The effect of phosphorylation and site-specific mutations in the immunodominant epitope of the human ribosomal P proteins.
The immunodominant epitope recognized by lupus antiribosomal P protein antibodies (anti-P antibodies) is located within the 11 C-terminal residues common to the three P proteins. This epitope contains a potential phosphorylation site for casein kinase II and clusters of acidic and hydrophobic amino acids. To determine the role of each of these features in antigen recognition, lupus anti-P sera were tested for binding to phospho- and dephospho- forms of the P proteins and to synthetic peptide antigens in which site-specific modifications had been introduced. Immunoblot analysis revealed that anti-P antibodies specific for the phospho- form of the P proteins represented only a minor population of anti-P antibodies and, in many cases, were absent altogether. In contrast, when charged substitutions were introduced into either the acidic 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 substitution of either Ser-105 or the C-terminal Asp-115 resulted in a < 35% reduction 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 optimal binding to synthetic peptide antigens. The remarkable degree of specificity demonstrated by these antibodies supports the view that anti-P autoantibodies result from a highly specific (at the B cell level) immune response to self antigen.
Hasler
P
P
Hospital for Special Surgery, Cornell University Medical College, New York, New York 10021.
Brot
N
N
Weissbach
H
H
Danho
W
W
Blount
Y
Y
Zhou
J L
JL
Elkon
K B
KB
D000595:Amino Acid Sequence; D000818:Animals; D000941:Antigens / Q000276:immunology; D001323:Autoantibodies / Q000378:metabolism; D001483:Base Sequence; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D006801:Humans; D016056:Immunodominant Epitopes / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000276:immunology; D010766:Phosphorylation; D015800:Protozoan Proteins; D012269:Ribosomal Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*
D000941:Antigens; D001323:Autoantibodies; D016056:Immunodominant Epitopes; C064601:L12E protein, Trypanosoma cruzi; D010455:Peptides; D015800:Protozoan Proteins; D012269:Ribosomal Proteins