Antibody specificity of human glomerular basement membrane type IV collagen NC1 subunits. Species variation in subunit composition.
NC1 subunits were purified from gel filtration pools of acid-extracted, collagenase-digested human glomerular basement membranes (hGBM). This methodology, which enriches 28-kDa monomers (M28) in the total digest, allowed purification of these monomers and 24-kDa (M24) and 26-kDa (M26) monomers free from dimers. Reactivity of these subunits with Goodpasture autoantibodies using immunoblotting of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional nonequilibrium pH gradient electrophoresis gels showed strong reactivity with the purified M28 subunits. Inhibition enzyme-linked immunosorbent assay, used to quantitate the reactivity of the purified NC1 subunits, indicated that M28 had a greater than 10-fold increase in ability to inhibit binding to NC1 than NC1 itself. Comparison of hGBM NC1 components were made with those obtained from collagenase digests of salt and acid-extracted bovine and sheep GBM and Englebreth-Holm-Swarm tumor similarly purified by gel filtration and reverse-phase high performance liquid chromatography. Two-dimensional gel analysis of these NC1 isolates revealed absence of the very cationic M28 monomers. Reactivity with antibodies eluted from diseased kidneys of sheep immunized with hGBM (Steblay nephritis) was compared with Goodpasture autoantibody reactivity by immunoblotting two-dimensional gels of hGBM NC1. We conclude that a very cationic M28 monomer (M28 ) found only in hGBM is the probable target in Goodpasture syndrome, that the epitope is present on most NC1 components from extracted and unextracted hGBM, and is exposed by urea denaturation which is enhanced by acid treatment. A weakly cationic M28 monomer (M28+) is present in GBM from other species and is the probable target in Steblay nephritis. Differential recognition of the two M28 components by these antibodies points to different genetic origins or possibly distinct post-translational modifications for these components. This is supported by their presence or absence in different species and tissues, as well as biochemical differences from the M24/26 monomers which presumably are derived from alpha 1(IV) and alpha 2(IV) collagen chains.
D000596:Amino Acids / Q000032:analysis; D000936:Antigen-Antibody Complex / Q000032:analysis; D001323:Autoantibodies; D001485:Basement Membrane / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D003094:Collagen / Q000032:analysis* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007678:Kidney Glomerulus / Q000032:analysis*; D046911:Macromolecular Substances
D000596:Amino Acids; D000936:Antigen-Antibody Complex; D001323:Autoantibodies; D046911:Macromolecular Substances; D003094:Collagen