Structure/Function Relationship of The Lumican Gene

Lumican基因的结构/功能关系

• 批准号: 7486855
• 负责人: WINSTON W KAO
• 金额: $ 41.89万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7486855
• 关键词: Accounting; Affinity; Alberta province; Amino Acid Sequence; Amino Acid Substitution; Antibodies; Binding; Biological Assay; C-terminal; Cell Adhesion; Cell Line; Cell Surface Receptors; Cell physiology; Cell surface; Chimeric Proteins; Circular Dichroism; Collagen; Collagen Fibril; Core Protein; Cornea; Corneal Injury; DNA; Diabetes Mellitus; Disease; Electron Microscopy; Epithelial; Epithelial Cells; Experimental Designs; Extracellular Matrix; Family; Fibroblasts; Figs - dietary; GAG Gene; Gene Expression; Genes; Hybrids; Immune; Immunohistochemistry; In Vitro; Individual; Inflammation; Injection of therapeutic agent; Label; Leucine-Rich Repeat; Link; Membrane Proteins; Mesenchyme; Modification; Molecular; Mus; Mutagenesis; N-terminal; Neoplasm Metastasis; Northern Blotting; Plasmids; Play; Post-Translational Protein Processing; Precipitation; Process; Protein Binding; Proteins; Proteoglycan; Recombinant Proteins; Recombinants; Regulation; Role; Site; Stromal Cells; Structure; Structure-Activity Relationship; Surface Plasmon Resonance; Testing; Thick; Transgenic Mice; Universities; Variant; Western Blotting; Wound Healing; cell motility; cell type; design; disulfide bond; extracellular; fibrillogenesis; improved; in vivo; injured; keratan sulfate proteoglycan; lens; lumican; mRNA Expression; macrophage; migration; mutant; neoplastic cell; northern hybridization; osteoinductive factor; plasmid DNA; receptor; research study; tumorigenesis; tyrosine O-sulfate

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.

描述：角膜透明度需要高水平的基质胶原蛋白纤维。众所周知，蛋白聚糖在形成这种构成角膜强度和透明度的良好组织胶原蛋白基质中起着至关重要的作用。角膜角膜硫酸盐蛋白聚糖，即Lumican（Lum），角膜（Kera）和Mimecan（Mime）属于富含Leucine富含Leucine的重复蛋白聚糖（SLRP）家族。我们先前研究的结果表明，单个KSPG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也是胶原纤维生成的调节剂。越来越多的证据表明，Lumican是一种与细胞表面受体结合的基金，其对多种细胞功能的作用，例如上皮细胞迁移和角膜伤口愈合过程中肿瘤细胞转移，诱导上皮 - 质质转变（EMT）的诱导含囊室和调节性的keraTocan genee（kera）的表达（EMT）的诱导（EMT）。我们假设分子机制负责调节基质胶原基质的形成和细胞功能，这是由核心蛋白特定域的独特氨基酸序列引起的。拟议的研究旨在进一步确定Lumican在胶原蛋白原纤维生成，KERA表达以及分离和表征Lumican受体（S）方面的结构/功能关系。具体目的1a，1b和1c将通过基质内腔内质粒DNA注入体内定义Lumican在体内的结构/功能关系，即PSECLUMY20F（Y20残基由F），Y22F，Y22F，Y20FY22F，Y20FY22F，以及C41S和C295S和C295S，以及N888T，N1660T和N1660T和N1660T。这种Lumican突变蛋白的合成和分泌将通过Western印迹和免疫组织化学分析确定，以确定N-和C末端结构域和Lumican分子在VIVO中胶原纤维纤维发生上的KS-GAG链的作用。 AIM 1D是用上述质粒编码的纯化的重组蛋白在体外进一步定义Lumican结合胶原蛋白的结构/功能。具体目标2是确定能够通过基质 - / - 小鼠的基质注射Lum-/ - 小鼠来用编码融合Lumican/ceratocan结构域来增强始终性表达的Lumican结构域。在特定的目标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，可以测试被确定在这些多种功能中活跃的Lumican的域，以便在治疗涉及Lumican的疾病中的潜在用途，例如持续和过度炎症，受伤的角膜的角膜透明度，改善糖尿病的伤口愈合，等等。等等。