The Goodpasture Autoantigen

Abstract

The Goodpasture (GP) autoantigen has been identi-fied as the ␣3(IV) collagen chain, one of six homologous chains designated ␣1–␣6 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 auto-epitope(s) that has been previously localized to the non-collagenous (NC1) domain of ␣3(IV) chain. Fourteen ␣1/␣3 NC1 chimeras were constructed by substituting one or more short sequences of ␣3(IV)NC1 at the corre-sponding positions in the non-immunoreactive ␣1(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 inhi-bition enzyme-linked immunosorbent assay. Two chime-ras, C2 containing residues 17–31 of ␣3(IV)NC1 and C6 containing residues 127–141 of ␣3(IV)NC1, bound au-toantibodies, as did combination chimeras containing these regions. The epitope(s) that encompasses these sequences is immunodominant, showing strong reactiv-ity with all GP sera and accounting for 50 –90% of the autoantibody reactivity toward ␣3(IV)NC1. The confor-mational nature of the epitope(s) in the C2 and C6 chi-meras was established by reduction of the disulfide bonds and by PEPSCAN analysis of overlapping 12-mer peptides derived from ␣1-and ␣3(IV)NC1 sequences. The amino acid sequences 17–31 and 127–141 in ␣3(IV)NC1 have thus been shown to contain the critical residues of one or two disulfide bond-dependent conformational au-toepitopes that bind GP autoantibodies. Goodpasture (GP) 1 autoimmune disease is characterized by pulmonary hemorrhage and/or rapidly progressing glomerulo-nephritis (1). Tissue injury is mediated by anti-basement mem-brane antibodies that bind alveolar and glomerular basement membranes. The target autoantigen of basement membranes has been identified as the ␣3(IV) collagen chain, one of six homologous chains designated ␣1–␣6 that comprise type IV collagen (2). In the glomerular basement membrane, the ␣3(IV) chain exists in a supramolecular network along with the ␣4(IV) and ␣5(IV) chains (3). The ␣3(IV) chain is composed of a long collagenous domain of 1410 amino acids and a non-collagenous (NC1) domain of 232 residues at the carboxyl terminus (4). The GP autoepitope(s) has been localized to the NC1 domain of the ␣3(IV) chain (5, 6). Antibodies that bind to the NC1 domain of other ␣(IV) chains may be found in some Goodpas-ture patients (7, 8), but they only account for about 10% of autoreactivity (9). The autoepitope(s) in the ␣3(IV)NC1 domain appears to be conformational, because reduction of disulfide bonds abolishes most of the binding (9 –11). The identification of the precise amino acid residues that constitute this epitope(s) is important for understanding the etiology and pathogenesis of the GP disease and for the development of diagnostic and therapeutic agents. Several groups have at-tempted to map the location of the autoepitope(s) by using short linear peptides (9, 11–14) or by site-directed mutagenesis of the ␣3(IV)NC1 domain expressed in Escherichia coli (15). Although linear sequences have been identified that bind GP antibodies, these findings are at variance with each other. Moreover, prior studies have not addressed whether these lin-ear sequences constitute the major conformational, disulfide bond-dependent epitope(s). The aim of this study was to identify the ␣3(IV)NC1 amino acid sequences that form the thus far elusive conformational GP epitope(s). To circumvent the limitations of previous ap-proaches, we pursued an epitope mapping strategy based on chimeric proteins. This approach has been specifically devel-oped and successfully used to map conformational epitopes (16) or autoepitopes (17). We hypothesized that the ␣3(IV)NC1 re-gions most likely to form the autoepitope(s) are those most divergent from the other homologous ␣(IV) chains. A series of chimeric ␣1/␣3(IV)NC1 domains were constructed in which these candidate ␣3(IV)NC1 sequences replaced the correspond-ing sequences in the non-immunoreactive ␣1(IV)NC1. The chi-meras were expressed in mammalian cells for correct protein folding and disulfide bond formation. We report that two spe-cific sequences, ␣3(IV)NC1 residues 17–31 and 127–141, con-tain the critical residues of one or two disulfide bond-dependent conformational GP autoepitopes within the ␣3(IV)NC1 domain.