Keratan sulfate proteoglycans, such as lumican and keratocan, play an important role in corneal morphogenesis by modulating collagen fibrillogenesis during embryonic development. We examined the role of a highly conserved 37-CX₃CXCX₉C-53 domain adjacent to the N-terminus of lumican in collagen fibrillogenesis, using site-specific mutagenesis to prepare plasmid DNA encoding for wild type (CX₃CXCX₉C) and C/S mutant (Cx₃SXCX₉C) lumican. cDNAs were cloned into a expression vector. Cultures of MK/T-1 cells, an immortalized cell line from mouse keratocytes expressing human telomerase reverse transcriptase were transfected with the cDNAs. Stable transfectants were selected and cloned in the presence of zeocin. The stable transfectants maintain a dendritic morphology similar to the parental MK/T-1 cells. Western blot analysis with antibodies against c-Myc-tag and lumican detected a 42 kDa lumican protein from the culture medium of the wild type and C/S mutant transfectants. Ultrastructural analyses by transmission electron microscopy (TEM) showed that both cell lines generated a multi-layered stroma
in vitro. However, the matrix assembled by the two cell lines differed. Compared to the mutant cell line, the wild type cells assembled a more organized matrix with regions containing orthogonal collagen fibrils. In addition, the fibrils in the extracellular matrix formed by the mutant cell line showed apparent alterations in fibril packing and structure. The results indicate that the lumican C/S mutant may interfere with collagen fibrillogenesis and stromal matrix assembly, potentially due to an alteration in lumican folding.
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