AGGRECAN SUBSTRATE CONSTRUCTS FOR AGGRECANASE CATABOLISM

聚集蛋白聚糖是软骨细胞外基质的一种大的杂合蛋白聚糖，对软骨的功能特性至关重要。 我们构建了编码全长牛聚集蛋白聚糖的表达载体，并证明了聚集蛋白聚糖在瞬时转染的COS-7细胞中的表达。 我们建议构建一系列诱变的聚集蛋白聚糖表达载体，其可用于分析聚集蛋白聚糖酶介导的聚集蛋白聚糖催化剂，例如通常发生在发育和成熟过程中，以及在衰老和骨关节炎过程中加速。 哺乳动物聚集蛋白聚糖中的球间结构域（IGD）含有易受基质金属蛋白酶切割的位点以及被称为聚集蛋白聚糖酶的酶活性切割的另外位点。 在聚集蛋白聚糖的CS-2结构域中已经描述了聚集蛋白聚糖酶介导的切割的其他位点。 有证据表明，N-连接或O-连接的糖基化可能会影响聚集蛋白聚糖酶位点对切割的敏感性，并且糖基化模式可能随年龄而变化。 因此，聚集蛋白聚糖周转率可能受聚集蛋白聚糖酶切割位点附近的糖基化模式调节。使用上述的聚集蛋白聚糖表达构建体，我们向这些人提出这样的假设：这些高度保守的蛋白酶切割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聚糖周转的调节中是重要的. 了解最易受蛋白水解裂解的位点将增强对抗骨关节炎软骨中聚集蛋白聚糖降解的努力。为了解决这一假设，我们提出了以下目标：具体目标1：构建诱变全长聚集蛋白聚糖表达载体。 我们将对IGD内的聚集蛋白聚糖酶切割位点和CS-2结构域内的聚集蛋白聚糖酶位点进行诱变，以进行实验，以确定它们各自对聚集蛋白聚糖周转的贡献。 我们将产生切割位点突变体，保守的糖基化位点突变，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蛋白聚糖。 具体目标2：开发使用特异性诱变底物分析聚集蛋白聚糖催化剂的测定法。 将使用在琼脂糖中培养的RA刺激的RCS细胞作为聚集蛋白聚糖酶来源，将这些细胞中产生的重组聚集蛋白聚糖作为底物进行测试。 裂解产物将通过使用裂解新表位特异性抗血清的蛋白质印迹分析来表征。

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.