Characterization of a human corneal metalloproteinase inhibitor (TIMP-1).
The gradual corneal thinning seen in keratoconus may be due to altered degradation of the corneal extracellular matrix. Studies have shown that human keratocytes produce matrix metalloproteinase-2 (MMP-2) and two proteins (28 kDa and 21 kDa) that are capable of inhibiting the activity of MMP-2. In the present study, the 28 kDa inhibitor from keratoconus keratocyte cultures has been characterized as it may be important to the elevated MMP-2 activity seen in these cultures. Biochemical analyses indicated that this keratoconus corneal inhibitor was similar to TIMP-1 from other sources. Oligonucleotides to the reported sequence of human tumor cell TIMP-1 were used for reverse-transcriptase PCR to generate a 700 bp clone of the 28 kDa inhibitor from keratoconus keratocyte cytoplasmic RNA. Sequence analysis verified that the clone was nearly identical to the reported human TIMP-1 with a single base substitution that did not affect the predicted amino acid sequence. In addition, protein translated from the clone corresponded to the expected size. This data suggests that the elevated levels of gelatinolytic activity in these keratoconus keratocyte cultures is not due to a primary alteration of the TIMP-1 molecule. Protein expression studies of the TIMP-1 clone are currently underway.
Opbroek
A
A
Department of Surgery, Cedars-Sinai Research Institute, Cedars-Sinai Medical Center, UCLA Medical School Affiliate 90048.
Kenney
M C
MC
Brown
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D
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D002478:Cells, Cultured; D003319:Corneal Stroma / Q000378:metabolism*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D018093:Gelatinases / Q000378:metabolism; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007640:Keratoconus / Q000378:metabolism*; D008297:Male; D020778:Matrix Metalloproteinase 2; D061965:Matrix Metalloproteinase Inhibitors; D008666:Metalloendopeptidases / Q000378:metabolism; D008969:Molecular Sequence Data; D009841:Oligonucleotides; D016133:Polymerase Chain Reaction; D012313:RNA / Q000378:metabolism; D019714:Tissue Inhibitor of Metalloproteinases
D017931:DNA Primers; D006023:Glycoproteins; D061965:Matrix Metalloproteinase Inhibitors; D009841:Oligonucleotides; D019714:Tissue Inhibitor of Metalloproteinases; D012313:RNA; D018093:Gelatinases; D008666:Metalloendopeptidases; D020778:Matrix Metalloproteinase 2