Structure/Function Relationship of The Lumican Gene

描述：角膜透明需要高水平的间质胶原原纤维组织。众所周知，蛋白聚糖在这种组织良好的胶原基质的形成中起着至关重要的作用，胶原基质可以解释角膜的强度和透明度。角膜硫酸角蛋白聚糖，即lumican （Lum）、keratocan （Kera）和mimecan (Mime)，属于富含亮氨酸的小重复蛋白聚糖（SLRP）家族。我们之前的研究结果表明，单个KSPGs在维持角膜透明度方面具有不同的作用。像其他SLRP蛋白一样，lumican作为胶原纤维形成的调节剂。越来越多的证据表明，lumican是一种与细胞表面受体结合的基质因子，其作用于角膜创面愈合过程中上皮细胞的迁移和增殖、肿瘤细胞的转移、损伤晶状体上皮-间质转化（EMT）的诱导以及角化蛋白基因（Kera）表达的调控等多种细胞功能。我们假设lumican调节基质胶原基质形成和细胞功能的分子机制源于核心蛋白特定结构域的独特氨基酸序列。本研究拟进一步确定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。特异性Aim 1A， 1B和1C将通过瘤内注射lumican质粒DNA，即pSecLumY20F （Y20残基被F取代），Y22F, Y20FY22F， C41S和C295S，以及N88T， N160T和N252T，在Lum-/-小鼠体内定义lumican的结构/功能关系。这类lumican突变蛋白的合成和分泌将分别通过western blot和免疫组织化学分析来确定lumican分子的N端结构域和c端结构域以及KS- GAG链修饰在体内胶原纤维形成中的作用。目的1D是用上述质粒编码的纯化重组蛋白进一步确定体外lumican结合胶原蛋白的结构/功能。特异性目的2是通过基质注射Lum-/-小鼠，确定能够通过编码融合lumican/keratocan结构域的质粒增强keratocan表达的lumican结构域。在Specific Aim 3中，我们将从角质细胞和巨噬细胞中分离和表征lumican受体。确定在这些不同功能中具有活性的lumican结构域，然后可以测试其在治疗涉及lumican的疾病中的潜在用途，例如，持续和过度炎症、受伤角膜的角膜透明、改善糖尿病伤口愈合等。

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.