Identification of the Goodpasture antigen, alpha 3(IV) NC1, and four other NC1 domains of type IV collagen, by amino-terminal sequence analysis of human glomerular basement membrane separated by two-dimensional electrophoresis.
Two-dimensional (2-D) electrophoresis of collagenase-digested human glomerular basement membrane (GBM), containing the Goodpasture antigen, revealed a range of monomeric (24-30 kD) and dimeric (43-56 kD) subunits present across a pI range of 3-10. Five distinct alpha(IV)-chains were identified by amino-terminal sequence analysis of 18 of these components transferred to polyvinylidene difluoride membrane. The positions of the well-characterised 26-kD alpha 1(IV)-chain and 24-kD alpha 2(IV)-chain were confirmed. A highly cationic 28-kD monomer was identified as the alpha 3(IV)-chain, while more neutral 28-kD monomers were found to contain the alpha 4(IV)-chain. Sequences from neutral 26-kD monomers corresponded to the known cDNA sequence of the alpha 5(IV)-chain. The presence of charge isoforms of the alpha 1(IV)- and alpha 4(IV)-chains was confirmed by identification of several monomers with different pI but the same sequence. Sequence analysis of dimeric components demonstrated homodimers of alpha 1(IV), alpha 2(IV) and alpha 4(IV), and suggested the presence of heterodimers of alpha 3/alpha 5 and alpha 1/alpha 5. 2-D Western blots of human GBM, with anti-GBM autoantibodies, a monoclonal antibody (P1) to the Goodpasture antigen and a monoclonal antibody to the alpha 3(IV)-chain, demonstrated that the major autoantigenic epitope was localised to the alpha 3(IV)-chain, but that there was also reactivity with the alpha 4(IV)-chain.
Derry
C J
CJ
Department of Medicine, Royal Postgraduate Medical School, London, UK.
Pickering
M
M
Baker
C
C
Pusey
C D
CD
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D001323:Autoantibodies / Q000276:immunology; D001324:Autoantigens / Q000032:analysis* / Q000276:immunology; D001485:Basement Membrane / Q000737:chemistry / Q000276:immunology / Q000648:ultrastructure; D015153:Blotting, Western; D003094:Collagen / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D024141:Collagen Type IV; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000032:analysis / Q000276:immunology; D006801:Humans; D007525:Isoelectric Focusing; D007536:Isomerism; D007678:Kidney Glomerulus / Q000737:chemistry* / Q000648:ultrastructure; D008969:Molecular Sequence Data
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D001324:Autoantigens; D024141:Collagen Type IV; D000939:Epitopes; C057112:type IV collagen alpha3 chain; D003094:Collagen