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 1 A, 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 1 D 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. ? ? ? ?
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