Reduction of Type V Collagen Using a Dominant-Negative Strategy Alters the Regulation of Fibrillogenesis and Results in the Loss of Corneal-Specific Fibril Morphology

• Published in: The Journal of cell biology Vol. 135; no. 5; pp. 1415 - 1426
• Main Authors: Marchant, Jeffrey K., Hahn, Rita A., Linsenmayer, Thomas F., Birk, David E.
• Format: Journal Article
• Language: English
• Published: United States Rockefeller University Press 01.12.1996; The Rockefeller University Press
• Subjects: Animals; Base Sequence; Cell lines; Cells; Cells, Cultured; Cellular biology; Chick Embryo; Cloning, Molecular; Collagen - biosynthesis; Collagen - genetics; Collagen - metabolism; Collagen - ultrastructure; Collagens; Cornea; Cornea - metabolism; Cornea - ultrastructure; Fibroblasts; Gels; Gene Expression; Genes, Reporter; Genes, Synthetic; Genetic Vectors; Lysosomes - metabolism; Messenger RNA; Molecular chains; Molecular Sequence Data; Molecules; Proteins; Reticuloendotheliosis virus - genetics; Reticuloendotheliosis virus - physiology; Solar fibrils; Viruses
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.135.5.1415

A number of factors have been implicated in the regulation of tissue-specific collagen fibril diameter. Previous data suggest that assembly of heterotypic fibrils composed of two different fibrillar collagens represents a general mechanism regulating fibril diameter. Specifically, we hypothesize that type V collagen is required for the assembly of the small diameter fibrils observed in the cornea. To test this, we used a dominant-negative retroviral strategy to decrease the levels of type V collagen secreted by chicken corneal fibroblasts. The chicken α1(V) collagen gene was cloned, and retroviral vectors that expressed a polycistronic mRNA encoding a truncated α1(V) minigene and the reporter gene LacZ were constructed. The efficiency of viral infection was 30-40%, as determined by assaying β-galactosidase activity. To assess the expression from the recombinant provirus, Northern analysis was performed and indicated that infected fibroblasts expressed high steady-state levels of retroviral mRNA. Infected cells synthesized the truncated α1(V) protein, and this was detectable only intracellularly, in a distribution that colocalized with lysosomes. To assess endogenous α1(V) protein levels, infected cell cultures were assayed, and these consistently demonstrated reductions relative to control virus-infected or uninfected cultures. Analyses of corneal fibril morphology demonstrated that the reduction in type V collagen resulted in the assembly of large-diameter fibrils with a broad size distribution, characteristics similar to fibrils produced in connective tissues with low type V concentrations. Immunoelectron microscopy demonstrated the amino-terminal domain of type V collagen was associated with the small-diameter fibrils, but not the large fibrils. These data indicate that type V collagen levels regulate corneal fibril diameter and that the reduction of type V collagen is sufficient to alter fibril assembly so that abnormally large-diameter fibrils are deposited into the matrix.