REGULATION OF AGGRECAN CATABOLISM

描述（由申请人提供）：聚集蛋白聚糖是一种大的杂合蛋白聚糖， 软骨细胞外基质，这是至关重要的软骨功能 特性.哺乳动物聚集蛋白聚糖中的球间结构域（IGD）含有 聚集蛋白聚糖酶和基质金属蛋白酶切割位点，以及另外的位点 对于聚集蛋白聚糖酶介导的切割，已经在CS-2结构域中描述。我们 构建了第一个完整的聚集蛋白聚糖表达载体， 以前用作蛋白聚糖的较小聚集蛋白聚糖亚结构域构建体的局限性 可诱变的聚集蛋白聚糖酶底物。我们提出假设，高度 聚集蛋白聚糖中保守的蛋白酶切割位点在调节 聚集蛋白聚糖营业额。我们进一步假设软骨细胞可以调节 通过改变聚集蛋白聚糖上的糖基化模式 衬底为了解决这些假设，我们提出了以下具体目标： 具体目标1：确定识别和识别所需的氨基酸残基， 通过聚集蛋白聚糖酶裂解。比较已知的聚集蛋白聚糖酶切割位点， 聚集蛋白聚糖和短蛋白聚糖揭示了侧翼蛋白簇的保守性， 残基我们将确定这些侧翼残基是否是 通过替换保守残基的聚集蛋白聚糖酶切割， 在每个位点上的分裂。具体目标2：确定功能 聚集蛋白聚糖中切割位点之间的关系。在MMP位点的裂解， IGD已显示抑制IGD内的聚集蛋白聚糖酶切割， 提示聚集蛋白聚糖酶底物中G1结构域中的基序参与 识别.我们将对这些G1基序进行诱变并测定聚集蛋白聚糖酶 在IGD位点的裂解。也有证据表明聚集蛋白聚糖的裂解 C-末端位点先于，并且可能是在细胞内切割的先决条件。 IGD。我们将对CS-2结构域内的聚集蛋白聚糖酶位点进行诱变， 直接检测IGD处的裂解是否受到抑制。具体目标3：确定 对聚集蛋白聚糖酶切割的敏感性是否可以由已知的和 聚集蛋白聚糖的KS取代的潜在位点。我们将确定最佳的 表达含KS聚集蛋白聚糖的细胞类型。我们会进行诱变 在IGD和CS-2结构域的“节点”区域中的苏氨酸残基，以防止 KS取代和聚集蛋白聚糖酶敏感性的测定。野生型和突变型 聚集蛋白聚糖构建体将在分离自软骨的软骨细胞中表达 不同年龄的动物，以确定是否有一个特定的年龄模式， 影响聚集蛋白聚糖酶敏感性的糖基化。

DESCRIPTION (provided by applicant): Aggrecan is a large hybrid proteoglycan of the cartilage extracellular matrix, which is critical to cartilage functional properties. The interglobular domain (IGD) in mammalian aggrecan contains aggrecanase and matrix metalloproteinase cleavage sites, and additional sites for aggrecanase-mediated cleavage have been described in the CS-2 domain. We have constructed the first full-sized aggrecan expression vector to surmount the limitations of smaller aggrecan subdomain constructs used previously as mutagenizable aggrecanase substrates. We propose the hypothesis that highly conserved proteinase cleavage sites in aggrecan are important in regulation of aggrecan turnover. We further hypothesize that the chondrocyte can regulate aggrecan catabolism by altering glycosylation patterns on the aggrecan substrate. To address these hypotheses, we propose the following Specific Aims: Specific Aim 1: To determine amino acid residues required for recognition and cleavage by aggrecanase. Comparison of known aggrecanase cleavage sites in aggrecan and brevican have revealed conservation of clusters of flanking residues. We will establish whether these flanking residues are required for cleavage by aggrecanase by replacing conserved residues and assaying for cleavage at each site. Specific Aim 2: To determine the functional relationships between cleavage sites in aggrecan. Cleavage at the MMP site in the IGD has been shown to inhibit aggrecanase cleavage within the IGD, suggesting the involvement of motifs in the G1 domain in aggrecanase substrate recognition. We will mutagenize these G1 motifs and assay for aggrecanase cleavage at the IGD site. There is also evidence that cleavage of aggrecan C-terminal sites precedes, and may be a prerequisite for cleavage within the IGD. We will mutagenize aggrecanase sites within the CS-2 domain and determine directly whether cleavage at the IGD is inhibited. Specific Aim 3: To determine whether susceptibility to aggrecanase cleavage may be regulated by known and potential sites for KS substitution of aggrecan. We will determine the optimal cell type for expression of KS-containing aggrecan. We will mutagenize threonine residues in the IGD and "nodal" region of the CS-2 domain to prevent KS substitution and assay for aggrecanase susceptibility. Wild-type and mutant aggrecan constructs will be expressed in chondrocytes isolated from cartilage of different aged animals, to determine if there is an age-specific pattern of glycosylation that influences aggrecanase susceptibility.