title: The critical role of arginine residues in the binding of human monoclonal antibodies to cardiolipin.
abstract: Previously we reported that the variable heavy chain region (VH) of a human beta2 glycoprotein I-dependent monoclonal antiphospholipid antibody (IS4) was dominant in conferring the ability to bind cardiolipin (CL). In contrast, the identity of the paired variable light chain region (VL) determined the strength of CL binding. In the present study, we examine the importance of specific arginine residues in IS4VH and paired VL in CL binding. The distribution of arginine residues in complementarity determining regions (CDRs) of VH and VL sequences was altered by site-directed mutagenesis or by CDR exchange. Ten different 2a2 germline gene-derived VL sequences were expressed with IS4VH and the VH of an anti-dsDNA antibody, B3. Six variants of IS4VH, containing different patterns of arginine residues in CDR3, were paired with B3VL and IS4VL. The ability of the 32 expressed heavy chain/light chain combinations to bind CL was determined by ELISA. Of four arginine residues in IS4VH CDR3 substituted to serines, two residues at positions 100 and 100 g had a major influence on the strength of CL binding while the two residues at positions 96 and 97 had no effect. In CDR exchange studies, VL containing B3VL CDR1 were associated with elevated CL binding, which was reduced significantly by substitution of a CDR1 arginine residue at position 27a with serine. In contrast, arginine residues in VL CDR2 or VL CDR3 did not enhance CL binding, and in one case may have contributed to inhibition of this binding. Subsets of arginine residues at specific locations in the CDRs of heavy chains and light chains of pathogenic antiphospholipid antibodies are important in determining their ability to bind CL.
authors.affiliation: Centre for Rheumatology, Department of Medicine, University College London, UK. i.giles@ich.ucl.ac.uk
authors.forename: Ian
authors.initials: I
authors.lastname: Giles
authors.forename: Nancy
authors.initials: N
authors.lastname: Lambrianides
authors.forename: David
authors.initials: D
authors.lastname: Latchman
authors.forename: Pojen
authors.initials: P
authors.lastname: Chen
authors.forename: Reginald
authors.initials: R
authors.lastname: Chukwuocha
authors.forename: David
authors.initials: D
authors.lastname: Isenberg
authors.forename: Anisur
authors.initials: A
authors.lastname: Rahman
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D017153:Antibodies, Anticardiolipin / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D000974:Antibodies, Antinuclear / Q000235:genetics / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D000937:Antigen-Antibody Reactions; D016736:Antiphospholipid Syndrome / Q000276:immunology; D001120:Arginine / Q000737:chemistry*; D001327:Autoimmune Diseases / Q000276:immunology; D019556:COS Cells; D002308:Cardiolipins / Q000276:immunology* / Q000378:metabolism; D002417:Cattle; D002522:Chlorocebus aethiops; D022801:Complementarity Determining Regions / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D006023:Glycoproteins / Q000276:immunology; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D013329:Structure-Activity Relationship; D053482:beta 2-Glycoprotein I
chemical_list: D017153:Antibodies, Anticardiolipin; D000974:Antibodies, Antinuclear; D000911:Antibodies, Monoclonal; D002308:Cardiolipins; D022801:Complementarity Determining Regions; D006023:Glycoproteins; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D011993:Recombinant Fusion Proteins; D053482:beta 2-Glycoprotein I; D012694:Serine; D001120:Arginine