Interaction of Receptors on Lectins with Membrane Erythrocyte Surfaces

The interactions of human genotype AO erythrocytes (red blood cells) (RBCs) with N-acetylgalactosamine-reactive lectins isolated from Helix pomatia (HPA)and from Dolichos biflorus (DBA) were studied. Binding curves obtained with the use of tritium-labeled lectins showed that the maximal numbers of lectin molecules capable of binding to human genotype AO RBCs were 3.8 x 105 and 2.7 x 105 molecules/RBC for HPA and DBA, respectively. The binding of one type of lectin may influence the binding of another type. HPA was found to inhibit the binding of DBA, but not vice versa. The binding of HPA was weakly inhibited by a /~-D-galactose-reactive lectin isolated from Ricinus communis (designated RCA1). Limufus polyphemus lectin (LPA), with specificity for N-acetylneuraminic acid, did not influence the binding of HPA but enhanced the binding of DBA. About 80% of LPA receptors (N-acetylneuraminic acid) were removed from RBC surfaces by neuraminidase treatment. Neuraminidase treatment of RBCs resulted in increases of binding of both HPA and DBA, but through different mechanisms. An equal number (7.6 x 105) of new HPA sites were generated on genotypes AO and GO RBCs by neuraminidase treatment, and these new sites accounted for the enhancement (AO cells) and appearance (OO cells) of hemagglutinability by HPA. Neuraminidase treatment did not generate new DBA sites, but increased the DBA affinity for the existing receptors; as a result, genotype AO cells increased their hemagglutinability by DBA, while GO cells remained unagglutinable. The use of RBCs of different genotypes in binding assays with 3H-labeled lectins of known specificities provides an experimental system for studying cell-cell recognition and association. Cell-cell interaction in multicellular organisms is a fundamental phenomenon which includes cell-cell recognition, communication, adhesion, and rearrangement, etc. Most of these interactions are mediated by the cell surface (1-3), which contains a number of surface membrane proteins or glycoproteins responsible for cellular recognition or junction formation (4, 5). The cell surface membrane-bound glycosyltransferases and the sugar acceptors have been implicated in dynamic intercellular adhesion and dissociation (6-8). Lectins are a group of carbohydrate-binding proteins or glycoproteins isolated from plants and animals capable of recognizing specific sugar residues and their linkages. Different types oflectins have been used extensively to study the topology of cell surfaces (2, 9), including cells during differentiation (10, 11) and after transformation (12). Many lectins can agglutinate human erythrocytes (red 646 blood cells) (RBCs) 1 (2). Indeed, RBC agglutination is the usual method for detecting the presence oflectins in a solution and has contributed to the discovery of many lectins (13). A considerable number of lectins are blood group-specific, and some recognize A, B, H, Le b, and N determinants (9, 14). In the present investigation, two blood group A-specific lectins, obtained from Helix pomatia (15-18) and Dolichos biflorus (19-21), were used to react with genotype AO human RBCs, with the aim of studying the interaction of these molecules Abbreviations used in this paperDBA, Dolichos biflorus agglutinin or lectin; GalNAc, N-acetylgalactosamine; HPA, Helb: pomatia agglutinin or lectin; LPA, Limulus polyphemus agglutinin or lectin; RBC, erythrocyte of human, genotype AO, unless stated otherwise; RCA1, an agglutinin isolated from Ricinus communis; TSA, Trisbuffered saline containing 2.5 mg/ml of bovine serum albumin, pH 7.4. THE JOURNAL OF CELt BIOLOGY . VOLUME 101 AUGUST 1985 646-651 © The Rockefeller University Press • 0021-9525/85/08[0646/06 $1.00 on D ecem er 3, 2017 jcb.rress.org D ow nladed fom with their receptors on the cell surface membrane and elucidating this specific recognition process of cell-cell association and dissociation. Helix pomatia lectin (HPA, A standing for agglutinin), which was isolated from the albumin glands of the edible garden snail (Helix pomatia), consists of six subunits, each containing one carbohydrate binding site (15-17). Dolichos biflorus lectin (DBA), which was extracted from the seeds of horse gram (Dolichos biflorus) (18), consists of four subunits, two type I and two type II; only the type I subunits bind carbohydrates (22). Both HPA and DBA resemble the anti-A antibodies in that they agglutinate human group A but not B or O RBCs, and that they precipitate with blood group A substance and group C streptococcal polysaccharides ( 16, 19). DBA is even more restricted in its affinity toward blood group substances; it can distinguish subgroup A~ from other subgroups (23). Both lectins were found to have combining sites specific for terminal nonreducing a-linked N-acetylgalactosamine (GalNAc) (16, 19, 24). These two lectins have overlapping but not identical specificities (25, 26), with HPA lectin having a broader sugar range than DBA. In the present study, HPA and DBA labeled with [3H] acetic anhydride were used to determine the equilibrium binding of HPA and DBA and the number of their receptor sites on the genotype AO RBC surface. Experiments were designed to evaluate and analyze the factors that may influence the interactions of lectins with their receptors on RBCs, including the competition between lectins for common receptor sites, the possible roles of steric hindrance and charge interference by other lectin molecules, and the effect of removal of N-acetylneuraminic acids. The analysis of these experimental data has provided a molecular basis for understanding the mechanism of hemagglutination induced by these lectins under different conditions. MATERIALS AND METHODS Materials: [3H] acetic anhydride (5.4 Ci/mM) was purchased from Amersham Corp. (Arlington Heights, IL). DBA (molecular weight = 110,000), Limulus polyphemus lectin (LPA) (=400,000), HPA (=79,000), and an agglutinin isolated from Ricinus communis (RCA 1) (= 120,000) were purchased from E.Y. Laboratory (San Mateo, CA). HPA was also purified in E.A. Kabat's Laboratory, with polyleucyl hog A + H blood group substances as previously described (15). The purity of these lectin preparations is shown in the SDS PAGE pattern (Fig. 1). PAGE was run according to the method of Fairbank et