Changes in rheumatoid factor and monoclonal IgG antibody specificity after site-specific mutations in antigenic region of beta 2-microglobulin.
Antigenic epitopes on the SKDWSFY region (positions 57-63) of human beta 2-microglobulin (beta 2m) reacting in ELISA with polyclonal and monoclonal human IgM rheumatoid factors (RF) as well as with monoclonal mouse IgG anti-beta 2m antibodies were studied after site-specific mutation of single or double beta 2m amino acid residues. Single site-specific mutations at lysine 58 and serine 61 produced a major decrease (50-100%) in reactivity of polyclonal IgM RF with beta 2m. Mutations at serine 57, tryptophane 60, and phenylalanine 62 also induced substantial decrements (25-49%) in IgM RF reactivity. beta 2m mutants containing double amino acid mutations including one residue outside the SKDWSFY region along with four different mutations at tryptophane 60 also often showed marked decrements in IgM RF anti-beta 2m reactivity. Site-specific mutations at virtually all residues within the SKDWSFY beta 2m region produced a major decrease in reactivity of two of three monoclonal human IgM RF. Results with nine murine IgG anti-beta 2m mAbs often showed an entirely different profile. Site-specific mutation of aspartic acid at position 59 produced a 50-100% decrease in binding of nine mAbs. Serine at position 61 and tyrosine at 63 also appeared to represent important residues for antigenic determinants reacting with anti-beta 2m mAbs. Results of mapping discrete epitopes using glycine substitution within overlapping 7-mers of beta 2m linear sequence compared with site-specific mutation of single beta 2m residues were similar in many instances, but also often showed marked differences. A marked change in antigenic contribution of single amino acid residues to beta 2m antigenic epitopes frequently depended on what residues were mutated to. Our findings emphasize that single residues within a small seven amino acid, solvent-accessible antigenic stretch of primary beta 2m sequence vary considerably in their contribution to the reactive antigenic structures for IgM RF as well as monoclonal IgG antibodies.
van Eyndhoven
W G
WG
Department of Medicine, University of Florida, Gainesville 32610.
Malone
C C
CC
Williams
R C
RC
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D001172:Arthritis, Rheumatoid / Q000276:immunology; D001483:Base Sequence; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D007075:Immunoglobulin M / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D012217:Rheumatoid Factor / Q000276:immunology*; D001613:beta 2-Microglobulin / Q000235:genetics / Q000276:immunology*
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D001613:beta 2-Microglobulin; D012217:Rheumatoid Factor