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

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.