September 1998
Volume 39, Issue 10
Free
Articles  |   September 1998
Isolation and characterization of human cathepsin V: a major proteinase in corneal epithelium.
Author Affiliations
  • W Adachi
    Institute for Molecular and Cellular Biology, Osaka University, Suita, Japan.
  • S Kawamoto
    Institute for Molecular and Cellular Biology, Osaka University, Suita, Japan.
  • I Ohno
    Institute for Molecular and Cellular Biology, Osaka University, Suita, Japan.
  • K Nishida
    Institute for Molecular and Cellular Biology, Osaka University, Suita, Japan.
  • S Kinoshita
    Institute for Molecular and Cellular Biology, Osaka University, Suita, Japan.
  • K Matsubara
    Institute for Molecular and Cellular Biology, Osaka University, Suita, Japan.
  • K Okubo
    Institute for Molecular and Cellular Biology, Osaka University, Suita, Japan.
Investigative Ophthalmology & Visual Science September 1998, Vol.39, 1789-1796. doi:
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    • Get Citation

      W Adachi, S Kawamoto, I Ohno, K Nishida, S Kinoshita, K Matsubara, K Okubo; Isolation and characterization of human cathepsin V: a major proteinase in corneal epithelium.. Invest. Ophthalmol. Vis. Sci. 1998;39(10):1789-1796.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

PURPOSE: To isolate and characterize a novel cathepsin gene, as part of the systematic isolation of genes uniquely active in corneal epithelium. METHODS: For the isolation of a full-length cDNA clone, a probe was selected from a set of expressed sequence tag clones classified as unique to corneal epithelium. Inserted cDNA was introduced into insect cells using a baculovirus expression system, and the secretion of recombinant protein was identified using antisera against a synthetic peptide. Proteolytic activity was determined using bovine serum albumin (BSA) as substrate. The expressions of the novel cathepsin in human cornea and other tissues were examined by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The full-length cDNA clone encoded a peptide of 334 amino acids with 82% identity with bovine cathepsin L and 77% identity with human cathepsin L when aligned. The recombinant protein produced in the baculovirus expression system cleaves BSA, and its activity was inhibited by the cysteine proteinase inhibitors E-64 and leupeptin, but not by pepstatin A, phenylmethylsulfonyl fluoride, and EDTA. By RT-PCR, a low level of expression was observed in some other epithelial tissues of ectodermal origin, but only in cornea was it higher than cathepsin L, which is known to be a general lysosomal cathepsin. Cathepsin V protein was detected in human corneal epithelium by western blot analysis, but not in tear fluid. CONCLUSIONS: The amino acid homology and proteolytic activity of the recombinant protein indicate that the novel gene is a new member of the cathepsins that have features of cysteine proteinase. Its uniquely high expression in corneal epithelium strongly implies an important role in corneal physiology.

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