The long-term objectives of this research proposal are to understand the biologic role of a group of related novel collagen chains with particular attention to the structure of specialized renal basement membranes and the changes related to certain diseases especially, Alport familial nephritis and other renal diseases. (1) A crucial role is the characterization of alpha5(IV) protein and its relationship to the Alport antigen. The latter, a 26 kD peptide, is defined by antibody probes that discriminate X-linked Alport syndrome and in common with alpha3(IV) NC1 and alpha4(IV) NC1 is not detected in Alport glomerular basement membrane (GBM). The gene for alpha5(IV) (COL4A5) has been assigned to the Xq22 locus of the X chromosome. We have shown that an alpha5(IV) NC1 peptide codistributes with the Alport antigen in normal GBM and is missing from Alport GBM. We propose to characterize this molecule, define its distribution, and determine its relationship to the Alport antigen, and its interaction with other novel chains in dimers and helices. The various methods used will involve biochemical and immunochemical techniques to isolate monomers and dimers, the development of monoclonal and polyclonal antibody probes to peptides and NC1 and the use of immunohistochemical techniques. (2) The sequencing of cDNA and genomic DNA of the Alport antigen, alpha4(IV) and alpha3(IV) is an important aspect of this proposal since it will define the genetically-discriminating Alport peptide and allow us to understand the interrelationships among these molecules. Since this component is missing from most males with Alport syndrome and present in a mosaic pattern in affected females, the definition of whether it is the same as alpha5(IV) NC1 or is an homologous related chain-is crucial. Screening of a library constructed from mRNA of transformed bovine endothelial cells (a line that makes alpha3(IV) NC1 and the Alport antigen) will be carried out with our antibody probes, and subsequent nucleotide sequencing, Northern analysis,and genomic analysis. Similarly, the cDNA of alpha4(IV) NCI will be sought-initially using nucleotides developed from N-terminal sequences of the isolated chains as well as antibody screening. (3) A major component of this research is to define further the genetic defect in Alport syndrome for mutations in novel collagen genes; to characterize further the relationship of these mutations to Alport phenotypes, and the occurrence of post-transplant anti-GBM nephritis. This will include investigations of the mutations involving the gene encoding alpha5(IV) NC1, the Alport antigen, and alpha4(IV) NC1. Further, we will address the hypothesis that patients with Alport syndrome that develop anti-GBM nephritis following transplantation may have unique mutations that prevent expression of certain epitopes and the development of immunologic tolerance during fetal life. These studies will involve a number of a molecular biologic techniques. (4) The recognition that these novel chains of type IV collagen in contrast to alpha1(IV) and alpha2(IV) have common distributions in renal basement membranes, late ontogenic expression, exhibit temporal and spatial segregation in disease-strongly suggest the presence of molecular interactions in a network and functional specialization. Using biochemical and immunochemical techniques, we will study the interactions of these chains in cultured cells and isolated basement membranes.
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