Basement membranes are extracellular matrices located between epithelial or endothelial cells and underlying connective tissues. They function as selective barriers and are of critical importance in maintaining normal tissue function and integrity. The major components of basement membranes are proteins designated Type IV collagens, non-collagenous glycoproteins (laminin) and proteoglycans. The goal of the research proposed here is to determine the complete covalent structure of human Type IV collagens which are thought to be the main structural component of basement membranes. To accomplish this goal we will use two different approaches: (a) We will synthesize complementary DNA (cDNA) to Type IV collagen-specific mRNA isolated from a human tumor cell line (American Type Culture Collection, HT-1080 Cells). The cDNA will be inserted into pBR322 and grown in E. coli strain HB101. DNA sequencing will be carried out and the sequence will be analyzed to yield an amino acid sequence. (b) We will isolate intact Type IV collagens from cultured HT-1080 cells and collagen fragments solubilized by limited pepsin digestion from human placental tissue. Post-translational modifications such as hydroxylation of prolyl and lysyl residues, glycosylation of hydroxylysyl and asparaginyl residues, location of disulfide bonds and cleavage sites of processing enzymes will be determined by direct protein sequencing. The long term objective of this research is to understand the structures and its relation to the function of basement membranes in normal and diseased states. As a first step in this direction, we will determine the complete covalent structure of basement membrane collagens using a dual approach: protein and DNA sequencing. Together these complementary approaches provide the most effective and rapid means of achieving the research goal. This work will provide a solid basis for further structural studies and will provide specific probes for investigating changes in basement membranes caused by pathological conditions such as are found in diabetes.
