Matrilysin cleavage of corneal collagen type XVIII NC1 domain and generation of a 28-kDa fragment.
OBJECTIVE
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.
Lin
H C
HC
Department of Ophthalmology, Massachusetts Eye and Ear Infirmary and the Schepens Eye Research Institute, Boston, Massachusetts, USA.
Chang
J H
JH
Jain
S
S
Gabison
E E
EE
Kure
T
T
Kato
T
T
Fukai
N
N
Azar
D T
DT
D000595:Amino Acid Sequence; D020533:Angiogenesis Inhibitors / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D000917:Antibody Formation; D001485:Basement Membrane / Q000378:metabolism; D015153:Blotting, Western; D003094:Collagen / Q000737:chemistry / Q000378:metabolism*; D043170:Collagen Type XVIII; D003315:Cornea / Q000187:drug effects* / Q000378:metabolism; D003886:Descemet Membrane / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D043169:Endostatins; D019573:Epithelium, Corneal / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D020783:Matrix Metalloproteinase 7 / Q000276:immunology / Q000494:pharmacology*; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D011817:Rabbits
D020533:Angiogenesis Inhibitors; D043170:Collagen Type XVIII; D043169:Endostatins; D010446:Peptide Fragments; D003094:Collagen; D020783:Matrix Metalloproteinase 7