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. 2001 Oct;42(11):2517-24.

Matrilysin cleavage of corneal collagen type XVIII NC1 domain and generation of a 28-kDa fragment

H C Lin  1 , J H Chang, S Jain, E E Gabison, T Kure, T Kato, N Fukai, D T Azar
Affiliations
  • PMID: 11581192

Matrilysin cleavage of corneal collagen type XVIII NC1 domain and generation of a 28-kDa fragment

H C Lin et al. Invest Ophthalmol Vis Sci. 2001 Oct.

Abstract

Purpose: To localize endostatin and collagen type XVIII in human corneas and to characterize the enzymatic action of matrix metalloproteinases (MMPs) in the cleavage of collagen type XVIII and generation of endostatin in the cornea.

Methods: Anti-endostatin and anti-hinge antibodies were generated using peptide fragments corresponding to the endostatin region and the adjacent nonendostatin hinge region of collagen XVIII noncollagenous (NC)1 domain, respectively. Confocal immunostaining was performed to localize collagen XVIII in human corneas. SV40-immortalized corneal epithelial cells were immunoprecipitated and incubated with active MMP-1, -2, -3, -7, or -9, and Western blot analysis was performed to study collagen XVIII cleavage. Incubation with MMP-7 was performed at various concentrations (0, 2, 4, and 6 microg/ml) and time intervals (0, 1, 5, and 12 hours). Purified recombinant NC1 fragment of collagen XVIII was also digested with MMP-7, and the cleavage product was sequenced.

Results: Collagen XVIII was immunolocalized to the human corneal epithelium, epithelial basement membrane, and Descemet membrane. Western blot analysis demonstrated a 180- to 200-kDa band corresponding to collagen XVIII. MMP-7 (but not MMP-1, -2, -3, and -9) cleaved corneal epithelium-derived collagen XVIII to generate a 28-kDa endostatin-spanning fragment in a time- and concentration-dependent fashion. MMP-7 cleaved purified recombinant 34-kDa NC1 fragment of collagen XVIII in the hinge region to generate a 28-kDa fragment.

Conclusions: Collagen XVIII is present in human cornea. MMP-7 cleaves the collagen XVIII NC1 domain to generate a 28-kDa fragment in the cornea.

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