A circulating factor found in some patients with focal segmental glomerulosclerosis is associated with recurrent disease after renal transplantation and may be responsible for initiating the renal injury.

The ultrafiltration function of the glomerular basement membrane (GBM) of the kidney is impaired in genetic and acquired diseases that affect type IV collagen. The GBM is composed of five (␣1 to ␣5) of the six chains of type IV collagen, organized into an ␣1⅐␣2(IV) and an ␣3⅐␣4⅐␣5(IV) network. In Alport syndrome, mutations in any of the genes encoding the ␣3(IV), ␣4(IV), and ␣5(IV) chains cause the absence of the ␣3⅐␣4⅐␣5 network, which leads to progressive renal failure. In the present study, the molecular mechanism underlying the network defect was explored by further characterization of the chain organization and elucidation of the discriminatory interactions that govern network assembly. The existence of the two networks was further established by analysis of the hexameric complex of the noncollagenous (NC1) domains, and the ␣5 chain was shown to be linked to the ␣3 and ␣4 chains by interaction through their respective NC1 domains. The potential recognition function of the NC1 domains in network assembly was investigated by comparing the composition of native NC1 hexamers with hexamers that were dissociated and reconstituted in vitro and with hexamers assembled in vitro from purified ␣1-␣5(IV) NC1 monomers. The results showed that NC1 monomers associate to form nativelike hexamers characterized by two distinct populations, an ␣1⅐␣2 and ␣3⅐␣4⅐␣5 heterohexamer. These findings indicate that the NC1 monomers contain recognition sequences for selection of chains and protomers that are sufficient to encode the assembly of the ␣1⅐␣2 and ␣3⅐␣4⅐␣5 networks of GBM. Moreover, hexamer formation from the ␣3, ␣4, and ␣5 NC1 monomers required co-assembly of all three monomers, suggesting that mutations in the NC1 domain in Alport syndrome may disrupt the assembly of the ␣3⅐␣4⅐␣5 network by interfering with the assembly of the ␣3⅐␣4⅐␣5 NC1 hexamer.

Glomerular basement membrane (GBM) plays a crucial function in the ultrafiltration of blood plasma by the kidney. This function is impaired in Alport syndrome, a hereditary disorder that is caused by mutations in the gene encoding type IV collagen, but it is not known how the mutations lead to a defective GBM. In the present study, the supramolecular organization of type IV collagen of GBM was investigated. This was accomplished by using pseudolysin (EC 3.4.24.26) digestion to excise truncated triple-helical protomers for structural studies. Two distinct sets of truncated protomers were solubilized, one at 4°C and the other at 25°C, and their chain composition was determined by use of monoclonal antibodies. The 4°C protomers comprise the ␣1

The Goodpasture (GP) autoantigen has been identified as the alpha3(IV) collagen chain, one of six homologous chains designated alpha1-alpha6 that comprise type IV collagen (Hudson, B. G., Reeders, S. T., and Tryggvason, K. (1993) J. Biol. Chem. 268, 26033-26036). In this study, chimeric proteins were used to map the location of the major conformational, disulfide bond-dependent GP autoepitope(s) that has been previously localized to the noncollagenous (NC1) domain of alpha3(IV) chain. Fourteen alpha1/alpha3 NC1 chimeras were constructed by substituting one or more short sequences of alpha3(IV)NC1 at the corresponding positions in the non-immunoreactive alpha1(IV)NC1 domain and expressed in mammalian cells for proper folding. The interaction between the chimeras and eight GP sera was assessed by both direct and inhibition enzyme-linked immunosorbent assay. Two chimeras, C2 containing residues 17-31 of alpha3(IV)NC1 and C6 containing residues 127-141 of alpha3(IV)NC1, bound autoantibodies, as did combination chimeras containing these regions. The epitope(s) that encompasses these sequences is immunodominant, showing strong reactivity with all GP sera and accounting for 50-90% of the autoantibody reactivity toward alpha3(IV)NC1. The conformational nature of the epitope(s) in the C2 and C6 chimeras was established by reduction of the disulfide bonds and by PEPSCAN analysis of overlapping 12-mer peptides derived from alpha1- and alpha3(IV)NC1 sequences. The amino acid sequences 17-31 and 127-141 in alpha3(IV)NC1 have thus been shown to contain the critical residues of one or two disulfide bond-dependent conformational autoepitopes that bind GP autoantibodies.

The noncollagenous domain hexamer of collagen IV from bovine alveolar basement membrane was excised with bacterial collagenase, purified under nondenaturing conditions, and characterized. The hexamer is comprised of four distinct subunits [alpha 1(IV)NC1, alpha 2(IV)NC1, alpha 3(IV)NC1, and alpha 4(IV)NC1]. Each subunit exists in both monomeric and dimeric (disulfide-crosslinked) form, and both monomers and dimers have charge isoforms. Certain dimers also contain nonreducible crosslinks. The alpha 3(IV)NC1 subunit, in both the monomeric and dimeric form, reacts with Goodpasture (GP) antibodies. The GP epitope is sequestered within the hexamer and becomes reactive with antibody upon exposure with protein denaturants. These results reveal that the alveolar basement membrane hexamer is identical to the hexamer from glomerular basement membrane with respect to subunit composition, identity of subunits reacting with GP antibodies, and sequestration of the GP epitope but differs greatly in the relative amount of the GP-reactive subunit and the degree of disulfide and nondisulfide crosslinking of subunits. This study leads to the conclusion that pulmonary hemorrhage associated with GP syndrome is mediated by the same autoantibody that mediates the glomerulonephritis, namely anti-collagen [alpha 3(IV)] antibody.