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INTRODUCTION |
The vertebrate pancreas synthesizes and secretes a subset of zinc
carboxypeptidases that hydrolyze alimentary proteins and peptides from
their COOH-terminal ends. These are traditionally classified into the A
types (with a preference for apolar COOH-terminal residues) and the B
types (with a preference for basic COOH-terminal residues) (1, 2). In
contrast, actinomycetes (bacteria) produce carboxypeptidases with a
dual A + B specificity toward both neutral and basic substrates and the
distinct vertebrate A and B types presumably arose from such a
precursor (3, 4). Why or when in the course of evolution a single
carboxypeptidase with both A and B specificities was abandoned in favor
of multiple enzymes with more limited specificities is not known. The
classification of carboxypeptidases into the A and B forms has been
further expanded with the identification of the A1 and A2 isoforms in
rat and humans. Carboxypeptidase A1 preferentially catalyzes aliphatic
COOH-terminal residues of peptide substrates, while the A2-type
selectively acts on the bulkier aromatic COOH-terminal residues, being
the only isoform that shows specificity toward tryptophan (2, 5, 6).
Carboxypeptidase A2 is apparently absent from bovine pancreas, so in
contrast the single carboxypeptidase gene has a relatively broad
substrate specificity (2). A number of non-digestive zinc
carboxypeptidases involved in hormone and neuropeptide processing, bioactive peptide activation or inactivation, or functional modulation of regulatory proteins have also been reported in the literature expanding the field of interest in metallocarboxypeptidases (1). Aside
the actinomycete enzymes (7) there is very limited information on the
categories and genealogies of carboxypeptidases present in species
other than a few animals. To date among fungi, almost all studies have
focused on serine carboxypeptidases and enzymes from
Aspergillus spp. (8), Penicillium (3),
Mucor racemosus, (9) and yeasts (4) have been thoroughly
characterized with regard to physiochemical properties and specificity.
However, an open reading frame predicted to encode a
metallocarboxypeptidase (YHT2) was identified as part of the
Saccharomyces cerevisiae genome project (10) suggesting that
these enzymes may also play a role in protein processing in fungi.
Given the limited information about metallocarboxypeptidases, isolation
and characterization of carboxypeptidases from species intermediate
between bacteria and vertebrates might help to confirm their
differential character and evolutionary pathways and to understand the
molecular reasons for their specific functional properties. We report
here the cDNA cloning of a carboxypeptidase A (MeCPA) that is
secreted by an insect pathogenic fungus Metarhizium anisopliae into infected tissues. M. anisopliae
produces a very large number of isoforms of different peptidases that
often hinders the rapid purification of the correct protein (11).
Consequently, Pro-MeCPA was overexpressed in an insect cell line to
produce soluble catalytically active protein in quantities amenable to functional analysis and free of any possible fungal contaminants. Similarly human carboxypeptidase A2 was expressed in Pichia
pastoris in order to characterize its activation pathway and
facilitate its potential for biotechnology (12).
The expressed MeCPA resembled mammalian enzymes in demonstrating an
A-type specificity toward aromatic and bulky aliphatic P1 side chains,
and contrasted with bacteria in possessing only marginal B-type
activity against charged residues. This substrate specificity was
associated with hydrophobic amino acids in the S1 subsite that are
mostly either the same, or conserved substitutions with those in A-type
carboxypeptidases. Thus, the separation into distinct A- and B-types
appears to have occurred early in the evolution of the eukaryotes, and
despite distant phylogenetic relationships, hypotheses concerning
structural determinants contributing to the A-type specificity of
mammalian enzymes (2) are backed in the fungal counterpart.
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EXPERIMENTAL PROCEDURES |
Organism and Growth--
M. anisopliae strain ARSEF
2575 (formerly ME-1) was maintained on potato dextrose agar.
Preparation and Analysis of Culture Filtrates of M. anisopliae--
Standardized mycelial inocula (5 g wet weight) from
48-h Sabouraud dextrose broth cultures were incubated with shaking (100 rpm) for 14 h in 100 ml of minimal media (0.05%
KH2P04, 0.01% MgSO4, pH 6)
supplemented with insect cuticle or alternate carbon and/or nitrogen
sources at 1% (w/v) (13). Clean samples of cuticle from 3-day-old
fifth instar Manduca sexta larvae were prepared as described
previously (14). Cuticle was obtained from the giant cockroach
(Blaberus giganteus) by extracting soft tissue from
homogenized insects with sodium tetraborate (15). Samples of culture
for enzyme assay were clarified by filtration through Whatman 1 filter
paper and centrifugation (1,800 × g for 10 min at
4 °C).
Enzymatic Reactions--
The rate of hydrolysis against a range
of carboxypeptidase substrates was continuously measured
spectrophotometrically in 50 mM Tris-HCl, 0.5 M
NaCl, pH 7.5, at 25 °C. The wavelengths used to monitor the various
reactions were as follows: Bz-Gly-Arg, 254 nm;
Cbz1-Gly-Gly-Phe,
Cbz-Gly-Gly-Leu, Cbz-Gly-Gly-Ala, and Cbz-Gly-Phe, 225 nm; Cbz-Gly-Trp
and Cbz-Gly-Tyr, 235 nm (2). Initial rates were obtained at substrate
concentrations bracketing the Km value whenever
possible. The values of the kinetic parameters, kcat and Km, were obtained by
direct fit of 8 points using a nonlinear least squares regression
analysis (16). Bovine carboxypeptidase (Type 1) and enzyme substrates
were supplied by Sigma.
Cloning and DNA Sequencing of MeCPA--
Differential display
was used to identify genes that are induced when M. anisopliae grows on insect cuticle (17). Total RNA from M. anisopliae cultures grown in the presence or absence of insect
cuticle was isolated with Tri-Reagent (Molecular Research Center) from
finely homogenized fungal mycelia and analyzed using the RNAimage
mRNA Differential display kit (GenHunter) as described (17). The
454-base pair MeCPA PCR band differentially expressed in
cuticle grown cultures (Fig. 1) was isolated, re-amplified, and ligated
into pCRII (Invitrogen) and transformed into One Shot competent cells
(Invitrogen). The plasmid DNA was purified using the Plasmid Mini Kit
(Qiagen). Purified plasmid DNA fragments were sequenced using a 373 Stretch DNA Sequencer (Applied Biosystems) on both strands.
The PCR fragment was 32P-labeled by random primer and used
to screen a
ZAP II cDNA library constructed from mycelia grown on insect cuticle (17). We screened a total of 10,000 plaques at high
stringency using standard procedures (13) and identified 10 positive
plaques that were rescued as Bluescript (Stratagene) plasmids for
sequence analysis. The sequence data were analyzed in Gene Bank using
BLASTX and FASTA searches and the tree was drawn using MEGALIGN in the
DNASTAR program. Southern and Northern blots were performed as
described previously (13).
Construction and Purification of a Baculovirus Expression Vector
(BEV) for MeCPA (BEV-MeCPA)--
The MeCPA gene was
isolated as a 1.6-kilobase DNA fragment from a
Bluescript/MeCPA and ligated into the pBacPAK8 transfer vector plasmid (CLONTECH Laboratories, Inc., Palo
Alto, CA). Spodoptera frugiperda cells (Sf-21), were
co-transfected with AcMNVP DNA (1.5 µg) and the
pBacPAK8/MeCPA plasmid DNA (3 µg) using Lipofectin reagent
(Life Technologies, Inc., Gaithersburg, MD) as described (18).
Recombinant virus (AcMNVP/MeCPA in which the polyhedrin gene was
replaced with the MeCPA gene was detected by the absence of
polyhedra in infection loci of tissue culture plaques using Sf-21 cells
(19). The progeny recombinant virus was purified with four sequential
plaque isolations.
To express MeCPA in insect cells (designated BEV-MeCPA),
Hi-5 cells (Invitrogen) were infected with AcMNVP/MeCPA at a
multiplicity of infection of 10 plaque forming units per cell. MeCPA
activity versus Cbz-Gly-Gly-Phe was first detected in the
tissue culture medium 36 h after infection and reached a maximum
titer of 24.2 mg/liter at 84 h post-infection. Harvested medium
was concentrated, and the MeCPA protein was separated from proteins in
the cell culture medium by affinity chromatography. A viral suspension prepared by infecting Hi-5 cells with Ac-E10 under the same conditions was used as a control. Ac-E10 is a mutant of AcMNVP which contains neither a functional polyhedrin gene nor a foreign gene insert (20).
Purification of BEV-MeCPA--
Media samples (50 ml aliquots)
were removed from infected insect cell cultures and concentrated by
lyophilization. The residual solids were dissolved in 15 ml of
distilled water and equilibrated with 20 mM MES, 0.1 M NaCl (pH 6.0) using Centriprep-10 ultrafiltration units
(Amicon, Danvers, MA). The sample was applied to a carboxypeptidase affinity column (1.5 × 9 cm2) of immobilized
glygyl-L-tyrosyl-azo-benzylsuccinate (21). (Pierce Chemical
Co.). The column was washed with 20 mM MES (pH 6.0), 0.1 M NaCl and enzyme eluted with a linear gradient of
NaCl in 20 mM Tris-HCl (pH 7.5).
Preparation of Antibodies and Immunoblotting--
MeCPA
expressed in insect cells and purified by affinity chromatography was
analyzed by SDS-polyacrylamide gel electrophoresis. After
electrophoresis, the gel was stained with 0.05% Coomassie Blue, and
the single 35-kDa protein band was excised with a scalpel. The gel
slices were lyophilized, ground into a powder, and resuspended in a
volume of water equal to one-half of the original volume. The
suspension was divided into three aliquots, mixed with Freund's complete adjuvant, and injected at 14-day intervals into New Zealand White rabbits. Antibodies were affinity purified on a Affi-Gel column
(Bio-Rad) and used to detect MeCPA protein by Western blot.
For Western blot analysis, proteins were transferred from gels to
nitrocellulose and immunoblotted as described previously (14). Blots
were developed with the ProtoBlot Western blot AP system (Promega).
NH2-terminal sequence analysis of proteins blotted on nylon
membranes followed previously used procedures (15).
Immunogold Labeling--
Cuticles from fifth-instar M. sexta larvae were excised, soaked in a saturated solution of
phenylthiourea (to inhibit phenoloxidase) inoculated with conidia, and
following incubation for up to 48 h, processed for electron
microscopy as described previously (15) using antibodies to MeCPA.
Ultrathin sections of LR White-embedded tissue were placed in blocking
solution (15) for 30 min, treated with a 10
2 dilution of
Protein A-gold (particle diameter, 10 nm; Sigma) in phosphate-buffered
saline containing Tween 20 (0.05%) and bovine serum albumin (0.02%)
for 1.5 h. The sections were then washed in distilled water and
stained for contrast in 4% (w/v) uranyl acetate in 50% (v/v) ethanol
for 20 min. Observations were made with a Zeiss EM10 transmission
electron microscope.
Specificity of the labeling was determined by: (i) incubation of the
sections with serum obtained before the rabbits were immunized, (ii)
omission of primary antisera, and (iii) treatment with Protein A prior
to treatment with Protein A-gold.
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RESULTS |
cDNA Cloning and Sequence Analysis--
A PCR product of a
M. anisopliae transcript specifically expressed during
growth on insect host cuticle was isolated using differential display
(Fig. 1). Approximately 0.1% of the
clones in a
ZAP II cDNA library constructed from mycelia grown
on insect cuticle hybridized to the radiolabeled PCR product.
Complementary DNA from 10 hybridization positive clones was isolated at
the preparative level and the longest clone, called MeCPA,
was entirely sequenced in both directions. The nucleotide sequence and
the corresponding amino acid sequence of the protein (MeCPA) are
shown in Fig. 2.

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Fig. 1.
Close-up of a part of the acrylamide gel
obtained by DD-PCR on an autoradiograph. Arrow points
to the band showing the MeCPA PCR fragment.
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Fig. 2.
The nucleotide sequence of MeCPA and deduced
amino acid sequence. An in-frame start codon and stop codon (TAA)
are shown by underlines. , show cysteine residues with
the potential of forming two disulfide bonds as in pancreatic
carboxypeptidases. Arrows indicate potential cleavage sites
for the signal and pro-sequence sites of MeCPA. The nucleotide sequence
was submitted to GenBank data base (accession number U76003).
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The analyzed full-length cDNA insert contained standard 5'- and
3'-flanking regions and an open reading frame of 1254 base pairs coding
for 418 amino acids. Analysis of the NH2-terminal amino
acid sequence of the primary translation product suggests the existence
of a hydrophobic 23-amino acid signal peptide which is presumably
cleaved during expression of the inactive zymogen. The format of the
signal includes a charged residue (Arg), a core of hydrophobic residues
and a helix breaking residue (Pro), five residues before a peptidase
cleavage site (SPV). This is consistent with the empirical rules of
fungal presecretory sequences and is similar to the
NH2-terminal sequence of M. anisopliae
subtilisin proteinase (Pr1) (13). The NH2-terminal of the
BEV-expressed protein was determined by microsequencing to be GTIQAYAA.
This sequence was located 99 residues from the NH2 terminus
of the 418 amino acid sequence (Fig. 2). The predicted
Mr of the mature protein is 35,151.
Southern analysis with MeCPA produced a single approximately
2-kilobase fragment (Fig. 3), indicating
that it is a single copy gene as this band is too small to contain more
than one gene copy.

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Fig. 3.
Southern blot analysis with MeCPA.
Genomic DNA (5 µg) prepared from mycelia was digested with the
indicated enzymes and separated by electrophoresis on a 1% agarose
gel. The fragments were transferred to GeneScreen Plus membranes (NEN
Life Science Products Inc.) and probed with a 32P-labeled
random primed MeCPA cDNA fragment under high stringency
conditions (28).
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Sequence Comparisons--
That MeCPA belongs to the A group of
digestive metallocarboxypeptidases is suggested by the comparison of
its overall amino acid sequence with proteins of other organisms. The
most closely related proteins are human carboxypeptidase A2 (37%
identity) and carboxypeptidase A1 (35% identity), rat carboxypeptidase
A2 (35% identity), bovine carboxypeptidase (34% identity), and the hypothetical yeast carboxypeptidase YHT2 (33% identity). YHT2 showed
31% identity to human carboxypeptidase A2 (Fig.
4). By contrast, bacterial
carboxypeptidases show about 25% identity to the pancreatic enzymes
(22). The fungal carboxypeptidases show much less overall sequence
similarities to the regulatory enzymes (Fig.
5). In fact MeCPA and human
carboxypeptidases share only a 15% amino acid identity with the
non-digestive mammalian carboxypeptidases.

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Fig. 4.
Amino acid alignment of MeCPA with those of
bovine procarboxypeptidase A (accession number EMBL Z33906),
human procarboxypeptidase A1 (accession number SwissProt P15085), human
procarboxypeptidase A2 (accession number SwissProt P48052), and yeast
putative carboxypeptidase (accession number SwissProt P38836). The
numbering system is made according to the bovine A enzyme (17).
Identical amino acids are boxed together.
Asterisks are placed over the functionally important
residues, which are discussed in the text. Open triangles
above the alignments identify the cysteine residues that form
disulfide bonds in pancreatic enzymes.
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Fig. 5.
Position of MeCPA in a phylogenic tree of the
metallocarboxypeptidase superfamily. The nodes have been placed
according to the distance between sequence pairs. The units on the
bottom scale indicate the number of substitution events.
Accession number: rat CPB1 (rat carboxypeptidase A1), SwissProt P00731;
human CPA1 (human pancreatic carboxypeptidase A1), SwissProt P15085;
BosCPBA (bovine carboxypeptidase A), EMBL Z33906; human CPA2 (human
carboxypeptidase A2), SwissProt P48052; ratCPB2 (rat carboxypeptidase
A2), SwissProt P19222; yeast YHT2 (putative carboxypeptidase-nucleotide
sequence in S. cerevisiae chromosome VIII), SwissProt
P38836; ThermCPT (Thermoactinomyces vulgaris
carboxypeptidase T), SwissProt P29068; StrepCPA (Streptomyces
griseus carboxypeptidase), SwissProt P39041; human CPN (human
carboxypeptidase N), SwissProt P15169; human CPM (human
carboxypeptidase M), SwissProt P14384.
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The fungal carboxypeptidases (MeCPA and YHT2) show highest local
identity with the domains of zinc carboxypeptidases that contain
residues important for catalysis and for the delineation of the active
site (22-24). These include residues involved in zinc binding
(His69, Glu72, and His196),
catalysis (Glu270 and Arg127), and substrate
anchoring and positioning (Arg71, Asn144,
Arg145, Pro205, Ile247,
Tyr248, and Asp256). However,
Asn144 and Arg145 are replaced by Asp and His
in YHT2 so these particular residues can no longer be regarded as
strictly invariant in zinc carboxypeptidases. The S2
(Arg71, Ser197, Tyr198, and
Ser199) and S3 (Phe279) subsites involved in
binding and torsion of peptide substrates are the same in pancreatic
enzymes, MeCPA and YHT2, although they are variable among the
regulatory zinc carboxypeptidases (22).
Variations between carboxypeptidases occur at residues that form the S1
specificity pocket (positions 194, 203, 243, 250, 253, 255, and 268)
and are responsible for the observed differences in primary specificity
(2, 22, 24). In Table I substitutions in
these positions are compared for MeCPA, YHT2, and zinc
carboxypeptidases with different specificity types. The B-type
specificity for positively charged residues is determined by a
negatively charged residue (Asp) in positions 253 or 255, i.e. at the center of the binding pocket of the catalytic
domain (22, 25). MeCPA and A-type carboxypeptidases have no equivalent
Asp residue; they possesses a hydrophobic Ile residue at position 255 (Leu in the yeast enzyme) that is believed to be a critical parameter
in determining a specificity for hydrophobic substrates (25).
Conversely, the presence of an Asp residue at position 194 in MeCPA and
YHT2, replacing a hydrophobic residue in A-types, will decrease the
hydrophobic character of the active site. MeCPA resembles the human
A-type enzymes in possessing the flexible Met at position 203, and also resembles the A2 isoform and bovine carboxypeptidase in possessing small Gly and Ser residues at positions 253 and 254 (Fig. 4). These
substitutions should provide a stronger hydrophobic character and favor
the enlargement of the specificity cavity to facilitate the recognition
of substrates with bulkier aromatic residues (23). However, while also
adding to the pockets hydrophobic character, the Val250 in
MeCPA will reduce the pocket size versus the A2 form. MeCPA resembles most other carboxypeptidases in possessing a Thr residue at
268, in place of Ala in carboxypeptidase A2.
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Table I
Amino acid variations in S1-subsite of Zn-carboxypeptidases
Except for BosCPBB (bovine carboxypeptidase B, P00732) enzyme
abbreviations are defined in the legend to Fig. 5. Numeration is given
according to BosCPBA sequence.
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Kinetic Properties of Bovine Carboxypeptidase and MeCPA--
To
confirm that the protein encoded by MeCPA is a
carboxypeptidase, we expressed MeCPA cDNA in insect
cells and compared the kinetic properties of affinity purified enzyme
(Fig. 6) with bovine carboxypeptidase
against typical substrates for carboxypeptidases (Table
II). For both enzymes the most
effectively hydrolyzed substrates contained P1 phenylalanine, and
substituting with leucine, tyrosine, or tryptophan reduced activity.
The catalytic efficiencies
(kcat/Km) of MeCPA toward
Cbz-Gly-Phe and Cbz-Gly-Trp substrates is higher than bovine
carboxypeptidase by 1.8- and 2.0-fold, respectively, reflected
primarily in increased Km values exhibited by MeCPA.
The catalytic efficiency of bovine carboxypeptidase toward the Tyr
substrate is 3.3-fold higher than MeCPA reflected in both
Km and Kcat values. The
Kcat values of MeCPA and bovine
carboxypeptidase are approximately the same for Cbz-Gly-Gly-Phe and
Cbz-Gly-Gly-Leu, although the Km values of MeCPA are
lower than bovine carboxypeptidase by 1.7- and 4.5-fold, respectively. MeCPA showed 100-fold less activity against typical substrates for
carboxypeptidase B (Hippuryl-Arg, Cbz-Gly-Arg) compared with Cbz-Gly-Phe, and no activity against aminopeptidase
(p-nitroanilide derivatives of Ala, Leu, and Pro) or
endoprotease (Hide protein azure, casein) substrates.

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Fig. 6.
Affinity chromatography of M. anisopliae carboxypeptidase expressed in an insect cell
line. The Gly-Gly-L-tyrosyl-azo-benzylsuccinate
affinity column was equilibrated and washed with 20 mM MES,
0.1 M NaCl (pH 6.0) until the A280
had receded to base line. Enzyme was eluted with a linear gradient of
Tris-HCl (20 mM, pH 7.5)-buffered NaCl (0.1-0.4
M). The flow rate was 15 ml h 1 and 5-ml
fractions were collected. Symbols are: square,
carboxypeptidase activity versus Cbz-Gly-Gly-Phe; ,
absorbance at 280 nm; - - -, NaCl concentration.
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Table II
Kinetic constants for substrate hydrolysis by M. anisopliae
carboxypeptidase and bovine carboxypeptidase
Assayed in aqueous buffer (50 mM Tris-HCl, 0.5 M NaCl, pH 7.5) as described in the text.
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Production of MeCPA in Culture--
Antibodies to MeCPA were
raised in rabbit, affinity purified, and found to recognize recombinant
35-kDa MeCPA and a 35-kDa band in culture filtrates of M. anisopliae grown on proteins (Fig. 7). Passing culture filtrates through a
carboxypeptidase affinity column eliminated the immunoreactive band
confirming that it is MeCPA. The immunoreactive band and CPA activity
versus CBZ-Gly-Gly-Phe were produced in cultures in
unsupplemented minimal medium indicating that limitation of carbon and
nitrogen source derepressed CPA secretion, but the presence of host
cuticle or proteins was required for secretion of high levels of MeCPA.
Secretion was repressed when NH4Cl was added to media
containing protein showing that nitrogen repression overrides the
enhancing effect of polymeric substrates. Consistent with regulation at
the level of transcription, a 1.2-kilobase MeCPA transcript
is present in very low abundance in a peptone medium but is highly
expressed during growth on insect cuticle (Fig.
8).

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Fig. 7.
Examination of secreted MeCPA activity and
demonstration by immunoblotting that antibody raised against MeCPA in a
baculovirus expression vector reacts with a same sized protein in
culture filtrates of M. anisopliae. Proteins in
0.5 ml of crude culture filtrates exhibiting MeCPA activity
versus CBZ-Gly-Gly-Phe were concentrated 100-fold and
separated by SDS-polyacrylamide gel electrophoresis, transferred to
nitrocellulose, and tested with antibodies raised against BEV-MeCPA
(lane 1). Molecular weight markers are as indicated. MeCPA
activity in culture filtrates is expressed as the amount of enzyme that
released 1 µmol of CBZ-Gly-Gly-Phe/min at 25 °C.
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Fig. 8.
Detection of the MeCPA
mRNA during growth of M. anisopliae on
cockroach cuticle. Poly(A)+ RNA isolated from mycelium
of M. anisopliae grown for 32 h in SDB and transferred
for 12 h to minimal media supplemented with 1% cockroach cuticle
(+) or 1% peptone ( ).
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Localization of the MeCPA in Vivo--
Conidia of M. anisopliae germinated to form appressoria on the surface of
M. sexta and penetrated cuticles within 40 h of inoculation as described previously (15, 14). Using ultrathin sections
and immunogold cytochemistry we explored the ultrastructural localization of MeCPA during these infection processes. The labeling of
sections from cuticles at 24 and 48 h post-infection with
antibodies to MeCPA revealed labeling in and over the appressorial wall
confirming production by prepenetration fungal structures (Fig.
9). Production continued during
penetration with the growth of hyphae through the cuticle (Fig.
10). The enzyme remained localized in
the cell wall and the immediate vicinity of the fungal structures. Very few gold particles were found intracellularly.

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Fig. 9.
Protein A-gold labeled MeCPA produced by a
M. anisopliae appressorium on excised M. sexta cuticle (24 h after inoculation). AP,
appressorium; Ep, epicuticle; Pc, procuticle.
Bar, 0.5 µm.
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Fig. 10.
Protein A-gold labeled
MeCPA produced by M. anisopliae
during penetration of excised M. sexta cuticle
(48 h after inoculation). CW, cell wall; Pc,
procuticle; PH, penetrant hyphae. Bar, 0.5 µm.
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Control sections (see "Experimental Procedures") showed only a very
low level of nonspecific binding of gold particles (0.08 ± 0.03 gold particles per µm2), with the results for all
controls being negative (data not shown), confirming that use of
antisera to the overexpressed carboxypeptidase in conjunction with the
Protein A-gold complex is a valid labeling method for detecting the
enzyme in insect cuticle.
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DISCUSSION |
The significant number of partially or completely known sequences
of mammalian and bacterial procarboxypeptidases has permitted the
formulation of some evolutionary hypotheses. The calculated time of
divergence between bovine carboxypeptidase A and
Thermoactinomyces carboxypeptidase (about 1300 millions
years ago) led Rawlings and Barrett (7) to suggest an endosymbiont
origin for the family in eukaryotes. The discovery of a
metallocarboxypeptidase from a "primitive" (fungal) eukaryote opens
up new perspectives in this field by providing insights into when in
the course of evolution the different forms of carboxypeptidase diverged.
A detailed evolutionary tree has not yet been drawn for the
metallocarboxypeptidases, and we do not assume that the tree of contemporary sequences we present (Fig. 5) is an evolutionary one.
However, it confirms the structural classification of the fungal
enzymes inside the digestive group rather than the regulatory group.
The bacterial enzymes are also in the digestive group. However, the
degree of similarity between bacterial and animal digestive enzymes
(25%) is less than that between fungal and animal enzymes consistent
with their greater phylogenic distance. The bacterial enzymes also
differ from the eukaryote digestive carboxypeptidases in their dual A
and B specificity. It is proposed that an ancestor of bovine
carboxypeptidases gave rise to separate A and B forms before the
radiation of mammals (1). The restricted A-type specificity of MeCPA
for neutral residues suggests that this specialization occurred during
the early development of eukaryotes and perhaps was already present in
the originating endosymbiont. The A-type specificity of mammalian
enzymes is associated with Ile or Leu in positions 243 and 255 (22).
MeCPA possesses the equally hydrophobic Val243 and
Ile255, whereas in the A + B bacterial enzymes the same
positions are alternatively occupied by Ala-Ser243 and
Thr/Asp. The substrate specificity of the yeast enzyme (YHT2) has not
been determined but with Ala243 and Leu255 it
may represent an intermediate stage. The ability to cleave off
positively charged residues always depends on the presence of an Asp
residue, but as shown by the two related bacterial enzymes (Table I),
its position is not exactly fixed (22). MeCPA showed only marginal
activity against Arg residues so Asp194 cannot provide
B-type specificity.
MeCPA shows similar overall amino acid identity to human
carboxypeptidase A1 (35%) and A2 (37%). Even though there are no reports of A2 in bovine, and taking into account the broad specificity of the known enzyme, it has been proposed that mammalian
carboxypeptidases A1 and A2 are derived from the duplication of an
ancestor of the bovine carboxypeptidase gene before the radiation of
mammals (23). MeCPA provides the first direct evidence for a common
ancestral gene as it has a specificity that is intermediate between A1
and A2. Using primary specificity for aromatic (Phe) amino acids as a
criteria the similarity is closer to A2, but MeCPA differs from A2 in
also hydrolyzing a Leu-containing substrate for A1; a broader specificity more closely resembling bovine carboxypeptidase. The different preferences of human A1 and A2 forms for aliphatic or aromatic amino acids are a consequence of amino acid substitutions in
positions 253 and 268 (2). MeCPA shows close local identity with
regions of mammalian enzymes known to be critical for folding and
delineation of the active site, indicative of similar three-dimensional structures. However, the role of position 268 in MeCPA and YHT2 specificity is ambiguous as the insertion of two residues between 266 and 267 (or their deletion in the mammalian enzymes), could displace
268 at the active site and influence specificity of these enzymes. In
addition, the broader specificity demonstrated by MeCPA as compared
with the mammalian enzymes could be a consequence of subtle
substitutions at several positions. Thus MeCPA shares Gly253 with A2 and Thr268 with both A1 and
bovine carboxypeptidase. The Ala
Thr substitution is linked to the
broader substrate specificity of the bovine enzyme as compared with A2
carboxypeptidases (2). The pocket size in the bacterial (A + B) enzymes
is reduced compared with B-type carboxypeptidases by large residues in
positions 243, 250, 253, and 255. This facilitates A-type specificity
by allowing tighter fixation and more appropriate positioning of
hydrophobic P1' residues (22). Likewise, apparent pocket size reduction
by Val250 in MeCPA might explain the dual specificity for
branched aliphatic (Leu) residues and Phe. Carboxypeptidase A2 is
23-fold less active toward a leucine containing substrate than is
bovine carboxypeptidase (2), while activity by MeCPA is 4-fold higher
as compared with the bovine enzyme (Table II). Conversely, bovine
carboxypeptidase is more active against Tyr, also implying that it
possesses a larger active site pocket than MeCPA. However, any
reduction of available space in MeCPA does not allow significant
activity against an Ala-containing substrate for carboxypeptidase A1
(Table II). MeCPA is also approximately 3-fold more active than bovine
carboxypeptidase against a Trp-containing substrate that is also
hydrolyzed more efficiently by rat carboxypeptidase A2, but is not
hydrolyzed by carboxypeptidase A1 (2).
It has been suggested that as both the A1 and A2 types are expressed in
the rat pancreas, these enzymes have been allowed to diverge from one
another with respect to substrate specificity, resulting in
complementary preferences (2). Genomic studies have revealed only a
single gene for metallocarboxypeptidase in yeast (10) and Southern
analysis indicates the same in M. anisopliae, suggesting
that these genes may have been under selection pressure to maintain a
relatively broad substrate specificity. The fungal enzymes can be
proposed as intermediates between the bacterial and mammalian enzymes
being positioned after the origin of separate A- and B-types of
carboxypeptidase, but before the A-type gene duplicated and
differentiated into separate A1 and A2 isoforms.
Aside from the fungal enzymes, only the pancreatic carboxypeptidases,
mast cell carboxypeptidase, human plasma carboxypeptidase B, and the
bacterial enzymes are encoded and synthesized with an
NH2-terminal activation segment, as procarboxypeptidases
(1). These function by binding to the enzyme active site as an
inhibitor and are required for proper folding and secretion of the
enzyme (26). Although there is a coincidence in size, there are few sequence similarities between the fungal and mammalian activation segments which suggests that they may have evolved independently. This
supports a previous hypothesis that the pro-regions appeared at a late
stage of evolution of proteases (27). One aim of our study was the
development of an efficient recombinant expression system for MeCPA.
Pancreatic carboxypeptidases are difficult to express in native and
soluble forms in E. coli, but the P. pastoris heterologous system has been used to study the proteolytic activation and maturation pathway of human Pro-CPA2 in vitro (12). The resulting recombinant human Pro-CPA2 required activation by treatment with trypsin which cleaves within the pro-region of the protein. No
such trypsin-promoted maturation was required to obtain active MeCPA
using an insect cell line. Correctly processed propeptides are commonly
observed using baculovirus expression systems (28) and there was no
evidence from protein microsequencing for heterogeneity at the
NH2 terminus of the secreted MeCPA that would have
resulted from different cleavage points in the processing of the
Pro-MeCPA prosegment. It remains to be determined whether this system
would correctly cleave within the pro-region of mammalian
enzymes. However, in view of their similarity with MeCPA we believe our
results could facilitate efforts directed toward expressing
active vertebrate carboxypeptidases.
Pancreatic carboxypeptidases exhibit roles in digestion. Since the
MeCPA transcript is not detected when the fungus is grown on
nutrient-rich conditions, the high expression of MeCPA with insect
cuticle or other proteins as sole carbon and nitrogen source suggests a
function in procuring nutrients during protein digestion; an equivalent
function to digestive pancreatic enzymes. In previous studies we
identified a subtilisin-like protease which rapidly degrades cuticle
and can be used to genetically enhance virulence by transformation with
multiple copies of the gene under altered regulation (29). The
specificity of MeCPA appears to be adapted to complement that of the
protease, as the protease shows a clear preference for cleavage of
bonds COOH-terminal to aromatic residues, particularly Phe and to a
lesser extent amino acids containing long hydrophobic side chains (16).
This action would release peptides terminating in Phe or other amino
acids with hydrophobic side groups, that would then be further degraded
by the carboxypeptidase to single amino acids. The protease is the
major protein secreted into host cuticle during invasion (30). The
secretion of carboxypeptidase into host cuticle as shown by the
immunogold study indicates that MeCPA probably functions after the
protease solubilizes cuticle proteins to peptides to supply amino acids
for nutrition.
In conclusion, we report here the properties of MeCPA, the first fungal
metallocarboxypeptidase to be characterized. From data on primary
structure and substrate specificity it is suggested that MeCPA is a
prototype of the two mammalian enzymes carboxypeptidase 1 and 2. Development of the baculovirus expression system will further studies
on the structural and functional determinants of its activation,
behavior, and role in fungal pathogens. As baculoviruses are themselves
insect pathogens we will also be able to determine by baculovirus
bioassays the extent to which expression of the fungal protein enhances
the speed of killing insect hosts. Finally, the expression system will
facilitate determination of whether the microbial enzyme can supplement
mammalian enzymes in their current and hypothesized future
biotechnological applications.