Cleavage of human corneal type VI collagen alpha 3 chain by matrix metalloproteinase-2.
The human cornea contains significant amounts of type VI collagen and matrix metalloproteinase-2 (MMP-2), but there has been no established relation between these two components. The objective of this study was to determine whether corneal type VI collagen was susceptible to digestion by MMP-2. Human corneas were frozen and then pulverized in liquid nitrogen. The type VI collagen was isolated by sequential extractions with sodium chloride buffer, guanidinium chloride solution, and guanidinium chloride/dithiothreitol solution. Visualization of type VI collagen alpha 3(VI) chain was made by using Western blots with specific monoclonal antibodies. The extracts were incubated up to 24 h with isolated, activated MMP-2. Within 4 h of incubation, two lower molecular weight bands (approximately 190 and 170 kDa) appeared. These bands increased in intensity with time but were not further digested into smaller fragments. This cleavage activity was inhibited by ethylenediaminetetraacetic acid (EDTA). Because type VI collagen represents approximately 25% of the corneal dry weight, its degradation properties may be important for the integrity of the stroma in scarring episodes and corneal diseases, such as keratoconus.
Myint
E
E
Department of Surgery, Cedars-Sinai Research Institute, Cedars-Sinai Medical Center, University of California at Los Angeles Medical School Affiliate 90048, USA.
Brown
D J
DJ
Ljubimov
A V
AV
Kyaw
M
M
Kenney
M C
MC
D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D003094:Collagen / Q000302:isolation & purification / Q000378:metabolism*; D003315:Cornea / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D019084:Fluorescent Antibody Technique, Indirect; D018093:Gelatinases / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D020778:Matrix Metalloproteinase 2; D008666:Metalloendopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D013552:Swine
D000911:Antibodies, Monoclonal; D003094:Collagen; D018093:Gelatinases; D008666:Metalloendopeptidases; D020778:Matrix Metalloproteinase 2