©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Carboxypeptidase A Isoforms Produced by Distinct Genes or Alternative Splicing in Brain and Other Extrapancreatic Tissues (*)

(Received for publication, May 10, 1995; and in revised form, June 14, 1995)

Emmanuel Normant Claude Gros Jean-Charles Schwartz

From the Unité de Neurobiologie et de Pharmacologie de l'INSERM, Centre Paul Broca, 2ter rue d'Alésia, 75014 Paris, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

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.


INTRODUCTION

A number of metallopeptidases were evidenced first in the digestive tract or kidney tissues in which their function is related to the release of amino acids for their absorption or reabsorption. The same or closely related metallopeptidases were more recently evidenced in other tissues, namely in brain, where they participate in signaling events, i.e. biosynthesis or inactivation of neuronal or hormonal peptide messengers(1, 2, 3) . However, only a restricted number of metallopeptidases were characterized so far in brain. As an example peptidyl-L-aminoacid hydrolase i.e. carboxypeptidase A (EC 3.4.17.1; CPA) (^1)is considered as a purely pancreatic enzyme.

CPA activity was first identified in bovine pancreatic extracts by Waldschmidt-Leitz and Purr(4) , and the enzyme was crystallized later by Anson(5) . Since then, pancreatic CPA has been one of the most extensively studied enzymes regarding structure and function. It is a Zn-dependent exopeptidase which specifically catalyzes the hydrolysis of esters and peptides in which the terminal residue has a free COOH-group and a branched aliphatic side chain or aromatic group in an L-configuration (for review, see (6) ). CPA structure has been extensively studied by a wide range of techniques that include crystallography and x-ray analysis(7, 8) , molecular cloning(9) , or site-directed mutagenesis(10, 11) .

The occurrence of multiple forms of pancreatic CPA in mammalian species is due to allelic polymorphism, diverse processing during activation, and the propensity of CPA to associate with chymotrypsin- and/or elastase-like enzymes, giving rise to complexed as well as free forms (12) . In addition, a second CPA gene was detected in rat pancreas by cDNA cloning and a novel CPA, termed CPA2, was identified from the predicted amino acid sequence, isolated and characterized(13, 14) . Recently these two isoenzymes, CPA1 and CPA2, were separated from human pancreas by HPLC(15) . In addition, a third CPA has been described in mast cells, being initially detected in rats(16) , and its cDNA cloned from mouse peritoneal connective tissue mast cells (17) and human skin or lung mast cells(18) .

These three CPAs are synthesized under inactive precursor forms, the procarboxypeptidases A (see (19) for review) corresponding to the 307-residue mature enzyme with a 94-amino-acid NH(2)-terminal tail covering the active site.

CPA seems to be absent from brain tissues. Neither CPA1 nor CPA2 mRNAs were detected therein. In addition, no typical CPA activity, sensitive to inhibition of synthetic thiol-containing compounds (20) or the peptide inhibitor from potato(21) , could be detected, even using a highly sensitive radioassay performed with a novel I-labeled substrate. (^2)The same assay, however, detected a CPA-like activity poorly sensitive to the same inhibitors.

In the present work, we have reassessed the presence of CPA mRNAs and catalytic activity in a number of extrapancreatic tissues, particularly in rat brain.


EXPERIMENTAL PROCEDURES

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. S. Biochemical Corp. Carboxypeptidase A, trypsin (from bovine pancreas), alpha-chymotrypsin (from porcine pancreas), and protease (from Streptomyces griseus) were supplied by Sigma. [P]dATP, [P]dCTP, [S]dATP, [^3H]phenylalanine, and I-Na were obtained from Amersham. All other chemicals were of the highest purity available.

Northern Blot Analysis

Total RNA was isolated as described (22) . Briefly, tissues from male Sprague-Dawley rats (Iffa-Credo) were dissected out, rapidly frozen in liquid nitrogen, and homogenized in 5 M guanidinium thiocyanate, 2 M CsCl, 0.5% Sarcosyl, 25 mM sodium acetate, pH 7.4, 1.6% beta-mercaptoethanol, in diethyl pyrocarbonate (DEPC) treated-water. Homogenates were centrifuged in a 7.5 M CsCl solution for 20 h, at 140,000 g. The pellet was resuspended in 0.4 ml of diethyl pyrocarbonate water and RNA extracted using the phenol/chloroform method. The total RNA present in the resulting 0.4-ml aqueous phase was then precipitated at -20 °C overnight. The pellet was resuspended in 0.5 ml of diethyl pyrocarbonate water and poly(A) mRNA isolated using oligo(dT) chromatography.

Poly(A) mRNAs (10 µg) from different tissues were electrophoresed on a 1% agarose gel containing 1 M formaldehyde, blotted onto a nitrocellulose membrane, and subsequently immobilized by heating at 80 °C for 2 h. Prehybridizations were at 42 °C for 3 h in 40 or 60% formamide, 2 Denhardt's solution, 50 mM Tris-HCl, pH 7.4, 4 saline sodium citrate buffer (1 SSC is 150 mM NaCl, and 150 mM trisodium citrate), 0.1% sodium pyrophosphate, 1% SDS, denatured salmon sperm DNA (100 µg/ml), and yeast tRNA (50 µg/ml). Hybridizations were carried out overnight at 42 °C in prehybridization solution. The RT-PCR products used to hybridize the blots were electrophoresed, purified using a purification kit (Geneclean, BIO 101), and P-labeled using the nick translation kit. Washes were achieved twice in 2 SSC, 0.1% SDS at 42 °C for 15 min, followed by 0.2 SSC containing 0.1% SDS, 2 15 min at 42 °C. Dehybridization of blots was carried out with boiling 10 mM Tris, 1 mM EDTA solution (3 15 min).

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) Amplification, Southern Blot Analysis, and Sequencing

Oligonucleotide primers were designed from the published sequence of CPA1 (14) and CPA2 (13) (Table 1) and synthetized using a PCR-Mate 391 A (Applied Biosystems). 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 µM antisense primers in 20 µl of 50 mM Tris-HCl buffer, pH 8.3, 40 mM KCl, 6 mM MgCl(2), 2.5 mM dNTP, 2.5 mM dithiothreitol, and 50 units of RNase inhibitor (Boehringer). Double-stranded cDNAs were synthesized and amplified using Taq polymerase (2.5 units, Cetus) and 80 nM sense and antisense primers in 0.1 ml of 10 mM Tris-HCl buffer, pH 8.3, 50 mM KCl, 2.5 mM MgCl(2), 0.5 mM dNTP, 0.5 mM dithiothreitol, and 0.01% gelatin.



Extension of the 5` region was carried out with the modified rapid amplification of cDNA ends (RACE)-PCR method (23) using the Amplifinder kit (Clontech).

PCR products were electrophoresed in 1% agarose gels and blotted onto nitrocellulose filters. Oligonucleotides used as probes (0.3 µg) were P-labeled using the terminal transferase kit (Boehringer). Prehybridizations were performed at 42 °C for 3 h and hybridizations at 42 °C for 16 h in 6 SSC, 1 Denhardt's, 25 µg/ml yeast tRNA, 0.05% sodium pyrophosphate and probe at 5 10^5 dpm/ml. Filters were washed twice for 20 min at 42 °C in 2 SSC, 0.1% SDS, and the same time at the same temperature in 0.2 SSC, 0.1% SDS.

The SmaI-digested pGEM-4Z vector (Promega), blunt ligated with phosphorylated PCR products of interest, was amplified in JM 109 competent cells after transformation using the Hanahan technique(24) . It was used as the template for sequencing(25) .

Carboxypeptidase A Activity Determination

CPA activity was determined as described elsewhere.^2 Briefly, samples were incubated in 150 mM phosphate buffer, pH 7.4, 500 mM NaCl, 0.05% Tween 20, in the presence of 3-(p-hydroxy m-I-phenyl)propionyl-L-arginyl-L-phenylalanine, i.e. the radioiodinated Bolton-Hunter reagent derivative of the dipeptide RF ([I]BRF), obtained by iodination of BRF according to the Hunter and Greenwood method(26) . The reaction mixture containing [I]BRF, the released [I]BR, and phenylalanine was loaded onto a Pasteur pipette containing 100 mg of polystyrene beads (Porapak Q, Millipore Corp.) equilibrated with 20% ethanol. Columns were washed with 1 ml of 20% ethanol and elution of [I]BR was carried out by 1 ml of 20% ethanol, the eluate being counted in a gamma counter.

Another method, involving hippuryl-[^3H]phenylalanine ([^3H]HF) as substrate was also carried out. In the same way, [^3H]phenylalanine formed under the action of CPA was separated on a polystyrene bead column and the radioactivity eluted with 1 ml of water was quantified by liquid scintillation spectrometry. In all cases specific CPA activity was defined as the hydrolyzing activity inhibited by 1 µM of 2-benzyl-3-mercaptopropanoic acid (BMPA), a potent CPA inhibitor(20) .

Carboxypeptidase A Expression in Escherichia coli

A cDNA encoding a shorter variant of the CPA2 was synthesized by RT-PCR using pancreas mRNA as template. Artificial PstI and EcoRI restriction sites were introduced at the 5` and 3` ends of the cDNA, respectively, by incorporation of the appropriate sequences within the PCR primers (sense primer: 5`-TTCTGCAGCCATGGATAACCTCGTGGCTGAG-3` and antisense primer: 5`-TTGAATTCCTAATAGGGGTGGTCTCGTAC-3`). After restriction enzyme digestion of the PCR products, the cDNA was ligated into the prokaryotic expression plasmid pUC9 (Sigma). In the latter, the start codon is situated 30 bases upstream the first ATG of the insert. The recombinant plasmid was used to transform E. coli (JM109 competent cells), and sequenced(25) . Bacteria were incubated at 37 °C for 18 h, centrifuged, and the resulting pellet sonicated (3 30 s) in 150 mM phosphate buffer, pH 7.4, 500 mM NaCl, containing 1 mM phenylmethylsulfonyl fluoride, and leupeptin (10 µg/ml). Expression of a carboxypeptidase activity was followed by using [I]BRF as substrate.

Purification of CPA1 and CPA2 from Rat Pancreas

Rat pancreata were homogenized in 10 volumes of 50 mM phosphate buffer, pH 7.4, and the homogenate centrifuged for 20 min at 46,000 g. The resulting supernatants were incubated for 1 h in presence of trypsin (100 µg/ml) and submitted to chromatofocusing on a PBE94 column, elution being performed with PB74 buffer (Pharmacia). The resulting active fractions were loaded onto a phenyl-Sepharose column equilibrated in 1 M ammonium sulfate that was thereafter washed with the same buffer and, then, eluted with 50 mM phosphate buffer. Active fractions were tested for purity by SDS-PAGE, and were used to generate antibodies. Fractions eluted at pH 5.5-5.0 and 4.8-4.4 corresponded to CPA2 and CPA1, respectively, as determined by NH(2)-terminal microsequencing(27) .

Preparation of Antibodies against CPA1 and CPA2

New Zealand female rabbits received, subcutaneously in multiple sites, an initial injection of 200 µg of purified CPA2 or CPA1 emulsified in Freund complete adjuvant, followed by three 100 µg injections at 10-day intervals in incomplete adjuvant. Additional boosters were performed monthly. Several bleedings were selected on the basis of their specificity against each isoform of CPA by Western immunoblotting visualized with a goat anti-rabbit antibody radioiodinated as described (26) .

Preparation of Tissue Extracts

Rat tissues were homogenized in 150 mM phosphate buffer, pH 7.4, 500 mM NaCl, and activities were determined on supernatants obtained after centrifugation at 46,000 g and pretreatment by 100 µg/ml trypsin for 1 h. For Western blot and activity studies, brain supernatant was precipitated by ammonium sulfate (60% saturation). The resulting pellet was redissolved in 1 M ammonium sulfate and concentrated by phenyl-Sepharose chromatography as described above.

Western Blots

Pretreated tissue extracts were diluted in 50 mM Tris-HCl, pH 6.8, containing 2% SDS, 1% 2-mercaptoethanol, and 5% glycerol and submitted to SDS-PAGE. Proteins were then transferred onto nitrocellulose by standard methods and probed with 1:1000 dilution of antiCPA2 serum. Bound antibodies were visualized as described above.

Gel Filtration Chromatography

Supernatant of brain homogenate, displaying a carboxypeptidase A inhibitory activity, was loaded through a Superdex 75 HR 10/30 column (Pharmacia), equilibrated, and eluted with Tris-HCl, 50 mM, pH 7.4, at room temperature. The column was calibrated using bovine pancreas CPA (34.5 kDa) and the potato carboxypeptidase inhibitor (4.2 kDa) revealed through their catalytic and inhibitory activity, respectively.

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).


RESULTS

Characterization of CPA1 mRNA in Various Tissues

Starting from the rat CPA1 sequence(14) , probes and primers (Fig. 1A, and Table 1) were prepared and used to characterize CPA1 mRNA by Northern blotting or PCR (Fig. 1B and 2). Northern blot analysis performed with the AC probe revealed a single band of 1.35 kb in various tissues (Fig. 1B). A similar Northern blot pattern was obtained using either the JC or UJ probes (data not shown). The hybridization signal was the highest in pancreas, with which it was obtained using only 5 ng of poly(A) mRNA, and was stronger than that obtained using 1 µg of jejunum mRNA. Strong signals were also obtained using 10 µg of mRNA from stomach or colon and clear, although less intense signals using the same amounts of brain or heart mRNA. In contrast, hybridization signals were weak or absent with other tissue mRNAs.


Figure 1: Northern blot analysis of CPA1 gene transcripts from various tissues. A, structure of the CPA1 gene and positions of probes and primers used to analyze the transcripts. The numbered boxes correspond to the exons (14) . The square box in exon 1 represents the coding region for the signal peptide. The open and stippled boxes refer to regions coding for the activation peptide and the mature CPA1, respectively. The positions of amplimers are indicated by arrows. The positions of P-labeled probes used to hybridize the Southern blot are denoted E and W. The theoretical size corresponding to each primer combination is indicated between brackets. B, poly(A) mRNAs (10 µg/lane, except for pancreas and jejunum) were electrophoresed. The AC probe shown in A was used at 5 10^6 dpm/ml. The same pattern was obtained with the JC probe (data not shown). The blots were exposed at -80 °C for 2 days with intensifying screens. Molecular sizes (kb) are indicated.



The presence of CPA1 mRNA in brain was confirmed by RT-PCR analysis of the same mRNAs using the shifted amplimers JK, JC, AC, and UJ (Fig. 2). Southern blot analysis of the amplification products using either an E probe, corresponding to a sequence common to JK, JC and AC, or a W probe for the UJ amplification, showed the presence of bands of the expected sizes (798, 648, 430, and 357 bp for JK, JC, AC, and UJ, respectively) in the three tissues analyzed, i.e. pancreas, jejunum, and brain. These products did not hybridize with the G probe, encompassing residues 130-137 of the CPA2 sequence (13) (not shown), and no signal was detected using probe E when amplifications were performed in the absence of mRNA (Fig. 2, lanes 4). The sequence of the UJ amplification product, obtained with brain mRNA as template, was established and found to match exactly with the corresponding sequence of the rat pancreas CPA1(14) .


Figure 2: RT-PCR Southern blot analysis of CPA1 gene transcripts from various tissues. RT-PCR amplification was carried out with mRNAs from brain (2 µg, lane 1), jejunum (1 µg, lane 2), pancreas (0.1 µg, lane 3), or no mRNA (lane 4) as template and the four sets of primers (JK, JC, AC, and UJ) described in Fig. 1; products were electrophoresed onto an agarose gel and blots hybridized with P-labeled E probe for JK, JC, and AC, or W probe for UJ. Blots were exposed for 1 h at -80 °C with intensifying screens. Molecular sizes are indicated.



Characterization of CPA2 mRNA in Various Tissues

Using probes from the CPA2 sequence (Fig. 3A and Table 1)(13) , mRNA from various tissues was analyzed by Northern blotting (Fig. 3B). Northern blot hybridized with the BD probe encompassing the CPA2 nucleotide sequence corresponding to residues Ser-88 to Lys-231 revealed a single 1.35-kb signal in a number of tissues, with the relative abundance of the message being pancreas jejunum = stomach > colon together with a hardly detectable message in kidney and heart. In brain, lung, and testis, however, a signal of 1.1 kb was detected (Fig. 3B). This 1.1-kb signal was still present when hybridization was performed under high stringency (60% instead of 40% formamide) or using the HI hybridization probe (not shown). In contrast, the MH probe hybridization revealed the same 1.35-kb signal in pancreas, but failed to detect the 1.1-kb mRNA in brain (Fig. 3B). This suggested that the shorter transcript (1.1 kb) corresponded to a deletion at the 5`-end of the sequence. This was confirmed by carrying out a modified 5`-RACE, i.e. rapid amplification of cDNA 5`-end(23) , using the P antisense amplimer (corresponding to the B antisense sequence) and testis mRNA (2 µg) as template in the reverse transcription step. The resulting cDNA was ligated with an oligonucleotide anchor, and successive amplifications were carried out with S and Q antisense primers (Fig. 3A and Table 1) and a sense primer matching with the previously ligated anchor. This led to a 245-bp PCR product whose sequence (Fig. 4) corresponded only partially to that of the 5`-end of the CPA2 mRNA: whereas the 3` part of the sequence (61 bp) matched with the initial portion of exon 4(13) , the 5` part (184 bp) sequence did not correspond to either the fourth intron or 3b exon and did not share any significant homology with any Genbank sequence. The existence of this short transcript was confirmed by RT-PCR analysis of testis mRNA using the two A`N and B`N (sense primers A` and B` see Fig. 4) amplimer pairs (Fig. 3A) which led to DNA fragments of the expected sizes, stained with ethidium bromide (not shown), and revealed with the Q probe (Fig. 5a). RT-PCR analysis of the same mRNA using iN pair, in which i is an intronic sequence located in the 3`-end of the intron 4, did not result in any signal detection following hybridization with the Q probe. Brain mRNA, tested under the same experimental procedures, using A`P as amplimer pair, allowed the obtention of the fragment of the expected size (405 bp, data not shown). Control PCR experiments omitting the reverse transcriptase step and conducted with testis mRNA or water, and A`Q amplimer pair (Fig. 5b) did not reveal any signal using B` as probe, whereas a control DNA (corresponding to a 245-bp A`Q sequence inserted in pGEM-4Z) led to a signal of the expected size.


Figure 3: Northern blot analysis of CPA2 gene transcripts from various tissues. A, the positions of primers are indicated by arrows, those of probes (G, Q, S) by heavy lines, and the theoretical sizes of amplification products are given between brackets. The higher part of the figure depicts the structure of the full coding sequence of CPA2 gene (15) whereas the lower part shows, for comparison, that of a shorter variant. B, poly(A) mRNAs from indicated tissues (10 µg/lane, except for pancreas and jejunum) were analyzed. 1, RNAs from pancreas (1 µg total RNA) and brain, hybridized with the MH probe. 2 and 3, RNAs hybridized with the BD probe (1 µg and 0.01 µg of mRNA for jejunum and pancreas, respectively). Probes were used at 5 10^6 dpm/ml and exposures were for 7 days for 1 and 2, and 2 days for 3 with intensifying screens at -80 °C. Molecular sizes (kb) are indicated.




Figure 4: Nucleotide sequence derived from a 5`-RACE experiment using testis mRNA and Q and S amplimers in exon 4 of CPA2. The underlined A` and B` sequences refer to amplimers used in further PCR studies shown in Fig. 5. The asterisk corresponds to a stop codon whereas the ATG initiation codon corresponds to the Met-22 of the CPA2 sequence. The numbers refer to the sequence of Ref 13. Bold characters correspond to the 5`-end of exon 4.




Figure 5: RT-PCR Southern blot analysis of CPA2 gene transcripts. a, Southern blot analysis of the RT-PCR products obtained with testis mRNA as template using two exonic (A`N and B`N) and one intronic (iN, the i amplimer is located at the 3`-end of the intron 4 sequence) sense amplimers. The blot was hybridized with Q probe (1 10^6 dpm/ml) and exposed for 10 h at -80 °C. b, PCR without reverse transcriptase. The experiment was carried out with testis mRNA (1 µg), pGEM-4Z containing insert DNA (control +), and H(2)O (control -) as template and A`Q as amplimer pair (245 bp). The blot was hybridized with B` probe (1 10^6 dpm/ml), and exposed for 5 h at -80 °C.



Characterization of a Short CPA2 Isoform after Expression in E. coli and Comparison with Brain and Pancreas CPA2

A cDNA encoding the short CPA2 isoform, consisting of 289 amino acids, i.e. lacking the 18 amino-terminal residues of the published mature CPA2 sequence(13) , was synthesized using amplimers (described under ``Experimental Procedures'') and testis mRNA or pancreas total RNA as template. The nucleotide sequence of both products was identical to that published (13) with four exceptions, i.e. Leu-38 (CTC) Ile (ATC), Asn-84 (AAC) Asn (AAT), Ala-156 (GCC) Ala (GCT), and Ala-275 (GCT) Gly (GGT), i.e. minor changes presumably reflecting allelic polymorphisms. After PstI and EcoRI restriction enzyme digestion, the short CPA2 cDNA (from the first ATG in exon 4 to the TAG stop codon) was inserted in a pUC9 prokaryotic expression vector. The vector in which the start codon was that of the pUC9, situated 30 bases upstream the insert first ATG, was used to transform JM109 cells. The transformed bacteria were found to express a CPA2-like catalytic activity ( Table 2and Fig. 6) and a 33-kDa protein recognized on Western blots by the CPA2 polyclonal antibodies (Fig. 7), whereas the control, i.e. bacteria transformed with a non-inserted plasmid, did not exhibit any signal. The properties of the recombinant enzyme, in terms of both Western blot analysis (taking into account the 10 amino acids addition from the vector) and sensitivity to various inhibitors, was found similar to those of the partially purified CPA2 activity from brain (obtained through ammonium sulfate precipitation and phenyl-Sepharose chromatography). In contrast, these properties were distinct from those of a semi-purified pancreatic enzyme (Table 2, Fig. 6and Fig. 7). These three enzymes were able to hydrolyze either [^3H]HF or [I]BRF (not shown).




Figure 6: Inhibition of [I]BRF degrading activities from various sources by BMPA and the potato inhibitor. Solid and open symbols represent inhibition by BMPA and potato inhibitor, respectively.




Figure 7: Western blot analysis of brain and pancreas CPA2 and of a recombinant NH(2)-terminal truncated isoform of CPA2. Extracts (2 µl) from rat brain and pancreas or from transformed E. coli were analyzed by SDS-PAGE. Lane 1, 1.5 µg of pancreas pretreated extract. Lane 2, 15 µg of recombinant CPA2 short isoform obtained from a transformed E. coli broth, (pUC9+). Lane 3, 40 µg of brain pretreated extract as described. Lane 4, 15 µg of control of the E. coli strain transformed with a non-inserted expression vector (pUC9--). After transfer, the nitrocellulose membrane was incubated with anti-CPA2 antibodies and revealed with a radioiodinated second antibody and exposed to film for 18 h. The prestained molecular weight markers are: lysosyme, 20,700; soybean trypsin inhibitor, 28,600; carbonic anhydrase, 33,300; ovalbumin, 47,800; and bovine serum albumin, 86,800.



Characterization of an Endogenous CPA Inhibitor

In contrast with the presence of CPA1 gene transcript in brain, no typical CPA activity (inhibited by BMPA in nM concentrations) could be detected in brain extracts, using either [^3H]HF or [I]BRF, even after pretreatment with trypsin (not shown). In order to check whether this could be due to an endogenous inhibitor, brain (and other tissues) extracts were added in increasing concentrations to purified bovine pancreas CPA. CPA activity was progressively inhibited (Fig. 8) with the various tissues displaying different inhibitory potencies (Table 3). The inhibitory activity in the brain extract was not affected by N-ethylmaleimide, enhanced by preincubation, decreased by Pronase, and suppressed by heating (Table 4), whereas 1 mM dithiothreitol, trypsin (100 µg/ml), or chymotrypsin (100 µg/ml) were without effect (not shown). Gel filtration chromatography of a brain homogenate supernatant (Fig. 9) indicated that the inhibitory activity and CPA exhibited a similar elution pattern.


Figure 8: Inhibition of purified bovine CPA activity by various tissue extracts. A bovine CPA solution (10 ng/ml) was added to various tissue extracts, and the resulting activities tested with [I]BRF as substrate.








Figure 9: Gel filtration chromatography analysis of a carboxypeptidase-inhibitory activity from rat brain. The column was calibrated with the bovine CPA (34.5 kDa), and the potato carboxypeptidase inhibitor (4.2 kDa). Each fraction activity and inhibitory activity were tested as described under ``Experimental Procedures.'' The inhibitory activity from the brain extract is detected in fractions 17-19 (hatched box).




DISCUSSION

In the present work we have characterized CPA1 and CPA2 gene transcripts in brain and several other tissues. The presence of these transcripts, however, was not accompanied with typical CPA catalytic activity, even using a highly sensitive radioassay^2 to detect it. We have therefore investigated the reasons for this apparent discrepancy and come to ascribe it to the presence of an endogenous carboxypeptidase inhibitor and a short isoform of CPA2 with modified catalytic activity.

Since members of the carboxypeptidase gene family display significant sequence homology, e.g. 65% nucleotide sequence identity in the case of CPA1 and CPA2 genes(13) , it was important to design selective hybridization probes and amplimers for a reliable characterization of the corresponding transcripts. Those used in Northern blot and RT-PCR analysis were selected to display only limited homology with corresponding sequences of other members of the family, i.e. 30% identity at maximum ( Fig. 1and Fig. 3).

A single CPA1 mRNA of 1.35 kb, i.e. the same size as in pancreas, was detected on Northern blots from brain, heart, stomach, and intestine, and its identity was confirmed in a series of RT-PCR studies with shifted amplimers as well as by sequencing a PCR-amplified fragment. As judged from the Northern blot, the abundance of the CPA1 gene transcript in brain was approximately 10^4 times lower than in pancreas, which reflects its selective expression in a restricted population of cerebral neurons, as revealed by in situ hybridization. (^3)This low abundance of the transcript in brain and several other tissues, relative to that in pancreas, is not inconsistent with a failure to detect it in a previous study with mRNAs of the same tissues in which the detection level was apparently set to 1% of pancreas level(30) . It it important to underline that the full CPA1 mRNA sequence, including those of the signal peptide and NH(2)-terminal pro-piece or activation segment, was apparently expressed. This suggests that, in these tissues, the carboxypeptidase may be present, as in pancreas, in a vesicular structure and that inactive precursor form requires the cleavage of the activation segment by a trypsin-like enzyme to acquire full catalytic activity. Nevertheless, it was reported that proCPA retains some activity (about 10%) toward short peptides, i.e. dipeptides or tripeptides(19) , and additional studies are required to establish any role of CPA1 in brain and other extrapancreatic tissues.

In the case of CPA2 mRNA, Northern blot analysis suggested also expression in a number of previously unsuspected tissues, such as brain, lung, and testis, and at an abundance which was by four orders of magnitude lower than in pancreas. Interestingly, however, the apparent size of the transcript in these tissues (1.1 kb) was significantly lower than in pancreas and other parts of the digestive tract (1.35 kb). Northern blot analysis using probes corresponding to various parts of the CPA2 sequence suggested that the shorter CPA2 gene transcript, designated here CPA2(S), differed from the full-length pancreatic transcript (CPA2(L)) by deletion of a short sequence at the NH(2) terminus of the pre-proprotein. This was confirmed by a series of RT-PCR studies performed with brain or testis mRNA as templates and various amplimers. In addition, the 5`-end sequence of the short transcript, established via 5`-RACE(23) , was shown to correspond to a deletion of exons 1 to 3b, indicating that its production was presumably the result of alternative splicing of CPA2 pro-mRNA. Accordingly, in this sequence, the 5`-end of exon 4 is preceded by a previously unknown nucleotide sequence comprising a stop codon, and Met-22 of CPA2, corresponding to the first ATG of exon 4, is likely to represent the initiation methionine of CPA2(S). This view is strengthened by the observation that antibodies raised against purified CPA2 identified on Western blots from rat brain a single band of 32 kDa (Fig. 7), i.e. of nearly the size expected for the shorter CPA2 isoform taking into account its mRNA sequence. This size is also consistent with that of the active form of pancreatic CPA2 (34 kDa) derived from SDS-PAGE studies after trypsin treatment of pro-CPA2, i.e. cleavage of the Arg-Glu bond(12) .

Hence, the CPA2(S) mRNA encodes a protein lacking a sequence corresponding to the signal peptide and activation peptide of the pre-proenzyme(13) . On the other hand, this protein contains all the residues held responsible for substrate anchoring, e.g. those of the ``hydrophobic pocket'' or S`(1) subsite such as Ile-255, Tyr-248, Arg-145, and Asn-144 or those implicated in catalysis (Glu-270 and Arg-127) and coordination of the active site zinc (His-69, Glu-72, and His-196).

Taken together these observations suggested that the short CPA2 isoform is no longer associated with vesicles (or membranes), retains catalytic activity, and does not require activation by cleavage of a propeptide. In agreement with these predictions, expression of the protein in E. coli transformed by a pUC9 expression vector in which the CPA2(S) cDNA had been inserted was associated with the appearance of a [I]BRF-hydrolyzing activity displaying properties similar to those detected in a cytosolic fraction of rat brain. HPLC analysis of [I]BRF hydrolysis products indicated that the Arg-Phe amide bond of the radioactive substrate was cleaved by the recombinant enzyme (not shown). Interestingly, however, both recombinant CPA2(S) and native CPA activity from brain differed from pancreatic CPA2(L) by sensitivity to inhibitors (Table 2). In agreement, the synthetic thiol-containing inhibitor BMPA (20) and the carboxypeptidase potato inhibitor (21) were less potent (by about three orders of magnitude) on CPA2(S) than on pancreatic CPA2(L) whereas the dipeptide Met-Tyr was approximately 10 times less potent (Table 2).

It seems, therefore, that the role of the activation segment, which is absent in CPA2(S) is not only to inhibit CPA catalytic activity by obstructing the active site of the enzyme as shown by x-ray crystallography (31) but also, perhaps, to facilitate the appropriate folding of the protein in a given conformation to which typical CPA2(L) catalytic activity is associated. A somewhat similar observation was reported for recombinant mouse mast-cell CPA (32) and human chymase (33) which were completely devoid of catalytic activity when expressed as proteins lacking their activation peptides. In the case of CPA2, the alternative splicing of the pro-mRNA in extrapancreatic tissues leading to CPA2(S) is associated with the appearance of a distinct enzyme activity with a distinct subcellular localization; hence, a role distinct from that CPA2 plays in digestion.

In the case of CPA1, the presence of a complete mRNA sequence in brain and other extrapancreatic tissues not associated with any typical CPA activity could not be explained by incorrect folding due to the absence of the activation segment. Incubation of brain extracts in presence of trypsin to cleave this segment did not result in the appearance of any detectable [I]BRF hydrolyzing activity. A low level of CPA1 expression, reflected by the low abundance of the corresponding mRNA, is not likely to account for the failure to detect the CPA catalytic activity since the radioassay used was sensitive enough to detect CPA enzyme activities 10^5 times lower than in pancreas.

The apparent paradox was resolved by identifying in brain, as well as other tissues in which the CPA1 gene transcript is not associated with typical CPA activity, a CPA-inhibitory activity. The sensitivity of the latter to Pronase and analysis of brain extracts by gel chromatography suggest that this inhibitor is a protein of 30 kDa (Fig. 9). We have recently confirmed this view by purifying the protein to homogeneity, cloning its cDNA, and expressing the recombinant protein to confirm that the latter is a so far unknown, potent inhibitor of rat CPA1 and CPA2(L) activities.^4


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The abbreviations used are: CPA, carboxypeptidase A; bp, base pair(s); kb, kilobase pairs; RT-PCR, reverse transcriptase-polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; 5`-RACE, rapid amplification of cDNA 5`-end; BMPA, 2-benzyl-3-mercapto-propanoic acid; HPLC, high performance liquid chromatography; dpm, disintegrations/min.

(^2)
E. Normant, C. Gros, and J. C. Schwartz, manuscript submitted for publication.

(^3)
P. Facchinetti, E. Normant, C. Gros, and J. C. Schwartz, manuscript in preparation.

(^4)
E. Normant, M. R. Martres, J. C. Schwartz, and C. Gros, manuscript submitted for publication.


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