Cell surface receptors for wheat germ agglutinin and limulin in baby hamster kidney cells and ricin resistant variants

MGmoires originaux

BIOCItlM1E, 1981, 63, 169-175.

Cell surthee receptors for wheat germ agglutinin and limulin in baby hamster kidney cells and ricin resistant variants.

(Regu le 30-7-1980, accept~ aprds r6vision le 8-12-1980).

* Centre de Biophysique Mol~culaire, Centre National de la Recherche Scientifique, 45045 Orleans Cedex. France. ** National Institute for Medical Research, Mill Hill, London N W 7 1 A A , U.K.

Rdsumd.

Summary.

Les glycocon]ugu~s membranaires des cellules de rein d'hamster nouveau-n~ ( B H K 21 C13) et des quatre lign~es r~sistantes gtla ricine (toxine des graines de Ricinus communis) ont dtd ~tudi& en utilisant la ricine marquee ?t l'iode 125, et des lectines substitutes par la fluoresc~ine : la limuline (lectine du Limule, Limulus polyphemus) qui est affine du dioside NeuAc-~2-3(6)-GaINAc, la lectine du germe de bl~ ( W G A , Triticum vulgare) qui [ixe les oligosides contenant un r~sidu de N e u A c ou un r~sidu de G I c N A c en position terminale ou subterminale, et la lectine du germe de bl~ succinyl~e qui reconna~t Ies r~sidus de G t c N A c mais ne fixe pas ceux de NeuAc. Des diff&ences importantes ont pu Otre mises en 6vidence en ce qui concerne le nombre d'osides accessibles ~ la surface des cellules de la lign~e 21 C 13 et des lign~es r#sistantes ?~ la ricine. Les rdsultats sont discutds sur la base des structures glucidiques connues et de la spdcificit~ des lectines et de la ricine. Cette ~tude a permis de mettre en 6vidence des diffOrences d'une lignde ~t l'autre, soit au niveau des glycopeptides ?t asparagine soit au niveau des glycopeptides ?t hydroxy amino-acides.

The cell surface glycoconjugates of Baby Hamster Kidney cells and o[ four ricin resistant variants were investigated by the use o[ l~U-substituted ricin (Ricinus communis toxin) which binds galactose residues, and by the use of fluorescein labelled lectins which bind N-acetylneuraminic acid and~or N-acetylglucosamine : Limulin (Limulus polyphemus agglutinin), wheat germ agglutinin (Triticum vulgare agglutinin) and succinylated wheat germ agglutinin. Striking differences in the number o[ lectin and~or ricin receptors were found between the cell surface o[ wild type cells and that of ricin resistant variants. The results are discussed on the basis of the main glycopeptide structure, and of the specificity of the sugar binding proteins used. The ricin resistance o[ variant cells is concommitant to modifications o[ the concentration of certain glycoconjugate structures which are accessible to the sugar binding proteins. Depending on the variants, N-asparaginyt glycopeptide types and~or O-glycosidic glycopeptide types are affected.

Angble OBRENOVITCH * Claude SENI~ * Annie Claude ROCHE *, Michel MONSIGNY * o, Peter VISHER ** and R. COLIN HUGHES **

Key-words: lectins / membrane glycoconjugates / cell cultures / cell surface, sialic acids.

Mots-cl~s : lectines / glycoconjugu~s membranaires / culture cellulaire / aeides sialiques.

Introduction. Cell surface glycoconjugates are involved in many biological processes [1, 2]. They are also involved in the binding and uptake of various lec-

To whom all correspondence should be addressed.

tins and toxins such as ricin which block cytoplasmic and mitochondrial protein biosynthesis. Various resistant variants were selected by growth of BHK cells in the presence of ricin [3, 4]. Several of the resistant variants are grossly deficient in the ricin binding peripheral sequences of complex GIcNAc- Asn glycans [1, 4] and lack specific glycosyltransferases responsibles for their

170

A . Obrenovitch and coll.

assembly [3, 5]. Other variants exhibit elevated sialyl transferase activity [5] which also leads to altered glycoprotein synthesis a n d a reduced comp l e m e n t of celle surface ricin receptor. I n order to further study the cell surface glycoconjugates of B H K cells a n d of their ricin resistant variants, we d e t e r m i n e d the n u m b e r of accessible cell surface receptors to wheat germ agglutinin, succinylated wheat germ agglutinin, l i m u l i n and ricin (Ricinus c o m m u n i s toxin). W h e a t germ agglutinin binds N-acetylglucosamine a n d glycoconjugates bearing accessible N - a c e t y l g l u c o s a m i n e [6, 7] a n d also binds N-acetyl n e u r a m i n i c acid and N-acetyl n e u r a m i n y l glycoconjugates [8-11]. Succinylated wheat germ agglutinin only binds N-acetylglucosamine a n d derivatives [12]. L i m u l i n binds N - a c e t y l n e u r a m i n y l - N - a c e t y l g a l a c t o s a m i n e units [13-15]. R i c i n u s c o m m u n i s toxin specifically binds galactose or N-acetyl galactosamine [16]. T h e use of limulin, wheat germ agglutinin a n d succinylated wheat germ agglutinin allows us to estimate the total n u m b e r of N-acetyl n e u r a m i n i c acid a n d N - a c e t y l n e u r a m i n y l - N-acetylgalactosamine units accessible o n the cell surface, a n d to correlate them with the resistance of variants to the b i n d i n g and the cytotoxicity of ricin.

Materials and Methods. 1) Cells. Baby Hamster Kidney fibroblasts (BHK 21 C 13) wild type and their ricin resistant variants (Ric R) isolated from mutagenized cells [41 were grown at 37°C in Glasgow modified essential medium supplemented with 10 per cent (v/v) foetal bovine serum (Rehatuin F-3, Armor, IBF Reactifs Pha~-mindustrie, Villeneuve- La Garenne, France), tryptose phosphate broth, NaHCO~ (2 g/l), and gentamycin (50 .~g/,mt). Cells were removed from culture vessels by incubation for 1-2 min at 37°C in the presence of 0.02 per cent (w/v) disodium ethylenediamine tetraacetate (EDTA) in phosphate buffered saline, pH 7.4. 2) Lectins. Wheat germ agglutinin (M. W. 36,000), prepared as previously described [17] and succinylated wheat germ agglutinin [12] were purchased from IBF - Reactifs Pharmindustrie, Villeneuve-La Garenne, France. Limulin (Limulus polyphemus agglutinin) (M.W. 340,000) was purified by affinity chromatography as previously described [13]. Ricinus communis toxin (Ricin) (M.W 60,000) was purehassed from Miles Laboratories, Stoke Pages, U.K.. Fluorescein substituted lectins were purchassed from IBF-Reactifs or prepared as previously described [18]; fluorescein content was estimated spectrophotometrically.

BIOCHIMIE, 1981, 63, n ° 3.

a) Fluorescent lectins [18] sub-confluent cells were rea) Fluorescent lectins [18] Sub-confluent cells were removed from culture flasks by incubation for 2 rain at 37°C in the presence of EDTA in phosphate buffered saline, pH 7.4. Cells were suspended three times in phosphate buffered saline and spun down (1000 x g) for experiments with fluoresceinyl-wheat germ agglutinin and fluoresceinyl-succinylated wheat germ agglutinin. Cells were washed three times in 0.05 M triethanolamine, 0.1 M NaC1, 0.01 M CaCh buffer, pH 8.5 for experiments with fluoresceinyl limulin. Incubation of cells (2 × 106 cells/ml) with fluoresceinyl lectins was carried out at different concentrations of fluoresceinyl lectins varying from 0.5 to 100 M/ml for 1 h at 4°C. At the end of the incubation, cells were centrifuged (1000 × g ; 5 min) and the concentration of free fluoresceinyl lectins in the supernatent was estimated by fluorescence measurements. Labeled cells were washed three times at room temperature with phosphate buffered saline or with triethanolamine buffer. Specifically bound lectin was released by incubating the cells for 1 h at 4°C with 1 ml of N-acety!glucosamine (0.3 M) in experiments with wheat germ agglutinin and succinylated wheat germ agglutinin, or with 1 ml of 0.01 M EDTA, 0.1 M NaC1 buffer, pH 8.5 in experiments with limulin. Concentration of fluoresceinyl lectins released from the cells was calculated from fluorescence emission intensity measurements; standard curves were determined under the same experimental conditions with a large range of fluoresceinyl lectin concentrations. Fluorescence measurements were performed using a high resolution differential spectrofluorimeter Fica MK II (Fica, France) equipped with a Houston 2000 recorder. Fluorescein derivatives wee excited at 495 nm and their emission was detected a 520 nm. All the measurements were carried out at 25°C. The base line was adjusted at 650 nm. The apparent number of binding sites per cell was evaluated from a Scatchard plot [18]. b) Radioactive lectin binding. Ricin was labeled with 125I by chloramine T or lactoperoxidase-catalyzed iodination in the presence of 10 mM lactose and purified by affinity chromatography [4, 20]. Binding to cells suspended with EDTA-phosphate pH 7.4 was carried out in small tubes previously soaked in 1 per cent (w/v) bovine serum albumin in phosphate buffered saline to eliminate non-specific attachment of cells to the tube walls. Cells (2-4 x 106/ml) and lzSI-lectin (0-200 Ixg, 1000 cpm/Ixg) were mixed (1 ml) and kept at room temperature for 60 min. An other series of tubes contained cells and a25I_ lectin plus 10 mM lactose. The cells were recovered and washed by centrifugation and the proportion of radioactivity bound to the cells estimated in a Packard "cspectrometer. Results. I n the c o n c e n t r a t i o n range used for wheat germ agglutinin a n d succ!nylated wheat germ agglutinin, the Scatchard plots (figure 1) were biphasic re-

Binding of lectins to cell surface glycoconjugates. gardless of the n a t u r e of the cells [12, 18]. Striking differences were o b s e r v e d b e t w e e n the n u m ber of binding sites of w h e a t g e r m agglutinin and of succinylated w h e a t germ agglutinin (table I). A

0

xlO "2 ~

B

10 15 jJgl2 x l O 6 c e l l s

2C) b

FIG. 1. - - Scatchard plot.s of the specific binding o] fluoresceinyl derivatives oJ (A) wheat germ agglutinin and of (B) succinylated wheat germ agglutinin to • • wild

171

tinin a n d of succinylated w h e a t g e r m agglutinin : W h e a t g e r m agglutinin binds to g l y c o c o n j u g a t e s containing either N - a c e t y l n e u r a m i n i c acid or N a c e t y l g l u c o s a m i n e [11], a n d succinylated wheat g e r m agglutinin only binds to g l y c o c o n j u g a t e s containing N - a c e t y l g l u c o s a m i n e [12]. T h e r e f o r e the difference in n u m b e r s of binding sites for w h e a t g e r m agglutinin a n d for succinylated w h e a t g e r m agglutinin refers to the n u m b e r of accessible N - a c e t y l n e u r a m i n i c acid surface r e c e p t o r s (table II). T h e n u m b e r of binding sites for wheat agglutinin on wild type cells and R i c R 17, R i c R 19 a n d R i c 21 cells are very similar (table I). H o w ever, the n u m b e r of binding sites for w h e a t g e r m agglutinin on R i c R 14 cells is at least twice as high as t h a t on wild type cells (table I). T h e n u m b e r of binding sites for succinylated w h e a t germ agglutinin on R i c R 19 and R i c R 21 cells is only half the n u m b e r of those on wild type cells (table I), while on R i c R 14 ce!ls this n u m b e r is 2.5 times higher.

type BHK cells, I1--11 Ric R 14 cells, rq__t2 Ric R 19 cells, b : amount of fluoresceinyl lectin bound to 2 × 106 cells and specifically released in the presence of 0.3 M N-acetylglucosamine. f : concentration of free fluoresceinyl lectin. TABI,E I.

Total number o[ binding sites oJ wheat germ agglutinin and succinylated wheat germ agglutinin on B H K cells.

0.25

lectins cells

wild type Ric R 14 Ric R 17 Ric R 19 Ric R 21

WGA nxl0 -~ sites/cell 62 146 71 51 66

-WGA nxl0-'~ sites/cell 8 20 5 3,5 4

The total number of binding sites per cell (n) were evaluated by Scatchard plot following the conditions described in Materials and Methods. Molecular weight of wheat germ agglutinin (WGA) and of succinylated wheat germ agglutinin (suc-WGA) was taken as 3.6 × 104 for all calculations. Data are the mean values of experiments in triplicates. T h e n u m b e r of binding sites for w h e a t germ agglutinin is m u c h higher t h a n that of succinylated w h e a t g e r m agglutinin. These differences come f r o m the binding specificities of w h e a t germ agglu-

BIOCHIMIE, 1981, 63, n ° 3.

0

5

10

15

pg 2/x IOec el Is

FIG. 2. - - Scatchard plots o/ the specific binding o] tluoresceinyl derivative of limulin to A----• wild type BHK cells ; I1--11 Ric R 14 cells ; ©--© Ric R 17 cells ; n__D R i c R 19 cells ; V -V R i c R 2 1 cells ; b : amount of fluoresceinyl limulin bound to 2 × 106 cells and specifically released in the presence of 0.01 M EDTA. f : concentration of free fluoresceinyl limulin. I n the c o n c e n t r a t i o n range used, the binding of limulin to B H K cells and to ricin resistant m u t a n t s was f o u n d to be c o o p e r a t i v e (figure 2). T h e origin of positive c o o p e r a t i v i t y is n o t yet understood, but, m a y be related either to the d y n a m i c p r o p e r t i e s of m e m b r a n e glycoconjugates [21, 22], or to a dissociation p h e n o m e n o n of limulin at low concentration. L i m u l i n is a d o d e c a m e r i c p r o t e i n at

172

A. Obrenovitch and coll.

pH 8.5 and at concentrations down to 0.2 mg/ml, but no data are available on the oligomeric state of limulin at the low concentrations (0.1 to 100 p.g/ml) used in the binding experiments. If limulin at low concentration undergoes dissociation, the binding of limulin to its specific ligand should induce a reassociation [21], as was recently shown with wheat germ agglutinin at acid pH [23] and with Con A [24]; consequently, a cooperative Scatchard plot would be expected. The number of limulin binding sites on wild type BHK cells (7 X i0n/ceU table II) is similar to the approximate numbers of additional ricin (4-6 X I0n/cell) and peanut agglutinin (6-9 X 10n/cell) binding sites exposed on these cells by neuraminidase treatment as calculated from Rosen and Hughes [25]. This result is consistent with the binding of all three lectins to O-glycans (fig. 3b) since peanut lectin is specific for the GaI-~3-1,3GalNAc sequence and binds poorly to Gal-~3-1AGIcNAc [31]. In Ric R 14 cells, there is a two-fold increase in limulin binding sites. The Ric R 19 cells binds limulin poorly, indicating a reduction in accessible surface NeuAc-~-2-6-GalNAc units of the O-Ser (Thr)-glycopeptides. The other two mutant cell lines examined, Ric R 17 and Ric R 21 appear to be very similar to wild type BHK cells in the binding of limulin and of wheat germ agglutinin. The number of ricin binding sites on Ric R 14 and Ric R 21 cells is about ten times lower on wild type cells ; the number on Ric R 19 cells is twice as high and the number on Ric R 17 cells is slightly lower than on wild type cells. These results are closely similar to those reported earlier for cells grown in monolayer cultures [4]. Discussion. The results of binding experiments using wheat germ agglutinin, succinylated wheat germ agglutinin, and limulin are discussed : (i) on the basis of the ricin resistance of the variants and of the ricin sensitivity of wild type BHK cells ; (ii) on the basis of the two main types of glycopeptides containing N-acetyl neuraminic acid (fig. 3); and (iii) on the basis of the specificities of the lectins. Wheat germ agglutinin binds N-acetyl neuraminic acid [8-11] and N-acetyl glucosamine in a non-reducing terminal or subterminat position [26, 27]. Succinylated wheat germ agglutinin does not

BIOCHIMIE, 1981, 63, n ° 3.

bind N-acetyl neuraminic acid but binds N-acetylglucosamine in a non-reducing terminal or subterminal position [12]. Limulin binds N-acetyl neuraminic acid-N-acetylgalactosamine units but does not bind or only slightly binds N-acetyl neuraminic acid-galactose units [13-15]. Ricin binds to both the main types of glycans shown in figure 3. However, the binding to O-glycans (fig. 3b) is greatly inhibited by the presence of sialic acid residues while binding to N-glycans (fig. 3a) is less affected [16, 28]. The ricin-resistant cell line Ric R 14 lacks [5] a key enzyme in the assembly of N-gtycans of the type shown in figure 3a. In the absence of this specific N-acetylglucosaminyl transferase (fig. 3a) the carbohydrate chains are terminated by mannose residues and attachment of the peripheral sequences NeuAc-Gal-GlcNAc is prevented. The significance of the profound loss [5] of sialyl transferase activity responsible for the final steps in N-glycans assembly (fig. 3a) is presently unclear since in the absence of the specific N-acetylglucosaminyl transferase activity it is unlikely that an appropriate substrate for the sialyl transferase accumulates in Ric R 14 cells. The demonstrated enzymic lesions in Ric R 14 cells satisfactorily explain the loss of ricin binding sites on the surface of these cells (table II) and also is consistent with the increased binding of succinylated-WGA by these cells (table I). Presumably, the premature termination of complex N-glycans (fig. 3a) allows an increased accessibility of the N,N'-diacetylchitobiose unit of the core region of these chains expressed on cell surface glycoproteins, particularly since addition of fucose to the core region (fig. 3a) requires the prior action of the specific N-acetylglucosaminyl transferase [321. In view of the enzyme defects in Ric R 14 cells, it is surprising that our present results show a greatly increased number of total sialic acid residues on the surface of these cells (table II). Presumably, this increase reflects either an increased sialylation of O-glycans of the general structure shown in figure 3b and/or an increased synthesis of these chains. An alternative explanation of an increased expression of gangliosides on the cell surface of Ric R 14 cells has been ruled out by direct glycolipid analysis (A. Gargas and R. C. Hughes, unpublished results). An increased sialylation of the O-glycans of cell surface glycoproteins would contribute to the decreased affinity of ricin for the resistant cells from the model binding experiments of Baenziger and Fiete [16]. An increase in the surface expression of NeuAc-GalNAc

Binding of lectins to cell surface glycoconjugates. sequences on Ric R 14 cells is indeed supported by a two-fold increase in limulin binding to these cells (table II). However, there is a considerab!e excess c o m p a r e d to wild type cells of surface siaiic acid in addition to the increased surface content

173

complex than the simple tetrasaccharide structure shown in figure 3b (D. Stojanovic, P. Vischer and R. C. Hughes, unpublished results), is substantially increased in the Ric R 14 mutant cell line. A n interesting analogy is the increased content of

TABLE II. Lectins binding sites on wild type and ricin resistant B H K ceils. Lectins Receptors

Cells

wild type Ric R 14 Ric R 17 Ric R 19 Ric R 21

(WGA)- (suc-WG)A (NeuAc) nx 10 ~ sites/cell

Limulin (NeuAc-GalNAc) nx 10 ~ sites/cell

Ricinus eomrnunis (Gal or GalNAc) nx 10-'~ sites cell (1)

54 126 66 47 62

7 15 8,5 2 8

4,3 0,4 2,9 7,9 0,3

Binding studies were performed under the same conditions as those described under table I for wheat germ agglutinin and in Materials and Methods. The molecular weights of limulin and of Ricinus communis toxin were assumed to be 340,000 and 60,000, respectively for calculation of numbers of binding sites. Footnote 1 : without neuraminidase treatment of cells. This value is increased to 8-10 × 106 sites/cell after neuraminidase treatment [2,5].

(Neukc-ct6 ~ - O a l -2l ~3-

1-4- GlCNAC-B- 1-2- Man-a- 1- 3 ~

[ j M~n- !B-I-4-GIcNAc-IB-1-4-G1cNAc-~-ksn

NeuAc-a-2-3[8)-Gal-B-1-4-GIcNkc-B-1-2-Man-~-1-6 /

1

l

(Fuc-°t-l-6)O

I

r,r 1

R1 R1 = (NeuAc-~-2-3(6)-Oal-~-l-4-GlcNkc-~-l)o

or I

Lactos~mlnyl type o~ asparaginyl glycopeptide.

[NeuAc-a-2-3(6)-Gal-B-1-3)O or I

®

|

! I [T h r) NeuAc-a-2- 3 (6) - GalNAc-O-Ser

I FIG. 3. - -

O-serine [or threonine] glycopeptide Basic structure oJ sialoglycopeptidex : a) L a c t o s a m i n y l type of O l c N A c - A s n gly-

copeptides ; b) O-Serine (or Threonine) glycopeptides. Tbe complete loss of a specific N-acetyl-

glucosaminyl transferase and the partial (~ 80 per cent) loss of sialyl transferase activities in ricin resistant cell line Ric R 14 affecting synthesis of the sequences underlined are discussed elsewhere [5]. of N e u A c - G a l N A c residues in these cells as indicated by the binding data obtained with W G A and succinylated W G A (table II). Evident!y, the sialic acid content of O-glycans of B H K cell surface proteins, which appear to be longer and more

BIOCH1MIE, 1981, 63, n ° 3.

sialylated carbohydrate chains in the band 3 glycoprotein of h u m a n erythrocyte m e m b r a n e in rare variants lacking the normal sialylated giycoprotein, glycophorin [30]. Evidently, a high content of sialic acid is maintained on the cell surface despite

174

.4. O b r e n o v i t c h

genetic events that prevent synthesis of the normally most heavily sialylated components of the cell surface membrane. Although the total number of surface N-acetyl neuraminic acids on Ric R 19 cells and wild type B H K cells are similar (table I), the number of accessible N-acetyl neuraminyl-N-acetyl galactosamine units (fig. 3b) is much lower on Ric R 19 cells than on wild type cells (table II). These results suggest that there is no direct correlation between the increased activity of sialyltransferase activity [5] in Ric R 19 cells and the lectin accessible sialoglycoconjugates. However, the activity of sialyltransferase was determined [5] using glycoconjugates having GlcNAc-Asn glycopeptides and is shown to be different to sialyltransferase activities responsible for the assembly of O-Ser (Thr)-glycopeptides. Accordingly, we may suggest that on Ric R 19 cells ricin binds to glycoproteins bearing O-Ser(Thr)-glycopeptides which are not sialylated as indicated by the decreased numbers of limulin binding sites and these glycoproteins are not able to internalize the bound ricin to affect cytoplasmic protein synthesis; the glycoproteins bearing O-Ser(Thr)-glycopeptides which are not volved in the internalization of ricin in wild type cells either are fully sialylated or lack the terminal carbohydrate chain in Ric R 19 cells. Finally, the significantly low numbers of succinylated wheat germ agglutinin receptors on Ric R 17, Ric R 19 and Ric R 21 cells (table I) suggest that the glycoconjugates bearing accessible N-acetylglucosamine are less numerous than on wild type cells. This result, together with a normal or slightly lower amount of total N-acetyl neuraminic acid residues as indicated by W G A binding (table I), suggests that the number of incomplete GlcNAc-Asn glycans is lower than in wild type cells, thus contributing to the reduced binding of ricin to the Ric R 17 and Ric R 21 cell lines. In summary, the joint use of limulin, wheat germ agglutinin and succinylated wheat germ agglutinin shows striking differences in the glycoprotein structures at the cell surface of ricin-resistant variants: an increase of accessible chitobiosyl sequences and of N e u A c - G a l N A c units in Ric R 14 cells and a decreased sialylation of O-glycan units in Ric R 19 cells. Confirmation of these conclusions by direct structural analysis of the surface glycoproteins of B H K ceils and ricin-resistant mutants in conjugation with the comparative activities of specific transferases in these cells [5] will contribute to knowledge of the control of B I O C H I M I E , 1981, 63, n o 3.

a n d coll.

O- and N-glycan assembly and also to the exact specificity of lectin binding to complex carbohydrate chains expressed on cell surfaces. Acknowledgements. We thank Mrs. Mich~le Mitterand [or valuable technical assistance. This work was supported by grants ,4CC 77.0252 and ,4CC : 78 71086 from Ddl~gation gdndrale d la Recherche Scienti]ique et Technique. ,4.0. and A.C.R. are <.

REFERENCES.

1. Hughes, R. C. (1976) in <> (Butterworth, London and Boston). 2. Monsigny, M., Roche, A. C. ~ Kieda, C. (1978) in <~Structure and fonction o] Biological Membranes. Molecular Aspects >>. Centre de Biophysique Mo-

3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

16culaire, ed. pp. 161-234. Commision of the European Communities, Brussels. Meager, A., Ungkitchanukit, A., Nairn, R. & Hughes, R. C. (1975) Nature, 257, 137-139. Meager, A., Ungkitchanukit, A. & "Hughes, R. C. (1976) Biochem. J., 154, 113-124. Vischer, P. & Hughes, R. C. (1979) in <>(R. Schauer et al. eds.) pp. 283-284, Georg Thieml, Stuttgart. Burger, M. M. & Goldberg, A. R. (1967) Proc. Nalt. Acad. Sci. USA, 57, 359-366. Allen, A. K., Neuberger, A. ~ Sharon, N. (1973) Biochem, J., 131, 155-162. Greenaway, P~ J. & LeVine, D. (1973) Nature, N e w Biology, 241, 191-192. Bhavanandan, V. P. & Katlic, A. W. (1979) J. Biol. Chem., 254, 4000-4008. Peters, B. P., EbJsu, S. Goldstein I. J. & Flashner, M. (1979)Biochemistry, 18, 5505-5511. Monsigny, M., Roche, A. C., Sen6, C., Maget-Dana, R. ~ Delmotte, F. (1980) Eur. J. Biochem., 104, 147-153. Monsigny, M., Sen6, C., Obr~novitch, A., Roche, A. C., Delmotte, F. s: Boschetti, E. (1979) Eur. J. Biochem., oo.8,39-45. Roche, A. C., Schauer, R. a Monsigny, M. (1975) FEBS Letters, 57, 245-249. Roche, A. C. & Monsigny, M. (1979) in <

E. Cohen, ed. pp. 603-616, Alan R. Liss, New York. 15. Maget-Dana, R., Roche, A. C. ~: Monsigny, M. (1979) in <> (Limulidae) E. Cohen, ed. pp. 567578, Alan R. Liss, New York. 16. Baenziger, J. U. ~= Fiete, D. (1979) J. Biol. Chem., 254, 9795-9799.

Binding ol lectins to cell sur[ace glycoconiugates. 17. Bouchard, P., Moroux, Y., Tixier, R., Privat, J. P. & Monsigny, M. (1976) Biochimie, 58, 1247-1253. 18. Monsigny, M., Sen6, C. & Obr6novitch, A. (1979) Eur. J. Biochem., 96, 295-300. 19. Scatchard, G. (1949) Ann. N. Y. Acad. Sci., 51, 660672. 20. Butters, T. D. & Hughes, R. C. (1978) Carbohydr. Res., 61, 159-168. 21. Cuatrecasas, P. (1973) Biochemistry, 12, 1312-1322. 22. Cann, J. R. (1978) Met. Enzymol., 48, 299-307. 23. Roche, A. C. & Monsigny, M. (1979) in <~Glycocon]ugates, (Schauer, R. et al., eds.) pp. 130-131 (Georg Thieme, Stuttgart). 24. Gordon, J. A. ,~ Young, R. K. (1979) J. Biol. Chem., 254, 1932-1937. 25. Rosen, S. W. ~ Hughes, R. C. (1977) Biochemistry, 16, 4908-4914.

BIOCH1MIE, 1981, 63, n ° 3.

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26. Goldstein, I. J., Hamarstr6m, S. & Sundblad, G. (1975) Biochim. Biophys. Acta, 405, 53-61. 27. Monsigny, M. (1979) in ~ A/finity Chromatography and molecular interactions ~ (I. M. Egly, ed.), 86, 207-216, INSERM, Paris. 28. Debray, H., Decout, D., Strecker, G., Montreuil, J. ,~ Monsigny, M. (1980) Protides of Biological Fluides. 27th Colloquium. Peeters, H. ed., pp. 451454. 29. Pena, S. D. J., Mills, G. s: Hughes, R. C. (1979) Biochim. Biophys. Acta, 550, 100-109. 30. Tanner, M. J. A. (1978) Current Topics in Membranes and Transport (F. Bronner & A. Kleinzeller, ed.), 11, 279-323. (Acad. Press, New York). 31. Pereira, M. E. A., Kabat, E. A., Lotan, R. e Sharon, N. (1976) Carbohyd. Res., 51, 107-118. 32. Wilson, J. R., Williams, D. s, Schachter, H. (1976) Biochim. Biophys. Acta, 72, 909-915.