GENETIC ANALYSIS OF TYPE I COLLAGEN FUNCTION

点击翻译按钮获取中文摘要

Modifications of specific amino acids in the pro alpha chains of type I collagen are thought to be major contributor to the biochemical, physical, and biomechanical properties of the type I collagen fibrillar architecture, dictating specialized functions in mineralized and nonmineralized tissues. In particular covalent intermolecular crosslinks between collagen molecules are postulated to be principal determinants of the stability, biomechanical and physiochemical properties of the collagen fibrillar matrix. Much has recently been elucidate concerning the location and chemical nature of type I collagen crosslinks, but very little is understood about their function. We propose to define the functional role of specific type I collagen crosslinks by altering the amino acid residues at specific molecular loci in the proalpha2(I) collagen chain and evaluating the effects on type I collagen structure/function in vivo and in vitro in various tissues. To do this high efficiency proalpha2(I) collagen expression genes will be created and amino acid substitutions: lysine-5N (amino-telopeptide: major skeletal crosslink), histidine-92 (helix: skin-specific crosslink), and hydroxylsine-87 (helix: skeletal and soft tissue crosslink). These constructs will be microinjected into fertilized oim (homozygous for null COLIA2 allele) mouse oocytes to generate transgenic mice, oim-a2(I) [transgenic wildtype] and cross-link minus [oim-alpha2(I)-lys-5N, oim- alpha2(I)-his-92, and oim-alpha2(I)-hyl-87)]. The effects of these transgenes in vivo will be characterized by several parameters: gross morphology, tissue specific expression, crosslink formation, rate of bone formation, and biomechanical strength of skin, bone and tendon. To evaluate in vitro the tissue specific biochemical role of crosslinks primary osteoblast (mineralizing) and skin fibroblast (non-mineralizing) cultures from these transgenic oim mice will be established and their ability to accumulate matrix, crosslink, and mineralize collagenous matrix will be determined. Understanding the role of specific crosslinks in different tissues is essential to understanding the structure/function of collagen in the fibrillar architecture may play a significant role in our understanding the pathogenesis of certain collagen disorders, and aging of the musculoskeletal system.