Identification of mouse CPX-2, a novel member of the metallocarboxypeptidase gene family: cDNA cloning, mRNA distribution, and protein expression and characterization.
A novel member of the metallocarboxypeptidase gene family was identified from its homology with carboxypeptidase E and has been designated CPX-2. The cDNA of 2500 nucleotides encodes a protein of 764 amino acids that contains an N-terminal signal peptide-like sequence, a 158-residue discoidin domain, and a 400-residue carboxypeptidase domain. The 400-residue metallocarboxypeptidase domain has 59% amino acid identity with a protein designated AEBP-1; 44% to 46% identity with carboxypeptidases E, N, and Z; and lower homology with other members of the metallocarboxypeptidase gene family. The discoidin domain of CPX-2 has 22% amino acid identity with the carbohydrate-binding domain of discoideum-I, 29% to 34% identity with the phospholipid-binding domain of human factors V and VIII, and 59% identity with the discoidin-like domain on AEBP-1. CPX-2 is missing several of the predicted active-site residues that are conserved in most other members of the metallocarboxypeptidase gene family and which are thought to be required for enzyme activity. Expression of CPX-2 using the baculovirus system produced several forms of protein, from 80 to 105 kDa, but no detectable activity toward a variety of carboxypeptidase substrates. A shorter 50-kDa form of CPX-2, which contains the carboxypeptidase domain but not the discoidin domain, was also inactive when expressed in the baculovirus system. CPX-2 is able to bind to Sepharose-Arg; this binding is blocked by 10 mM Arg. Northern blot analysis showed CPX-2 mRNA in mouse brain, liver, kidney, and lung. In situ hybridization analysis of brain revealed a broad distribution. Areas that are enriched in CPX-2 include the hippocampus, cerebral cortex, median eminence, and choroid plexus. Taken together, these data suggest a widespread function for CPX-2, possibly as a binding protein rather than an active carboxypeptidase.
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Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
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Dong
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Fricker
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D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D001483:Base Sequence; D001921:Brain / Q000201:enzymology*; D002268:Carboxypeptidases / Q000235:genetics*; D043422:Carboxypeptidases A; D002352:Carrier Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular; D020224:Expressed Sequence Tags; D006801:Humans; D017403:In Situ Hybridization; D008666:Metalloendopeptidases / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D005810:Multigene Family; D009928:Organ Specificity; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera; D014158:Transcription, Genetic; D014162:Transfection
D002352:Carrier Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D002268:Carboxypeptidases; C115512:Cpxm2 protein, mouse; D043422:Carboxypeptidases A; D008666:Metalloendopeptidases