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Nephrol Dial Transplant (1994) 9: 355-361
© 1994 European Renal Association-European Dialysis and Transplant Association

research-article

Tissue-specific distribution of the Goodpasture antigen demonstrated by 2-D electrophoresis and Western blotting

C. J. Derry and C. D. Pusey

Renal Unit, Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital London, UK

Correspondence and offprint requests to: Correspondence and offprint requests to: C. J. Derry, Renal Unit, Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London W12 ONN, UK

The target of autoantibodies in Goodpasture's disease, the Goodpasture antigen has recently been characterized as the NC1 domain of the {alpha}3 chain of type IV collagen. In order to study the Goodpasture antigen in different organs, NC1 domains were isolated from basement membranes (BM) of human glomeruli (GBM), tubules (TBM), alveoli (ABM), placenta (PBM) and aorta (VBM). NC1 preparations were separated by 2-D electrophoresis, and silver stained or immunoblotted to determine the subunit structure and antigenicity of different basement membranes. All basement membranes contained monomeric components of MW 26 kDa and 24 kDa, and associated dimers, corresponding to the 2-D location of {alpha}1(IV) and cc2(IV) chains respectively. However, GBM, ABM, and to a lesser extent TBM possessed an extra set of monomeric components of MW 28 kDa and associated dimers corresponding to the proposed location of {alpha}3(IV) and {alpha}4(IV) chains. 2-D-separated polypeptides were Western blotted with autoantibodies from patients with Goodpasture's disease, a monoclonal antibody to the Goodpasture antigen (P1) and a monoclonal antibody to the bovine {alpha}3(IV) chain. The predominant binding of all these reagents was to cationic 28 kDa monomers of GBM, ABM and TBM, corresponding to the 3(IV) chain, although autoantibodies and P1 also bound to neutral 28 kDa monomers, corresponding to the {alpha}4(IV) chain. Autoantibodies bound weakly to more neutral components of PBM and VBM, but neither monoclonal antibody bound to these basement membranes. This study suggests that there are two separate type IV collagen networks: one containing the {alpha}l(IV) and {alpha}2(IV) chains appears to be present in all basement membranes, and the other containing the {alpha}3(IV) and {alpha}4(IV) chains has a tissue specific distribution. The latter network bearing the Goodpasture antigen is expressed in the basement membranes of both kidney and lung, the organs involved in Goodpasture's disease.

Keywords: autoantigen; Goodpasture's disease; 2-Delectrophoresis; Western blotting; glomerular basement membrane


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