IDENTIFICATION OF ANTIGENIC EPITOPES IN TYPE-IV COLLAGEN BY USE OF SYNTHETIC PEPTIDES

Peptides representing potential antigenic regions of the NC-1 and 7-S domains of the human alpha1 and alpha2, and bovine alpha3 chains of type IV collagen were synthesized either chemically or by the recombinant DNA technique and tested by ELISA using antibodies raised in rabbits against the whole type IV collagen or the NC-1 domain.1 Sera from patients with Goodpasture syndrome (GP) or with acute poststreptococcal glomerulonephritis (APSGN) were also tested. The location of antigenic determinants was predicted from the primary and secondary structure of the chains, that is, aromaticity, hydrophilicity and presence of beta-turns. All synthetic peptides reacted with the antiserum to type IV collagen (anti-Col IV). Whereas all peptides arising from the NC-1 domain reacted with anti-NC-1, intact 7-S or peptides of the alpha1 or alpha2 chain of the 7-S domain did not react. However intact 7-S reacted with anti-Col IV. Two synthetic peptides from the NC-1 domain of alpha1, (a.a. 71-90 and a.a. 176-190), one from the alpha2 (a.a. 70-83) and four from the alpha3 chain (a.a. 72-89, a.a. 104-117, a.a. 133-145, a.a. 185-203) reacted with anti-NC-1 and anti-COL IV. The above peptides, except alpha3 (72-89) and alpha3 (185-203), were tested and found to be reactive with sera from patients with GP. Intact 7-S reacted with several sera from patients with GP; however, only two synthetic peptides from the 7-S domain of the alpha2 chain reacted with two of the GP sera. One serum from a patient with GP reacted with intact 7-S but not with NC-1. More than the presence of beta-turns and hydrophilicity, the single most consistent predictor of immunoreactivity of synthetic peptides from the NC-1 domain was the presence of aromatic amino acids. Sera of patients with APSGN reacted exclusively with the 7-S domain. This reactivity resided in the synthetic peptide alpha2(IV) 1-18 from the 7-S domain. The results show that this approach can be used to define more precisely the nature of antigenic epitopes that may be involved in the mediation of immunologic injury in renal disease.