Immunochemical studies of the Alport antigen

Abstract

The Alport antigen, a component of normal glomerular basement membranes (GBM) which is absent in Alport familial nephritis, is characterized as a 26 kD non-collagenous (NC1) peptide identified by a monoclonal antibody (Mab A7) and an Alport alloantibody. Both antibodies discriminate X-linkage of the Alport defect using indirect immunofluorescence of hemizygous and heterozygous Alport GBM and epidermal basement membrane (EBM). Immunoblotting of SDS-PAGE gels of collagenase-digested Alport renal BM shows absence of monomeric and dimeric components of the Alport antigen, α3(IV) NC1, and α4(IV) NC1. By immunoprecipitation experiments with specific antibodies, the Alport antigen is distinct from the 26 kD NC1 peptide derived from α1(IV). The monoclonal antibody to the Alport antigen and rabbit antiserum to a non-consensus sequence of α5(IV) NC1 react similarly by immunofluorescence with normal kidney and both fail to bind to Alport renal BM. Two dimension Western blots of collagenase-digested BM show that the anti-Alport antigen and the ant-α5(IV) NC1 react similarly with monomeric and dimeric components of BM collagen. These studies are consistent with the likelihood that the Alport antigen and α5(IV) NC1 are the same or are highly homologous molecules. The precise relationship will require characterization of α5(IV) NC1 protein and determination of the nucleotide sequence of the Alport antigen. The associated absence of α3(IV) NC1 and a4(IV) (NC1) from Alport BM is consistent with other observations for a molecular association of these chains in a novel collagen network.