July 1988
Volume 29, Issue 7
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Articles  |   July 1988
Corneal collagen fibrils: dissection with specific collagenases and monoclonal antibodies.
Author Affiliations
  • J M Fitch
    Department of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston, Massachusetts 02111.
  • D E Birk
    Department of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston, Massachusetts 02111.
  • A Mentzer
    Department of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston, Massachusetts 02111.
  • K A Hasty
    Department of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston, Massachusetts 02111.
  • C Mainardi
    Department of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston, Massachusetts 02111.
  • T F Linsenmayer
    Department of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston, Massachusetts 02111.
Investigative Ophthalmology & Visual Science July 1988, Vol.29, 1125-1136. doi:
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      J M Fitch, D E Birk, A Mentzer, K A Hasty, C Mainardi, T F Linsenmayer; Corneal collagen fibrils: dissection with specific collagenases and monoclonal antibodies.. Invest. Ophthalmol. Vis. Sci. 1988;29(7):1125-1136.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

To investigate the relationship of collagen types I and V within corneal fibrils, and the collagenolytic mechanisms potentially involved in corneal development and remodeling, we have incubated cryostat sections of avian corneas with collagenases that specifically degrade collagen types I or V to digest selectively the collagen in situ. These preparations were then analyzed by immunofluorescence histochemistry and immunoelectron microscopy using anti-collagen type-specific monoclonal antibodies. Digestion of corneal sections with the type I-specific collagenase ("I'ase") exposed antigenically masked type V collagen, indicating that epitopes on type V collagen in heterotypic fibrils are inaccessible to the antibody due to their interaction with type I collagen. Sections digested with the type V collagen-degrading enzyme ("V'ase") showed no removal of type V collagen. However, after the fibrillar structure was disrupted by acetic acid treatment before enzyme digestion, the type V collagen was then degraded. Likewise, prior digestion of type I collagen by I'ase also rendered type V collagen susceptible to digestion by V'ase. These results suggest that the cleavage sites on type V collagen also are buried within heterotypic fibrils and therefore inaccessible to the enzyme. They also document, for the first time, V'ase activity against type V collagen in situ. Electron microscopic observations of sections partially digested with the I'ase revealed many short striated fibrillar segments from which smaller filaments protrude. Both the striated regions and some of the filaments were labeled by an antibody against type I collagen; anti-type V antibody reacted preferentially with the thin filaments. Thus avian corneal fibrils contain type I collagen, in which filaments of type V collagen are embedded. Complete removal of the fibrillar stromal matrix in the course of normal or pathological remodeling requires at least two different collagenases acting in concert.

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