COLLAGEN-PROTEOGLYCAN INTERACTION IN CONNECTIVE TISSUE

描述（由申请人提供）：I型胶原蛋白中超过300种突变与人类遗传性结缔组织（CT）疾病相关，其特征为基质组装、稳定性和功能改变。蛋白聚糖（PG），包括核心蛋白聚糖、角质蛋白聚糖和光蛋白聚糖，存在于各种组织中的I型胶原纤维上，并被提出调节纤维组装和侧向缔合。我们认为，这种PG-I型胶原蛋白的相互作用是健康组织中基质结构的关键调节因子，它们的破坏可能是某些CT疾病的潜在机制。我们特别绘制了配体结合位点和I型胶原上突变位置之间的空间关系（DiLullo等人，2002，J. Biol. Chem，277，4223），并报道PG可影响许多I型胶原蛋白功能。尽管如此，它们相互作用的地点仍然不明确。此外，我们的地图显示，一些突变的成骨细胞瘤和其他疾病的共同定位与推定的PG结合位点，三螺旋区，缺乏报告的突变，进一步暗示PG-胶原蛋白的相互作用作为基质结构的关键调节。因此，为了更好地确定胶原-PG相互作用对健康组织和CT疾病的贡献，我们将：1）合成携带候选PG结合序列的I型胶原模拟三螺旋肽（THP），并使用它们构建胶原肽微阵列; 2）纯化与I型胶原蛋白结合的PG，并利用肽芯片筛选其在I型胶原蛋白中的结合位点，和3）产生携带突变的PG-结合区的重组人Ope I胶原，并评估对PG-胶原相互作用和胶原的影响。 纤维形成这项工作将：1）增加我们对PG在I型胶原组装、功能和人类疾病中的作用的理解，2）探索胶原肽微阵列用于胶原活性配体的功能筛选的可行性，3）引导开发用于探测PG-I型胶原相互作用的体内功能的转基因模型。

DESCRIPTION (provided by applicant): Over 300 mutations in type I collagen are associated with heritable connective tissue (CT) disorders in humans, characterized by altered matrix assembly, stability, and function. Proteoglscans', (PGs) including decorin, keratocan and lumican reside on type I collagen fibrils in various tissues, and are proposed to regulate fibril assembly and lateral association. We propose that such PG-type I collagen interactions are key regulators of matrix structure in healthy tissues, and that their disruption may be the underlying mechanism of certain CT diseases. We specifically mapped the spatial relationships between ligand-binding sites and mutation positions on type I collagen (DiLullo et al., 2002, J. Biol. Chem, 277, 4223), and reported that PGs may impact upon a number of type I collagen functions. Despite this, their sites of interaction remain poorly defined. Moreover, our map revealed that some mutations for osteogenesis imperfecta and other diseases co-localize with putative PG-binding sites, as do triple helical regions that lack reported mutations, further implicating PG-collagen interactions as key regulators of matrix structure. Thus, to better define the contribution of collagen-PG interactions to healthy tissues and to CT diseases, we will: 1) Synthesize type I collagen mimetic triple helical peptides (THPs) carrying candidate PG-binding sequences, and use them to construct a collagen peptide microarray; 2) Purify type I collagen-binding PGs and screen for their binding sites in type I collagen through use of the peptide microarray, and by affinity coelectrophoresis; and 3) Generate recombinant human Ope I collagens carrying mutated PG-binding regions and assess the effect on PG-collagen interactions and collagen fibrillogenesis. This work will: 1) increase our understanding of the role of PGs in type I collagen assembly, function, and in human diseases, 2) explore the feasibility of collagen peptide microarrays for the functional screening of collagen-intcractive ligands, and 3) lead the way to developing transgenic models for probing the in vivo function of PG-type I collagen interactions.