Abstract
The presence of carboxypeptidase A (EC 3.4.17.1; CPA) gene transcripts and corresponding catalytic activity was investigated in brain and other extradigestive rat tissues in which presence of the pancreatic enzyme had not been reported so far. Transcripts of two known CPA genes, CPA1 and CPA2, were identified in extremely low abundance in brain and several other extrapancreatic tissues using Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Whereas the CPA1 gene transcripts in brain, heart, stomach, or colon had a size similar to that in pancreas (1.35 kilobases), the CPA2 gene transcripts in brain, testis, or lung were of a smaller size (1.1 kilobases). Northern blot analysis using various probes, RT-PCR, and 5'-rapid amplification of cDNA 5'-end (5' RACE analysis) all indicated that this smaller size of the brain transcript was attributable to production by alternative splicing of the pro-mRNA. This process corresponds to deletion of the first four exons, leading to a mRNA encoding a protein in which the signal peptide and activation peptide of prepro-CPA2 are absent but the active site remains. The prediction that the shorter CPA2 isoform, designated CPA2(S), should correspond to a cytoplasmic metallopeptidase that does not require tryptic activation was verified by characterization of the recombinant protein and comparing it with the native CPA-like activity in brain. Both recombinant CPA2(S) generated in Escherichia coli and a soluble protein from brain displayed similar sizes on Western blots (32 kDa to be compared to 34 kDa for pancreatic CPA2). Recombinant CPA2(S) and a soluble CPA-like activity from brain displayed similar sensitivity to a series of inhibitors, contrasting with that of the pancreatic enzyme. It is concluded that alternative splicing produces a truncated CPA2 with distinct subcellular localization and modified catalytic activity. In spite of the presence of the CPA1 mRNA, no corresponding CPA activity could be detected in brain extracts, even after tryptic activation. This apparent discrepancy seems attributable to the presence of an endogenous peptide inhibitor which remains to be identified.
Highlights
From the Unite de Neurobiologie et de Pharmacologie de I'INSERM, Centre Paul Broca, 2ter rue d'Alesia, 75014 Paris, France
The presence of carboxypeptidase A (EC 3.4.17.1; CPA) gene transcripts and corresponding catalytic activity was investigated in brain and other extradigestive rat tissues in which presence of the pancreatic enzyme had not been reported so far
Whereas the CPA1 gene transcripts in brain, heart, stomach, or colon had a size similar to that in pancreas (1.35 kilobases), the CPA2 gene transcripts in brain, testis, or lung were of a smaller size (1.1 kilobases)
Summary
Materials-Restriction enzymes, avian myeloblastosis virus-reverse transcriptase, ribonuclease (RNase) inhibitor, and nick translation kit were purchased from Boehringer Mannheim, Taq polymerase from Perkin Elmer Cetus, and Sequenase sequencing kit from U. Avian myeloblastosis virus-reverse transcriptase (25 units, Boehringer) was used to synthesize (2 h, 42°C) a single-stranded cDNA from rat brain, pancreas, jejunum, and testis mRNA in the presence of 0.4 /LM antisense primers in 20 /LI of 50 mM Tris-HCI buffer, pH 8.3, 40 mM KCI, 6 rnsr MgCI2, 2.5 mM dNTP, 2.5 mM dithiothreitol, and 50 units of RNase inhibitor (Boehringer). Preparation ofTissue Extracts-Rat tissues were homogenized in 150 mM phosphate buffer, pH 7.4, 500 mM NaCI, and activities were determined on supernatants obtained after centrifugation at 46,000 X g and pretreatment by 100 /Lg/mltrypsin for 1 h. Gel Filtration Chromatography-Supernatant of brain homogenate, displaying a carboxypeptidase A inhibitory activity, was loaded through a Superdex'P' 75 HR 10/30 column (Pharmacia), equilibrated, and eluted with Tris-HCI, 50 mM, pH 7.4, at room temperature. Computer Analysis-Sequence comparisons were performed using the Kanehisa et al (28) algorithm with the help of BISANCE facilities (29) at the Centre Inter-universitaire de Traitement de l'information (CITI 2, Paris)
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