Structure/Function Relationship of The Lumican Gene

描述：角膜透明需要基质胶原纤维高度组织化。众所周知，蛋白聚糖在形成这种组织良好的胶原基质中起关键作用，这种胶原基质是角膜强度和透明度的原因。角膜硫酸角质素蛋白聚糖，即，Lumican（Lum）、keratocan（Kera）和mimecan（Mime）属于小亮氨酸重复蛋白聚糖（SLRP）家族。我们以前的研究结果表明，个人KSPGs在维持角膜透明度有不同的作用。与其他SLRP蛋白一样，Lumican充当胶原原纤维形成的调节剂。越来越多的证据表明，Lumican是一种基质因子，可与细胞表面受体结合，对多种细胞功能发挥作用，如角膜伤口愈合过程中上皮细胞的迁移和增殖、肿瘤细胞的转移、诱导受损透镜的上皮-间充质转化（EMT）以及调节角膜蛋白聚糖基因（Kera）的表达。我们假设负责调节基质胶原蛋白基质形成和细胞功能的分子机制由lumican核心蛋白的特定结构域的独特氨基酸序列产生。拟定的研究旨在进一步确定Lumican在胶原纤维形成、Kera表达方面的结构/功能关系，并分离和表征Lumican受体。具体目标1A、1B和1C将通过基质内Lumican质粒DNA注射来定义Lumican在体内的结构/功能关系，即，pSecLumY20F（Y20残基被F取代）、Y22F、Y20FY22F、和C41S和C295S、以及N88T、N160T和N252T，对Lum-/-小鼠的免疫反应。将通过蛋白质印迹和免疫组织化学分析来确定这种Lumican突变蛋白的合成和分泌，以分别确定N-和C-末端结构域以及Lumican分子的KS-GAG链的修饰对体内胶原原纤维形成的作用。目的1D利用上述质粒编码的重组蛋白进一步确定Lumican结合胶原蛋白的结构和功能。具体目的2是通过Lum-/-小鼠的基质注射确定能够用编码融合Lumican/keratocan结构域的质粒增强keratocan表达的Lumican结构域。在具体目标3中，将分离和表征来自角膜细胞和巨噬细胞的lumican受体。然后可以测试被鉴定为在这些不同功能中有活性的Lumican的结构域在治疗涉及Lumican的疾病中的潜在用途，例如，持续性和过度炎症，损伤角膜的角膜透明度，改善糖尿病的伤口愈合等。

DESCRIPTION: Corneal transparency requires a high level of organization of stromal collagen fibrils. Proteoglycans are well known to play a critical role in the formation of such a well organized collagen matrix that accounts for corneal strength and transparency. Corneal keratan sulfate proteoglycans, i.e., lumican (Lum), keratocan (Kera) and mimecan (Mime), belong to small leucine-rich repeat proteoglycans (SLRP) family. Results of our previous studies suggest that individual KSPGs have distinct roles in maintaining corneal transparency. Like other SLRP proteins, lumican serves as a regulator of collagen fibrillogenesis. Ever increasing evidence indicate that lumican is a matrikine that binds to cell surface receptor(s) to exert its effects on multiple cellular functions such as epithelial cell migration and proliferation during corneal wound healing, metastasis of tumor cells, induction of epithelial-mesenchyme transition (EMT) of injured lens, and regulation of keratocan gene (Kera) expression. We hypothesize the molecular mechanism accountable for regulating stromal collagen matrix formation and cellular functions by lumican arises from unique amino acid sequences of specific domains of the core protein. The proposed studies are to further determine the structure/function relationships of lumican in respect to collagen fibrillogenesis, Kera expression, and to isolate and characterize lumican receptor(s). Specific Aim 1A, 1B and 1C will define structure/function relationship of lumican in vivo by intrastromal lumican Plasmid DNA injection, i.e., pSecLumY20F (Y20 residue substituted by F), Y22F, Y20FY22F, and C41S and C295S, and N88T, N160T and N252T, to Lum-/- mice. The synthesis and secretion of such lumican mutant proteins will be determined by western blot and immunohistochemistry analysis to determine the roles of N- and C-terminal domains and modification of KS- GAG chain of lumican molecules on collagen fibrillogenesis in vivo, respectively. Aim 1D is to further define structure/function of lumican binding collagen in vitro with purified recombinant proteins encoded by the above plasmids. Specific Aim 2 is to determine lumican domains that are capable of enhancing keratocan expression with plasmids encoding fusion lumican/keratocan domains by stroma injection of Lum-/- mice. In Specific Aim 3, lumican receptor(s) from keratocytes and macrophages will be isolated and characterized. The domains of lumican identified to be active in these diverse functions can then be tested for potential use in treating diseases involving lumican, e.g., persistent and excessive inflammation, corneal transparency of injured cornea, improved wound healing of diabetes, etc.