COLLAGEN-PROTEOGLYCAN INTERACTION IN CONNECTIVE TISSUE

结缔组织中胶原蛋白-蛋白聚糖的相互作用

• 批准号: 6795455
• 负责人: James D SanAntonio
• 金额: $ 11.78万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-20 至 2006-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6795455
• 关键词: binding sites; collagen; connective tissue development; connective tissue disorder; decorin; electron microscopy; electrophoresis; extracellular matrix; laboratory rat; ligands; microarray technology; peptide library; polymerase chain reaction; protein binding; protein protein interaction; proteoglycan; recombinant proteins; transfection

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.

描述(申请人提供)：超过300个I型胶原突变与人类遗传性结缔组织(CT)疾病有关，其特征是基质组装、稳定性和功能改变。蛋白扫描蛋白(PGs)包括核心蛋白聚糖、角化蛋白聚糖和LUMICAN，它们存在于各种组织的I型胶原纤维上，被认为是调节纤维组装和侧向结合的物质。我们认为，这种PG-I型胶原相互作用是健康组织中基质结构的关键调节因素，它们的破坏可能是某些CT疾病的潜在机制。我们专门绘制了I型胶原上配体结合位点和突变位置之间的空间关系(DiLullo等人，2002，J.Biol。Chem，277,4223)，并报道了PGs可能影响I型胶原的一些功能。尽管如此，他们的互动网站仍然定义不清。此外，我们的图谱显示，成骨不全和其他疾病的一些突变与假定的PG结合位点共定位，以及缺乏已报道突变的三螺旋区域，进一步暗示PG-胶原相互作用是基质结构的关键调节因素。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型胶原模拟三螺旋多肽(THPS)，并利用它们构建胶原肽微阵列；2)提纯I型胶原结合PG，并通过亲和电泳法筛选其在I型胶原中的结合部位；以及3)制备携带突变的PG结合区域的重组人Ope I型胶原，并评估其对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PGs在I型胶原组装、功能以及在人类疾病中的作用的了解，2)探索胶原肽芯片用于胶原激活配体功能筛选的可行性，3)领导建立转基因模型来探索Pg-I型胶原相互作用的体内功能。