A novel rat carboxypeptidase, CPA2: characterization, molecular cloning, and evolutionary implications on substrate specificity in the carboxypeptidase gene family.

S J Gardell,C S Craik,E Clauser,E J Goldsmith,C B Stewart,M Graf,W J Rutter

doi:10.1016/s0021-9258(19)77910-1

S J Gardell, C S Craik + Show 5 more

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https://doi.org/10.1016/s0021-9258(19)77910-1

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Abstract

A new member of the carboxypeptidase gene family, carboxypeptidase A2 (CPA2), has been identified from the predicted amino acid sequence of a rat pancreatic cDNA clone. In vivo recombination and in situ hybridization techniques employing the CPA2 cDNA resulted in the isolation of two genomic clones spanning the 25-kilobase pair rat CPA2 gene. Evolutionary trees built from the amino acid sequences of the known pancreatic carboxypeptidases show that CPA2 and carboxypeptidase A1 (CPA1) are the products of genes which duplicated before the mammalian radiation, and that bovine CPA is of the A1 type. The substrate specificities of CPA1 and CPA2 isolated from rat pancreas are similar to bovine CPA in that carboxyl-terminal amino acids with aromatic or branched aliphatic side chains are preferred. However, the substrate preference of rat CPA1 is skewed toward smaller amino acids, while that of rat CPA2 is skewed toward bulkier amino acids as compared to bovine CPA. The differences in the substrate specificities of these three carboxypeptidases are compatible with the nature of the amino acid replacements in their binding pockets for the carboxylterminal amino acid of the substrate.

Highlights

A Novel Rat Carboxypeptidase,CPAB: Characterization, Molecular Cloning, and Evolutionary Implications on SubstratSepecificity in the Carboxypeptidase Gene Family*
CPAB is apparently absentfrom bovinepancreas. This are compatible with the nature of the amino acid re- novel carboxypeptidase has been purified from rat pancreas placements in their binding pockets for the carboxyl- and its substratpereference compared to rat CPAl abnodvine terminal amino acid of the substrate
XcA210, contained a 1.3-kb insert, cA2-10, which was sequenced in its entirety3 and shown to exhibit 65 and 54% identity tothe rat CPAl and carboxypeptidase B (CPB) cDNAs, respectively

Summary

11 Present address

Howard Hughes Medical Institute, University of Texas, Health Sciences Center, Dallas, T X 75235. ** National Science Foundation Postdoctoral Research Fellow in Environmental Biology (BSR-8700192). Positive plaques were purified and bacteriophage X DNA was prepared as described in Craik et al (1984) One of these clones, XcA2-10, contained a 1.3-kb cDNA insert, cA2-10, which was subcloned in both orientationsinto theEcoRI site of M13mp (Messing and Vieira, 1982).The nucleotide sequence of cA2-10 wasdetermined by the dideoxy chain-terminating method (Sanger et al, 1977) with the use of the M13 universal primer and a setof oligonucleotides (18-20-mers) that were designed from the sequence of the CPA2 cDNA. Rat CPA2 Gene-The bacteriophage X Charon 4A rat genomic DNA library was screened for the CPAZ gene by the in uiuo recombination and selection system developed by Seed (1983) For this purpose, 7120 was inserted into the PstI site of TAN-7, a miniplasmid which carries the Escherichia coli tyrosine tRNA amber-suppressor supF gene. Lipscomb) were used to obtain predictions regarding substrate specificity differences

RESULTS

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DISCUSSION