REGULATION OF AGGRECAN CATABOLISM

描述(申请人提供)：Aggrecan是一种大分子杂交蛋白多糖 软骨细胞外基质，这是软骨功能的关键 属性。哺乳动物aggrecan的球间结构域(IGD)包含 聚集葡聚糖酶和基质金属蛋白酶的裂解位点，以及其他位点 对于Aggrecanase介导的切割，已在CS-2结构域中描述。我们 构建了首个全长aggrecan表达载体 以前使用的更小的aggrecan子域结构的限制 可致突变的阿奇卡那酶底物。我们提出了这样一个假设，即 聚集素中保守的蛋白水解酶切割位点在调控中起重要作用 阿格利康成交额。我们进一步假设软骨细胞可以调节 通过改变聚集素的糖基化模式实现聚集素的分解代谢 底物。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 被阿特卡纳酶切割。已知的Aggrecanase切割位点的比较 Aggrecan和Brivican揭示了侧翼集群的保存 残留物。我们将确定这些侧翼残留物是否需要 通过取代保守残基和检测Aggrecanase切割 每个部位都有卵裂。具体目标2：确定功能 Aggrecan中裂解位点之间的关系。基质金属丝裂蛋白的切割 IGD已被证明抑制IGD内的侵袭性聚糖酶切割， 提示Aggrecanase底物中G1结构域中的基序参与 承认。我们将对这些G1基序进行突变，并对其进行聚集聚糖酶的检测 IGD位点的卵裂。也有证据表明，aggrecan的卵裂 C-末端位于前面，并且可能是内切割的先决条件。 IGD。我们将突变CS-2结构域中的聚集酶位点，并确定 直接决定IGD上的切割是否被抑制。具体目标3：确定 Aggrecanase裂解的敏感性是否可能受已知和 Aggrecan的KS替代的潜在位点。我们将确定最优的 表达含KS聚集素的细胞类型。我们会诱变 Ig D和CS-2结构域“节点区”中的苏氨酸残基可预防 KS替代和阿克葡聚糖酶敏感性测定。野生型和突变型 Aggrecan结构将在分离自软骨的软骨细胞中表达 以确定是否存在特定于年龄的模式 影响侵袭性葡聚糖酶敏感性的糖基化作用。

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.