Isolation and characterization of human cathepsin V: a major proteinase in corneal epithelium.
OBJECTIVE
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.
Adachi
W
W
Institute for Molecular and Cellular Biology, Osaka University, Suita, Japan.
Kawamoto
S
S
Ohno
I
I
Nishida
K
K
Kinoshita
S
S
Matsubara
K
K
Okubo
K
K
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western; D056668:Cathepsin L; D002403:Cathepsins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000032:analysis; D010450:Endopeptidases; D019573:Epithelium, Corneal / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D005136:Eye Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013666:Tears / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D015853:Cysteine Proteinase Inhibitors; D017931:DNA Primers; D018076:DNA, Complementary; D005136:Eye Proteins; D011994:Recombinant Proteins; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; C534281:CTSL protein, human; D056668:Cathepsin L