Preparation of overlapping peptides of bovine retinal S-antigen and their localization by immunoblotting with peptide-specific antibodies.
Bovine retinal S-antigen was cleaved by three chemical cleavage procedures including o-iodosobenzoic acid (IBA), mile acid and cyanogen bromide. The resultant peptides were used to study antibody-defined epitopes. Treatment with IBA, which cleaves primarily at tryptophanyl peptide bonds, produced at least 4 major fragments and several minor fragments. The peptides have been identified by their migration on SDS-PAGE and tested for their immunoreactivity to several affinity-purified anti-CNBr-peptide antibodies and to affinity-purified anti-IBA peptide antibodies. The presence of a single tryptophan residue 194 residues from the amino-terminus should result in 2 fragments of approximately 23,000 and 26,000 molecular weight based on the known size of intact S-antigen. The additional fragmentation is due to the presence of acid labile bonds and cleavage at IBA-sensitive tyrosyl residues associated with a side reaction. Western immunoblots using affinity-purified antibodies against the various IBA and CNBr peptides have allowed location of these peptides within the intact molecule. Specifically, IBA23K and IBA21K are overlapping fragments on the carboxy end, mutally exclusive of all other peptides. IBA15K and IBA5.6K overlap, and IBA18K and IBA10K overlap within IBA26K which comprises the N-terminal half of S-Ag. Additionally, IBA10K contains an antibody epitope destroyed by CNBr cleavage of the methionyl residue between CB53 and CB56. Further characterization of these IBA peptides will expedite the location of possible additional uveitogenic epitopes in the amino-terminal half of S-Ag as well as epitopes lost by other peptide generating techniques.
Fling
S P
SP
Department of Microbiology, University of Minnesota, Minneapolis 55455.
Knospe
V
V
Gregerson
D S
DS
D000818:Animals; D000906:Antibodies / Q000032:analysis / Q000276:immunology*; D000918:Antibody Specificity; D002417:Cattle; D003488:Cyanogen Bromide / Q000276:immunology; D007158:Immunologic Techniques; D007463:Iodobenzoates / Q000276:immunology; D008213:Lymphocyte Activation; D010455:Peptides / Q000276:immunology*; D012160:Retina / Q000276:immunology; D014269:Trifluoroacetic Acid
D000906:Antibodies; D007463:Iodobenzoates; D010455:Peptides; C008154:2-iodosobenzoic acid; D014269:Trifluoroacetic Acid; D003488:Cyanogen Bromide