REGULATION OF AGGRECAN CATABOLISM

描述（由申请人提供）：聚集蛋白聚糖是一种大型杂合蛋白聚糖 软骨细胞外基质，对软骨功能至关重要 属性。哺乳动物聚集蛋白聚糖中的球间结构域 (IGD) 包含 聚集蛋白聚糖酶和基质金属蛋白酶切割位点以及其他位点 聚集蛋白聚糖酶介导的切割已在 CS-2 结构域中进行了描述。我们 构建了第一个全尺寸聚集蛋白聚糖表达载体来克服 先前使用的较小聚集蛋白聚糖子域构造的局限性 可诱变的聚集蛋白聚糖酶底物。我们提出这样的假设：高度 聚集蛋白聚糖中保守的蛋白酶切割位点对于调节 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来进行聚集蛋白聚糖分解代谢 基材。为了解决这些假设，我们提出以下具体目标： 具体目标1：确定识别和识别所需的氨基酸残基 被聚集蛋白聚糖酶切割。已知聚集蛋白聚糖酶切割位点的比较 聚蛋白聚糖和短蛋白聚糖揭示了侧翼簇的保守性 残留物。我们将确定这些侧翼残基是否需要 通过替换保守残基并测定聚集蛋白聚糖酶的切割 每个位点都有裂解。具体目标 2：确定泛函 聚集蛋白聚糖中切割位点之间的关系。 MMP 位点的裂解 IGD 已被证明可以抑制 IGD 内的聚集蛋白聚糖酶裂解， 表明聚集蛋白聚糖酶底物的 G1 结构域中的基序参与 认可。我们将对这些 G1 基序进行诱变并进行蛋白聚糖酶测定 IGD 位点的裂解。还有证据表明聚集蛋白聚糖的裂解 C 端位点位于前面，并且可能是 C 端位点内裂解的先决条件。 IGD。我们将诱变 CS-2 域内的聚集蛋白聚糖酶位点并确定 直接判断 IGD 处的裂解是否受到抑制。具体目标 3：确定 对聚集蛋白聚糖酶裂解的敏感性是否可以通过已知和 聚集蛋白聚糖 KS 替代的潜在位点。我们将确定最优的 用于表达含有 KS 聚集蛋白聚糖的细胞类型。我们将诱变 IGD 和 CS-2 结构域的“节点”区域中的苏氨酸残基可防止 KS 替代和软骨聚集蛋白聚糖酶敏感性测定。野生型和突变型 聚集蛋白聚糖构建体将在从软骨中分离的软骨细胞中表达 不同年龄的动物，以确定是否存在特定于年龄的模式 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.