Production and characterization of recombinant Goodpasture antigen in insect cells.
The Goodpasture antigen is the target of anti-basement membrane autoantibodies in Goodpasture's disease, a severe human autoimmune disease characterized by glomerulonephritis and lung hemorrhage. It has been identified as the NC1 domain of the alpha 3 chain of type IV collagen (alpha 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(IV)NC1 produced in Escherichia coli has been insoluble and poorly recognized by patients' autoantibodies. We have used the baculovirus expression system to produce the antigen as a soluble product in Sf9 cells. A transfer vector was constructed from cDNAs encoding the leader peptide, NH2 terminus, 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 human alpha 3 chain under the control of the polyhedrin promoter. It therefore encodes a hybrid "mini"-collagen chain from which the majority of the central triple helical region has been deleted. The recombinant antigen was seen on SDS-polyacrylamide gel electrophoresis and Western 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 binding of autoantibodies to human GBM, and bound two monoclonal antibodies known to recognize human alpha 3(IV)NC1. A common alternatively spliced 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 was not recognized by autoantibodies. Other NC1 domains could be expressed in the same way. These recombinant molecules should prove invaluable for the in vitro study of the immunopathogenesis of Goodpasture's disease, and the approach provides a means by which interactions between the different type IV collagen chains found in GBM could be studied in vitro.
Turner
N
N
Department of Medicine, Royal Postgraduate Medical School, London, United Kingdom.
Forstová
J
J
Rees
A
A
Pusey
C D
CD
Mason
P J
PJ
D000818:Animals; D001323:Autoantibodies / Q000276:immunology; D001324:Autoantigens / Q000235:genetics*; D001483:Base Sequence; D001485:Basement Membrane / Q000276:immunology; D002460:Cell Line; D003094:Collagen / Q000235:genetics*; D024141:Collagen Type IV; D017364:Collagenases / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D006801:Humans; D007678:Kidney Glomerulus / Q000276:immunology; D008969:Molecular Sequence Data; D009036:Moths; D011487:Protein Conformation; D011994:Recombinant Proteins
D001323:Autoantibodies; D001324:Autoantigens; D024141:Collagen Type IV; D017931:DNA Primers; D011994:Recombinant Proteins; C057112:type IV collagen alpha3 chain; D003094:Collagen; D017364:Collagenases