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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL042798-12
Application #
3361099
Study Section
Pathobiochemistry Study Section (PBC)
Project Start
1978-07-01
Project End
1993-11-30
Budget Start
1990-12-15
Budget End
1991-11-30
Support Year
12
Fiscal Year
1991
Total Cost
Indirect Cost
Name
University of Medicine & Dentistry of NJ
Department
Type
Schools of Medicine
DUNS #
622146454
City
Piscataway
State
NJ
Country
United States
Zip Code
08854
Anderson, D W; Edwards, T K; Ricketts, M H et al. (1996) Multiple defects in type III collagen synthesis are associated with the pathogenesis of abdominal aortic aneurysms. Ann N Y Acad Sci 800:216-28
Csiszar, K; Entersz, I; Trackman, P C et al. (1996) Functional analysis of the promoter and first intron of the human lysyl oxidase gene. Mol Biol Rep 23:97-108
Mauch, J C; Sandberg, L B; Roos, P J et al. (1995) Extensive alternate exon usage at the 5' end of the sheep tropoelastin gene. Matrix Biol 14:635-41
Boyd, C D; Mariani, T J; Kim, Y et al. (1995) The size heterogeneity of human lysyl oxidase mRNA is due to alternate polyadenylation site and not alternate exon usage. Mol Biol Rep 21:95-103
Sechler, J L; Sandberg, L B; Roos, P J et al. (1995) Elastin gene mutations in transgenic mice. Ciba Found Symp 192:148-65;discussion 165-71
Schwartz, E; Feinberg, E; Lebwohl, M et al. (1995) Ultraviolet radiation increases tropoelastin accumulation by a post-transcriptional mechanism in dermal fibroblasts. J Invest Dermatol 105:65-9
Wydner, K S; Sechler, J L; Boyd, C D et al. (1994) Use of an intron polymorphism to localize the tropoelastin gene to mouse chromosome 5 in a region of linkage conservation with human chromosome 7. Genomics 23:125-31
Boyd, C D; Pierce, R A; Schwarzbauer, J E et al. (1993) Alternate exon usage is a commonly used mechanism for increasing coding diversity within genes coding for extracellular matrix proteins. Matrix 13:457-69
Mariani, T J; Trackman, P C; Kagan, H M et al. (1992) The complete derived amino acid sequence of human lysyl oxidase and assignment of the gene to chromosome 5 (extensive sequence homology with the murine ras recision gene). Matrix 12:242-8
Pierce, R A; Alatawi, A; Deak, S B et al. (1992) Elements of the rat tropoelastin gene associated with alternative splicing. Genomics 12:651-8

Showing the most recent 10 out of 14 publications