After 30 years of research, the pathogenesis of experimental allergic orchitis (EAO), an autoimmune disease of the testis, remains elusive. This is due, in part, to the lack of sufficient purified aspermatogenic antigen required for precise immunologic analysis of the disease. We recently isolated an aspermatogenic protein, AP3, capable of inducing EAO at a submicrogram dose. The biological activity of AP3 has been shown to resist denaturation, including reduction and alkylation, autoclaving and treatment with 1.0 N HCl, 1.0 N NaOH, 8.ON urea and 1.0% SDS. Thus, the disease-inducing activity of AP3 appears to reside in a linear (sequential) determinant(s) rather than in a conformational determinant(s). In the proposed work we will identify the minimal peptide sequence that constitutes the aspermatogenic determinant(s) of AP3. In addition, the complete amino acid sequence of AP3 will be determined. To accomplish this goal, we will subject AP3 to the following immunochemical studies: 1) site specific chemical modifications of specific amino acid residues within AP3; 2) assessment of aspermatogenic activity following chemical derivatization; 3) fragmentation by site specific cleavage into AP3-peptides; 4) purification of the resultant peptides followed by chemical characterization and assessment of aspermatogenic activity; 5) additional site specific modifications and fragmentations of the aspermatogenic AP3-peptide(s) obtained from the initial cleavage, followed by purification and biological and chemical characterization; and 6) based on the amino acid sequence of the individual AP3-peptides, establish the amino acid sequence of AP3. Also, heterologous and homologous antibodies to AP3 will be prepared, and with these antisea, determine the antigenic and auto-(allo-)antigenic determinants of AP3 by their reactivity with the AP3-peptides and chemically modified AP3-peptides in a radioimmunoassay. Finally, we will localize AP3 in/on guinea pig spermatozoa and testicular cells by immunofluorescence. Information gained from this work will allow for the generation of an organically synthesized peptide(s) analogous to the disease-inducing peptide(s) of AP3. Synthetic peptides of AP3, when available, will permit an in-depth analysis of the pathogenetic mechanisms in EAO. In addition, the complete amino acid sequence of aspermatogenic testicular peptides will be established for the first time.
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