Structure/Function Relationship of The Lumican Gene

描述：角膜透明需要高水平的基质胶原纤维组织。众所周知，蛋白多糖在形成这样一种组织良好的胶原基质中起着关键作用，这种基质解释了角膜的强度和透明度。角膜硫酸角蛋白多糖，即LUM、KERA和MIME，属于富含亮氨酸的重复蛋白多糖家族。我们先前的研究结果表明，个体KSPG在维持角膜透明度方面有不同的作用。与其他SLRP蛋白一样，LUMICAN是胶原纤维形成的调节因子。越来越多的证据表明，鲁米肯是一种与细胞表面受体(S)结合的基质因子，在角膜创伤愈合过程中对角膜上皮细胞的迁移和增殖、肿瘤细胞的转移、诱导损伤晶状体上皮细胞间充质转化、调节角蛋白聚糖基因(KERA)的表达等多种细胞功能发挥作用。我们假设LUMICAN调节间质胶原基质形成和细胞功能的分子机制来自于核心蛋白特定结构域的独特氨基酸序列。本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，并分离和鉴定LUMICAN受体(S)。特异性靶点1A、1B和1C将通过在Lum-/-小鼠体内注射Lum-/-小鼠的间质粒DNA，即pSecLumY20F(Y20残基被F取代)、Y22F、Y20FY22F、C41S和C295S以及N88T、N160T和N252T来确定Lum-in的结构/功能关系。这些突变蛋白的合成和分泌将通过蛋白质印迹和免疫组织化学分析来确定LUMICA分子的N-末端和C-末端结构域以及LUMICA分子KS-GAG链的修饰在体内胶原纤维形成中的作用。目的1D以纯化的重组蛋白为模板，在体外进一步确定LUMICAN结合胶原的结构和功能。特异目的2是通过基质注射Lum-/-小鼠，确定能够通过编码融合Lum-can/Kkeratocan结构域的质粒增强角化蛋白聚糖表达的Lumican结构域。在具体目标3中，将分离角质形成细胞和巨噬细胞的鲁米肯受体(S)并对其进行鉴定。然后，可以测试在这些不同功能中活跃的LUMICAN结构域在治疗涉及LUMICA的疾病方面的潜在用途，例如，持续和过度的炎症、受损角膜的角膜透明度、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.