COLLAGEN-PROTEOGLYCAN INTERACTION IN CONNECTIVE TISSUE

描述（由申请人提供）：I型胶原蛋白中超过300个突变与人类遗传性结缔组织（CT）疾病相关，其特征是基质组装、稳定性和功能改变。蛋白蛋白扫描蛋白（PGs）包括decorin， keratocan和lumican存在于各种组织的I型胶原原纤维上，并被认为可以调节原纤维的组装和横向结合。我们认为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子，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型胶原的配体结合位点和突变位置之间的空间关系（DiLullo et al., 2002， J. Biol.）。化学，277,4223)，并报道pg可能影响一些I型胶原蛋白的功能。尽管如此，它们相互作用的地点仍然不明确。此外，我们的图谱显示，成骨不全症和其他疾病的一些突变与假定的pg结合位点共定位，缺少突变的三螺旋区域也是如此，进一步暗示pg -胶原相互作用是基质结构的关键调节因子。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（THPs），并利用它们构建胶原肽微阵列；2)利用肽芯片和亲和共电泳纯化I型胶原结合蛋白pg，筛选其在I型胶原中的结合位点；3)生成携带突变pg结合区域的重组人Ope I胶原，并评估其对pg -胶原相互作用和胶原蛋白的影响

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.