GENETIC ANALYSIS OF TYPE I COLLAGEN FUNCTION

I型胶原蛋白功能的遗传分析

• 批准号: 2081358
• 负责人: CHARLOTTE L PHILLIPS
• 金额: $ 6.95万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-01-01 至 1994-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2081358
• 关键词: collagen; connective tissue development; crosslink; gene expression; genetically modified animals; laboratory mouse; protein structure function; site directed mutagenesis

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.

在I型α链中的特定氨基酸修饰 胶原蛋白被认为是生化的主要贡献者 I型胶原蛋白原纤维的物理和生物力学特性 建筑，指示矿化和 非矿物组织。 特别是共价分子交联 胶原蛋白分子之间假定为主要决定因素 稳定性，生物力学和生理化学特性 胶原蛋白纤维基质。 最近有很多关于 I型胶原蛋白交联的位置和化学性质，但非常 对它们的功能知之甚少。 我们建议定义 特定I型胶原蛋白交联的功能作用通过改变 proalpha2（i）中特定分子基因座的氨基酸残基 胶原蛋白链并评估对I型胶原蛋白的影响 体内的结构/功能在各种组织中。 为此 将创建高效率proalpha2（i）胶原蛋白表达基因 和氨基酸取代：赖氨酸5N（氨基 - 甲基肽：主要 骨骼交叉链接），组氨酸-92（螺旋：皮肤特异性交叉链接）和 羟甲氨酸-87（螺旋：骨骼和软组织交联）。 这些 构建体将被微注射到受精的OIM中（纯合为null colia2等位基因）小鼠卵母细胞生成转基因小鼠，OIM-A2（i） [转基因野生型]和交叉链接减去[oim-alpha2（i）-lys-5n，oim- alpha2（i）-His-92和Oim-Alpha2（i）-Hyl-87）]。 这些影响 体内转基因将以几个参数为特征：总体 形态，组织特异性表达，交联形成，骨骼速率 皮肤，骨骼和肌腱的形成和生物力学强度。 到 在体外评估交联的组织特异性生化作用 原代成骨细胞（矿化）和皮肤成纤维细胞（非矿物化） 这些转基因OIM小鼠的培养物将建立 积累矩阵，交联和矿化胶原式的能力 矩阵将被确定。 了解特定的交联的作用 在不同的组织中，对于理解结构/功能至关重要 纤维化建筑中的胶原蛋白的作用可能在 我们了解某些胶原蛋白疾病的发病机理，以及 肌肉骨骼系统的老化。