The goals are to define the mechanisms involved in remodeling of the extracellular matrix (ECM) with focus on the roles of collagenases and their mechanisms of proteolysis in resorption of interstitial collagens. In order to further understand the roles of collagenase action directed at different cleavage sites (helical and N-telopeptide) in type I as well as type II collagens and to determine how collagenases function during embryonic development compared to postnatal life, we propose a series of studies designed to alter collagenase cleavage of these interstitial collagens. The information derived from these studies will be utilized to identify mutations to target to the collagenase genes. Genetic targeting of the enzyme (collagenase), and substrates, (cleavage sites in type I collagen [tendons, ligaments, dermis and bone] and type II collagen [growth plate and articular cartilage]), will then be employed. Examination of the affected mice, particularly with respect to remodeling of cartilage and bone, will help to define the roles of collagenase in degradation of the most abundant specific tissue collagens.
Specific Aim 1 : Determine structural requirements for proteolytic cleavage by collagenases by introduction of mutations into collagenase genes and identify the determinants of collagenase cleavage in the helical and N-telopeptide domains of type I collagen. Sequences in the catalytic domain of mouse collagenase that control N- telopeptidase activity will be identified and replaced with sequences from the corresponding domain of human MMP-1 which does not have N- telopeptidase activity. The mutated collagenases expressed in E. coli or insect cells will be assayed on type I collagen substrates extracted from plus/plus mice and mice (r/r) resistant to cleavage in the helical domain. This information will be used for the design of targeted mutations in Specific Aim 2.
Specific Aim 2 : Manipulate the murine interstitial collagenase genome by: (a) introduction of null mutations in order to delete all collagenase activity (knockout) and (b) targeting subtle mutations in the gene in order to abrogate N-telopeptidase activity. These separate mutations will be introduced by homologous recombination in embryonic stem cells, insertion into blastocysts and expression in the germline to create mice expressing these mutations in both alleles of the collagenase gene.
Specific Aim 3 : Target mutations that encode collagenase resistance into the murine type II collagen (Col2a-1) gene.
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