left (Becker et al., 1975).

each is surrounded by an octahedral coordination shell consisting of four ligands from the protein and two water molecules. A drawing of these sites is given by Becker et al. (1975). The saccharide-binding site is perhaps the most interesting and yet controversial feature of the Con-A structure. Con-A crystals are found to have a deep, narrow cavity formed by portions of the back and front pleated sheets which opens at the lower right in Fig. 2; it extends 18 A into the core of the molecule and is roughly 5 A wide and 7.5 A high. This cavity is easily large enough to accommodate the a-D-mannose and a-Dglucose groups for which Con A is specific, and Becker et al. (1971) originally proposed that this cavity is the place where saccharides are ordinarily bound. Studies on Con-A-saccharide interactions in solution, however, have not supported this view. For example, Brewer et al. (1973) used pulsed 13Cnuclear magnetic resonance (NMR) spectroscopy to study the interaction of 13C-enricheda-methylD-glucopyranoside (a-MG) with Con A. The techniques employed allowed these workers to calculate the distance between the transition metal (MnZ+)site and individual carbon atoms in bound a-MG. Their results showed clearly that a-MG was too close to Mn2+for the sugar to be bound in the major cleft. For example, the C-3 and C-4 carbon atoms ofa-MG were found to be at a mean distance of no more than 10A from Mn2+,while they would have to be more than 20 A from Mn2+if aMG were bound in the major cleft.

LECTINS

29 1

Recent x-ray crystallographic studies of Con A containing specifically bound saccharides have been in overall agreement with the NMR studies. Methyl-a-D-mannopyranoside (Hardman and Ainsworth, 1976) and an iodine-containing analog (2-deoxy-2-iodomethyla-Dmannopyranoside) of a-D-mannose (Becker et al., 1976) were found to bind to Con A at a shallow pocket near the metal ion sites but slightly to the left and in front of them, as shown in Fig. 1. Since the metal ions are required to stabilize this site, the results neatly explain why demetallized Con A has no specific saccharide-binding site. 2. Other Lectins It is clear that our extensive knowledge of Con-A structure is due primarily to the growth of suitable crystals of this lectin and to their analysis by x-ray diffraction techniques. Crystals that promise to be equally useful for structural studies have been prepared from two other lectins, WGA (Wright, 1974; Wright et al., 1974) and favin (Wang et al., 1974),and analysis of these crystals is currently in progress. In the case of WGA an electron-density map has been obtained at 2.2 A resolution by the isomorphous replacement method (C. Wright, personal communication). This map has confirmed previous studies on WGA in solution, which showed that the overall WGA molecule is a dimer of two identical subunits each of which has a molecular weight of about 17,500 (Nagata and Burger, 1974; Rice and Etzler, 1974). In the crystal these two monomers appear in close association with each other across an exact twofold axis. Each monomer contains an assembly of four spatially distinct but structurally similar domains of approximately 41 amino acids each. The domains are related to each other in such a way as to maximize the exposure of amino acid side chains to the solvent; nearly 75%of the WGA side chains have access to the surrounding solvent. Four intradomain disulfide bonds bridge the polypeptide chain within each domain, and this accounts for the unusually high content of cysteine in WGA (approximately 32 of 164 amino acid residues). The WGA molecule contains no regular secondary structure such as an a-helix or a P-pleated sheet. However, each domain contains one irregular a-helical turn and numerous hydrogen bonds between neighboring regions of backbone. The specific binding sites for [(D-gkNAc),], of which there are two per WGA monomer, have recently been identified (Wright, 1977). Studies on the structure of favin were undertaken because it has the same saccharide specificity as Con A. These studies may clarify some of the unresolved questions about Con-A mitogenesis, and it will be of interest to compare the saccharide-binding sites of these two lectins.

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Much relevant structural information about other well-studied lectins can be found in the review by Lis and Sharon (1973b).

111. Lectin-Induced Lymphocyte Mitogenesis A. NATURE OF

THE

MITOGENIC RESPONSE

One of the most remarkable effects of certain lectins is their ability to stimulate a mitogenic response in normal lymphocytes. This property was first discovered in 1960 by Nowell, working with human lymphocytes and red kidney bean agglutinin (PHA). When lymphocytes are treated with a mitogenic lectin, they first undergo “blast” transformation; they increase progressively in size, endocytosis is stimulated, cytoplasmic vacuoles appear, and many metabolic processes are stimulated including DNA, RNA, protein, and lipid synthesis. Certain ion transport activities also increase. Cells ultimately divide and continue to do so as long as the lectin is present. After many cell divisions in the presence of the lectin, cell division becomes independent of the lectin and cells can be cultured indefinitely. This strategy has been extensively employed for establishing continuously growing lines of lymphoid cells from many sources, and it is also routinely employed to expand blood lymphocyte populations for karyotyping purposes. Not all lectins are mitogenic, and it is difficult to predict in advance whether a lectin will be mitogenic or not. The mitogenicity of several well-known lectins is indicated in Table IV. Studies of Wands et al. (1976) have suggested that the multiple valence of Con A is required for its mitogenicity, (but see Fraser et al., 1976)and this may be true of other mitogenic lectins as well. Whereas some lectins are mitogenic for both T and B lymphocytes, others are mitogenic for T lymphocytes only. No known lectin is mitogenic for B lymphocytes only, although other structures such as bacterial lipopolysaccharides have this property. Some instances of specific mitogenicity are indicated in Table IV along with the concentrations of lectin required for optimal mitogenesis. In contrast to their soluble form, insolubilized or aggregated T-cell-specific lectins including Con A and PHA may be mitogenic for B as well as for T cells (Andersson et al., 1972; Greaves and Bauminger, 1972).

B. MECHANISM OF LECTIN-INDUCED MITOGENESIS The mechanism of lectin-induced mitogenesis has now been widely studied, because of its similarity to specific antigen-mediated

293

LECTINS TABLE IV MITOGENICITY OF SELECTED LECTINS ~

Lectin Mitogenic Con A Lentil lectin Lima bean lectin Pokeweed mitogen PHA Ulex europeus agglutinin I Vicia faba agglutinin (favin)

~~

Mitogenic concentration (pg/ml)

Specificity

5 50

T cells only

5 5 -

T and B cells T cells only T and B cells Unknown

-

Nonmitogenic Dolichos bijloms lectin RCAI and RCAI, Soybean lectin WCA

lymphocyte activation and because it offers a convenient model system for studying the regulation of cell growth and proliferation. PHA and Con A are the lectins most frequently examined, and they are used in conjunction with peripheral blood lymphocytes from human or other mammalian sources; the uptake of radioactively labeled thymidine into DNA is measured as an overall index of the mitogenic response. When sensitive lymphocyte cultures are treated with a lectin, they reach and maintain a maximal rate of DNA synthesis between 36 and 72 hours after the lectin is applied. This experimental system has now been quite thoroughly studied in an effort to understand the chain of molecular events involved in the induction of mitogenesis. The system has also been adapted for clinical use to identify patients with impaired immunological function.

1. Lectin Binding to the Lymphocyte Cell Surface It is clear that the first step in mitogenesis is binding of the lectin to carbohydrate-containing groups (glycoproteins or glycolipids) on the cell surface; if the lectin is prevented from binding, no mitogenesis occurs. However, Stobo et al. (1972) and Inbar et al. (1973b)showed that relatively few (6-25%) of the potential Con-A-binding sites on the surface of rat or mouse lymphocytes need to be occupied to induce an optimal mitogenic response. Furthermore, there is now good evidence (Anderson et al., 1972; Greaves and Bauminger, 1972) that some lectins, including Con A, PHA, and pokeweed mitogen, can in-

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JAY C. BROWN A N D RICHARD C. HUNT

duce mitogenesis without entering the cell. Lectins covalently attached to large, insoluble supports such as plastic culture dishes or Sepharose beads are found to be mitogenic under conditions in which only a negligible amount of the lectin is lost from the support. This result strongly suggests that plasma membrane events may be of crucial importance in the initial phases of mitogenesis and draws attention to the need for detailed studies of the cell surface receptors for mitogenic lectins. Progress in this area is discussed in Section VII. The differential response of T and B lymphocytes to Con A and PHA cannot be explained by a difference in the total amount of lectin bound to the cell surface (Greaves et aZ., 1972), since both mouse T and B cells bind the same amount (approximately 10’ molecules) of Con A (Stobo et al., 1972). The difference must involve events that occur after lectin binding. However, migration of lectin-lectin receptor complexes on the cell surface to form large “patches” or a “cap” does not appear to be required for mitogenesis (Edelman, 1974). 2. Commitment to Mitogenesis Gunther et al. (1974) intensively studied the time course with which mouse lymphocytes become committed to DNA synthesis after they have bound Con A. Sensitive lymphocytes are exposed to a mitogenic concentration of Con A for various periods of time and then washed free of lectin with a-methyl-smannopyranoside. The mitogenic response is determined by measuring the total thyn~idine-~H uptake between 48 and 72 hours after the addition of lectin and by auH the same period. toradiography of cells exposed to t h ~ m i d i n e - ~for The results show that lymphocyte cultures must be exposed to Con A for 20 hours before a maximal mitogenic response is observed; shorter periods of exposure result in less total DNA synthesis (Novogrodsky and Katchalski, 1971; Lindahl-Kiessling, 1972). Cell autoradiographic experiments have demonstrated that this is due to the fact that lymphocytes are heterogeneous with respect to the time of exposure to Con A required to induce them to become committed to mitogenesis. That is, different lymphocytes become committed to DNA synthesis at different times during the 20-hour interval of exposure to Con A. Once a cell has become committed to mitogenesis, however, DNA is synthesized at a rate independent of the amount of time the cell was exposed to Con A. The interpretation of these results is not entirely clear. It is possible, for example, that resting lymphocytes in the GI phase of the cell cycle are actually undergoing a secondary or subcell cycle within GI and that they are sensitive to Con-A mitogenesis only during a portion

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295

of this subcell cycle. On the other hand, it may be that Con-A-treated lymphocytes accumulate a metabolite required for mitogenesis at different rates and that commitment to DNA synthesis results when a critical concentration of this (unknown) metabolite is reached. Further experimentation will be required to settle this issue, but it is clear that the heterogeneity of lymphocyte response to Con A must be considered in attempts to interpret the biochemical results described in the following discussion.

3. Biochemical Changes Accompanying Mitogenesis Numerous biochemical studies have been undertaken in an attempt to account for the crucial molecular events that follow lectin binding to lymphocytes and which result in mitogenesis. Although the problem has not yet been solved, much relevant information is now available. For example, one of the earliest changes detectable in human peripheral blood lymphocytes treated with Con A or PHA is a dramatic increase in the intracellular concentration of 3',5'-cyclic GMP (cGMP). The concentration increases 10- to 50-fold in the first 30 minutes following cell exposure to mitogen and then returns to the original level after approximately an hour (Hadden et al., 1972; Watson, 1975).No corresponding change is observed in the intracellular concentration of 3',5'-cyclic AMP (CAMP)and, in fact, CAMP has been found to inhibit mitogenesis (Hadden et al., 1970; Watson, 1975).The mitogenic effects of cGMP (Watson, 1975) and of phorbol myristate acetate (Goldberg et al., 1973; Mastro and Mueller, 1974; Wang et al., 1975b), which increases the intracellular concentration of cGMP, suggest that the transitory rise in cGMP concentration may be functionally involved in mitogenesis. If so, then cGMP must serve as a signal or trigger to induce subsequent events, because its level is not elevated throughout the mitogenic response. A significant transitory increase in the fluidity of human lymphocyte membranes following the treatment of cells with Con A or PHA has been found to follow the same time course as the rise in intracellular cGMP concentration (Barnett et d . , 1974), and these two phenomena may be related. A dramatic increase in the metabolic turnover of phosphatidylinositol, but not other membrane phospholipids, has also been shown to take place within the first 30 minutes after stimulation of human lymphocytes with PHA (Fisher and Mueller, 1971). Quastel and Kaplan (1970a,b) carried out experiments which indicate that an increased intracellular Concentration of K+ may be necessary for mitogenesis. A two- to threefold increase in the rate of K+ influx is observed beginning approximately 1 hour after treatment of

296

JAY C. BROWN AND RICHARD C. HUNT

human lymphocytes with PHA. No corresponding increase in the rate of K+ efflux is observed, so the intracellular level of K+ probably rises slightly. A similar increase in K+ influx is observed in quiescent 3T3 cells stimulated to divide by the addition of serum (Rozengurt and Heppel, 1975). Increased K+ transport is found to be due to an increased number of K+ carriers on the lymphocyte cell surface rather than to an increased rate of functioning by a fixed number of carriers (Wright et al., 1973).The increased rate of K+ influx is probably due to an increased number of membrane-associated Na,K-ATPase molecules (Dahl and Hokin, 1974), since ouabain, a specific inhibitor of the Na,K-ATPase, blocks K+ uptake. Ouabain is also a potent inhibitor of mitogenesis; it can act in a reversible fashion at any stage of the mitogenic response. High concentrations of K+, however, can overcome all effects of ouabain (Quastel and Kaplan, 1970a). This argues strongly that continued functioning of the Na,K-ATPase is required throughout the mitogenic response. Significant evidence also exists suggesting that increased Ca2+ transport may be functionally involved in lectin-induced mitogenesis. A twofold increase in the rate of Ca2+uptake is observed within an hour after PHA stimulation of human lymphocytes, and this increase persists throughout the period of blast transformation and DNA synthesis (Allwood et al., 1971; Whitney and Sutherland, 1972,1973). No mitogenesis is observed in the absence of Ca2+.The increased rate of Ca2+uptake is found to be due to an increased affinity of membrane carriers for Ca2+(Whitney and Sutherland, 1973). A functional role for Ca2+in the induction of mitogenesis is suggested by the fact that the specific Ca2+ionophore A23187 can stimulate blast transformation and mitosis; it also potentiates PHA mitogenesis (Maino et al., 1974; Luckasen et al., 1974).Although sensitive lymphocytes must be exposed to the ionophore for a considerable length of time (several hours), a low M) of ionophore can induce blastogenesis in a subconcentration ( stantial fraction (50%) of lymphocytes. The requirement for Ca2+ in mitogenesis may be related to the proposed involvement of intracellular filamentous elements. Both microtubules and microfilaments have been implicated in mitogenesis by the observation that both vinca alkaloids (Edelman, 1974),which disrupt microtubule structure, and cytochalasin B (Yoshinagaet al., 1972), which inhibits microfilament formation, are potent inhibitors of lectin-induced mitogenesis. Lectin stimulation of lymphocytes leads to an increased uptake of several other cellular metabolites including glucose (Peters and Hausen, 1971)and uridine (Cooper, 1972).Compared to K+ and Ca2+,however, stimulation of these transport activities occurs relatively late (10

LECTINS TABLE V METABOLIC CHANGESOBSERVED DURING THE LECTIN-INDUCED MITOGENESIS OF LYMPHOCYTES Biochemical response

Greatest effect observed

Increased cGMP con20 minutes centration Increased membrane flu- 30 minutes idity Increased K+ transport 2 hours

Required continuously

No No Yes

Increased lipid turnover

2 hours

Increased uridine and glucose concentration Increased Ca2+concentration

10-20 hours

Yes

48 hours

Yes

48 hours

Yes

DNA synthesis

Probably

Reference Hadden et al. (1972); Watson (1975) Barnett et al. (1974) Quastel and Kaplan (1970a,b) Fisher and Mueller (1971) Cooper (1972); Peters and Hausen (1971) Whitney and Sutherland (1973); Allwood et al. (1971) Gunther et al. (1974)

to 20 hours) after the exposure of cells to lectin. This suggests that increased glucose and uridine uptake are probably secondary consequences of earlier mitogenic events and not directly involved in the induction of commitment to blastogenesis. A summary of some of the metabolic changes observed in lectin-stimulated lymphocytes is given in Table V. It is clear from these results that we are a long way from the goal of understanding the complete sequence of molecular events, which begins with the binding of lectin to the cell surface and ends with DNA replication and cell division. However, a most promising start has been made on this project, and there is every reason to believe that further experimental effort will not be in vain.

IV. Selective Agglutination of Transformed Cells A.

CORRELATIONOF LECTINAGGLUTINABILITY WITH TRANSFORMATION

1. Situations in which a Positive Correlation Exists Although lectins were originally identified by their ability to agglutinate erythrocytes, most are found to clump other animal cell types as well. In fact, a great many cell types are agglutinable by lectins, and

298

JAY C. BROWN AND RICHARD C. HUNT

this property has been thoroughly studied in experiments designed to probe molecular events taking place at the cell surface. These studies have revealed striking changes in lectin agglutinability accompanying neoplastic transformation, infection of cells with some nononcogenic viruses, and certain developmental processes. By far the best studied of these phenomena is the change in lectin agglutinability that accompanies neoplastic transformation; transformed cells are usually found to be agglutinated at a much lower concentration of lectin than is required for the corresponding normal, untransformed cell type. This difference has been extensively studied in an effort to identify the particular features of transformed cells that render them more agglutinable than normal cells. Studies on this topic have been the subject of previous reviews by Burger (1970a, 1971a,b), Rapin and Burger (1974), Nicolson ( 1 9 7 4 ~1976b), ~ and Poste (1975). Selective lectin agglutination of transformed cells was first observed by Aub et al. (1963), who found that, when a crude preparation of wheat germ lipase was added to suspensions of isolated mouse lymphoma or ascites tumor cells, the cells agglutinated, while normal, untransformed cells remained dispersed. The properties of the agglutination reaction suggested that the active factor in producing aggregation was not the lipase itself, but a contaminant in the preparation. This contaminant was later purified and called WGA (Burger and Goldberg, 1967). In the relatively short period since these original observations were made, many lectins have been examined and found to agglutinate selectively the transformed, but not the normal, cells from a variety of sources, including cells of both fibroblastic and lymphoid origin. References to some of the relevant literature are given in Tables VI and VII. These studies have shown clearly that the preferential agglutination of transformed cells by lectins is independent of the means by which transformation was achieved. For example, whereas normal mouse, rat, and hamster fibroblasts show no agglutination at 500 pg/ml Con A, considerable agglutination is observed after transformation with SV-40 virus, or polyoma virus, x rays, or chemical carcinogens such as dimethylnitrosamine and benzopyrene (Inbar and Sachs, 1969a; Inbar et al., 1972a; Weber, 1973) at Con-A concentrations as low as 1 pg/ml. Furthermore, early studies with normal and transformed spleen cells, and with normal and regenerating liver cells, indicated that agglutination by WGA is not the result of cellular alterations associated with rapid growth, but rather with the acquisition of transformation-specific functions (Aub et al., 1965). More recent studies have served to confirm the correlation between

TABLE VI ENHANCED LECTIN-MEDIATED AGGLUTINATION

Lectin Con A

Cell type Mouse 3T3

Mouse 3T3

Mouse 3T3 Hamster BHK

Chick embryo fibroblasts

Hamster fibroblasts

OF

TRANSFORMED CELLS

Transforming agent or transformed cell type

References

SV-40 virus

Inbar and Sachs (1969a,b); Ben-Bassat et al. (1970); AmdtJovin and Berg (1971); Cline and Livingston (1971); Nicolson (1971); Ozanne and Sambrook (1971a,b); Culp and Black (1972); Tomita et al. (1972a); Van der Noorda et al. (1972); Yin et al. (1972); Noonan et al. (1973a); Van Nest and Grimes (1974); Poste et al. (1975~); Poste and Nicolson (1976) Eckhart et al. (1971); Polyoma virus Inbar et al. (1971); DePetris et al. (1973); Noonan and Burger (1973a); Sakiyama and Robbins (1973) Van Nest and Grimes Murine leukemia (1974) virus Amdt-Jovin and Berg Polyoma virus (1971); Cline and Livingston (1971); Ozanne and Sarnbrook (1971a); Poste (1972); Weber (1973); Nicolson et al. (1975a); Poste et al. (1975b) Biquard and Vigier RSV (1972); Burger and Martin (1972); Kapeller and Doljanski (1972); Lehman and Sheppard (1972) Ben-Bassat et al. (1971); Various agents inInbar et al. (1972a); cluding polyoma Poste (1972) viruses and SV-40 and RSV (Continued)

300

JAY C. BROWN AND RICHARD C. HUNT

TABLE VI (Continued)

Lectin Con A

Cell type

Transforming agent or transformed cell type

Mouse 3T3

SV-40 virus

Inbar and Sachs (1969a) DeSalle et al. (1972); Ben-Bassat et al. (1974, 1976) Borek et al. (1973); Becker (1974); Nakamura and Terayama (1975) Salzberg and Green (1972); Vesely et al. (1972); Noonan e t al. (1976) Tomita et al. (1972a)

Rat hepatocytes Mouse 3T3 Rat and human fibroblast-type cells Mouse 3T3

Hepatoma SV40 virus RSV

Borek et al. (1973) Tomita et al. (1972a) Veselg et al. (1972)

Lymphocytes

Leukemic cells

Hepatocytes

Hepatoma cells

Sarcoma and leuVarious fibrokosis viruses blast-type cells Lentil lectin and PHA Pea lectin

RCA

Soybean lectin

WGA

References

Hamster BHK Mouse 3T3 Hamster and rat fibroblast-type cells Mouse 3T3

Mouse 3T3

Tomita et al. (1972a); Nicolson (1973a); Nicolson and Lacorbiere (1973) Polyoma virus Nicolson e t al. (1975a) Inbar et al. (1971) S V 4 0 virus Sela et al. (1970, 1971); Various agents ineluding SV40 and Inbar et al. (1972a) polyoma viruses and RSV SV-40 virus Burger (1969, 1970a); Pollack and Burger (1969); Inbar et al. (1971); Ozanne and Sambrook (1971a); Sheppard (1971); Sivak and Wolman (1972); Tomita et al. (1972a) Polyoma virus Benjamin and Burger (1970); Biddle et al. (1970); Eckhart e t al. (1971);Inbar et al. (1972a); Sakiyama and Robbins (1973); Weber (1973) SV40 virus

30 1

LECTINS

TABLE VI (Continued)

Lectin

Cell type

WGA

Transforming agent or transformed cell type

Hamster BHK

Polyoma virus

Chick embryo fibroblasts

RSV

Lymphocytes Hepatocytes

Leukemic cells Hepatoma cells

References Hakomori and Murakami (1968);Burger (1969, 1970a); Ozanne and Sambrook (1971a); Nicolson et al. (1975a) Burger and Martin (1972); Kapeller and Doljanski (1972); Lehman and Sheppard (1972) Aub et al. (1965) Borek et al. (1973)

transformation and sensitivity to lectin-mediated agglutination. For example, Pollack and Burger (1969) showed a good correlation between saturation density (a measure of relative malignancy) reached by mouse 3T3 cells or their transformed derivatives growing in tissue culture and the WGA concentration causing agglutination. Using SV-40 virus-transformed 3T3 cells, 3T12 (spontaneously transformed) TABLE VII LECTIN AGGLUTINATION OF MOUSE 3T3 AND SV40-TRANSFORMED 3T3 CELLS ~~

Lowest lectin concentration for agglutination (pg/ml)

Lectin

3T3

Con A"

1000

Lentil lectinb Pea lectinb PHAb RCAb Soybeanlectin" WGA"

2000 2000 500 250 500 500

a

Purified lectin. Crude extract.

Trypsinized 3T3 62 500 250 62.5 31.25 100 6

SV-40 3T3

Trypsinized SV40 3T3

62

-

500 250 31.25 31.25 20 12

250 250 7.8 7.8 -

-

Reference Ozanne and Sambrook (1971a,b) Tomita et al. (1972a) Tomita et al. (1972a) Tomita et al. (1972a) Tomita et al. (1972a) Sela et al. (1970) Ozanne and Sambrook (1971a,b)

302

JAY C. BROWN AND RICHARD C. HUNT

cells, 3T3E cells (which lack thymidine kinase) and F1-SV101 cells (“flat” revertants of SV-40-transformed 3T3 cells), these investigators showed that the WGA concentration required for agglutination falls as the cell saturation density increases. Weber (1973) and Inbar et al. (1972a) also observed an inverse relationship between the concentration of lectin required for agglutination and the saturation density to which many normal and transformed cell lines grow in culture. Nontransforming mutants of polyoma virus fail to produce the same increase in lectin-mediated agglutination observed after infection of 3T3 cells with transforming viruses (Benjamin and Burger, 1970).Further, when a mutant of polyoma virus temperature-sensitive for producing the transformed cell phenotype (ts 3) was used to infect BALB/3T3 or BHK cells, it was found that agglutinability by WGA or Con A occurred only at the permissive temperature for transformation (Eckhart et al., 1971). Renger and Basilico (1972) and Noonan et al. (1973a) obtained similar results using a temperature-sensitive mutant of SV-40-transformed3T3 cells. Similar studies have been carried out with a mutant of Rous sarcoma virus (T5) which transforms chick embryo cells in such a way that cells express the transformed phenotype at 36°C but not at 41°C. These cells exhibit enhanced agglutinability by Con A and WGA at the low but not at the high temperature (Burger and Martin, 1972; Biquard and Vigier, 1972). However, the correlation of agglutinability and some morphological aspects of transformation does not always exist in this system. For example, when cells are shifted from 41” to 36”C, it is possible to inhibit expression of the morphological aspects of transformation by blocking protein synthesis with puromycin or cycloheximide. This treatment, however, does not prevent the increase in lectin agglutinability (Biquard, 1973) associated with transformation. Further evidence supporting the correlation between transformation and lectin agglutinability has been obtained from studies of mutant cell lines called revertants, which are derived from transformed cells but which lack many of the properties of the parent transformed cells. Inbar et al. (1969) examined the properties of revertant polyoma virus-transformed hamster embryo cells that retained the polyomaspecific nuclear (T) antigen. They found a partial or complete loss of Con-A agglutinability. Flat revertants of SV-40-transformed 3T3 cells have been produced by treating SV-3T3 cells with 5-fluoro-2’-deoxyuridine, a compound that selectively kills actively dividing cells and which is employed to select for cells subject to contact or densitydependent inhibition of growth (Pollack et al., 1970; Culp et al., 1971). Revertants of this type were found to have lost their trans-

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303

formed phenotype; they had regained sensitivity to growth control, the normal 3T3 cell morphology had reappeared, and the cells had lost their agglutinability at low lectin concentrations. At the same time, these cells had retained the viral genome, as demonstrated by the fact that SV-40 T antigen was still expressed and SV-40 virus could be recovered from the revertants by fusing them with permissive msnkey cells. Similarly, revertants of SV-40-transformed 3T3 cells have been selected by growing these cells in the presence of WGAand Con A (Ozanne and Sambrook, 1971b; Culp and Black, 1972; Ozanne, 1973). Although these revertants were found to retain the SV-40 genome, they acquired many of the properties of untransformed cells including loss of sensitivity to agglutination by Con A and WGA. The treatment of transformed fibroblasts with dibutyryl cAMP has now been extensively studied as a method for phenotypically reverting these cells to an apparently normal state. The randomly oriented, multilayered transformed cell cultures are converted by dibutyryl CAMP into elongated, parallel, fibroblast-like cells which have regained density-dependent inhibition of growth and other characteristics of untransformed cells. These changes take place over a period of hours in culture, and they are completely reversible when dibutyryl cAMP is removed (Hsie and Puck, 1971; Sheppard, 1971,1972; Johnson et al., 1971). In oncornavirus-transformed mouse cells, a fall in Con A-mediated agglutinability accompanies this phenotypic reversion to the untransformed state (Hsie et al., 1971; Kurth and Bauer,

1973).

2. Exceptions to the Rule Although there are now many instances in which cell transformation correlates well with agglutinability at low lectin concentration, this is not always the case. There are many normal cell types that are agglutinated at low lectin concentration, including erythrocytes, cells of embryonic origin (see Section VI), sperm (see Section VI), rat lung cells, monkey kidney cells (Sivak and Wolman, 1972), mouse spleen cells, and mouse bone marrow cells (Liske and Franks, 1968). Moreover, there are instances in which normal cell lines are more susceptible to lectin-induced agglutination than the transformed cells derived from them. For example, normal rat liver cells are agglutinable by lentil lectin, but rat hepatoma cells are not (Borek et d., 1973),and Con-A agglutinates mouse lymphocytes to a greater extent than L1210 leukemic cells (Burger, 1973). Con A also agglutinates malignant mouse mammary cells to a lesser extent than nonmalignant variants (Hozumi

304

JAY C. BROWN AND RICHARD C. HUNT

et al., 1972). In a survey of one nonneoplastic and five neoplastic cell lines, Gantt et al. (1969) found that the nonneoplastic and two of the five neoplastic lines agglutinated at the same WGA concentration. Similarly, Ukena et al. (1976) identified several established lines of

mouse and hamster cells in which Con-A agglutinability did not correlate with the ability of cells to escape from density-dependent inhibition of growth in culture. No correlation was observed between transformation and RCA or Con-A agglutinability among several different types of human lymphoid cells (Glimelius et al., 1974, 1975; Maca, 1976),and a similar lack of correlation between Con-A or WGA agglutinability and transformation of several hamster cell lines has been reported (Berman, 1975). Thus, in spite of the frequent association of lectin agglutinability and transformation, the two do not always go together; agglutinability is not an invariant feature of the transformed cell phenotype. Indeed, from Table VI it can be seen that most investigators who have concluded that transformed cells are more agglutinable than normal cells have utilized only a few normal cell types, predominant among which are 3T3, BHK, and CEF cells. Furthermore, in most cases one of three transforming viruses (SV-40,polyoma, or RSV) has been employed. It is possible, therefore, that the correlation of agglutinability with transformation would become much less clear if a wider range of cells were investigated. In monolayer cultures, transformed fibroblasts are found to differ in their lectin agglutinability, depending upon whether cultures are sparse or confluent. For example, Inbar et al. (1971) and Ben-Bassat et al. (1971) found that 1 day after subculturing transformed hamster cells were not agglutinable b y Con A. Maximum agglutination was attained after 4 days of growth, when cultures had become confluent. Noonan and Burger (1973b) made similar observations with polyomatransformed 3T3 cells. Agglutination of rat c6 glioma cells is also density-dependent; as cells grow to a higher density in culture, their sensitivity to Con-A-mediated agglutination increases. This density-dependent agglutination appears to be related to the anchorage of cells to a solid surface, since cells grown in suspension do not show the same effect. It has been proposed that the extracellular matrix produced by c6 cells may be involved in this phenomenon (Skehan and Friedman, 1975). In the case of spontaneously transformed mouse 3T6 cells, however, there is a decrease in Con-A-mediated agglutinability when the cells reach saturation density (Goto et al., 1972).Agents that decrease the saturation density in culture also decrease the Con-Amediated agglutinability. Similar results were obtained with 3T3 and SV-40-transformed3T3 cells when both Con A and RCA agglutinability were examined as a function of cell density (Nicolson and Lacor-

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biere, 1973; Nicolson, 1974a). It was noted that, while RCA agglutinability decreases in 3T3 cells at confluence, more RCA is bound at this time. B. LECTINAGGLUTINATION AND TUMOFUGENICITY The generally satisfactory correlation between lectin agglutinability and transformed cell growth in tissue culture has motivated several studies designed to determine whether lectin agglutinability also correlates well with cell tumorigenicity in vivo. In some cases a positive correlation has been observed. For example, DeMicco and Berebbi (1972) examined Chinese hamster ovary (CHO) fibroblast-like cells and found that the most tumorigenic cells were also best agglutinated by Con A. Similar studies with mouse 3T3 cells showed that Con-A agglutinability correlates well with tumorigenicity but does not predict which cells will form progressively growing tumors which will eventually kill the host (Van Nest and Grimes, 1974). In other cases, however, no correlation is observed between lectin agglutinability and tumorigenicity. Sakiyama and Robbins (1973) and Berman (1975) found no correlation between Con A or WGA agglutinability and the ability to form tumors in vivo among several different hamster cell lines including N1L 2 and BHK-21. Dent and Hillcoat (1972) and Gantt et al. (1969) made similar observations with mouse lymphoid cells, while Glimelius et al. (1975) showed that neoplastic character does not correlate with Con A or RCA agglutinability in several human lymphoid cell types. In fact, two studies, one involving mouse mammary tumor cells (Hozumi et al., 1972) and the other using mouse lymphoid cells (Smets and Broekhuysen-Davies, 1972), have indicated that transplantability of tumor cells in vivo is correlated with a low rather than a high sensitivity to agglutination by Con A; highly transplantable tumor cells were weakly agglutinable, while weakly transplantable or nontransplantable cells were agglutinable at low Con-A concentrations. It is clear therefore that, insofar as a correlation exists, it is between lectin agglutinability and the expression of transformed cell growth properties in culture. Agglutination does not correlate well with the ability to escape immunological attack and form a progressively growing tumor in uivo.

c.

TRANSIENT AGGLUTINABILITY OF UNTRANSFORMED CELLS

1. Agglutinability of Protease-Treated Cells Although untransformed cells are ordinarily resistant to agglutination by lectins, two situations exist in which normal cells become tran-

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siently agglutinable. One is when they are treated with proteolytic enzymes. Brief proteolysis renders normal cells as agglutinable as transformed cells with WGA (Burger, 1969; Fox et al., 1971), Con A (Inbar and Sachs, 1969a), RCA (Nicolson and Blaustein, 1972), and soybean agglutinin (Sela et al., 1970,1971), but it does not ordinarily affect the agglutinability of transformed cells. Representative results for Con A and trypsinized 3T3 cells are shown in Table VII. Only a very gentle proteolytic digestion is required to produce increased agglutinability in untransformed fibroblast-type cells. For instance, Burger (1969) found that untransformed BHK cells became as agglutinable with WGA as polyoma virus-transformed BHK cells after treatment with 25 pg/ml trypsin for 5 minutes. Similar results are observed with other cell types (Rapin and Burger, 1974). Trypsin treatment of revertants (Inbar et al., 1969) and temperature-sensitive mutants of transformed cells at the nonpermissive temperature (Eckhart et al., 1971) renders them as agglutinable as the corresponding transformed cell type. The changes in the normal cell surface produced by proteolytic digestion, however, are not permanent. Trypsinized normal cells revert to their nonagglutinable state after a few hours of growth in tissue culture. The similarity, as judged by lectin agglutinability, of transformed cells and normal cells treated with trypsin has motivated a wide variety of studies on the molecular details of how trypsin affects normal cell surfaces. These studies have indicated that trypsin treatment increases the agglutinability of untransformed fibroblasts without changing the total number of Con A or WGA binding sites available on the cell surface (Rosenblith et al., 1973; Cuatrecasas, 1973a; Inbar et al., 1971; Nicolson, 1973a). This is consistent with the fact, as discussed below, that normal and transformed fibroblast-type cells do not usually differ in the number of binding sites for Con A or WGA (Ozanne and Sambrook, 1971a; Arndt-Jovin and Berg, 1971; Cline and Livingston, 1971; Inbar et al., 1971; Phillips et al., 1974; Trowbridge and Hilborn, 1974; Nicolson et al., 1975a; but compare Noonan and Burger, 1973a,b). However, proteolytic treatment of normal cells does increase the ability of Con A and WGA receptors to move laterally in the plane of the plasma membrane (Nicolson, 1972; Huet and H e n berg, 1973; Rosenblith et al., 1973; Garrido et al., 1974) to the point where the receptors of treated normal cells come to exhibit the same high degree of lateral mobility found in transformed cells. Trypsin treatment of normal cells also results in a loss of intracellular actin cables (Pollack and Rifkin, 1975) and in the digestion of a high-molecular-weight cell surface protein called LETS (large, external transformation-sensitive glycoprotein; Hynes, 1974). Both of these structures

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are found in normal but not in transformed fibroblasts. Any of these factors, or a combination of them, may be responsible for the increased agglutinability of trypsinized compared to untreated normal cells. It is attractive in the present context, however, to speculate that the generation of extracellular proteolytic activity may be functionally involved in the natural expression of the transformed cell phenotype. Transformed cell surfaces may actually come to resemble those of trypsinized normal cells, because transformed cells themselves synthesize and secrete a proteolytic enzyme which has the same effect on the cell surface as exogeneously added trypsin. According to this hypothesis, a crucial step in the transformation process would be the induction of a proteolytic enzyme activity that could be expressed extracellularly. This view is supported by the fact that proteolytic treatment causes untransformed cells to lose their sensitivity to densitydependent inhibition of growth in culture (Burger, 1970b; Sefton and Rubin, 1970) and therefore to resemble transformed cells in this key property. Furthermore, many transformed fibroblasts are found to express an extracellular proteolytic activity (Unkeless et al., 1973; Goldberg, 1974). This proteolytic function is generated by a cellular plasminogen activator interacting with serum plasminogen to produce plasmin (Unkeless et al., 1974), a potent trypsin-like proteolytic enzyme. Functioning of this system appears to be required for expression of certain aspects of transformed cell growth (Ossowski et al., 1974; Schnebli, 1974; Weber, 1975; but compare Chou et al., 1974; McIlhinney and Hogan, 1974). It is therefore possible that added trypsin converts normal fibroblasts phenotypically to the transformed state by mimicking the effect of a proteolytic system normally involved in the expression of transformed cell behavior. Nicolson (1973a) observed that treatment of both normal and SV-40 virus-transformed 3T3 cells with neuraminidase increases their sensitivity to agglutination by RCA in much the same way that trypsin treatment increases agglutinability; similar results were obtained with normal and polyoma virus-transformed BHK cells (Nicolson et al., 1975a). At the same time, binding of RCA-lZ5Iincreased two- to threefold in both cell types. These observations are best explained by assuming that the removal of terminal sialic acids with neuraminidase exposes subterminal galactose groups which are then available for binding to RCA. This interpretation is supported by the observation that neuraminidase treatment is required before many cell surface glycoproteins will react with galactose oxidase (Gahmberg and Hakomori, 1973; Hunt and Brown, 1974). Unlike RCA-mediated agglutination, the agglutination of transformed cells by WGA (Burger and Gold-

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berg, 1967; Kapeller and Doljanski, 1972; Greenaway and Levine, 1973)and Con A (Nicolson et al., 1975a) is reduced by neuraminidase treatment of the cell surface. Associated with these changes is a decrease in WGA binding but no change in Con-A binding (Nicolson et al., 1975a). 2. Agglutinability during Mitosis A second situation in which untransformed cells become transiently agglutinable with lectins is during the process of cell division (the M phase of the cell cycle). For example, Shoham and Sachs (1974a,b) found that normal hamster fibroblasts become agglutinable with low concentrations of Con A and WGA specifically during mitosis, but that the cells revert to the nonagglutinable state during interphase. A similar situation applies to 3T3 (Collard et al., 1975) and BHK (Glick and Buck, 1973) cells that are found to be agglutinable only in M phase. Transformed cells also vary in their sensitivity to lectin agglutination during mitosis, but some become more, and others less, agglutinable. For example, hamster fibroblasts transformed by RSV or by dimethylnitrosamine are agglutinated by Con A or WGA during interphase but not during M phase (Shoham and Sachs, 1974a,b), while SV-40-transformed 3T3 cells (Collard et al., 1975) and Epstein-Barr virus-transformed human lymphocytes (Smets, 1973) are most agglutinable during M phase. As in the case of trypsinized compared to untreated normal fibroblasts, it is not yet known why normal cells in mitosis are more agglutinable than similar cells in interphase. Some studies have shown that mitotic normal cells bind more Con A and WGA than interphase cells when the lectin is present at a very low concentration (Fox et al., 1971; Shoham and Sachs, 1972, 1974b; Noonan and Burger, 1973a; Noonan et al., 1973b). At a high lectin concentration, however, cells in all phases of the cycle bind similar amounts of lectin (Shoham and Sachs, 1974b). It is not likely therefore that increased lectin agglutinability during mitosis can be explained by increased lectin binding. Increased agglutinability of normal cells in M phase is, however, consistent with many other observations suggesting that the surfaces of normal cells in mitosis bear an intriguing similarity to transformed cell surfaces. For instance, Glick and Buck (1973) found that the set of fucose-containing glycopeptides solubilized by trypsin treatment of mitotic BHK cells more closely resembled the set of glycopeptides obtained from transformed cells than the set obtained from interphase normal cells. Also, a study (Garrido, 1975) of Con-A and WGA receptor mobility in CHO cells revealed that, whereas these receptors are relatively immobile during interphase, they display the

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same high degree of lateral mobility during mitosis that one finds for these receptors in transformed cells. D. MECHANISM OF AGGLUTINATION

1. Role o f t h e Lectin The striking difference in lectin agglutinability between normal and transformed cells has motivated many studies on the molecular basis of this phenomenon. It is quite reasonable to suppose that, if one could rationalize the selective agglutinability of transformed cells, one would at the same time learn something significant about the role of the cell surface in aspects of transformed cell behavior such as uncontrolled growth, invasiveness, metastasis, and resistance to immunological attack. One expects that agglutination of cells by a multivalent lectin would involve binding of the lectin to carbohydrate groups on adjacent cells. This should result in the cross-linking or aggregation of cells, and the experimental studies carried out so far are fully consistent with this view. For example, when cells agglutinated with fenitin-labeled Con A are examined in the electron microscope, ferritin-Con-A molecules are found between agglutinated cells in the regions where their plasma membranes are attached (DePetris et al., 1973). Also, when the number of carbohydrate-combining sites (the valence) of Con A is reduced by succinylation (Gunther et al., 1973; Trowbridge and Hilborn, 1974) or by treatment with chymotrypsin (Steinberg and Gepner, 1973), its ability to agglutinate a wide variety of cells is also significantly reduced. Conversely, when the functional valence of soybean lectin is increased by cross-linking native molecules with glutaraldehyde to produce dimers, trimers, and tetramers, its ability to agglutinate erythrocytes and lymphocytes is increased 10fold or more (Lotan et al., 197313).The role of the saccharide-combining site is indicated b y the fact that the specific, but not other, saccharide haptens can inhibit cell agglutination by WGA (Burger, 1969) or Con A (Inbar and Sachs, 1969a).In fact, agglutination of trypsinized or SV-40-transformed 3T3 cells by Con A can be reversed by a-MG if the hapten is provided shortly (within approximately 5 minutes) after agglutination takes place (Burger, 1970a; Rottmann et al., 1974). If a lectin is to agglutinate cells by serving as a cross-link or bridge between two adjacent cells, it must do so by binding to carbohydratecontaining structures on the surface of both cells. Cell surface glycoproteins and glycolipids are most likely to serve as lectin receptors, and all cells sensitive to lectin agglutination possess receptors for the agglutinating lectin. For example, transformed fibroblasts have be-

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tween 1 x lo7and 4 x lo7binding sites for Con A and a similar number of sites for WGA. In the case of fibroblast and fibroblast-like cells, however, even normal, untransformed cells, which are resistant to lectin agglutination, still contain a significant number of lectin receptors. In fact, in spite of one group’s contrary results (Noonan and Burger, 1973a,b), there is now general agreement that normal cells do not differ significantly from transformed or trypsinized normal cells in the number of receptors for Con A, WGA, or RCA (Ozanne and Sambrook, 1971a; Cline and Livingston, 1971; Arndt-Jovin and Berg, 1971; Inbar et al., 1971; Nicolson and Lacorbiere, 1973; Phillips et al., 1974; Trowbridge and Hilborn, 1974; Nicolson et al., 1975a). Considerable controversy was involved in establishing this point, as previously described by Nicolson (1974~). It is now clear, however, that one cannot account for the difference in agglutinability between normal and transformed cells by assuming that untransformed cells are lacking or depleted in cell surface lectin-binding sites. A more complicated explanation must apply. 2. Receptor Distribution and Mobility It was suggested quite early in the development of this field that the difference in agglutinability between normal and transformed cells may be due not so much to the total number of lectin receptors as to the way they are arranged on the cell surface. In particular, it was suggested that, if transformed cell lectin receptors were organized into small islands or clusters while normal cell receptors were evenly distributed over the cell surface, and if cells could be cross-linked only in the regions of receptor clusters where multiple cross-links could be formed, one would be able to account for the selective agglutination of transformed cells (Burger, 1970a; Sela et al., 1971). Experimental support for this model was provided by Nicolson (1971), who examined electron micrographs of ferritin-labeled Con A (Con A-FT) bound to the spread membranes of 3T3 and SV-40-transformed3T3 cells. These micrographs were interpreted to support the view that Con-A receptors are aggregated into small patches or clusters on the transformed cell surface but evenly distributed in normal cells (Nicolson, 1971). Similar micrographs of cells agglutinated by Con A-FT showed that the cells were cross-linked preferentially at regions of the plasma membrane where lectin-receptor complexes were located in clusters (Nicolson, 1972). Other studies appeared to confirm these early results. For example, an electron microscope technique involving horseradish peroxidase coupled to Con A was employed to demonstrate clustering of surface Con-A receptors in adenovims-12-trans-

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formed (Rowlatt et al., 1973) and SV-40-transformed (Bretton et al., 1972; Garrido et al., 1974) hamster cells, Ehrlich ascites cells (Roth et al., 1974), chemically transformed rat liver cells (Roth, 1974; Roth et al., 1975), and erythroblasts in chickens infected with avian erythroblastosis virus (Barbarese et al., 1973),whereas Con-A receptors were found to be uniformly distributed in the normal cells from which all of these were derived. Subsequent studies have not confirmed these early results. It has now been clearly demonstrated that clustering or patching of surface lectin-binding sites in transformed cells is induced by the presence of the lectin and is not a property of the unperturbed cell membrane. Binding sites for Con A (Comoglio and Guglielmone, 1972; Inbar et al., 1973a,b; Inbar and Sachs, 1973; Nicolson, 1973b; Rosenblith et al., 1973; DePetris et al., 1973; Garrido et al., 1974; Marikovsky et al., 1974; Temmink et al., 1975; Ukena et al., 1974; Berlin, 1975), WGA (Garrido et al., 1974), and RCA (Nicolson, 1974a) were found to be evenly distributed on the cell surface when normal, trypsinized normal, or transformed fibroblasts were fixed prior to labeling with lectin, or if they were labeled with lectin at 4°C. Clustering of receptors in transformed or trypsinized normal cells was observed only when unfixed cells were exposed to lectin and incubated at 37°C. At this temperature lectin receptors of transformed cells are free to move laterally in the plane of the membrane, and they can be aggregated to form clusters by multivalent lectin molecules. The results of Inbar and Sachs (1973) illustrate this point clearly. These investigators bound a fluorescent (fluorescein isothiocyanate-labeled) derivative of Con A (F-Con A) to normal and transformed cells either with or without prior glutaraldehyde fixation. F-Con-A molecules were then localized on the cell surface by examining cells in the fluorescence microscope. The results as shown in Fig. 3 indicate that, whereas F-Con-A molecules are uniformly distributed on the surface of prefixed normal or transformed cells, they are aggregated in clusters in unfixed transformed cells. Although the Con-A receptors of most transformed or trypsinized normal fibroblasts can be aggregated into patches (for the few exceptions reported, see Martinez-Palomo et al., 1972; Francois et al., 1972), untransformed cells differ among themselves in this property. Some normal cells have Con-A receptors that can be aggregated into clusters (Smith and Revel, 1972; Roth et al., 1973; DePetris et al., 1973; Torpier and Montagnier, 1973; Huet, 1974; Huet and Bernhard, 1974; Marikovsky et al., 1974; Raff et al., 1974; Temmink and Collard, 1974; Collard et al., 1975; Temmink et al., 1975), while in other cases

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FIG. 3. Distribution of F-Con A on the surface membrane of normal and transformed cells. (A) Binding of F-Con A to lymphoma cells after fixation with 2.5% glutaraldehyde. Similar results were obtained with normal lymphocytes, normal fibroblasts, and transformed fibroblasts. The binding of F-Con A shows a completely uniform distribution on the cell surface. (B) Distribution of F-Con A of the type seen with unfixed normal fibroblasts. Little or no change from the uniform distribution is detectable. (C) Distribution of F-Con A of the type seen with unfixed transformed fibroblasts and lymphoma cells. The formation of clusters is clearly evident. (D) Cap formation in normal lymphocytes. Cells were incubated with 100 pg F-Con Nml for 15 minutes at 37"C, washed, and examined in a Leitz Ortholux microscope with transmitted ultraviolet light. x 2500.

the receptors remain dispersed at 37°C in the presence of lectin (Inbar and Sachs, 1973; Nicolson, 1973b; Rosenblith et aZ., 1973).In spite of the difference in ability to form receptor clusters, however, normal cells usually remain resistant to lectin agglutination. It is highly unlikely therefore that a patched distribution of Con-A receptors or the ability of receptors to be aggregated into patches by Con A is directly involved in the selective agglutination of transformed cells. In fact, a recent study devoted to reexamining the role of receptor mobility in the Con-A-induced agglutination of fibroblasts has confirmed the fact

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that untransformed cells may differ in their ability to form receptor patches without differing in agglutinability (Ukena et al., 1976).It was concluded that the clustering of Con-A receptor sites is neither necessary nor sufficient to account for the difference in agglutinability between normal and transformed cells. It is possible, however, that, although formation of larger clusters or patches of Con-A receptors is not involved in specific agglutination, some degree of lateral receptor mobility may be required. Rutishauser and Sachs (1974,1975) tested this possibility by examining the distribution of Con-A receptors on the surface of single lymphoid cells. Cells immobilized on nylon fibers were allowed to interact with free cells in the presence or absence of lectin. It was found that Con A agglutinated two lymphoma cells and, less frequently, one lymphoma cell and one lymphocyte. Agglutination of two lymphocytes was only rarely observed. Perhaps the most interesting observation was that, while agglutination was inhibited by rixation of both the immobilized and the free cells, fixation of only one of the cells actually enhanced agglutination. This clearly indicates that receptor mobility is necessary in only one of the t.wo cells involved in a cross-link. It is possible therefore that an important factor in the formation of stable lectin cross-bridges is the ability of the receptors on at least one of the cells to come into precise alignment with the receptors on the other cell. Thus only short-range lateral mobility in the unfixed cells is necessary to stabilize agglutination. 3. Effect of Temperature and Membrane Fluidity In contrast to agglutination by either WGA or soybean lectin, which is independent of temperature (Inbar et al., 1973b; Gordon and Marquardt, 1974; c. Huet, 1974; M. Huet, 1975; Horwitz et al., 1974), Con-A-mediated agglutination is inhibited by cooling cells to 4°C. Con A binds to transformed cells at 4°C in sufficient amounts to cause agglutination if the cells are subsequently warmed to 37°C (Inbar et al., 1971, 1973b; Noonan and Burger, 1973a,b; Gordon and Marquardt, 1974; c. Huet, 1974; M. Huet, 1975; Horwitz et al., 1974),but no agglutination takes place at 4°C. Since agglutination involves the interaction of a multivalent lectin with receptors on the surfaces of two cells, any change in agglutinability on changing the temperature could result from an alteration in either the lectin or the surface membrane. The available evidence suggests that both can occur in the case of Con-A-mediated agglutination. For example, cooling from 37" to 4°C results in the conversion of Con A from a tetramer to a dimer, but this temperature change has no effect on the valence of PHA, WGA,

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or soybean lectin (Gordon and Marquardt, 1974; C. Huet, 1974; M. Huet, 1975). Since reduction of the valence of Con A by succinylation or chymotrypsin digestion (Burger and Noonan, 1970; Gunther et al., 1973) has been shown to result in decreased agglutination (Trowbridge and Hilborn, 1974), this may explain the temperature-dependence phenomenon. Cooling also results in changes in the cell membrane. Current models of membrane structure envisage a lipid bilayer containing globular proteins dispersed throughout the lipid (Singer and Nicolson, 1972; Nicolson, 1976a).At physiological temperatures, the lipids are relatively fluid and proteins are free to move laterally in the plane of the membrane. However, on cooling the lipids become less mobile, and they eventually freeze into a paracrystalline state below their transition temperature. Electron and fluorescence microscopy of Con-A-Con-A receptor complexes on the surface of many transformed cell types has demonstrated that at 4°C the receptors behave similarly to fixed cells at 37°C; that is, they remain randomly distributed over the cell surface. On warming, however, the receptors are able to migrate in the presence of Con A to form clusters of Con-A-Con-A receptor complexes (Inbar et al., 1973a; Nicolson, 197313; Rosenblith et al., 1973; Ukena et al., 1974; Berlin, 1975). This temperature-dependent increase in receptor mobility most probably results from a change in the fluidity of the lipid continuum. Support for this idea is derived from studies on the effect of altering the membrane fatty acid composition. Con-A-mediated agglutination of LM- or SV-40-transformed 3T3 cells shows a temperature-dependent transition at about 14"- 18°C; above this temperature the cells are readily agglutinated, whereas below 14"- 18°C they are not. When cells are cultured in medium supplemented with unsaturated fatty acids, there is a change in the composition of the membrane such that membrane phospholipids come to have a higher content of unsaturated fatty acyl chains. This results in a lowering of the transition temperature for the lipid bilayer itself and also for Con-A-mediated agglutination. An increase in both transition temperatures is observed when cells are grown in medium supplemented with saturated fatty acids (Horwitz et al., 1974; Rittenhouse et al., 1974). Thus changes in the fluidity of membrane lipids with temperature may account for the difference in Con-A-mediated agglutinability at 0°C compared to 37°C. It is very unlikely, however, that a difference in bilayer fluidity could account for the difference in Con-A-mediated agglutinability between normal and transformed fibroblasts. The lipid composition of normal and transformed cells is quite similar (Quigley et al., 1971,1972; Gaffney et al., 1974; Micklem

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et al., 1976), and physical methods for measuring membrane fluidity have either failed to reveal a significant difference between normal and transformed cells (Robbins et al., 1974; Gaffney, 1975) or have shown greater fluidity in normal cell membranes (Fuchs et al., 1975; Edwards et al., 1976; Yau et al., 1976; but compare Barnett e t al., 1974). Furthermore, the mechanism for selective agglutination of transformed cells by lectins other than Con A must be even less dependent on membrane fluidity, since in these cases agglutination is relatively independent of temperature.

4. Role of Cytoskeletal Elements Neoplastic transformation affects both major intracellular cytoskeletal systems, the microtubules and the microfilaments. After transformation, cell surface-associated tubulin (Brinkley et al., 1975; Fine and Taylor, 1976) and actin (Wickus et al., 1975; Pollack and Rifkin, 1975; Pollack et al., 1975) are dramatically reduced in amount. Both fluorescence and electron microscopy (McNutt et al., 1971, 1973; Nicolson, 1975) show this reduction to be associated with less well-defined intracellular microtubule and microfilament structures. If these cytoskeletal systems are involved in lectin agglutinability, agents that disrupt microtubules or microfilaments should cause an increase in the agglutinability of untransformed cells. In fact, there is considerable evidence to support this expectation. Incubation of 3T3 cells with colchicine (which disrupts microtubule structure) and cytochalasin B (which affects microfilaments) together results in the clustering of Con A receptors and in enhancement of agglutinability. Agents such as colchicine and calcium ionophores, which disrupt only microtubules, have little effect on either parameter (Yin et al., 1972; Ukena et al., 1974; Poste and Nicolson, 1976). Trypsin treatment, which converts untransformed cells from the unagglutinable to the agglutinable state, is also found to disrupt the intracellular microfilament system severely in rat embryo cells (Pollack and Rifkin, 1975). Local anesthetics such as dibucaine, tetracaine, and procaine, which affect cell membrane fluidity, act on 3T3 and BHK cells in a manner similar to colchicine and cytochalasin B combined; that is, they lead to a redistribution of lectin receptors into clusters and to an increase in Con-Amediated agglutinability (Poste et al., 1975a,b,c).Although local anesthetics increase the fluidity of membrane lipids, and therefore probably allow receptors to become more mobile (see, e.g., Papahajopoulos et al., 1975), the increase in fluidity is small, and it has been proposed that the anesthetics may increase cell agglutinability by disrupting intracellular cytoskeletal elements rather than b y affecting membrane

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lipids. This site of action is supported by electron microscope studies showing that both intracellular microtubules and microfilaments are reduced in number in normal cells following incubation with local anesthetics (Nicolson et al., 1976). Compared to the situation with normal cells, the effects of microtubule- and microfilament-disrupting agents on transformed cells are much more difficult to interpret. Indeed, it would be expected that, as transformed cells appear to have a disorganized cytoskeletal system, these agents would have no further effect on agglutinability. In some cases this is so. Colchicine and cytochalasin B together have no effect on the Con-A-mediated agglutinability of SV-40-transformed 3T3 cells (Poste et al., 1975b), and colchicine alone has no effect on the agglutinability of CHO (Van Veen et al., 1976) or rat ascites tumor cells (Kaneko et al., 1973). Cytochalasin B has no effect on the agglutinability of CHO cells. However, cytochalasin B does have an inhibitory effect on the Con-A-mediated agglutinability of rat ascites tumor cells (Kaneko et al., 1973), and agglutination of several transformed cell types by Con A is altered by microtubule-disrupting agents. For example, incubation of SV-40-transformed 3T3 cells with colchicine or with calcium ionophores leads to the inhibition of Con-A-mediated agglutination (Yin et al., 1972; Poste et al., 1975b; Poste and Nicolson, 1976). Similarly, the agglutination of rat hepatoma cells is reduced by microtubule disruption (Nakamura and Terayama, 1975). LM cells treated with colchicine show either enhanced or decreased Con-A agglutinability, depending on the length of time they are exposed to the lectin (Rittenhouse et al., 1976). Morphological observations on SV-40-transformed 3T3 cells confirm that colchicine is able to alter the distribution of Con-A receptors on the surface of these cells. A clustered distribution of receptors is induced by Con A alone, and these clusters can be redistributed into a cap when cells are also incubated with colchicine. Presumably this capping is under direct control of microfilaments, because cells incubated with cytochalasin B plus colchicine are not capped (Ukena et al., 1974). Local anesthetics have no effect on the Con-A-mediated agglutination of SV-40-transformed 3T3 cells or on the distribution of Con-A receptors in these cells. Receptors remain clustered. in the presence of Con A plus anesthetics. Local anesthetics, however, reverse the inhibition of agglutination produced by colchicine (Poste et al., 1975b,c). Together these observations provide reasonable evidence that cytoskeletal systems are in fact involved in the sensitivity of cells to lectin-mediated agglutination. The overall effect of cytoskeletal involve-

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merit is to prevent or inhibit agglutination. Thus one observes agglutination only in cells whose cytoskeletal systems have been disrupted either as a result of neoplastic transformation or in the presence of drugs. In fact, it may be that the effect of microtubules and microfilaments will prove to be central to our understanding of why transformed but not normal cells are agglutinable. Since both normal and transformed cells possess abundant lectin receptor sites, it is more difficult to understand why normal cells are resistant to agglutination than why transformed cells are agglutinable. Therefore it may be that, if one could determine how cytoskeletal structures are involved in the inhibition of normal cell agglutination, one would have the key to the whole question of selective transformed cell agglutinability. Microtubules and microfilaments are most likely to exert their effects on agglutination by controlling the distribution of lectin receptor molecules on the cell surface. It is reasonable to assume that this control can be exerted b y a direct interaction inside the cell between cytoskeletal structures and “transmembrane” lectin receptor glycoproteins which are exposed both outside and inside the cell. The association between glycophorin and spectrin inside the human erythrocyte provides a model for how this type of interaction may occur (Nicolson and Painter, 1973), and there is evidence in lymphocytes for an association between cell surface structures and intracellular microtubules (Edelman, 1974). The results of experiments in which transformed cells are exposed to microtubule- and microfilament-disrupting drugs strongly suggest that, in spite of the fact that the cytoskeletal systems of transformed cells are quite abnormal, some degree of cytoskeletal control over cell surface properties still exists.

5. Microvilli An idea related to the involvement of cytoskeletal elements suggests that microvilli may be the key to selective agglutination of transformed cells. If transformed cells had more microvilli than untransformed cells, and if agglutination by lectins required cross-bridges in the regions of interdigitating microvilli, or if lectin receptors were concentrated in the microvilli, it would be reasonable to expect that transformed cells would be more agglutinable than normal ones (Boyde et al., 1972; Porter et al., 1973a; Willingham and Pastan, 1975; Borek and Fenoglio, 1976; Malick and Langenbach, 1976). Some experimental studies have been interpreted to support this view. For example, the incubation of mouse L-929 cells (which have numerous surface microvilli) with CAMPcaused a reduction both in the number of microvilli and in lectin-mediated agglutinability (Willingham and

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Pastan, 1975). In 3T3 cells trypsin treatment, which enhances agglutinability, was also found to increase the number of microvilli (Follett and Goldman, 1970; Willingham and Pastan, 1975). Furthermore, some of the effects of microtubule- and microfilament-disrupting agents on agglutinability may result from the loss of microvilli caused by the disruption of cytoskeletal elements (Loor, 1973; Loor and Hagg, 1975). However, there are many significant exceptions to the proposed correlation between increased number of microvilli and enhanced agglutinability in transformed cells. Temmink and his associates showed that normal 3T3 cells and normal lymphocytes have a greater number of microvilli than their more agglutinable transformed counterparts (Collard and Temmink, 1975, 1976; Temmink et al., 1976). Similarly, in a study of BALB/c 3T3 cells and several transformed lines derived from them, no correlation was observed between microvillus formation and tumorigenicity. Lines transformed by SV-40, murine sarcoma virus, or polyoma virus were largely free of microvilli, while a “spontaneous” transformant possessed numerous microvilli (Porter et al., 1973b). It seems most unlikely therefore that an increased number of microvilli in transformed cells is the only explanation for their increased lectin agglutinability. 6. Conclusion It is fair to say at the present time that, despite a great deal of effort, we really do not know why transformed cells are generally more agglutinable by lectins than their normal counterparts. We do know of some explanations that do not apply. One cannot account for the difference between normal and transformed fibroblast agglutinability with Con A or with WGA by assuming that transformed cells have a greater total number of receptors for these lectins. Experimental studies have shown that the total number of binding sites for Con A or for WGA does not differ significantly in normal compared to transformed cells (see Section IV,D,l). Similarly, one cannot account for the difference in agglutinability by assuming that lectin receptors are distributed differently on the surfaces of normal and transformed cells; a uniform distribution of Con-A receptors is found on the unperturbed surface of both normal and transformed fibroblasts. Although a small amount of lateral receptor mobility in the plane of the membrane may be required for Con-A agglutination, it is now clear that the migration of receptors to form large clusters or patches also cannot account cleanly for the selective agglutination of transformed cells. Some nonagglutinable cells are able to form receptor patches, while some agglutinable cells are not (Ukena et al., 1976).

LECTINS

319

Elimination of these earlier hypotheses has not entirely depleted our supply of good ideas to explain the selective agglutination of transformed cells. There are several proposals that have not yet been fully tested experimentally. For example, it may be that untransformed fibroblasts are not agglutinated by lectins because of their high surface negative charge (zeta potential); electrostatic repulsion between cells may prevent them from being cross-linked noncovalently by lectins. According to this hypothesis, transformed cells, which usually have a much lower density of surface negative charge, would be agglutinated because of the reduced electrostatic charge repulsion between cells. conversion of normal cells from the unagglutinable to the agglutinable state by trypsin treatment would be explained by the fact that proteolysis simply reduces the net cell surface negative charge. Ukena and Karnovsky (1976) recently proposed that selective agglutination of transformed cells occurs because transformed fibroblasts spontaneously, in the absence of lectin, undergo an aggregation process that is not found in untransformed cells or that takes place much more slowly in untransformed cells. According to this “spontaneous adhesion” hypothesis, the role of the lectin would simply be to accelerate a preexisting transformed cell property. This proposal clearly deserves further study. The same can be said of proposals that emphasize the role of cytoskeletal elements and microvilli in selective agglutination. Studies with drugs that disrupt microtubules and microfilaments have provided a strong case for the involvement of these structures in selective lectin-mediated agglutination (see Section IV,D,4), and it would be very surprising if microvilli, by which fibroblasts ordinarily make initial contact with each other, were not also somehow involved in the agglutination process. Despite the unsatisfactory state of our knowledge at the present time, the motivation for trying to understand the molecular basis of the selective agglutination of transformed cells remains as strong as ever. It may really be that a satisfactory explanation of this phenomenon will provide important new information about the role of the cell surface in transformed cell growth. The time may have come in the development of this field, however, when it would be more fruitful to pursue basic studies on membrane structure and function than to continue to attack the issue of selective agglutination directly.

V. Interaction of Lectins with Cells Infected by Nononcogenic Viruses Although neoplastic transformation of normal cells is by far the best studied situation in which changes in lectin agglutinability accom-

320

JAY C. BROWN AND RICHARD C. HUNT

pany biological variables, such changes are not restricted to transformation. Animal cells may also undergo changes in sensitivity to lectin agglutination during the productive growth of certain nononcogenic viruses. The growth process ordinarily involves intracellular replication of virus particles and their release either with or without lysis of the host cell. In the case of infection by viruses containing a membrane envelope, virus replication may involve insertion of virusspecific proteins into the host cell plasma membrane, followed by budding of the virus particle from the cell at this region of modified membrane. Lectin-detectable changes in the 'cell surface have been observed accompanying productive infection with a wide variety of both RNA- and DNA-containing animaI viruses, as shown in Table VIII. In their studies of myxoviruses, Becht et al. (1972) consistently observed an increase in the Con-A agglutinability of cells infected with these viruses. These investigators concluded that it was insertion of TABLE VIII

VIRUSES CAPABLE OF INDUCING INCREASED LECTIN AGGLUTINABILITY I N HOST CELLS DURING PRODUCTIVE INFECTION

Virus class RNA-containing viruses Orthomyxoviruses Paramyxoviruses

Arboviruses Rhabdoviruses DNA-containing viruses Pox viruses

virus

Influenza virus Fowl plague virus NDV Sendai virus, canine distemper virus, parainfluenza a, mumps virus, measles virus, respiratoq syncitial virus Simian virus 5 Sindbis virus Vesicular stomatitis virus

References

Becht et al. (1972) Fresen and Illiger (1974) Becht et al. (1972); Poste and Reeve (1972); Bubel and Blackman (1975) Poste (1975)

Becht et al. (1972) Birdwell and Strauss (1973) Becht et al. (1972)

Herpes viruses

Herpesvirus hominis type 1

Zarling and Tevethia (1971); Bubel and Blackman (1975) Tevethia et al. (1972); Poste

Adenoviruses

Adenovirus type 2

Salzberg and Raskas (1972)

Vaccinia

(1972)

LECTINS

32 1

virus-specific glycoproteins into the cell membrane that gave rise to enhanced Con-A-mediated agglutinability. They also noted that Con-A precipitated purified fowl plague and SV-5 viruses. Other investigators have suggested that the insertion of viral components into the cell membrane may not be necessary for the surface change that results in increased agglutinability. For example, Zarling and Tevethia (1971)reported that vaccinia virus causes rabbit kidney cells to become agglutinable with Con A early (2 hours after infection) in its replicative cycle, although no virus particles are detected until much later (6 hours after infection). Herpesvirus hominis type- 1 infection of chick embryo fibroblasts also leads to an early expression of Con-A agglutinability; in this case agglutinability is observed 4 hours before the expression of a virus-specific cell surface antigen (Tevethia et al., 1972). De no00 protein synthesis is required after virus infection before this increased agglutinability can be detected. The agglutinability of cells infected by Newcastle disease virus (NDV) has been more extensively studied than any other nononcogenic virus system (Poste, 1975; Reeve et al., 1975). A useful feature of NDV has been the availability of virus strains with varying degrees of virulence. The infection of cells by virulent strains causes an increase in Con-A- or WGA-mediated agglutinability. At 4-8 hours after infection, cell agglutinability rises and, at the same time, the thickness of the cell coat decreases. The decreased thickness of the cell coat results from shedding of surface material into the medium and is accompanied by the release of lysosomal enzymes. In contrast, after infection by an avirulent strain, no change in coat thickness or agglutinability is observed (Poste and Reeve, 1972; Poste et al., 1972; Reeve et al., 1972, 1975). Although the increases in agglutinability observed after virulent virus infection could be due simply to the insertion of virus-specific glycoproteins into the plasma membrane, this is not thought to be the case; no difference can be detected in the amount of labeled Con A bound to the cell surface before or after infection with virulent or with avirulent strains of NDV (Poste and Reeve, 1974). There is also no difference in the binding at 0" and 37°C (compare Noonan and Burger, 1973a). BHK cells labeled with fluorescent Con A after infection with a virulent strain of NDV reveal another similarity to transformed cells. The surface of these cells shows patches of fluorescence at 37"C, while uninfected cells or cells infected with an avirulent strain of the virus show uniform fluorescence. Glutaraldehyde fixation or cooling to 0°C inhibits patch formation (Poste and Reeve, 1974). The increase in Con-A agglutinability induced by infection with virulent NDV is

322

JAY C. BROWN AND RICHARD C. HUNT

therefore strikingly similar to the increase observed after neoplastic transformation.

VI. Interaction of Lectins with Developing Cells A. EGGSAND EMBRYOS Eggs and embryonic cells of several organisms resemble erythrocytes and transformed cells in that they are strongly agglutinated by lectins. Fertilized eggs are frequently more agglutinable than unfertilized eggs but, as the embryo develops, its cells generally become less agglutinable. The molecular mechanism by which agglutinability is gained and lost during development has yet to be elucidated, but it is reasonable to suppose that these changes may resemble those that take place after neoplastic transformation of normal cells. In fact, transformed and embryonic cells have many properties in common, including a high rate of growth, the ability to migrate in uiuo, and the ability to invade surrounding tissues. Whether or not these similarities have a common basis at the molecular level remains to be determined. Con A does not agglutinate unfertilized eggs of the sea urchin Paracentrotus liuidus, but it does inhibit the fertilization process. It also blocks formation of the fertilization membrane and cleavage (Lallier, 1972).One day after fertilization, dissociated sea urchin embryo cells are strongly agglutinated by Con A and RCA, but the effect of the lectins diminishes during subsequent development (Oppenheimer and Odencrantz, 1972; Krach et al., 1974). A most interesting observation is that the cells of sea urchin embryos at a later stage of development (the 32- to 64-cell stage) differ among themselves in their relative agglutinability by Con A. Cells that show migratory activity are both more agglutinable and have a greater degree of Con-A-induced receptor clustering than nonmigratory cells (Roberson and Oppenheimer, 1975; Neri et al., 1975).At no developmental stage does WGA agglutinate sea urchin cells that have not first been treated with trypsin. Studies on the agglutination of chick embryonic cells have shown that receptors for different lectins behave quite differently during development. At very early stages after fertilization (e.g., in eggs immediately after fertilization or in 22-hour embryos), Con A and WGA both agglutinate chick embryo cells. WGA agglutination is lost quickly, however, and by 8 days of incubation chick embryonic liver or neural retina cells are agglutinated only by Con A or RCA. WGA agglutination can be restored by trypsin digestion of the cell surface [Moscona,

LECTINS

323

1971; Kleinschuster and Moscona, 1972; Zalik and Cook, 1976; but compare McDonough and Lilien (1975) who were unable to demonstrate agglutination of early chick embryo cells by Con A]. At later stages of embryonic development Con-A-mediated agglutination progressively decreases, until by day 20 it is not present in mechanically dissociated chick cells. Cells remain fully agglutinable by RCA up to at least 20 days, and trypsinization restores full agglutinability by WGA and Con A at all stages up to this time (Kleinschuster and Moscona, 1972). The binding of 1251-labeledCon A to chick embryo cells shows that, contrary to what would be expected, decreasing agglutinability with age is accompanied by increased lectin binding. At all stages, lectin binding to mechanically dissociated cells is greater than to cells dissociated with trypsin, even though trypsin increases agglutinability. The lack of correlation between Con-A binding and agglutinability has been interpreted to imply that a difference in receptor mobility with age may be the basis for the decrease in agglutinability. In support of this proposal is the observation that, although binding of C O ~ - A - ' ~to~ Itrypsinized 19-day-old chick cells is not affected by lowering the temperature to 4"C, agglutination is completely abolished. By analogy with observations on transformed and normal cells it might be expected that glutaraldehyde fixation of chick embryo cells would inhibit their agglutination by Con A. Instead, fixation promotes agglutination not only of mechanically dissociated cells at 37°C but also of trypsin-dissociated cells at 4°C. This puzzling result is suggested to be the consequence of receptor cross-linking by the fixative (Martinozzi and Moscona, 1975), but further research will be necessary to determine whether this is in fact the case. In addition to the changes in Con-A agglutinability and in Con-A binding observed during the development of chick embryonic cells, Con A is also found to affect DNA synthesis differently as development proceeds (Kaplowitz and Moscona, 1973; Roguet and Bourrillon, 1975a,b). As in the case of chick cells, rodent embryo cells also exhibit changes in lectin agglutinability during development. For example, unfertilized mouse eggs lacking a zona pellucida (a glycoprotein layer around the egg plasma membrane which the sperm penetrates before fusion with the egg) are not agglutinated by Con A unless they have been pretreated with protease. However, after fertilization the eggs are strongly agglutinated (Pienkowski, 1974).The difference in agglutinability in the fertilized and unfertilized eggs may result from a difference in receptor distribution. With the use of fluorescent Con A, it

324

JAY C. BROWN AND RICHARD C. HUNT

has been demonstrated that the lectin binds to both fertilized and unfertilized mouse eggs, but that the pattern of fluorescence is different; whereas unfertilized eggs bound the lectin over only part of their surface, fertilized eggs bound lectin uniformly over their whole surface (Johnson et al., 1975).As in chick embryos, agglutination is reduced as the embryo develops, so that by the blastocyst stage high Con-A concentrations fail to agglutinate mouse embryo cells (Rowinski et al., 1976). In hamster eggs, the change in lectin agglutinability after fertilization is not as pronounced as in the mouse. Fertilized zona pellucidafree eggs are only slightly more agglutinated by Con A, lentil (Lens culinaris) lectin, and WGA than unfertilized eggs. RCA and D . biflorus agglutinin (DBA) agglutinate the zona pellucida of both fertilized and unfertilized eggs, and binding of fluorescent lectins to the zona pellucida does not change during maturation. In zona-free eggs the binding of fluorescent Con A, RCA, and WGA decreases as the egg develops (Yanagimachi and Nicolson, 1976). Pretreatment of hamster eggs with WGA, RCA, or DBA prevents fertilization and reduces the sensitivity of the zona pellucida to digestion by trypsin. These lectins cause a change in the zona pellucida which results in a greater degree of light scattering when the zona is viewed by dark-field illumination. Spermatozoa do not either penetrate the WGA-treated zona or even bind to its surface. Con A, however, does not block fertilization, nor does it cause the change in light scattering (Oikawa et al., 1973,1974). It has been speculated that lectins that inhibit fertilization do so by cross-linking their receptors and thereby block digestion of the zona pellucida by sperm-borne enzymes; according to this model, they inhibit fertilization by mimicking the action of a natural lectin contained within the egg and released after fertilization. This lectin reacts with zona pellucida glycoprotein to prevent multiple fertilization (Wyrick et al., 1974). Electron microscope analysis of hamster eggs after incubation with ferritin-labeled lectins has shown that RCA, Con-A, and WGA receptors are localized in the zona pellucida and in the underlying plasma membrane. RCA and WGA receptors are distributed asymmetrically throughout the zona pellucida, with the highest concentrations at the surface. Con-A receptors are located sparsely throughout this layer (Nicolson et al., 197%). The plasma membrane receptors for the three lectins are randomly distributed in fixed cells or in cells that have been incubated with the lectin at 4°C. However, Con-A and WGA receptors form clusters at 25°C (Nicolson et al., 197513).It is attractive to

LECTINS

325

speculate that the mobility of plasma membrane receptors may be important in sperm fusion but, as yet, there is no experimental evidence to support this possibility. Lectin interactions with the cell surface have been examined in several other developmental systems including human fetal intestine (Weiser, 1972), cultured muscle (Betschart and Burger, 1975), the slime mold Dictyostelium discoideum (Weeks and Weeks, 1975), spinal ganglions of chick embryos (Treska-Ciesielski et al., 1971), ascidian eggs (Monroy et al., 1973), and neural crest cells from frog (Johnson and Smith, 1976) and from the urodele Ambystoma mexicanum (Moran, 1974). B. MALE GERMINALCELLS The early germinal cells of the seminiferous tubule (the spermatogonia) in adult animals undergo waves of proliferation, giving rise to cells which differentiate into spermatozoa. This process of proliferation and differentiation takes place in a highly coordinated, synchronous manner. The spermatogonia, which adhere tightly to one another, give rise first to spermatocytes; these differentiate into spermatids and finally spermatozoa. During differentiation the germinal cells lose their mutual adhesion, and spermatozoa are secreted into the lumen of the tubule as a suspension of individual motile cells. Since glycoproteins have been implicated in intercellular adhesion and in information transfer during differentiation, it is not surprising that they have been the subject of experimental studies in the field of spermatogenesis. Lectins have been shown to agglutinate the sperm cells of several species, including clam (Venus mercenaria and Mytilus mytilus; see Bade1 and Brilliantine, 1969), rodent (Edelman and Millette, 1971; Nicolson and Yanagimachi, 1972, 1974; Nicolson et al., 1972; Gordon et al., 1974), bull (Kashiwabara et al., 1965), and human (Uhlenbruck and Hermann, 1972). Separation of the head and tail regions of rodent sperm revealed that Con-A receptors were predominantly located in the head region, and fluorescence microscope studies showed binding to the acrosome to be most visible (Edelman and Millette, 1971). Electron microscopy of sperm incubated with ferritin-RCA revealed clusters of receptors in the postacrosomal region only. At O'C, or after glutaraldehyde fixation, no clusters were observed (Nicolson and Yanagimachi, 1974). From these data, it is possible to conclude that lectin receptors cannot move freely over the entire sperm cell surface, even though the cell is surrounded by a continuous plasma membrane. It

326

JAY C. BROWN AND RICHARD C. HUNT

may be that the more mobile receptors in the region behind the acrosome are involved in the fusion of the sperm with the egg plasma membrane, as suggested by Nicolson and Yanagimachi (1974). Changes that take place during differentiation of spermatozoa are more difficult to study, as they necessitate the isolation of different cell types in the germinal cell differentiation pathway. The rat testis has been employed as a source of cell suspensions highly enriched in secondary spermatocytes, spermatids, spermatozoa, and nongerminal interstitial cells. A major lectin-detectable difference exists between the cells of the germinal differentiation pathway and the nongerminal cells. Spermatocytes, spermatids, and spermatozoa are strongly agglutinated by either Con A or WGA, but the interstitial cells are agglutinated by neither lectin. Trypsinization of the interstitial cells increases their agglutinability by WGA but not by Con A. Nevertheless, A to the interstitial there is no difference in the binding of 1251-C~n cells and to the spermatocytes (Hunt et al., 1977).

VII. Biochemistry of Cell Surface Lectin Receptors The cell-agglutinating and mitogenic effects of lectins have stimulated a considerable amount of research into the nature of the cell surface structures recognized by lectins. Although both glycolipids (Surolia et al., 1975)and glycoproteins may serve as lectin receptors, most studies have focused on the glycoprotein components only. These studies usually begin with purified membrane fractions or with cells whose surface structures have been radioactively labeled by one of the many techniques, such as the lactoperoxidase method, now available for this purpose. Cells or membrane fractions are then solubilized and subjected to affinity chromatography on lectin-Sepharose columns; cell lectin receptors are assumed to be among those glycoproteins that are bound to the column and eluted with the appropriate haptenic saccharide. Receptor glycoproteins prepared in this way are ordinarily characterized by their mobility on sodium dodecyl sulfatepolyacrylamide gels. Further studies are ordinarily required to prove that the lectin-binding glycoproteins isolated in this way actually serve as lectin receptors in vivo on the cell surface as well as in solubilized extracts. For example, the observation that a lectin can protect its receptor from digestion when cells are treated with proteolytic enzymes has been taken as evidence for a receptor function in vivo (Brown, 1973; Taylor et al., 1974). Since lectins bind to the carbohydrate portions of their cell surface receptors, it is clear that receptors for the same lectin on different cell types need not have the same bio-

327

LECTINS

logical function. The same or similar carbohydrate groups may exist on different protein species. Furthermore, the same glycoprotein species may contain receptor sites for more than one lectin. The experimental strategy outlined above has led to the isolation and purification of lectin receptors from several different cell types. Human erythrocytes, for instance, are found to have two cell surface glycoprotein components. One of these, the major glycoprotein or glycophorin, has receptor sites for WGA, PHA, and U . europeus lectin (Marchesi et al., 1972). Recent structural studies on the glycophorin molecule (Winzler, 1969; Tomita and Marchesi, 1975) have rationalized these receptor activities. Glycophorin (MW 31,000) has been shown to be composed of a polypeptide backbone containing 131 amino acid residues, as shown in Fig. 4; a portion of the polypeptide backbone spans the erythrocyte plasma membrane. Sixteen carbohy-

( (

20

t

50

60

70

80

90

too

110

I20

I30

FIG.4. Amino acid sequence of the major human erythrocyte glycoprotein (glycophorin). The sites of attachment for 0-glycosidically linked (squares) and N-glycosidically linked (hexagon) carbohydrates are indicated (Tomita and Marchesi, 1975).

328

JAY C. BROWN AND RICHARD C. HUNT

drate side chains of two different types are attached to the N-terminal portion of the polypeptide and extend outward from the external surface of the cell. Of the 16 side chains 15 are identical; they are O-glycosidically linked to serine or threonine residues, and they have the structure shown in Fig. 5a (Thomas and Winzler, 1969). These side chains are thought to have the receptor sites for PHA (N-acetylgalactosamine, galNAc). The remaining side chain is N-glycosidically linked to asparagine and has the structure shown in Fig. 5b (Kornfeld and Komfeld, 1970). It is thought to contain the receptor sites for WGA [(glcNAc),] and for U . europeus lectin [(glcNAc),]. Since this structure has the carbohydrate groups recognized by Con A (a-linked mannose) and RCA (galactose),it is surprising that glycophorin does not serve as a receptor for these lectins as well (Marchesi et al., 1972; Fukuda and Osawa, 1973). However, Fukuda and Osawa (1973) showed that Con-A and RCA receptor sites can be exposed after removal of the 0-glycosidically linked carbohydrate groups from the glycophorin molecule by alkaline sodium borohydride treatment. This suggests that a portion of the N-glycosidically linked carbohydrate group may be masked on the surface of the glycophorin molecule. The minor human erythrocyte cell surface glycoprotein, called component I11 or component a, has been extensively purified by affinity chromatography on a column of Con A-Sepharose (Findlay, 1974). It has a molecular weight of approximately 100,000, it spans the erythrocyte membrane (Bretscher, 1973), and it has recently been shown to be a major anion channel through the erythrocyte plasma membrane (Cabantchik and Rothstein, 1974a,b; Ho and Guidotti, 1975). Affinity chromatography on lectin-Sepharose columns has also been employed to purify receptors from several other cell types, including human platelets, mouse L cells, D.discoideum, rat brain, and Torpedo (a)

$1,3

0-glycosidic Serine or

or 4

1;1,3

or 4

p-glycosidic Asparagine

Threonine

FIG.5. Structures of the 0-glycosidically linked (a) and N-glycosidically linked (b) carbohydrate groups found in the major human erythrocyte glycoprotein (glycophorin). Adapted from Thomas and Winzler (1969) and from Kornfeld and Kornfeld (1970).

329

LECTINS

californicus electric organ. A summary of the properties of these receptors is given in Table IX. The rat brain lentil lectin receptor and the Torpedo Con-A receptor have been shown to possess the major cell surface acetylcholinesterase activities of these tissues. Similar studies have shown that bovine rhodopsin can serve as a Con-A receptor in isolated rod outer segments (Steinemann and Stryer, 1973) and that the insulin receptor of rat fat cells can also serve as a receptor for WGA and for Con A (Cuatrecasas, 1973b; Cuatrecasas and Tell, 1973). Other studies have resulted in the identification of one major (MW 60,000) and three minor lentil lectin receptors from human KB cells (Butters and Hughes, 1975),lentil lectin and WGA receptors from rat brain synaptic plasma membranes (Gurd and Mahler, 1974), and Con A, WGA, and lentil lectin receptors from L1210 cells (Hourani et al., 1973; Jansons and Burger, 1973). The isolation of lymphocyte cell surface lectin receptors has been undertaken in an attempt to understand the role of these structures in lectin-induced mitogenesis. So far these studies have produced a complicated set of results. All workers have reported multiple lymphocyte receptors for the lectins they have studied. For example, Henkart and Fisher (1975) identified at least five different glycoprotein receptors for Con A on the surface of human peripheral blood lymphocytes. Three of these have molecular weights of 68,000, 53,000, and 43,000, respectively. Mouse B lymphocytes have been shown to have at least TABLE IX PURIFIEDCELL SURFACELECTIN RECEPTORS _

Cell type Human erythrocyte Human erythrocyte

Human platelet Mouse L cells Dictyostelium discoideum Torpedo californicus electric organ Rat brain Rat thymocytes

Receptor for WGA and PHA Con A

_

_

~ ~

Molecular weight 31,000 100,000

Comment Glycophorin Component 111 Anion channel

Lentil lectin

80.000

LIS extraction LIS extraction Aggregation function? Acetylcholinesterase

Con A Lentil lectin

80,000 25,000

Acetylcholinesterase Thy-1 antigen

Con A Con A Con A

100,OOO 100,000 Unknown

Reference Winzler (1969); Tomita and Marchesi (1975) Findlay (1974) Cabantchik and Rothstein (1974a.b); Ho and Guidotti (1975) Nachman et al. (1973) Hunt et al. (1975) Wilhelms et al. (1974) Taylor et al. (1974) Wenthold et al. (1974) Letarte-Muirhead et al.

(1975)

330

JAY C. BROWN AND RICHARD C. HUNT

six different Con-A receptors (Hunt and Marchalonis, 1974), and pig lymphocytes have multiple receptors for both Con A (Allan et al., 1972) and for lentil lectin (Hayman and Crumpton, 1972). LetarteMuirhead et al. (1975) isolated a lentil lectin receptor from rat thymocytes in purified form and showed it to be identical to the serologically defined thy-1 antigen. Choi and Jenson (1974) employed a clever method to identify a Con-A receptor (MW 160,000) on the surface of chick spleen lymphocytes and, recently, R. Emmons and D. C. Benjamin (personal communication) demonstrated that rabbit thymocytes have at least three receptors for Con A (MW 200,000, 70,000, and 40,000) and one for PHA (MW 20,000). None of these studies has as yet determined which, if any, of the known receptors is involved in lectin-induced mitogenesis. Clearly, this must be regarded as a priority for future studies of lymphocyte lectin receptors. A second experimental strategy employed for the isolation and characterization of cell surface lectin receptors has been to solubilize cell surface glycopeptide fragments by treating intact cells with proteolytic enzymes. Solubilized glycopeptides are then separated from each other chemically and assayed for receptor activity by measuring their ability to inhibit lectin-induced cell agglutination. In this way one isolates glycopeptide fragments derived from the parent molecule and not the intact receptor molecule itself. The method has been successfully employed for the isolation and structural characterization of N- and of O-glycosidically linked carbohydrate chains derived from the glycophorin molecule, as shown in Fig. 5 (Thomas and Winzler, 1969; Kornfeld et al., 1971). Recent studies have resulted in the isolation of Con-A- and WGA-binding glycopeptides from Novikoff ascites hepatoma cells (Neri et al., 1974) and from AS-SOD rat ascites hepatoma cells (Smith et al., 1973). Earlier studies of this type have been adequately discussed in previous review articles (Lis and Sharon, 1973b; Nicolson, 1974~).

VIII. Lectin Toxicity In addition to their mitogenic and agglutinating properties, some lectins are found to be extremely toxic to animal cells. It has long been known, for example, that the seeds of the castor bean plant, R . communis, are poisonous. Their toxicity results from a lectin called ricin, and there has been considerable debate concerning whether this toxin and RCAII,which is isolated from castor beans by affinity chromatography (Nicolson and Blaustein, 1972; Kornfeld et al., 1974), are actually the same molecular species. Both ricin and RCA,, bind to cell

LECTINS

331

surface carbohydrates and to Sepharose (Tomita et al., 197213; Kornfeld et al., 1974; but contrast Lugnier and Dirheimer, 1973),both have hemagglutinating activity (Kabat et al., 1947; Nicolson and Blaustein, 1972; Kornfeld et al., 1974; Olsnes et al., 1974a; but contrast Ishiguro et al., 1964), and the two have similar molecular weights (Ishiguro et al., 1964; Nicolson and Blaustein, 1972).Like ricin, RCAIlis extremely toxic, and the toxicity of both RCAII and ricin is blocked by lactose (Kornfeld et al., 1974). RCAII and ricin, however, behave differently on ion-exchange chromatography and in some polyacrylamide gel systems (Lugnier and Dirheimer, 1973). The resolution of these conflicting results appears to be in a small structural difference between ricin and RCAII. Both ricin and RCAIl consist of two polypeptides, an A chain (MW 30,000) and a B chain (MW 35,000), which are linked by a disulfide bond in the intact molecule (Olsnes and Pihl, 1972b; 1973a,b). Whereas the B chain of RCAII has been shown by immunological criteria to be similar to the B chain of ricin, the A chains are clearly different (Olsnes et al., 1974b; Pappenheimer et al., 1974). Ricin and a similar toxic protein called abrin, which can be isolated from the seeds of Abrus pecatorius (McPherson and Rich, 1973; Olsnes et al., 1974a), inhibit protein biosynthesis (Lin et al., 1971, 1972; Onozaki et al., 1972, 1975; Olsnes, 1972; Olsnes and Pihl, 1972a; Montanaro et al., 1973; Sperti et al., 1973; Grollman et al., 1974; Kornfeld et al., 1974; Refsnes et al., 1974). It was originally reported that agarose-coupled RCA had toxic effects similar to those of the free lectin, and this was interpreted as showing that the toxic effect was exerted without the lectin entering the cell (Onozaki et al., 1972). However, this interpretation proved to be incorrect, probably because the lectin was able to detach from agarose beads and enter the cell. In fact, strong evidence now exists showing that ricin and abrin must enter the cell to exert their toxic effects. For example, a lag is observed between the binding of the lectin and the onset of inhibition of protein synthesis (Refsnes et al., 1974; Kornfeld et al., 1974; Nicolson et al., 1975~); this lag is of the order of 30 minutes at 37°C in Ehrlich ascites cells, whereas no lag is observed when cell lysates are employed (Refsnes et al., 1974). Various observations strongly suggest a multistep process consisting of binding to the cell surface, entry into the cell, and inhibition of protein synthesis. Lactose or antisera against the toxin can stop the inhibitory effects only during the initial part of the lag period (Refsnes et al., 1974; Nicolson, 1974b). Protein synthesis is not inhibited in whole reticulocyte cells (which have little endocytotic activity) when they are incubated with

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the toxin, but protein synthesis is inhibited in reticulocyte lysates (Refsnes et al.,.1974; Nicolson et al., 1975c; Sandviget al., 1976). Furthermore, electron microscope studies have shown that the toxin enters cells by endocytosis (Nicolson, 1974b; Gonatas et al., 1975). For the inhibition of protein synthesis to occur in a reticulocyte lysate, the A and B polypeptides of the toxin must be separated (Olsnes et al., 1976). In whole cells the B chain is thought to be involved in binding to the cell surface, since it binds to carbohydrates but is not toxic alone. The A chain, however, does not interact with carbohydrates but is toxic alone in lysates (Olsnes and Pihl, 1973a,b; Olsnes et al., 1976). The inhibition of protein synthesis by ricin and abrin occurs be-

cause these toxins stop the completion of nascent peptide chains already initiated on the ribosome (Olsnes, 1972; Olsnes and Pihl, 1973a,b). In vitro protein synthesis studies have demonstrated that ricin inhibits poly-U-directed incorporation of phenylalanine into polypeptide in an irreversible fashion. Ricin functions by interacting with the ribosome in such a way as to prevent the action of elongation factor 2, the translocase, probably by preventing it from binding to the ribosome surface (Montanaro et al., 1973; Carrasco et al., 1975). Since a single ricin molecule can inactivate many ribosomes, it is likely that ricin acts catalytically. Hybrid ribosomes consisting of control and toxin-treated subunits have been employed to localize the site of ricin action to the 60s ribosomal subunit (Sperti et al., 1973; Onozaki et al., 1975) and, within the 60s subunit, the toxin acts on an 8s complex which can be released from the ribosome by EDTA (Benson et al., 1975).This 8s complex contains the GTPase and ATPase activities associated with elongation factors 1 and 2. The toxin affects the site to which elongation factor 2 binds (Benson et al., 1975; Olsnes et al., 1975; Fernandez-Puentes et al., 1976; Sperti et al., 1976). The toxin may have a hydrolytic effect on either the ribosomal proteins or the rRNA, but polyacrylamide gel electrophoresis has failed to show any gross change in either of these components (Olsnes et al., 1975; Lugnier et al., 1976). In addition to the well-documented action of ricin and abrin on the ribosome, it has been suggested that both toxins may act indirectly to inhibit protein synthesis, by raising intracellular levels of RNase, and thereby disaggregate polysomes (Lin et al., 1972; Grollman et al., 1974). Unfortunately, the mechanism(s) b y which other lectins exert their toxic effects has not been elucidated as clearly or as elegantly as that of ricin and abrin. A few results, however, are worth noting. PHA has been found to impair DNA and RNA synthesis, but how it does so is not known (Caso, 1968; Dent, 1971; Dent and Hillcoat, 1972). Also,

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little is known of how Con A and WGA exert their toxic effects, except that WGA appears to be toxic to mouse L-929 cells only during the S phase of the cell cycle-that is, when cells are actively synthesizing DNA (P. Welch and J. Brown, unpublished observations). Not only are many lectins toxic, but this toxicity is selective. Transformed cells are frequently much more sensitive than normal cells (Shoham et al., 1970; Ozanne and Sambrook, 1971b; Ozanne, 1973). For example, while 3T3 cells were found to be resistant to the toxic effects of Con A at a concentration of 1 mg/ml over a period of 8 hours, 70% of the SV-40-transformed 3T3 cells were killed (Shoham et al., 1970). Thus it is not surprising that lectins have been employed in attempts to inhibit tumor growth in vivo. The injection of hamsters with Con A following injection with transformed cells caused a significant inhibition of tumor growth. This effect was abolished by simultaneous injection of a-MG (Shoham et al., 1970).Tumor inhibition by Con A has also been observed in other systems (Lin and Bruce, 1971; Gericke et al., 1971; Inbar et aZ., 197213; Friberg et al., 1972; Ralph and Nakoinz, 1973). Unfortunately, such high concentrations of Con A usually must be employed for tumor suppression that some control animals die from its toxic effects. Other lectins that have been employed in experiments on tumor cell growth suppression include Robinia pseudoacacia lectin (Aubery et al., 1972), PHA (Datta et al., 1969; Robinson and Mekori, 1972; Ralph and Nakoinz, 1973),RCA (Nicolson and Blaustein, 1972), and ricin and abrin (Lin et al., 1970). None of these experiments has led to any therapeutic use of lectins.

IX. The Biological Role of Lectins A fascinating aspect of lectin studies, and one that has received comparatively little attention, is the biological functions lectins may perform in the organisms from which they are isolated. What does Con A do for the jack bean? A lack of experimental results in this field has not inhibited speculation, and many possible functions have been suggested. These include a protective effect, because lectins have a superficial similarity to antibodies, an involvement in the selective binding of rhizobia during the initiation of root nodules in legumes, and facilitation of sugar transport within the plant or, alternatively, immobilization of sugars within a particular part of the plant (Boyd et al., 1958; Boyd, 1963). Only the first two of these suggestions are supported by existing experimental evidence. Although lectins have some properties in common with antibodies, their comparatively narrow range of specificities

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makes a precise correlation unlikely. Nevertheless, it is possible to see how the presence of a toxin or a precipitating agent could protect a plant against attack from a variety of organisms from viruses to herbivores. It has been long known, for example, that some plants are toxic to animals, and a protective function of PHA against beetle infestation of Phaseolus vulgaris has been demonstrated. Bruchid beetles are killed by a diet of black beans (P. uulgaris),but not by cowpeas (Vigna unguiculata) which are agglutinin-free (Janzen et al., 1976). These studies are of potential significance to agriculturalists, since the selection of a bean crop free of the agglutinin so as to reduce processing costs is likely to produce a crop with no beetle resistance. The elegant experiments of Shannon and his colleagues suggest that some lectins may protect plants against the spread of viral infections. It has been observed that certain plant viruses contain surface glycoproteins and that their presence correlates with the manner in which the virus is transmitted from plant to plant. For instance, barley stripe mosaic virus (BSMV) and cowpea mosaic virus are both transmitted via the seeds of the host plant, and both have surfaceassociated glycoproteins, while three non-seed-transmitted viruses studied (including a virus closely related to BSMV) do not contain carbohydrate (Partridge et al., 1974). Thus lectins (which are usually concentrated within the seed) may confer on the plant a selective advantage by protecting it against seed-transmitted viruses. In fact, barley has been found to contain a lectin which is specific for nonacetylated amino sugars. This lectin binds to and precipitates BSMV in uitro. Furthermore, virus treated in vitro with the lectin is noninfectious (L. Shannon, personal communication). It would be most interesting to determine the degree of resistance to BSMV infection of barley mutants that lack or produce low concentrations of the lectin. Although lectins are frequently concentrated in seeds, they can also be secreted from the roots of some plants, particularly legumes. It has therefore been suggested that a lectin may function in the binding of symbiotic rhizobia to form root nodules (Hamblin and Kent, 1973) and, indeed, some specificity seems to exist. Soybean lectin combines, for example, with most strains of Rhizobium japonicum, the soybean-nodulating bacterium, but not with strains of Rhizobium that fail to nodulate soybeans (Bohlool and Schmidt, 1974).Secreted lectin interacts with the bacterial 0-antigen-containing lipopolysaccharide and, when tested with a variety of lipopolysaccharides, lectins from a range of legumes have been found to bind only the lipopolysaccharide of their symboint Rhinobium (Wolpert and Albersheim, 1976). Most of the lectins that have been extensively studied come from

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plants, but proteins with similar properties are known in other organisms where their functions have aroused considerable interest. For example, surrounding the eggs of the African clawed toad Xenopus laevis is a jelly coat composed of glycoprotein (Yurewicz et al., 1975). On fertilization, the cortical granules of the egg fuse with the plasma membrane and release their contents. The granule contents, which have the properties of a lectin, bind to the components of the jelly coat to form the fertilization envelope. The precipitation of the fertilization envelope probably serves as a block to polyspermy, fertilization of the egg by more than one sperm cell (Wyrick et al., 1974). A lectinlike protein has also been isolated from rabbit liver. This protein agglutinates erythrocytes and is inhibited by several monosaccharides including N-acetyl-D-glucosamine. It has been named mammalian hepatic lectin, is a constituent of the plasma membrane of hepatocytes, and has been shown to be involved in the removal of desialylated plasma proteins from the blood (Stockert et al., 1974; Lunney and Ashwell, 1976). This lectin is also mitogenic for lymphocytes. There is a series of interesting lectinlike proteins which have been isolated from cellular slime molds (Rosen et al., 1973, 1974, 1975; Simpson et al., 1974).These proteins, called discoidin I and discoidin 11, are located on the surface of cohesive but not vegetative cells. It is therefore thought that they may function in intercellular adhesion (Chang et d., 1975; Reithennan et d., 1975; Frazier et d., 1975). Finally, Teichberg et al. (1975) identified a saccharide-binding protein called electrolectin in the electric organ of Electrophorus electricus. This lectin agglutinates trypsinized rabbit erythrocytes, and agglutination is inhibited by compounds containing P-D-galactose groups but not by a variety of other saccharides. A similar lectin activity has been identified in chick embryonic muscle cells (Den et al., 1976) and in the L6 line of rat myoblasts (Gartner and Podleski, 1975). This galactose-specific lectin is found on the cell surface in both cell types, but there are conflicting results regarding whether it is involved in the specific fusion of myoblast cells to form myotubes; 15 mM thiodigalactoside inhibits the fusion of L6 rat myoblasts, but not that of chick embryonic myoblasts. It is clear from the scarcity of our knowledge about lectin function that considerable experimentation and perhaps some more good ideas will be required before we will have a reasonable understanding of this subject. It is ironic in fact that, although lectins have been successfully employed to probe a wide variety of cell surface structures and activities, we still do not know what their normal, in vivo functions are.

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