Nature of the receptor sites for galactosyl-specific lectins on human lymphocytes

Experimental Cell Research 152 (1984) 486-492

Nature of the Receptor Sites for Galactosyl-specific on Human Lymphocytes ELISABETH

‘U 180 INSERM,

TURPIN,’ YVES GOUSSAULT,’ and NATHAN SHARON’

Facultk

HALINA

de Mkdecine, 75006 Paris, France, and ‘Biophysics Weizmann Institute, Rehovot, Israel

Lectins

LIS*

Department,

The nature of the receptors for four lectins specific for D-&tCtOSyl residues was examined in human lymphocytes. The cells were fixed with formaldehyde to avoid subsequent cell lysis, treated with pronase, sialidase and organic solvents, and the binding of the lectins to the treated cells measured. The results show that the bulk of the receptors for peanut agghrtinin (PNA) and ricin (RCA 60) are glycoproteins, whereas those for Ricinus communis agglutinin (RCA 120) and soybean agglutinin (SBA) are distributed nearly equally between membrane glycoproteins and glycolipids.

Human lymphocytes bind a variety of lectins, but little is known about the molecular nature of the lectin receptors on these cells. In the few cases that have been investigated, the receptors were shown to be glycoproteins [l, 21. Recently, neutral and acid glycolipids were also shown to act as receptors for lectins [3, 41, but the relative contribution of the two types of glycoconjugate to the binding to human lymphocytes is not known. To define the relative contribution of glycoproteins and glycolipids in the binding of lectins to human lymphocytes, we used four different lectins specific for D-galactosyl residues: soybean agglutinin (SBA), peanut agglutinin (PNA), Ricinus communis agglutinin (RCA 120) and ricin (RCA 60). To avoid cell lysis upon treatment with organic solvents, the cells were prefixed with formaldehyde, as described by Mehta [5]. Binding studies have shown that the receptors for PNA and RCA 60 are essentially glycoproteins, whereas those for SBA and RCA 120 included both glycoproteins and glycolipids.

MATERIALS

AND METHODS

Cells Human peripheral lymphocytes were purified from huffy coats by centrifugation on a FicollHypaque gradient according to Boyum [61. Contaminating red cells were eliminated by a 5 min treatment with 0.01 M Ttis-HCI, pH 7.4, containing 0.155 M NH&I, followed by centrifugation at 700 g. Cell viability was over 95% when tested by Trypan blue exclusion. Copyright @ 1984 by Academic Press, Inc. All rights of reproduction in any form rescned 0014427/84

$03.00

Lectin receptors on lymphocytes

481

Fig.1. Binding of RCA 120to untreated and formaldehyde-

treated lymphocytes. Cells were incubated for 1 h at 20°C with increasing amounts of [“‘CIRCA 120, 2-85 &5x106 cells/ml in a final volume of 400 td. Results were processed according to Scatchard [18]. 0, Treated cells; 0, untreated cells. Bound

Lymphocyte

RCA

M x 10’

Fixation

by Formaldehyde

Cells (1 x108/ml) were incubated for 18 h at room temperature in a 0.35% formaldehyde solution in 0.15 M NaCl. Under these conditions, cell aggregation was about 2% and the cell yield 80%. Higher concentrations of formaldehyde increased aggregation and reduced the yield (results not shown). Cells treated as described were stable in the cold for several weeks.

Treatment of Lymphocytes

after Fixation

Sialiduse. Cells (1 x lO?ml) were treated for 60 min at 37°C with 5 x 10e3 units of Vibrio cholerae sialidase (Behringwerke, W. Germany). The cells were then washed three times with a 0.15 M NaCl solution. No decrease in cell number nor cell aggregation were observed under these conditions. The sialic acid released was estimated by the thiobarbituric acid method [7]. Pronase. Untreated and sialidase-treated cells (5 x lO’/ml) were incubated for 60 min at 37°C with 100 &ml of pronase in 0.15 M NaCl (Calbiochem, USA). These conditions allowed maximum release of proteins, hexoses and sialic acid. Proteins and neutral hexoses were measured by the methods of Lowry et al. [S] and Lustig & Langer [9], respectively, with bovine serum albumine (BSA), lo-200 pg (Calbiochem, USA)., and 1: 1 galactose-mannose solution, lo-200 ug (Sigma, USA) as standards. Organic solvenrs.Cells treated with sialidase and/or pronase as well as untreated cells, were extracted first with 2 : 1 and 1: 2 chloroform-methanol mixtures (pooled in the Ll fraction), followed by 100, 50 and 25% methanol solutions in water (pooled in the L2 fraction), using 10 ml of solvent for 2x 10’ cells. Washing with methanol solutions of decreasing strength was necessary to prevent the cells from aggregating when they were resuspended in saline. Ll and L2 fractions were assayed for protein, hexoses, sialic acid, cholesterol (by the cholesterol C system, Boehringer, Mannhelm) and phospholipids (by the phospholipids B test, Wako, Japan). The amounts of sialic acid estimated in these fractions, especially in Ll, were probably overestimated because of unsaturated lipids which interfere in the assay [lo].

Preparation

of Radiolabeled

Lectins

RCA 120and RCA 60 were prepared according to Nicolson & Blaustein [l l] and labeled with [r4C]acetic anhydride according to Miller & Great 1121.The labeled lectins were repurified by gel filtration on Sephadex G 200 in 0.05 M Tris-HCl buffer, pH 7.4. Specific activities were 1050cpm/pg and 1400 cpm/ug, respectively. PNA and SBA were prepared by the methods of Lotan et al. [13] and Gordon et al. [14], respectively, labeled with “‘1 by the chloramine T procedure [15] and repurified by affinity chromatography. Specific activities were 4740 cpm/pg and 1550cpmlpg, respectively.

ExpCell Res 152 (1984)

488 Tut-pin et al. Table 1. Composition of the supernatants obtained after different treatments applied to fixed cells Supernatants

S

P

Ll

L2

Proteins Hexoses Sialic acid Total cholesterol Free cholesterol Phospolipids

8+2

880 +lOO 78.5f 11 3.2+ 0.7 0 0 0

16.2+ 6.2 7.3f 2.3 1.2-r- 0.5 54.3f 14 45.7+ 12.5 297 +112

23 + 8.2 9.52 3.2 2.2+ 0.9 0 0 122 590

-

Values are expressed in pg per 1x 10’ cells. Each value is the mean of three experiments. S, P, Supematants obtained after 60 min treatment with sialidase (5x lo-’ U/l X 10” cells/ml) or pronase (100 wg 5X 10’ cells/ml). Cell extracts obtained with Ll, chloroform-methanol mixtures; L2, methanol solutions.

Binding of Radiolabeled Lectins to Lymphocytes Binding for ‘251-labeled SBA and PNA was measured according to a previously described method [16] using fast centrifugation on a mixture of phtalates. Binding of [14C]-RCA 120 and RCA 60 was assayed as described earlier [17]. Non-specific binding was estimated by the same methods, but in the presence of 0.5 M D-palactose for SBA and PNA, and of 0.25 M lactose for RCA 120and RCA 60.

RESULTS The treatment with sialidase, pronase and organic solvents was carried out with lymphocytes, which have first been fixed with formaldehyde, to avoid cell lysis. The influence of fixation on lectin binding was examined with RCA 120, and no significant effect was found (fig. 1). The amounts of protein, neutral hexoses, sialic acid and phospholipids released from the fixed cells by the various treatments are given in table 1. The sum of the amounts of sialic acid released by pronase and organic solvents (3.2+2.2+ 1.2 pg/l x lo8 cells) was less than the quantity obtained by sialidase treatment alone (8 &I x IO8cells). Figs 2-5 show the extent of binding of each of the four lectins to lymphocytes after different treatments. The amounts of bound lectins are all expressed in clg/ 10’ cells. The amount of PNA (fig. 2) bound to untreated cells was very small (0.8 cLg>and even less to pronase-treated cells (0.4 vg), but larger with cells extracted with organic solvents (1.4 pg). The latter treatment seems to facilitate the access of the lectin to its receptors. Treatment of the cells with sialidase raised PNA binding to 8 pg. Lipid extraction had little effect on binding to sialidase-treated cells, whereas pronase treatment decreased binding by 75%. It appears thus that lymphocyte surface receptors for PNA are mainly or perhaps exclusively glycoproteins. Binding of SBA (fig. 3) was of the same order of magnitude (0.7 pg) as that of Exp CellRes 152 (1984)

Lectin receptors on lymphocytes

: :6

489

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.

:: c

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s a2

LL

P

z

: c

P

L

PL

s

SP SL SPL

c

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PL

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SP SL SR

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2-5. Binding of the four lectins to treated lymphocytes. Fig. 2, PNA. llO’ug/4.3x 10’ cells/ml; reaction volume, 230 pl;fig. 3, SBA. A, 110 &4.3x 10’ cells/ml; B, 440 l&4./4.3x10’ cells/ml. Reaction volume, 230 ul; fig. 4, RCA 120. A, 25 @1./1.25x10’ cells/ml; B, 50 ug/l.25~ 10’ cells/ml. Reaction volume, 400 ~1; fig. 5, RCA 60. A, 50 ug/2.5~10 cells/ml; B, 50 ug/l.25x107 cells/ml. Reaction volume, 400 ~1. The amounts of bound lectins are expressed’in l&O’ cells. Each value is the mean of two experiments. In all cases, incubation lasted for 1 h at 20°C. All cells were treated with formaldehyde; additional treatments were as follows: C, none (control cells); P, pronase; L, organic solvents; PL, pronase + organic solvents; S, sialidase; SP, sialidase + pronase; SL, sialidase + organic solvents; SPL, sialidase + pronase + organic solvents.

Figs

PNA at the same concentration of lectin. At the two concentrations studied (25 and 100 ug/107 cells), sialidase treatment enhanced about 3-fold the binding of SBA, which is much lower than the increase observed for the binding of PNA. Upon treatment of the cells with either pronase or organic solvents, SBA binding dropped by half, implying that glycoproteins and glycolipids each contribute to a similar extent to the binding of SBA to human lymphocytes. Binding of RCA 120 (fig. 4), which was studied at two lectin concentrations (20 and 40 ug/107 cells) was four times higher than that of SBA and PNA under the same conditions. The effect of pronase and organic solvents were slightly different at each lectin concentration. At the lower concentration, only pronase diminished binding, whereas at the higher concentration, both pronase and organic solvents produce a decrease in binding. As expected, sialidase treatment led to an increase in binding of the lectin. Lectin binding to sialidase-treated cells decreased to the same extent as a result of either pronase treatment or extraction Exp Cell Res 152 (1984)

490 Turpin et al. with organic solvents, indicating that the lectin receptors unmasked by sialidase are located both on glycoproteins and glycolipids. Binding of RCA 60 (fig. 5) was less than that of RCA 120, and only slightly higher than that of SBA and PI”JA. At both lectin concentrations tested (20 and 40 ug/107 cells), only pronase decreased RCA 60 bindings, so that the receptors for this lectin appear to be essentially glycoproteins. The increase in RCA 60 binding after treatment of the lymphocytes with sialidase was not different from that observed with RCA 120. DISCUSSION In this study we demonstrate that it is possible to assessthe relative contribution of membrane glycoproteins and glycolipids to the binding of lectins to cells. For this purpose, prior cell fixation by a cross-linking agent is necessary, since treatment of unfixed cells with organic solvents causes rapid cell lysis. By analogy with a method for red cell fixation [5], formaldehyde was used at a dilution sufficient to avoid cell aggregation. Binding of RCA 120 to lymphocytes was found not to be significantly affected by cell fixation (fig. I), and it is assumed that this treatment did not alter the binding to the cells of the other lectins used. The absence of lipid components from the supernatants obtained after pronase treatment (table 1) permits us to conclude that changes in lectin binding observed as a result of the above treatment are due to changes in cell surface glycoproteins. Treatment with organic solvents led to the release of neutral and acidic glycolipids, as shown by measurements of the lipid components in the various supernatants (table l), and this finding was confirmed by thin-layer chromatography (results not shown). However, very small amounts of proteins or peptides were also found in these supernatants. It is probable that only hydrophobic proteins would be soluble in such organic extracts. It should be noted that proteins soluble in organic solvents were isolated from other cells, such as bovine erythrocytes [ 191and calf thyroid cells 1201. The lectins used here are specific for D-galactosyl residues. In glycoproteins, sialic acid residues are often linked to galactose at the non-reducing end of the carbohydrate chains and sialidase treatment enables to estimate the effect of the removal of these terminal sialic acid residues on the binding of lectins to lymphocytes. The amount of sialic acid released (8 cl&/lo* cells) agrees with the results reported by Kornfeld for fresh human lymphocytes [21]. Formaldehyde treatment did not, therefore, affect the access of sialidase to the non-reducing end of the carbohydrate chains. As expected, binding of all four lectins tested was enhanced by sialidase treatment. The increase in binding varied from 50% for RCA 60 to 900% for PNA; the latter result is in agreement with the data reported earlier [22]. Kawaguchi & Osawa [23], on the other hand, did not find any change in the binding of RCA 120 to human lymphocytes after sialidase treatment. PNA bound principally to glycoproteins, since organic solvents had virtually no Exp CellRes 152 (1984)

Lectin receptors on lymphocytes

491

effect on its binding to the cells, whether or not they were treated with sialidase. This confirms earlier findings by Uhlenbruck et al. 1241who have shown that on the surface of human T and B lymphocytes, PNA receptors unmasked by sialidase reside in glycoproteins [25]. Although glycolipids (GM, and asialo-GM,), possessing the PNA-specific disaccharide ~-Gal @ 1+3 D-GalNac [13], were characterized on the surface of lymphocytes, chiefly of the B type, by means of specific antibodies [26], these glycolipids appear not to be accessible to PNA. The affinity of SBA for N-acetyl-D-galactosamine is 20 times higher than for Dgalactose [27]. Our results show that the receptors for this lectin on human lymphocytes are both glycoproteins and glycolipids. N-aCCtyl-D-galaCtOSaminC residues are not generally found at the non-reducing end of carbohydrate chains of cell surface glycoproteins. It has been shown that globoside, GalNac @1+3 Gal @l+4 Gal @1+4 Glc-&er is, despite its small quantity, the major glycolipid with terminal non-reducing N-acetylgalactosamine on human lymphocyte surface [28]. This structure might therefore constitute the glycolipid receptor for SBA. Globoside is the major, and perhaps sole, SBA receptor on human erythrocytes [29]. Receptors for RCA 120 are both glycoproteins and glycolipids, since pronase and organic solvents decreased lectin binding. It should be noted that the affinity constant previously found for the binding of lectin to low affinity sites on lymphocytes (K, = 5 x lo6 M-i) [17] differed little from the constant found for the binding of RCA 120 to liposomes containing the ganglioside GM, [30], which has a galactose residue at its non-reducing end. On sialidase-treated cells, the decrease in amount of bound lectin on the cells successively treated with pronase and organic solvents was much lower than the sum of the decrease obtained separately with pronase and organic solvents. This may perhaps be due to the release of lipophilic glycoproteins by extraction with organic solvents, as discussed above. Indeed, preliminary experiments seem to indicate that glycoproteins that bind to RCA 120 are found in the organic solvents extracts (unpublished results). Smaller amounts of RCA 60 than RCA 120 bound to the lymphocytes. This difference does probably not reflect a lower number of receptors but rather lower affinity constants, a possibility compatible with the difference between the respective affinities of RCA 60 and RCA 120for galactose [3 1,321. The receptors for RCA 60 were found to be essentially glycoproteins. Although RCA 60, in contrast to RCA 120, has the same affinity for N-acetylgalactosamine as for galactose 1331, it does not appear to interact with globoside. The present work has shown that receptors for PNA, SBA, RCA 120 and RCA 60 are glycoproteins and/or glycolipids. Although all these lectins are specific for D-gtiaCtOSy1 residues, they display certain differences as regards their affinities for galactose and its derivatives, and these differences are reflected in the nature of their respective receptors in human lymphocytes. The authors are very grateful to Dr Reviron (Centre de Transfusion sanguine, Hapital Saint-Louis) for supplying them with blood. This work was supported by grants from the INSERM (CRL 0,79-5162-3). Exp Ceil Res 152 (1984)

492

Turpin et al. REFERENCES

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Exp Cell Res 152 (1984)

F’rintcd

in Sweden