J. P. M. Langeveld

The goodpastiure autoantigen; mapping the major conformational epitope(s) of alpha3(IV) collagen to residues 17-31 and 127-141 of the NC-1 domain

The Goodpasture (GP) autoantigen has been identified as the α3(IV) collagen chain, one of six homologous chains designated α1–α6 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 α3(IV) chain. Fourteen α1/α3 NC1 chimeras were constructed by substituting one or more short sequences of α3(IV)NC1 at the corresponding positions in the non-immunoreactive α1(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 α3(IV)NC1 and C6 containing residues 127–141 of α3(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 α3(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 α1- and α3(IV)NC1 sequences. The amino acid sequences 17–31 and 127–141 in α3(IV)NC1 have thus been shown to contain the critical residues of one or two disulfide bond-dependent conformational autoepitopes that bind GP autoantibodies.

Glomerular basement membrane: Evidence for collagenous domain of the α3 and α4 chains of collagen IV

Abstract A collagenous component(s) of Mr = 60K was extracted from glomerular basement membrane with urea and was purified. Upon digestion, it yielded a collagenase-resistant fragment(s) of Mr = 23.5K. Both component and fragment showed immunochemical identity with the noncollagenous domains of the new α3 & α4 chains of collagen IV. The component is characterized by a collagenous domain of about 280 residues and a noncollagenous domain of about 250 residues. These findings further establish these new chains as distinct entities of collagen IV.