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Corneal collagen fibrils: dissection with specific collagenases and monoclonal antibodies
J M Fitch1, D E Birk, A Mentzer
1Department of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston, Massachusetts 02111.
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.
Insights
Corneal fibrils contain type I collagen with embedded type V collagen filaments. Specific collagenases are needed to degrade these fibrils during corneal remodeling.
Area of Science:
- Biochemistry
- Ophthalmology
- Connective Tissue Biology
Background:
- Collagen types I and V are key components of corneal fibrils.
- Understanding their interaction is crucial for corneal development and remodeling.
- Collagenolytic mechanisms in the cornea are not fully elucidated.
Purpose of the Study:
- To investigate the relationship between collagen types I and V within corneal fibrils.
- To explore the collagenolytic mechanisms involved in corneal development and remodeling.
Main Methods:
- Incubation of avian corneal sections with specific collagenases (type I and type V).
- Analysis using immunofluorescence histochemistry and immunoelectron microscopy.
- Selective enzymatic digestion and acetic acid treatment to assess collagen accessibility.
Main Results:
- Type I collagenase digestion exposed masked type V collagen, indicating its inaccessibility within fibrils.
- Type V collagen was degraded only after disruption of fibrillar structure or prior type I collagen digestion.
- Immunoelectron microscopy revealed type V collagen filaments embedded within a type I collagen matrix.
Conclusions:
- Corneal fibrils are composed of a type I collagen matrix with embedded type V collagen filaments.
- Cleavage sites of type V collagen are buried within heterotypic fibrils, requiring specific enzymatic action.
- Complete corneal matrix remodeling necessitates the coordinated action of at least two distinct collagenases.