Abstract
A novel cDNA, designated carboxypeptidase Z (CPZ), was identified based on its homology to known metallocarboxypeptidases. Northern blot analysis shows bands of 2.1 and/or 2.6 kilobases in all tissues examined. The major form of CPZ mRNA in human salivary gland encodes a protein with an open reading frame of 641 amino acids. In addition, three variants were found that presumably arise due to alternative intron splicing. The 641-amino acid protein contains an 18-residue signal peptide-like sequence, a 120-residue region that shows 23-29% amino acid identity with a Cys-rich domain found in frizzled proteins and in type XVIII collagen, and then a 390-residue carboxypeptidase domain with 49% amino acid identity to carboxypeptidases E and N. The 641-amino acid form of CPZ expressed in the baculovirus system cleaves 5-dimethylaminonaphthalene-1-sulfonyl (dansyl)-Phe-Ala-Arg, although the level of enzyme activity was approximately 10-fold lower than either carboxypeptidase E or D expressed using the same viral system. The CPZ activity is more active at neutral pH than at pH 5.5 and is inhibited by active site-directed inhibitors of metallocarboxypeptidases. In summary, CPZ is a novel metallocarboxypeptidase that is active toward substrates with C-terminal basic amino acids.
Highlights
Carboxypeptidases (CPs)1 perform many important functions in a variety of tissues, ranging from digestion of food to selective processing of bioactive peptides (CPE)
Human salivary gland mRNA was used since preliminary studies using reverse transription Polymerase Chain Reaction (PCR) showed that this tissue expressed carboxypeptidase Z (CPZ) mRNA
A major finding of the present study is that CPZ is a novel member of the CPE subfamily of metallocarboxypeptidases
Summary
Carboxypeptidases (CPs) perform many important functions in a variety of tissues, ranging from digestion of food (pancreatic CPA and CPB) to selective processing of bioactive peptides (CPE). One subfamily includes mast cell CPA, plasma CPB ( known as CPU), and pancreatic CPA1, CPA2, and CPB [1] Proteins in this subfamily are approximately 30 – 40 kDa and do not contain C-terminal extensions beyond the CP domain. Subfamily includes CPE ( known as CPH and enkephalin convertase), CPM, CPN, CPD, and a protein designated AEBP1 [2]. Members of this subfamily contain a CP core region that is slightly longer than the CP core of the CPA subfamily and contain C-terminal extensions beyond the CP domain. Recent subcellular localization studies have found CPD to be present mainly in the Golgi and/or trans Golgi network and not in the secretory vesicles where the majority of processing is thought to occur. it is possible that additional CPs are involved in peptide processing
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