内吞性胶原蛋白受体Endo180 的结构研究

The breakdown and remodeling of the extracellular matrix (ECM) and the basement membrane are important steps in embryonic growth and tissue rearrangements in the healthy body, and in invasive cancer growth. The collagens make up by far the most abundant component in ECM. Collagen turnover is a complicated process and involves different mechanisms and degradation pathways. The unique structure of collagens makes them resilient to most means of proteolytic attack and only relatively few proteases are known to degrade collagens in their native state. Most of these active collagenases are belong to the matrix metalloproteinase family. In addition to extracellular degradation, a pathway for intracellular degradation of collagen has been identified. This pathway involves collagen endocytosis mediated by Endo180/uPARAP (the urokinase plasminogen activator receptor-associated protein, uPARAP), a member of the mannose receptor family of endocytic receptors. Endo180 is expressed at sites with ongoing tissue remodeling and functions in delivering collagens for intracellular degradation in lysosomes. Recent research has revealed that Endo180 can exist in at least two conformations: an extended conformation with the N-terminal cysteine- rich domain pointing outwards from the cell membrane and a bent conformation where the N-terminal domains fold back to interact with C-type lectin-like domain 2 at the middle of the structure. The alternation between bent and extended conformations in the Endo180, regulated by differences in the pH, might regulate ligand binding. To date, higher resolution structural details for Endo180 function remains largely unexplored. In this project, we plan to apply X-ray crystallography, in combination with other biochemical or cellular functional assays, to study the structure of Endo180 and its complex with ligands under different pH. The study will provide detailed structural information at the atomic level for the understanding of how domain rearrangement under different pH regulate ligand binding, and will provide critical molecular insights into Endo180 function in bone development, fibrosis protection and tumor metastasis.

胶原蛋白作为细胞外基质的主要成分，通过与细胞外基质或细胞受体的相互作用，诱导上皮细胞等的增殖分化和迁移，起支撑器官和保护机体的重要机能。对胶原蛋白分解代谢的传统研究主要集中在少数特异性蛋白酶介导的胞外降解。然而，近年的研究表明，内吞及在胞内的进一步降解对胶原蛋白的分解代谢至关重要。胶原蛋白的内吞主要是通过内吞性胶原蛋白受体Endo180介导完成，而Endo180介导的配体内吞与其pH 诱导的结构域重排关系密切。迄今为止，尚无Endo180调控配体结合的详细结构信息。本项目拟采用X-射线晶体衍射技术为主要研究手段，结合其他生物化学和细胞实验开展Endo180多结构域蛋白及其与配体复合物在不同pH 条件下的结构研究，通过精细的结构信息解释Endo180不同生理条件下与配体结合或释放的分子机制，从分子水平上阐明Endo180在骨发育、纤维沉积清除及肿瘤迁移等方面发挥作用的机理。

尿激酶受体相关蛋白（Urokinase plasminogen activator receptor associated protein (uPARAP) ，或称Endo180)是甘露糖受体家族的一个主要成员，其通过与胶原蛋白和尿激酶受体等蛋白的相互作用，在细胞外基质重塑中起重要作用。本课题在执行期间，根据项目申请书中的研究内容，采用X-射线晶体衍射技术为主要研究手段，结合其他生物化学和细胞实验开展uPARAP及其与不同配体复合物结构研究。通过本项目，我们解析了uPARAP配体结合区在酸性、中性（有或无钙离子）及与糖配体的三维蛋白质晶体结构。对系列结构的解析、分析及对比表明，uPARAP配体结合区的四个结构域以L形排列。其中FnII domain的疏水性口袋（预测的胶原蛋白结合区）结合一个长链PEG分子，这个区域的关键氨基酸的突变大大降低了uPARAP对胶原蛋白的结合和内吞。CTLD1和CTLD2通过long loop region（LLR）相互作用。钙离子调节着CTLD LLR的构象变化，进而引起了CTLD1和CTLD2作用方式和相对位置的变化。而uPARAP配体结合区与糖配体的晶体结构显示，糖结合在uPARAP CTLD2的钙结合位点2附近，结合模式与MBP中糖分子的结合相似。糖的结合并没有引起uPARAP的构象变化。根据这些结果，我们推测在低pH和低钙离子条件下，uPARAP可能通过CTLD1和CTLD2的构象变化，进行结构域重排，从而释放配体。以上这些精细的结构信息解释uPARAP不同生理条件下与配体结合或释放的分子机制，完成了项目申请书中的研究计划。除此以外，我们还筛选到两种uPARAP胶原蛋白结合多肽，为今后进一步uPARAP介导的胶原蛋白内吞打下了良好的基础。