Structure-function studies of S-antigen: use of proteases to reveal a dominant uveitogenic site.
Retinal S-antigen induced experimental autoimmune uveitis (EAU) is a severe, predominantly T-cell mediated inflammatory disease of the uveal tract and retina of the eye. Pretreatment of LEW rats with the monoclonal antibody, MAbS2.4.C5, which defines an epitope in S-antigen, has been shown to effectively inhibit the subsequent induction of EAU with S-antigen. Using synthetic peptides and cyanogen bromide fragments of S-antigen we found the binding site of MAbS2.4.C5 to be located at the carboxy terminus of the molecule corresponding to amino acid positions 375 to 380. Limited Staphylococcus aureus V8 protease digestion yielded several polypeptide fragments including one large 43 kD fragment which retained antibody binding to a variety of both polyclonal and monoclonal antibodies which identify epitopes that span the length of the S-antigen. This treatment, however, completely destroys the MAbS2.4.C5 binding site and dramatically reduces uveitopathogenicity. Limited trypsin and papain digestion, on the other hand, had little effect on pathogenicity or on MAbS2.4.C5 binding to S-antigen or its peptide fragments. These results indicate that the carboxy-terminus of S-antigen plays a predominant role in the pathogenesis of EAU.
Dua
H S
HS
Department of Ophthalmology, University of Aberdeen, Medical School, Scotland, U.K.
Hossain
P
P
Brown
P A
PA
McKinnon
A
A
Forrester
J V
JV
Gregerson
D S
DS
Donoso
L A
LA
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000627:therapeutic use; D000941:Antigens / Q000737:chemistry / Q000187:drug effects / Q000276:immunology*; D019393:Arrestin; D001324:Autoantigens / Q000737:chemistry / Q000276:immunology*; D001327:Autoimmune Diseases / Q000276:immunology* / Q000517:prevention & control; D001666:Binding Sites, Antibody; D010450:Endopeptidases / Q000494:pharmacology; D000939:Epitopes / Q000276:immunology; D005136:Eye Proteins / Q000737:chemistry / Q000187:drug effects / Q000276:immunology*; D005260:Female; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology; D051381:Rats; D011917:Rats, Inbred Lew / Q000276:immunology; D013329:Structure-Activity Relationship; D014605:Uveitis / Q000276:immunology* / Q000517:prevention & control
D000911:Antibodies, Monoclonal; D000941:Antigens; D019393:Arrestin; D001324:Autoantigens; D000939:Epitopes; D005136:Eye Proteins; D010446:Peptide Fragments; D010450:Endopeptidases