Role of Cys41 in the N-terminal domain of lumican in ex vivo collagen fibrillogenesis by cultured corneal stromal cells.
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 (Cys(37)-Xaa(3)-Cys(41)-Xaa-Cys-Xaa(9)-Cys) and a Cys-->Ser (C/S) mutant (Cys(37)-Xaa(3)-Ser(41)-Xaa-Cys-Xaa(9)-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 42 kDa 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.
Carlson
Eric C
EC
Department of Ophthalmology, University of Cincinnati, 3223 Eden Avenue, Cincinnati, OH 45267, USA.
Mamiya
Kazuhisa
K
Liu
Chia-Yang
CY
Gendron
Robert L
RL
Birk
David E
DE
Funderburgh
James L
JL
Kao
Winston W-Y
WW
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D011508:Chondroitin Sulfate Proteoglycans / Q000235:genetics / Q000378:metabolism*; D003319:Corneal Stroma / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism*; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D024022:Fibrillar Collagens / Q000378:metabolism* / Q000648:ultrastructure; D007632:Keratan Sulfate / Q000235:genetics / Q000378:metabolism*; D000071234:Lumican; D051379:Mice; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009154:Mutation; D017434:Protein Structure, Tertiary; D016212:Transforming Growth Factor beta / Q000494:pharmacology
D011508:Chondroitin Sulfate Proteoglycans; D024022:Fibrillar Collagens; C000604307:Lum protein, mouse; D000071234:Lumican; D016212:Transforming Growth Factor beta; D007632:Keratan Sulfate; D003545:Cysteine