AGGRECAN SUBSTRATE CONSTRUCTS FOR AGGRECANASE CATABOLISM

聚集蛋白是软骨细胞外基质的一种大型杂化蛋白多糖，对软骨的功能特性至关重要。我们构建了全长牛聚合蛋白的表达载体，并在瞬时转染的COS-7细胞中证实了聚合蛋白的表达。我们建议构建一系列诱变的聚集蛋白表达载体，用于分析聚集酶介导的聚集蛋白分解代谢，如通常发生在发育和成熟过程中，并在衰老和骨关节炎过程中加速。哺乳动物聚集蛋白的球间结构域（IGD）包含一个易被基质金属蛋白酶裂解的位点，以及一个被称为聚集酶活性的附加位点。在聚集蛋白的CS-2结构域中，已经描述了聚集酶介导的裂解的其他位点。有证据表明，n -链或o -链的糖基化可能影响聚聚糖酶位点对裂解的敏感性，并且糖基化模式可能随年龄而变化。因此，聚集酶的周转可能受到聚集酶裂解位点附近的糖基化模式的调控。利用上述聚集蛋白表达结构，我们提出了这些假设，即这些高度保守的蛋白酶裂解位点，以及年龄特异性糖基化的侧翼位点，在正常软骨中聚集蛋白的转换调节中很重要。了解最易受蛋白水解裂解影响的位点将增强对抗骨性关节炎软骨中聚集蛋白降解的努力。为了解决这一假设，我们提出以下目标：具体目标1：构建诱变的全长聚合蛋白表达载体。我们将诱变IGD内的聚合酶切割位点和CS-2结构域内的聚合酶位点进行实验，以确定它们各自对聚合酶周转的贡献。我们将在大鼠软骨肉瘤细胞（RCS）或原代牛软骨细胞中产生裂解位点突变和保守糖基化位点突变，并瞬时表达重组聚集蛋白。具体目标2：开发利用特异性诱变底物分析聚集蛋白分解代谢的方法。在这些细胞中产生的重组聚集蛋白将作为底物进行测试，使用琼脂糖培养的ra刺激的RCS细胞作为聚集酶源。裂解产物将使用裂解新表位特异性抗血清进行Western blot分析。

Aggrecan is a large hybrid proteoglycan of the cartilage extracellular matrix, which is critical to cartilage functional properties. We have constructed an expression vector coding for full-length bovine aggrecan and have demonstrated expression of aggrecan in transiently transfected COS-7 cells. We propose to construct a series of mutagenized aggrecan expression vectors that can be used to analyze aggrecanase-mediated aggrecan catabolism such as normally occurs during development, and maturation, and which is accelerated during aging and osteoarthritis. The interglobular domain (IGD) in mammalian aggrecan contains a site susceptible to cleavage by matrix metalloproteinases as well as at an additional site by an enzyme activity referred to as aggrecanase. Additional sites for aggrecanase-mediated cleavage have been described in the CS-2 domain of aggrecan. There is evidence that N-linked or O-linked glycosylation may influence the susceptibility of aggrecanase sites to cleavage, and that glycosylation patterns may vary with age. Aggrecan turnover, therefore, may be regulated by the glycosylation pattern near the aggrecanase cleavage site. Using the aggrecan expression construct described above, we propose to these the HYPOTHESIS that these highly conserved proteinase cleavage sites, as well as flanking sites for age- specific glycosylation, are important in regulation of turnover of aggrecan in normal cartilage. An understanding of sites which are most susceptible to proteolytic cleavage will enhance efforts to antagonize aggrecan degradation in osteoarthritic cartilage. To address this hypothesis we propose the following aims: Specific Aim 1: Construction of mutagenized full-length aggrecan expression vectors. We will mutagenize aggrecanase cleavage sites within the IGD and aggrecanase sites within the CS-2 domain for experiments to ascertain their respective contributions to aggrecan turnover. We will generate cleavage site mutants, and mutations in conserved glycosylation sites, and transiently express recombinant aggrecan in rat chondrosarcoma cells (RCS) or in primary bovine chondrocytes. Specific Aim 2: Development of assays to analyze aggrecan catabolism using specifically mutagenized substrates. Recombinant aggrecan produced in these cells will be tested as a substrate using RA-stimulated RCS cells cultured in agarose as an aggrecanase source. Cleavage products will be characterized by Western blot analysis using cleavage neoepitope-specific antisera.