(Received for publication, May 10, 1995; and in revised form, June 14, 1995)
From the
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.
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) ()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-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. (
)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.
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).
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
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) .
Another method, involving
hippuryl-[H]phenylalanine
([
H]HF) as substrate was also carried out. In the
same way, [
H]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) .
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
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.
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
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
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
O (control
-) as template and A`Q as amplimer pair (245 bp). The blot was
hybridized with B` probe (1
10
dpm/ml), and exposed
for 5 h at -80 °C.
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-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.
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).
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 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 times lower
than in pancreas, which reflects its selective expression in a
restricted population of cerebral neurons, as revealed by in situ hybridization. (
)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
-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
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` 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
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.