Abstract
A cDNA encoding a novel member of the cysteine proteinase family of proteins has been cloned from a human breast carcinoma cDNA library, by using a polymerase chain reaction-based cloning strategy. The isolated cDNA contains an open reading frame coding for a polypeptide of 321 amino acids that has been tentatively called cathepsin O. This protein presents all the structural features characteristic of the different cysteine proteinases identified to date, including the active site cysteine residue that is involved in covalent intermediate formation during peptide hydrolysis. The cathepsin O cDNA was expressed in Escherichia coli, and after purification and refolding, the recombinant protein was able to degrade the synthetic peptides benzyloxycarbonyl-Phe-Arg-7-amido-4- methylcoumarin and benzyloxycarbonyl-Arg-Arg-7-amido-4-methylcoumarin widely used as substrates for cysteine proteinases. Cathepsin O proteolytic activity was abolished by trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64), an inhibitor of this subclass of proteolytic enzymes, thus providing additional evidence that the isolated cDNA codes for an authentic cysteine proteinase. Northern blot analysis of poly(A)+ RNAs isolated from a variety of human tissues demonstrated that cathepsin O is expressed in all examined tissues, which is consistent with a putative role of this protein as a proteolytic enzyme involved in normal cellular protein degradation and turnover.
Highlights
MOLECULAR CLONING FROM A BREAST CARCINOMA, PRODUCTION OF THE ACTIVE ENZYME IN ESCHERICHIA COLI, AND EXPRESSION ANALYSIS IN HUMAN TISSUES*
Proteinases identified to date, including the active site A comparison of structural characteristics of the different cysteine residue thaits involved in covalent intermedi- members of the cysteine proteinase family reveals that they are ate formation during peptide hydrolysis
Molecular Cloning and Nucleotide Sequencing of a cDNA Encoding Human Cathepsin 0-As a preliminarystepto search for new cysteine proteinases that could be produced by humanbreast carcinomas, two degenerate oligonucleotide primers based on highly conserved amino acid sequences among the different members of this proteinase family were synthesized
Summary
Molecular Cloning and Nucleotide Sequencing of a cDNA Encoding Human Cathepsin 0-As a preliminarystepto search for new cysteine proteinases that could be produced by humanbreast carcinomas, two degenerate oligonucleotide primers based on highly conserved amino acid sequences among the different members of this proteinase family were synthesized. Similar percentages were obtained when the remaining cathepsins werceompared between them.In addition, when this amino acid sequence comparison was extended to other cathepsinsfrom animal, plant, or parasitoerigin, similar degrees of identity were found, the largest one (34.1%) being with a cysteine proteinase from Leishmania mexicana (Mottram et al, 1992) Despite this overall limited sequence identity, the deduced amino acid sequence from the human cDNA isolated in thiswork, contains all the features characteristoicf the different members of the cysteine proteinase family of proteins (Fig. 2). It contains the active site Cys residue at position 132 that is transiently acetylated during peptide hydrolysis, as well as other residues proposed t o be important in catalysis including His-269 and Asn-289 (Kamphuisetal., 1984, 1985;Musil et aE., 1991). The presence of a stretch of hydropho- the remaining human cathepsins that contain glycosylation
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