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Hsin-Chiung Lin, Jin-Hong Chang, Sandeep Jain, Eric E. Gabison, Tomoko Kure, Takuji Kato, Naomi Fukai, Dimitri T. Azar; Matrilysin Cleavage of Corneal Collagen Type XVIII NC1 Domain and Generation of a 28-kDa Fragment. Invest. Ophthalmol. Vis. Sci. 2001;42(11):2517-2524.
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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
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 μg/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
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