PRODUCTION AND CHARACTERIZATION OF RECOMBINANT GOODPASTURE ANTIGEN ININSECT CELLS

The Goodpasture antigen is the target of anti-basement membrane autoan tibodies in Goodpasture's disease, a severe human autoimmune disease c haracterized by glomerulonephritis and lung hemorrhage. It has been id entified as the NC1 domain of the alpha 3 chain of type IV collagen (a lpha 3(IV)NC1), a minority component of glomerular basement membrane ( GBM). Protocols for obtaining pure human antigen are laborious and low yielding and require cadaver kidneys. Recombinant (alpha 3(TV)NC1 pro duced in Escherichia coil has been insoluble and poorly recognized by patients' autoantibodies. We have used the baculovirus expression syst em to produce the antigen as a soluble product in Sf9 cells. A transfe r vector was constructed from cDNAs encoding the leader peptide, NH2 t erminus, and 7 S domain of the human alpha 1 chain of type IV collagen and was joined inframe to the NC1 domain and COOH terminus of the hum an alpha 3 chain under the control of the polyhedrin promoter. It ther efore encodes a hybrid ''mini''-collagen chain from which the majority of the central triple helical region has been deleted. The recombinan t antigen was seen on SDS-polyacrylamide gel electrophoresis and Weste rn blots of supernatants at its predicted molecular size of 41 kDa and as dimers of 82 kDa. It reacted strongly with human autoantibodies by Western blotting and enzyme-linked immunosorbent assay, inhibited bin ding of autoantibodies to human GBM, and bound two monoclonal antibodi es known to recognize human alpha 3(IV)NC1. A common alternatively spl iced variant alpha 3(IV)NC1 mRNA, leading to a truncated NC1 domain of 60 amino acids, was expressed as a fusion protein with the same alpha 1 NH2-terminal sequence. It failed to be exported from the cell and w as not recognized by autoantibodies. Other NC1 domains could be expres sed in the same way. These recombinant molecules should prove invaluab le for the in vitro study of the immunopathogenesis of Goodpasture's d isease, and the approach provides a means by which interactions betwee n the different type IV collagen chains found in GBM could be studied in vitro.