Proposed are studies into the structure, expression, and function of a small group of structurally related genes likely to be important in matrix production and in normal, and perhaps abnormal, development of mammalian embryos. Studies include: 1) Further characterization of the gene (PCOLCE), whose protein product, the procollagen C-proteinase enhancer protein, is involved in biosynthesis of type l collagen, the major component of matrix. Studies will include: A) Determining temporal and spatial distribution of expression of PCOLCE during development and comparison to the distribution of expression of other genes with whose products the enhancer protein might interact (eg. the type J collagen gene COL1A1; the BMP-1/mTld gene, which may encode the type I procollagen C-proteinase; and a novel BMP-1/mTld-like gene recently discovered in the P.I's lab); B) Functional study of the mechanism whereby the enhancer protein stimulates the activity of C-proteinase and assaying whether the enhancer may also stimulate activation of TGF-beta-like proteins: C) Studying the possibility that PCOLCE is up-regulated by TGF- beta. Such studies should provide further insight into the role(s) of the enhancer protein and possible interactions with other proteins in developmental and physiological processes. 2) Definitive study of a gene (BMP-1/mTld) encoding alternatively spliced transcripts for a protein involved in organogenesis of bone (BMP-1) and for a mammalian homologue (mTld) of a protein important in dorsal/ventral patterning of Drosophila embryos (tolloid). Studies will: A) Determine the extent of differential expression of the various alternatively spliced RNA forms in embryonic extraembryonic and adult tissues and distribution of mTld in the central nervous system: B) Demonstrate that the protein products of the various alternatively spliced RNA forms exist and localization of these proteins in developing and adult tissues. and determine whether they may be associated with cell membranes: C) Confirm and extend the original observation that BMP-1 is. or is very closely related to the physiological type I procollagen C-proteinase: D) Confirm and extend the original observation that BMP-1 can directly activate TGF- beta-like molecules: E) Determine possible up-regulation of expression of BMP-1/mTld by TGF-beta; F) Investigate possible enzymatic activities of mTld. 3) Characterization of a new gene encoding a protein product with a domain structure identical to but sequence different than. that of mTld. This gene was recently isolated in the P.I.'s lab from a cDNA library constructed from mRNA of +/- knockout mouse embryos which no longer express BMP-1/mTld but which seem to express compensatory enzymatic activity. Characterization will include determining: A) Full-length coding sequences: B) Chromosomal locations in mouse and human genomes: C) Whether alternative splicing occurs: D) Spatial and temporal patterns of expression in adult and developing tissues; E) Possible functions of the protein product(s): F) Whether levels and/or distribution of expression are changed to compensate for loss of BMP-1/mTld in +/- knockout embryos: G) Role(s) of the new gene in development, determined through creation and study of knockout mice homozygous for null alleles of the new gene.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR043621-03
Application #
2683336
Study Section
Human Embryology and Development Subcommittee 1 (HED)
Project Start
1996-04-01
Project End
2000-03-31
Budget Start
1998-04-01
Budget End
1999-03-31
Support Year
3
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Pathology
Type
Schools of Medicine
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Takahara, Kazuhiko; Schwarze, Ulrike; Imamura, Yasutada et al. (2002) Order of intron removal influences multiple splice outcomes, including a two-exon skip, in a COL5A1 acceptor-site mutation that results in abnormal pro-alpha1(V) N-propeptides and Ehlers-Danlos syndrome type I. Am J Hum Genet 71:451-65
Uzel, M I; Scott, I C; Babakhanlou-Chase, H et al. (2001) Multiple bone morphogenetic protein 1-related mammalian metalloproteinases process pro-lysyl oxidase at the correct physiological site and control lysyl oxidase activation in mouse embryo fibroblast cultures. J Biol Chem 276:22537-43
Imamura, Y; Scott, I C; Greenspan, D S (2000) The pro-alpha3(V) collagen chain. Complete primary structure, expression domains in adult and developing tissues, and comparison to the structures and expression domains of the other types V and XI procollagen chains. J Biol Chem 275:8749-59
Mott, J D; Thomas, C L; Rosenbach, M T et al. (2000) Post-translational proteolytic processing of procollagen C-terminal proteinase enhancer releases a metalloproteinase inhibitor. J Biol Chem 275:1384-90
Scott, I C; Steiglitz, B M; Clark, T G et al. (2000) Spatiotemporal expression patterns of mammalian chordin during postgastrulation embryogenesis and in postnatal brain. Dev Dyn 217:449-56
Amano, S; Scott, I C; Takahara, K et al. (2000) Bone morphogenetic protein 1 is an extracellular processing enzyme of the laminin 5 gamma 2 chain. J Biol Chem 275:22728-35
Schwarze, U; Atkinson, M; Hoffman, G G et al. (2000) Null alleles of the COL5A1 gene of type V collagen are a cause of the classical forms of Ehlers-Danlos syndrome (types I and II). Am J Hum Genet 66:1757-65
Scott, I C; Imamura, Y; Pappano, W N et al. (2000) Bone morphogenetic protein-1 processes probiglycan. J Biol Chem 275:30504-11
Scott, I C; Blitz, I L; Pappano, W N et al. (1999) Mammalian BMP-1/Tolloid-related metalloproteinases, including novel family member mammalian Tolloid-like 2, have differential enzymatic activities and distributions of expression relevant to patterning and skeletogenesis. Dev Biol 213:283-300
Scott, I C; Clark, T G; Takahara, K et al. (1999) Assignment of TLL1 and TLL2, which encode human BMP-1/Tolloid-related metalloproteases, to chromosomes 4q32-->q33 and 10q23-->q24 and assignment of murine Tll2 to chromosome 19. Cytogenet Cell Genet 86:64-5

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