STRUCTURE/FUNCTION RELATIONSHIP OF LUMICAN

University Of Cincinnati, Cincinnati OH

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Abstract

DESCRIPTION: (Applicant's Abstract) Corneal strength and transparency rely on an organized stromal collagenous matrix. Lumican (Lum) a keratan sulfate proteoglycan (KSPG), belongs to the small leucine-rich proteoglycan family (SLRPs). Lumican and other corneal proteoglycans, i.e., keratocan and mimecan are important regulatory molecules that modulate the different steps in collagen fibrillogenesis during the development and in the maintenance of transparent corneas. This is best exemplified in lumican deficient mice (Lum-/-), which are characterized by cloudy cornea due to formation of irregular stromal collagenous matrix. It is hypothesized that lumican like members of SLRPs exert its effect on collagen fibrillogenesis via its bifunctional characters: 1) protein moiety binding collagen fibrils and 2) highly charged glycosaminoglycans regulating interfibrillar spacings. To examine this hypothesis, Lum+/- and Lum-/- mice have been produced via gene targeting techniques. Aim 1. To further characterize the lumican deficient mice, collagenous matrix in various connective tissues, e.g., cornea, sclera, heart, kidney and lung, will be examined by transmission electron microscopy. Preliminary data revealed that corneal epithelial cells expressed lumican during corneal wound healing. To examine whether lumican plays any role in epithelial cell migration, anti-lumican antibodies will be added to the corneas of epithelium debridements. Aim 2. To elucidate the domain functions, mutant lumicans with substitutions of Y -> F, C -> S, and N -> T at strategic loci will be purified from cultured Lum-/- fibroblasts derived from lumican deficient mice that are transfected with pCMV-Lum Y-F, pCMV-Lum C-S and pCMV-Lum N-T cDNA constructs, respectively. The mutant lumicans will then be used in in vitro collagen binding and fibrillogenesis assays. Stable transformants of Lum-/- fibroblasts will be used in studies of ex vivo fibrillogenesis. Thus, the domain functions of lumican can be defined. Aim 3. To examine the effects of mutant lumican in vivo appropriate genomic DNA constructs, e.g. LumY-F, LumE-S and LumN-T will be used to target deltaLumHPRT-E14TG2aES cells. The resulting homologous recombinant ES cells will be used to prepare knock-in mice, e.g., LumS/S, LumF/F and LumT/T. The phenotypic changes in ECM of such knock- in mice will be examined.