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Invest Ophthalmol Vis Sci. 1998 Sep;39(10):1789-96.

Isolation and characterization of human cathepsin V: a major proteinase in corneal epithelium.

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  • 1Institute for Molecular and Cellular Biology, Osaka University, Suita, Japan.

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.

PMID:
9727401
[PubMed - indexed for MEDLINE]
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