Goodpasture syndrome. Localization of the epitope for the autoantibodies to the carboxyl-terminal region of the alpha 3(IV) chain of basement membrane collagen.

The autoantibodies of patients with Goodpasture syndrome are primarily targeted to the noncollagenous (NC1) domain of the alpha 3(IV) chain of basement membrane collagen (Saus, J., Wieslander, J., Langeveld, J. P. M., Quinones, S., and Hudson, B. G. (1988) J. Biol. Chem. 263, 13374-13380). In the present study, the location of the Goodpasture epitope in human alpha 3NC1 was determined, and its structure was partially characterized. This was achieved by identification of regions of alpha 3NC1 which are candidates for the epitope and which are structurally unique among the five known homologous NC1 domains (alpha 1-alpha 5); amino acids that are critical for Goodpasture antibody binding, by selective chemical modifications; and regions that are critical for Goodpasture antibody binding, by synthesis of 12 alpha 3NC1 peptides and measurement of their antibody binding capacity. The carboxyl-terminal region, residues 198-233, was identified as the most likely region for the epitope. By experiment, lysine and cysteine were identified as critical amino acids for antibody binding. Three synthetic peptides were found to inhibit Goodpasture antibody binding to alpha 3NC1 markedly: a 36-mer (residues 198-233), a 12-mer (residues 222-233), and a 5-mer (residues 229-233). Together, these results strongly indicate that the Goodpasture epitope is localized to the carboxyl-terminal region of alpha 3NC1, encompassing residues 198-233 as the primary antibody interaction site and that its structure is discontinuous. These findings provide a conceptual framework for future studies to elucidate a more complete epitope structure by sequential replacement of residues encompassing the epitope using cDNA expression products and peptides synthesized chemically

Sequential Expression of Type IV Collagen Networks: Testis as a Model and Relevance to Spermatogenesis

The six α chains of type IV collagen are organized into three networks: α1/α2, α3/α4/α5, and α1/α2/α5/α6. A shift from the α1/α2 to the α3/α4/α5 network occurs in the developing glomerular basement membrane, but how the α1/α2/α5/α6 network fits into this sequence is less clear, because the three networks do not colocalize. Here, we studied the seminiferous tubule basement membrane of normal canine testis where all three networks do colocalize: the α1/α2 network is expressed from birth, the α1/α2/α5/α6 network by 5–6 weeks of age, and the α3/α4/α5 network by 2 months of age. A canine model of Alport syndrome allowed study of the absence of α3/α4/α5 and α1/α2/α5/α6 networks in testis. In Alport dogs, the seminiferous tubule basement membrane was thinner than in controls. Spermatogenesis began at the same time as with normal dogs; however, the number of mature sperm was significantly reduced in Alport dogs. Thus, it would appear that α3/α4/α5 and α1/α2/α5/α6 networks are not essential for onset of spermatogenesis, but long-term function may be compromised by the loss of one or both networks. This situation is analogous to the glomerular basement membrane in Alport syndrome. In conclusion, testis can serve as a model system to study the sequence of type IV collagen network expression