Specificity of Goodpasture autoantibodies for the recombinant noncollagenous domains of human type IV collagen.

Neilson EG, Kalluri R, Sun MJ, Gunwar S, Danoff T, Mariyama M, Myers JC, Reeders ST, Hudson BG
J Biol Chem. 1993 268 (12): 8402-5

PMID: 8473281

Type IV collagen has recently emerged as a family composed of five known chains (alpha 1-alpha 5), each of which contains a carboxyl-terminal noncollagenous domain (NC1) of approximately 230 amino acids. The NC1 domain of the alpha 3(IV) chain is the probable target for autoantibodies in patients with Goodpasture syndrome (GP), as evidenced from studies employing bovine type IV collagen. In the present experiments, the specificity of GP antibodies for the five NC1 domains of human type IV collagen was determined by using recombinant NC1 domains as the antigen. cDNAs encoding each NC1 domain were expressed in E. coli as fusion proteins with a 6-histidine amino-terminal leader. The recombinant NC1 monomers r alpha 1(IV), r alpha 2(IV), r alpha 3(IV), r alpha 4(IV), and r alpha 5(IV) were purified by affinity chromatography to the fusion protein using a nickel resin column, and then characterized by electrophoresis and immunoblot analysis using chain-specific peptide antibodies. The specificity of GP antibodies from four patients to these recombinant proteins was then further evaluated by immunoblot analysis and enzyme-linked immunosorbent assay measurements. The GP antibodies reacted strongly with the r alpha 3(IV) NC1 domain but were not reactive when tested against the other four recombinant monomers. In contrast, neither antisera from patients with two other forms of autoimmune disease (anti-tubular basement membrane disease and Wegener's syndrome) nor normal control sera bound to any of the recombinant NC1 moieties. These results unambiguously establish that GP antibodies are specifically targeted to the NC1 domain of the alpha 3(IV) chain of human type IV collagen. The findings also establish a methodology for large scale preparation of r alpha 3(IV) NC1 domain for use in diagnostic tests and development of therapeutic procedures and offer a strategy for the elucidation of a more complete GP epitope by site-directed mutagenesis.

MeSH Terms (15)

Amino Acid Sequence Anti-Glomerular Basement Membrane Disease Antibody Specificity Autoantibodies Autoantigens Base Sequence Collagen Collagen Type IV DNA Electrophoresis, Polyacrylamide Gel Enzyme-Linked Immunosorbent Assay Humans Immunoblotting Molecular Sequence Data Recombinant Proteins

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