THE EFFECT OF PHOSPHORYLATION AND SITE-SPECIFIC MUTATIONS IN THE IMMUNODOMINANT EPITOPE OF THE HUMAN RIBOSOMAL P-PROTEINS

The immunodominant epitope recognized by lupus anti-ribosomal P protei n antibodies (anti-P antibodies) is located within the 11 C-terminal r esidues common to the three P proteins. This epitope contains a potent ial phosphorylation site for casein kinase II and clusters of acidic a nd hydrophobic amino acids. To determine the role of each of these fea tures in antigen recognition, lupus anti-P sera were tested for bindin g to phospho- and dephospho- forms of the P proteins and to synthetic peptide antigens in which site-specific modifications had been introdu ced. Immunoblot analysis revealed that anti-P antibodies specific for the phospho- form of the P proteins represented only a minor populatio n of anti-P antibodies and, in many cases, were absent altogether. In contrast, when charged substitutions were introduced into either the a cidic or hydrophobic clusters and tested by ELISA, striking reductions of 64-86% were observed. Conservative Gly --> Pro substitutions also produced a 73% average reduction in anti-P binding whereas substitutio n of either Ser-105 or the C-terminal Asp-115 resulted in a <35% reduc tion in binding. These findings suggest that phosphorylation of the P proteins does not play a role in antibody recognition but that anti-P antibodies require both the acidic and hydrophobic clusters for optima l binding to synthetic peptide antigens. The remarkable degree of spec ificity demonstrated by these antibodies supports the view that anti-P autoantibodies result from a highly specific (at the B cell level) im mune response to self antigen. (C) 1994 Academic Press, Inc.