Identification and characterization of three members of the human metallocarboxypeptidase gene family.
Amino acid homology searches of the human genome revealed three members of the metallocarboxypeptidase (metallo-CP) family that had not been described in the literature in addition to the 14 known genes. One of these three, named CPA5, is present in a gene cluster with CPA1, CPA2, and CPA4 on chromosome 7. The cDNA encoding a mouse homolog of human CPA5 was isolated from a testis library and sequenced. The deduced amino acid sequence of human CPA5 has highest amino acid sequence identity (60%) to CPA1. Modeling analysis shows the overall structure to be very similar to that of other members of the A/B subfamily of metallocarboxypeptidases. The active site of CPA5 is predicted to cleave substrates with C-terminal hydrophobic residues, as do CPA1, -2, and -3. Using Northern blot analysis, CPA5 mRNA is detected in testis but not in kidney, liver, brain, or lung. In situ hybridization analysis shows that CPA5 is localized to testis germ cells. Mouse pro-CPA5 protein expressed in Sf9 cells using the baculovirus system was retained in the particulate fraction of the cells and was not secreted into the media. Pro-CPA5 was not enzymatically active toward standard CPA substrates, but after incubation with prohormone convertase 4 the resulting protein was able to cleave furylacryloyl-Gly-Leu, with 3-4-fold greater activity at pH 7.4 than at 5.6. Two additional members of the human CP gene family were also studied. Modeling analysis indicates that both contain the necessary amino acids required for enzymatic activity. The CP on chromosome 8 is predicted to have a CPA-like specificity for C-terminal hydrophobic residues and was named CPA6. The CP on chromosome 2 is predicted to cleave substrates with C-terminal acidic residues and was named CPO.
Wei
Suwen
S
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Segura
Sonia
S
Vendrell
Josep
J
Aviles
Francesc X
FX
Lanoue
Edith
E
Day
Robert
R
Feng
Yun
Y
Fricker
Lloyd D
LD
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002268:Carboxypeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers; D018076:DNA, Complementary; D006801:Humans; D017403:In Situ Hybridization; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D008969:Molecular Sequence Data; D005810:Multigene Family; D011487:Protein Conformation; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D018076:DNA, Complementary; D002268:Carboxypeptidases