Role of Cys41 in the N-terminal domain of lumican in ex vivo collagen fibrillogenesis by cultured corneal stromal cells
Open Access
- 1 February 2003
- journal article
- Published by Portland Press Ltd. in Biochemical Journal
- Vol. 369 (3), 461-468
- https://doi.org/10.1042/bj20020593
Abstract
The keratan sulphate proteoglycan lumican regulates collagen fibrillogenesis to maintain the integrity and function of connective tissues such as cornea. We examined the role of a highly conserved cysteine-containing domain proximal to the N-terminus of lumican in collagen fibrillogenesis using site-specific mutagenesis to prepare plasmid DNA encoding wild-type murine lumican (Cys37-Xaa3-Cys41-Xaa-Cys-Xaa9-Cys) and a Cys→Ser (C/S) mutant (Cys37-Xaa3-Ser41-Xaa-Cys-Xaa9-Cys). cDNAs were cloned into the pSecTag2A vector, and cultures of MK/T-1 cells (an immortalized cell line from mouse keratocytes) were transfected with the cDNAs. Stable transformants were selected and cloned in the presence of Zeocin. All stable transformants maintained a dendritic morphology and growth rate similar to those of parental MK/T-1 cells. Western blot analysis with anti-lumican antibody detected a 42kDa lumican protein secreted into the culture medium of both wild-type and C/S mutant lumican cell lines. Ultrastructural analyses by transmission electron microscopy showed both cell lines to form a multi-layered stroma ex vivo, but the matrix assembled by the two cell lines differed. Compared with 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 exhibited alterations in fibril packing and structure. Immunostaining analysed by confocal microscopy showed a further difference in this matrix, with the marked occurrence of lumican and collagen I co-localization in the lumican wild-type cells, but a lack thereof in the lumican C/S mutant cells. The results indicate that the cysteine-rich domain of lumican is important in collagen fibrillogenesis and stromal matrix assembly.Keywords
This publication has 27 references indexed in Scilit:
- MK/T-1, an immortalized fibroblast cell line derived using cultures of mouse corneal stroma.2001
- Role of Lumican in the Corneal Epithelium during Wound HealingJournal of Biological Chemistry, 2000
- Complete Chromogen Separation and Analysis in Double Immunohistochemical Stains Using Photoshop-based Image AnalysisJournal of Histochemistry & Cytochemistry, 1999
- Heterotypic Collagen Fibrils and Stabilizing CollagensAnnals of the New York Academy of Sciences, 1990
- Type v collagen synthesis and deposition by chicken embryo corneal fibroblasts in vitroJournal of Cell Science, 1989
- In vitro Formation of Hybrid Fibrils of Type v Collagen and Type I Collagen Limited Growth of Type I Collagen Into Thick Fibrils by Type V CollagenConnective Tissue Research, 1986
- Extracellular compartments in matrix morphogenesis: collagen fibril, bundle, and lamellar formation by corneal fibroblasts.The Journal of cell biology, 1984
- Proteoglycan changes during restoration of transparency in corneal scarsArchives of Biochemistry and Biophysics, 1983
- Development of the Vertebrate CorneaInternational Review of Cytology, 1980
- The hydration of proteoglycans of bovine corneaBiochimica et Biophysica Acta (BBA) - General Subjects, 1975