AGGRECAN SUBSTRATE CONSTRUCTS FOR AGGRECANASE CATABOLISM

聚集素是软骨细胞外基质的一种大分子杂交蛋白多糖，对软骨的功能特性起着至关重要的作用。我们构建了编码牛aggrecan全长的表达载体，并在瞬时转染的COS-7细胞中证明了aggrecan的表达。我们建议构建一系列突变的aggrecan表达载体，用于分析agrecanase介导的aggrecan分解代谢，如在发育和成熟过程中正常发生的，以及在衰老和骨关节炎过程中加速的。哺乳动物蛋白聚糖的球间结构域(IGD)包含一个易被基质金属蛋白酶切割的位置，以及一个额外的被称为agrecanase的酶活性的位置。在aggrecan的CS-2结构域中，已经描述了聚集聚糖酶介导的其他切割位点。有证据表明，N-连接或O-连接的糖基化可能影响Aggrecanase位点的切割敏感性，并且糖基化模式可能会随着年龄的变化而变化。因此，聚集素的周转可能受聚集聚糖酶裂解位点附近的糖基化模式的调节。利用上面描述的aggrecan表达结构，我们提出了一种假设，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调节正常软骨中aggrecan的周转方面是重要的。了解哪些部位最容易发生蛋白水解性切割，将会加强对抗骨性关节炎软骨中聚集素降解的努力。为了解决这一假设，我们提出了以下目标：具体目标1：构建诱变的全长aggrecan表达载体。我们将突变IGD内的agrecanase裂解位点和CS-2结构域内的agrecanase位点，以确定它们各自对aggrecan周转的贡献。我们将产生裂解位点突变和保守糖基化位点突变，并在大鼠软骨肉瘤细胞(RCS)或原代牛软骨细胞中瞬时表达重组聚集素。具体目标2：利用特定诱变底物开发分析聚集素分解代谢的分析方法。在这些细胞中产生的重组聚集素将作为底物进行测试，使用在琼脂糖中培养的RA刺激的RCS细胞作为聚集聚糖酶来源。裂解产物将用裂解新表位特异性抗血清进行Western印迹分析。

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.