Molecular Immunology, Vol. 32, No. 17/l& pp. 1399-1404, 1995 Pergamon 0161~5890(95)00096-8 Copyright 0 1996 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0161-5890/95 $9.50 + 0.00 HUMAN RECOMBINANT CD4 AND CD6DERIVED SYNTHETIC PEPTIDES AGGLUTINATE IMMUNOGLOBULIN- COATED LATEX PARTICLES. EVIDENCE THAT RESIDUES 25-28 AND 35-38 OF HUMAN CD4 FORM TWO SEPARATE IMMUNOGLOBULIN BINDING SITES PETAR LENERT,*t GORDANA LENERT* and MAURIZIO ZANETTI$ *Research Center, Notre-Dame Hospital, University of Montreal, Montreal, Quebec, Canada H2L 4M 1; IDepartment of Medicine and Cancer Center, 9500Gilman Drive, University of California, San Diego, CA 92093-0063, U.S.A. (First received 31 March 1995; accepted in revisedform 19 June 1995) Abstract-It has been shown previously that amino acid residues 21-49 of the first extracellular domain of human CD4 form the core of an immunoglobulin (Ig) binding site. Synthetic peptides of human CD4 that encompass this region also bind Ig and, with higher affinity, antigen/antibody complexes. Synthetic peptides also enhance binding of both monomeric and aggregated Ig to mon- ocytic U937 cells and Staphylococcus aureus Protein A. To better characterize the nature of the Ig binding site on CD4, we tested the ability of human recombinant CD4 (rCD4) to agglutinate polystyrene particles coated with Ig. Evidence is presented that soluble rCD4 and CD4 peptide (p)2149 were capable of specific agglutination of polystyrene particles coated with polyclonal Ig of either human or sheeporigin. Agglutination could be blocked by soluble human polyclonal IgG or F(ab’)z fragments. Both heparin and sulfated dextrans also inhibit agglutination, suggestingthat charged residues on rCD4 played an important role in aggluti- nation mediated by rCD4 or CD4 peptide. Similarly, aurintricarboxylic acid (ATA) also blocked agglutination of Ig-coated particles by rCD4. Agglutination mapping studies performed using trunc- ated peptides revealed the existence of two discrete, closely related Ig binding sites (residues 25-28 and 35-38). Key words: CD4, immunoglobulins, dextran sulfate, heparin. INTRODUCTION A few years ago we (Lenert et al., 1990; Lenert and Zanetti, 1991) and others (Lederman et al., 1990) dem- onstrated that human CD4 binds immunoglobulins (Ig) and, with greateravidity, antigen:antibody (Ag :Ab) com- plexes. We also demonstratedthat CDCderived synthetic peptides enhance the binding of monomeric and aggre- gated Ig to U937 cells and Staphylococcus aureus protein A (Lenert et al., 1992; Mehta et al., 1994). Similarly to CD4/MHC-Class II interaction, binding of Ig to CD4 is of relatively low affinity and could be inhibited by both sulfated dextrans and HIV-gp120 (Lenert et al., 1990; Lenert and Zanetti, 1991). Early mapping studies indi- cated that, on the Ig side, binding is contributed mainly by residuesin the Fd region of the heavy (H) chain. On the CD4 side, the critical Ig binding site was localized within residues 2149 of the first extracellular domain TAuthor to whom correspondenceshould be addressed. Abbreviations: ATA, aurintricarboxylic acid, HRP, horse-rad- ish peroxidase, M, molar, p, peptide, rCD4, soluble recom- binant CD4. (Dl) (Lenert et al., 1990). Furthermore, these experi- ments raised the question of whether there could exist two distinct binding sites for Ig. In order to verify this hypothesis, we designed experiments based on aggluti- nation of Ig-coated polystyrene particles by CD4. We reasoned that if two separateIg binding sites exist, then visible agglutination of Ig-coated polystyrene particles would occur. In this paper, we presentevidence that rCD4 and CD4- derived peptides specifically agglutinate Ig-coated poly- styrene particles. Agglutination occurred within seconds and was dependent on the concentration of rCD4 or its peptides. Agglutination was blocked by pre-incubation with human polyclonal IgG and their F(ab’), fragments specifically. Mapping studies using truncated peptides overlapping residues 21-38 of the first extracellular domain revealedthat agglutination occurred only when both clusters (residues 25-28 and 36-38) were present simultaneously. These results imply that CD4 expressed on T cells and cells of the monocyte/macrophagelineage may have an important function, not only in the rec- ognition and handling of Ig and immune complexes (Lenert et al., 1990; Lenert and Zanetti, 1991; Lenert et 1399