Activated HaCA42 carboxypeptidase hydrolysed a synthetic substrate for glutamate carboxypeptidases FAEE, C-ter-minal Glu, but did not hydrolyse substrates for carboxy-peptidase A or B FA
Trang 1Characterization of a digestive carboxypeptidase from the insect
towards C-terminal glutamate residues
David P Bown and John A Gatehouse
School of Biological and Biomedical Sciences, University of Durham, UK
Carboxypeptidases were purified from guts of larvae of corn
earworm (Helicoverpa armigera), a lepidopteran crop pest,
by affinity chromatography on immobilized potato
carb-oxypeptidase inhibitor, and characterized by N-terminal
sequencing A larval gut cDNA library was screened using
probes based on these protein sequences cDNA HaCA42
encoded a carboxypeptidase with sequence similarity to
enzymes of clan MC [Barrett, A J., Rawlings, N D &
Woessner, J F (1998) Handbook of Proteolytic Enzymes
Academic Press, London.], but with a novel predicted
spe-cificity towards C-terminal acidic residues This
carboxyp-eptidase was expressed as a recombinant proprotein in the
yeast Pichia pastoris The expressed protein could be
acti-vated by treatment with bovine trypsin; degradation of
bound pro-region, rather than cleavage of pro-region from
mature protein, was the rate-limiting step in activation
Activated HaCA42 carboxypeptidase hydrolysed a synthetic
substrate for glutamate carboxypeptidases (FAEE,
C-ter-minal Glu), but did not hydrolyse substrates for
carboxy-peptidase A or B (FAPP or FAAK, C-terminal Phe or Lys)
or methotrexate, cleaved by clan MH glutamate carboxy-peptidases The enzyme was highly specific for C-terminal glutamate in peptide substrates, with slow hydrolysis of C-terminal aspartate also observed Glutamate carboxyp-eptidase activity was present in larval gut extract from H.armigera The HaCA42 protein is the first glutamate-specific metallocarboxypeptidase from clan MC to be iden-tified and characterized The genome of Drosophila mel-anogaster contains genes encoding enzymes with similar sequences and predicted specificity, and a cDNA encoding a similar enzyme has been isolated from gut tissue in tsetse fly
We suggest that digestive carboxypeptidases with sequence similarity to the classical mammalian enzymes, but with specificity towards C-terminal glutamate, are widely distri-buted in insects
Keywords: clan MC metalloproteinase; digestive proteinase; glutamate carboxypeptidase; insect herbivore; proteinase activation
Carboxypeptidases are exopeptidases that remove a single
amino acid residue from the C-terminus of a protein or
peptide substrate They play an important role in protein
digestion in the guts of higher animals, acting to liberate free
amino acids from the peptides produced by endopeptidase
action, thus completing the digestive process and generating
molecules that can be absorbed by the gut, via amino acid
transporters Mammals contain three genes encoding
digestive carboxypeptidases, designated carboxypeptidases
A1, A2 and B [1] All three proteins are zinc-containing
metallopeptidases of clan MC [2]
The specificity of the digestive carboxypeptidases in mammals has been extensively investigated These enzymes show a specificity directed towards the C-terminal amino acid residue in their substrates Carboxypeptidases A1 and A2 prefer neutral amino acids, with A1 favouring smaller amino acid side chains, whereas A2 favours bulkier side chains [3] Carboxypeptidase B is highly specific for basic C-terminal residues (with arginine favoured over lysine [4]) Specificity is primarily determined by interaction of the side chain of the C-terminal amino acid of the substrate with a binding pocket on the enzyme, with amino acid 255 (human carboxypeptidase A1 numbering) at the bottom [5] The side chain of this residue interacts with the substrate side chain; in carboxypeptidase B amino acid 255
is negatively charged aspartic acid to interact with positively charged basic side chains, whereas in carboxypeptidase A1 and A2 it is isoleucine, to form a hydrophobic interaction with neutral side chains Amino acids Tyr248 (hydrogen bond to P1 amino group), Arg71 (hydrogen bond to P2 carbonyl oxygen), Asn144, Arg145 (bind C-terminal carb-oxylate group of substrate) and Tyr198 are also important for substrate binding [2,6]
The presence of digestive carboxypeptidases in insects was established by Ward [7] who partially purified an enzyme with carboxypeptidase A activity from gut
Correspondence to J A Gatehouse, School of Biological and
Biomedical Sciences, University of Durham, South Road, Durham,
DH1 3LE, UK Fax: + 44 191 334 1201, Tel.: + 44 191 334 1264,
E-mail: J.A.Gatehouse@durham.ac.uk
Abbreviations: FAAK, furylacryloyl-Ala-Lys; FAEE,
furylacryloyl-Glu-Glu; FAPP, furylacryloyl-Phe-Phe; PCI, potato
carboxypeptidase inhibitor; SKTI, soya bean Kunitz trypsin inhibitor,
ACE, angiotension 1 converting enzyme.
Enzymes: glutamate carboxypeptidases (EC 3.4.17).
Note: A website is available at http://silver-server.dur.ac.uk
(Received 4 February 2004, revised 19 March 2004,
accepted 24 March 2004)
Trang 2extracts of the webbing clothes moth, Tineola bisselliella.
Soluble carboxypeptidase activity was subsequently found
in gut extracts from larvae of both coleopteran
(mealworm; Tenebrio molitor [8]); and lepidopteran
(armyworm; Spodoptera frugiperda [9]); insect species In
addition to carboxypeptidase A activity, carboxypeptidase
B activity has been detected in corn earworm
(Heli-coverpa armigera) although at much lower levels than
carboxypeptidase A activity [10] The molecular
charac-terization of these enzymes has been carried out through
the identification of cDNA clones encoding them The
first putative insect digestive carboxypeptidase to be
cloned was from black fly (Simulium vittatum [11]),
followed by enzymes from corn earworm (H.armigera
[12]), mosquito (Anopheles gambiae [13]; Aedes aegypti
[14]), tsetse fly (Glossinia morsitans [15]), and bertha
armyworm (Mamestra configurata [16]) On the basis of
sequence similarity, the genes from black fly, mosquito
and corn earworm were predicted to encode proteins with
similar specificity to carboxypeptidase A, whereas the
cDNA from tsetse fly was predicted to encode a protein
showing similar specificity to carboxypeptidase B These
predicted enzyme activities have not been directly
demon-strated except in the case of a carboxypeptidase A-like
enzyme from corn earworm, which has been expressed as a
recombinant protein in insect cells using a baculovirus-based
expression system, and has been shown to hydrolyse a
synthetic substrate for carboxypeptidase A, but not a
synthetic substrate for carboxypeptidase B [10] Although
other insect carboxypeptidases have not been expressed as
recombinant enzymes, expression of putative digestive
carboxypeptidases has been shown to be strongly
gut-specific, and upregulated by feeding [11,13,14,17] Insects
also contain other types of metallopeptidases, such as
angiotensin I-converting enzyme (clan MA) [18], but these
have not been shown to be involved in digestion
Mammalian digestive carboxypeptidases are synthesized
as inactive proenzymes (after cotranslational removal of
signal peptides) which contain a long N-terminal
pro-region [19] Activation of the carboxypeptidase results
from cleavage of the peptide bond between the pro-region
and the mature enzyme, catalysed by an activating
proteinase (trypsin in mammals) The insect digestive
carboxypeptidases also show evidence of the presence of
pro-regions, on the basis of sequence similarity, but the
activation process has only been directly demonstrated
with the carboxypeptidase A-like enzyme from Helicoverpa
armigera[20]
The present paper describes the identification of further
digestive carboxypeptidases in corn earworm, which are
predicted to show differing specificities towards C-terminal
amino acid residues One of these enzymes is shown to
have a novel specificity towards C-terminal glutamate
residues
Experimental procedures
Materials
Cultures of H.armigera were obtained from Syngenta plc
(Jealott’s Hill Research Station, Bracknell, Berks, UK) and
were maintained at 25C, with a 16 h day length in a
licenced facility (DEFRA PHL 179/4428) Larvae were routinely reared on the standard artificial diet described by Bown et al [12]
Purification and characterization of carboxypeptidases fromH armigera larval gut extract
Gut extract was prepared from fourth instar lavae of H.armigera as described previously [10] Extract from 65 larvae (13.25 mL) was diluted with an equal volume of 2· loading buffer (1 · ¼ 50 mM Tris/HCl, 100 mMNaCl
pH 7.5), centrifuged at 10 000 g for 10 min, and filtered through a GF/C glass fibre disc (Whatman Biochemicals) followed by a 0.47 lm cellulose acetate membrane The extract was applied to a column of immobilized potato carboxypeptidase inhibitor (PCI) which had been prepared
by coupling 2 mg PCI (gift from F X Aviles, Institut de Biolotechnologia i de Biomedicina, Universitat Autonoma
de Barcelona, Spain) to a 1 mL Hi-Trap NHS-activated Sepharose column as described by the manufacturer (Amersham-Pharmacia Biotech) The column was washed successively with 6 mL portions of: loading buffer; 0.25M
NaCl; 2Mglycine/HCl pH 2.0; 0.25MNaCl; 0.1Mglycine/ NaOH, pH 12.0; 0.25MNaCl; and 6Mguanidine hydro-chloride in loading buffer Pooled fractions were acetone precipitated and analysed by SDS/PAGE N-terminal sequencing was carried out on proteins blotted onto poly (vinylidene difluoride) membrane after SDS/PAGE by standard Edman degradation procedures using an Applied Biosystems Model 477A Protein Sequencer
Isolation and characterization of cDNAs encoding
H armigera carboxypeptidase The construction of a cDNA library in the phage k vector Lambda Uni-ZAP XR (Stratagene) from RNA extracted from gut tissue of H.armigera larvae has been described previously [12] The library was screened (as described by Sambrook & Russell [21]) using PCR products as probes for carboxypeptidases The probes were generated by PCR amplification of the library with specific primers encoding carboxypeptidase N-terminal sequences: Band C Fig 1A, 5¢-ATIACITGGGA(C/T)ACITA(C/T)TA(C/T)(A/C)G-3¢; band D Fig 1A, 5¢-TT(C/T)GA(C/T)CA(A/G)ATITA (C/T)CA(C/T)C-3¢; and a generic vector primer (T7 primer), 5¢-GTAATACGACTCACTATAGGGCG-3¢ PCR prod-ucts were cloned in pCR2.1 using the TOPO cloning method (Invitrogen) and checked by DNA sequencing Clones identified in the primary screen of the library were plaque-purified, excised into pBluescript SK+, and characterized
by DNA sequencing as described previously [12] 5¢ RACE was carried out using a BD SMART RACE cDNA Amplification Kit, using the manufacturer’s protocol (http://www.bdbiosciences.com/clontech/) and the gene-specific primer: 5¢-CCTCGTCAATGGAGTACTCGTAG CCATCAG-3¢ The amplified product was cloned in pCR2.1
as described for DNA sequencing DNA sequences were determined by standard dideoxynucleotide sequencing pro-tocols as adapted for ABI automated DNA sequencers, carried out by the DNA Sequencing Service, School of Biological and Biomedical Sciences, University of Durham Both DNA strands were fully sequenced on overlapping
Trang 3fragments Sequences were assembled using SEQUENCHER
software (Genecodes; http://www.genecodes.com) running
on Apple MacOS computers Sequence analysis was carried
out using BLASTsearches to identify sequence similarities
(http://www.ncbi.nlm.nih.gov/BLAST/), and SIGNALP [22]
to identify signal peptides (http://www.cbs.dtu.dk/services/
SignalP-2.0/) Multiple sequence comparison and
phylo-genetic tree analysis was carried out using the Clustal method
in theMEGALIGNprogram (DNAStar LaserGene software;
www.dnastar.com)
Preparation of expression construct for recombinant
HaCA42 procarboxypeptidase
A complete ORF for the predicted HaCA42
procarboxy-peptidase (i.e., without the signal peptide) was produced by
PCR using primers designed to match the first 21 and last 21
bases of the coding sequence of the proprotein, which had
extra bases added to include PstI (N-terminal) and SalI
(C-terminal) restriction sites: Forward, 5¢-CGCGCTGCA
GGTCATGAGAAATATGAAGGA-3¢; Reverse, 5¢-GC
GCGTCGACTGAATAGTTTTGCAAGACGTACTG-3¢
They were designed to allow the amplified sequences
encoding the proproteins to be ligated into the pGAPZa
(Invitrogen Life Technologies) to form a continuous reading
frame from the a-factor secretion signal of the vector,
through the procarboxypeptidase sequence, and into the
6· His-tag and stop codon of the vector The PCR
products were first cloned into the pCR2.1 vector using a
TOPO cloning system (Invitrogen) After confirming the
identity of the intermediate clone, the coding sequence
fragment was excised by restriction with PstI and SalI, and
ligated to pGAPZa which had been restricted with the same
enzymes Vector constructs were transformed into
chemi-cally competent TOP10F¢ cells (Invitrogen) and maintained
on medium containing zeocin (Invitrogen) at a final
concentration of 50 lgÆmL)1 All expression constructs
were sequenced through the ligation sites and inserted
sequence to ensure that no errors had been introduced into
the expressed polypeptides by the PCR process, and that the
construct had been correctly assembled
Expression and purification of recombinant
HaCA42 procarboxypeptidase
Competent Pichia pastoris cells (protease-deficient strain
SMD1168H) were prepared using the Pichia EasyComp
Transformation Kit (Invitrogen) following the
manufac-turer’s protocol Cells were transformed using linearized
DNA (restricted with BlnI) from the expression construct
Transformed yeast cells were selected by plating on YPDS
agar medium (10 gÆL)1 yeast extract, 20 gÆL)1 peptone,
20 gÆL)1dextrose, 1M sorbitol, 20 gÆL)1agar) containing
zeocin at a final concentration of 100 lgÆmL)1 Selected
colonies were screened for the presence of the expression
construct by colony PCR Selected PCR-positive colonies
were screened for expression of the recombinant protein by
immuno dot-blot analysis [23] of culture supernatant from
small-scale (10 mL) cultures grown for 72 h at 30C in
YPD/zeocin medium (10 gÆL)1 yeast extract, 20 gÆL)1
peptone, 20 gÆL)1dextrose, 100 lgÆmL)1zeocin)
Recom-binant protein was detected with anti-His(C-term) primary
antibodies (Invitrogen) followed by horseradish peroxidase-linked goat antimouse secondary Ig (Bio-Rad) Bound peroxidase activity was visualized with a chemiluminesent ECL detection system (Amersham Biosciences)
The highest-expressing clone was grown in large-scale culture in a 2.5 L laboratory fermenter (BioFlo 3000, New Brunswick Scientific Co Inc.; www.nbsc.com) using the method described in Rogelj et al [24], but omitting the methanol induction step After pelleting the yeast cells by centrifugation at 8000 g for 30 min at 4C, NaCl was added to the resulting culture supernatant to a final concentration of 2M Recombinant protein was purified from this solution by hydrophobic interaction chromato-graphy on a column of phenyl-Sepharose (1 cm i.d., 25 mL volume), equilibrated in and washed with 2MNaCl The column was eluted with water, and the eluted peak of protein was pooled The pooled fractions were adjusted to
20 mMTris/HCl pH 7.8, 0.5MNaCl (buffer A) and 5 mM
imidazole by adding concentrated buffer solutions The recombinant protein was finally purified by nickel affinity chromatography on a Ni/nitriolitriacetic acid agarose (Qiagen) column (1 cm i.d., 5 mL volume) The column was washed with Buffer A plus 5 mM imidazole The recombinant 6· His-tagged protein bound comparatively weakly, and eluted with both Buffer A plus 20 mM
imidazole and Buffer A plus 300 mM imidazole These fractions were pooled and the protein was precipitated with ammonium sulphate to 90% saturation The precipitated protein was resuspended in a minimum volume of buffer and desalted by gel filtration Glycerol was added to the excluded peak and this material was stored frozen in aliquots at)20 C The frozen aliquots were thawed before use in all subsequent assays; no loss of activity occurred on storage under these conditions
Activation of HaCA42 carboxypeptidase with trypsin The HaCA42 procarboxypeptidase was activated by treat-ment with bovine trypsin in 50 mMTris/HCl pH 8 at 37C Both the molar ratio of trypsin/procarboxypeptidase and the time of incubation were varied Samples were removed and diluted 1 : 5 into ice-cold sodium borate buffer pH 8.5 Samples of diluted enzyme were assayed for activity against furylacryoyl-Glu-Glu (FAEE, see below) as a substrate, and the remaining protein was precipitated by acetone The protein pellet was redissolved in SDS sample buffer and analysed by SDS/PAGE
Carboxypeptidase assays and expression
of HaCA42 mRNA Carboxypeptidase assays using the synthetic substrates furylacryloyl-Phe-Phe (FAPP), furylacryloyl-Ala-Lys (FAAK) and FAEE and Northern blotting of RNA from larval gut tissue, were carried out as described previously [10]
Peptide digestion by HaCA42 carboxypeptidase Recombinant HaCA42 procarboxypeptidase was activated
by treatment with bovine trypsin as described above The activated enzyme was diluted into buffer containing 1 m
Trang 4benzamidine to inhibit trypsin, and the mixture was treated
with phenyl methylsulphonyl fluoride or
aminoethyl-ben-zene sulphonyl fluoride (1 mM) to inactivate the serine
proteinase Diluted carboxypeptidase was incubated with
peptide substrates at a concentration of 1–2 lM in 10 mM
Tris/HCl pH 7.5 for varying times up to 120 min at 30C,
routinely at an enzyme/substrate molar ratio of 1 : 200
Other ratios were used as required Reactions were sampled
and quenched by adding dithiothreitol to 20 mM, and
spotted onto Ciphergen H4 protein chips (www.ciphergen
com) Peptides were analysed by surface enhanced laser
desorption/ionization MS, using a Ciphergen instrument, as
described in the manufacturer’s literature Mass ion sizes
were estimated by calibrating the instrument with size
standards covering the range analysed
Results
Purification of carboxypeptidase enzymes
fromH armigera larval gut
In order to characterize the total complement of digestive
carboxypeptidases in larval corn earworm, a H.armigera
larval gut extract was subjected to affinity chromatography
using immobilized PCI as a ligand The gut extract was
applied to the column under nondenaturing conditions at
neutral pH, and the column was washed extensively prior
to elution under successively more denaturing conditions
Eluted protein fractions were pooled, concentrated and
analysed by SDS/PAGE (Fig 1A) No protein bands were
visible in the fraction eluted using buffer at pH 2 (data not
shown) Subsequent elution of the column with buffer at
pH 12 gave a fraction containing a number of discrete
polypeptides, with major bands at 25, 50 and
55 kDa Finally, the column was eluted under highly
denaturing conditions, using buffer containing 6M guani-dine hydrochloride; the eluted fraction contained three polypeptides, a major band at 35 kDa, and a closely spaced doublet of bands at 30 kDa
Proteins were identified by N-terminal sequencing of polypeptide bands blotted from gel electrophoretic separa-tions (Table 1) None of the major bands eluted at pH 12 contained N-terminal sequences similar to carboxypepti-dases present in the databases The two proteins migrating
at 50 and 55 kDa (bands A and B; Fig 1A) were identified from their N-terminal sequences as similar to a-amylase (accession no AAA17751) from silkworm (Bombyx mori) The 25 kDa polypeptide band (band F; Fig 1A) gave an N-terminal sequence which corresponded
to that predicted by a cDNA previously isolated from the H.armigera larval gut library [12] This cDNA, SR21 (accession No Y12274) encodes a protein with sequence similarity to serine proteases, but which appears to lack members of the catalytic triad required for enzyme activity Binding of these proteins to PCI may be a result of specific interactions between the inhibitor and the proteins them-selves, although this would not be expected on the basis of their functional properties and sequences, or may suggest that they are present in a tightly bound complex with carboxypeptidase(s) in vivo
In contrast to the bands eluted at pH 12, the polypeptides eluted by 6M guanidine hydrochloride had N-terminal sequences with similarity to, or identity with carboxypep-tidases The band estimated as 35 kDa (band C; Fig 1A) gave an N-terminal sequence which had 41% identity over
29 amino acid residues to the N-terminal region of a crayfish carboxypeptidase (P04069) The less strongly stained bands estimated as 30 kDa (bands D and E; Fig 1A) contained two different N-terminal sequences The lower molecular mass band of the doublet (band E; Fig 1) gave an N-terminal sequence identical over 32 amino acid residues
to the N-terminal region of carboxypeptidase A from H.armigera larval gut (sequence predicted by cDNAs AJ005176–8) The higher molecular mass band of the doublet (band D; Fig 1A) gave an N-terminal sequence of
20 amino acid residues which was similar to (47% identity), rather than identical with the N-terminal region of H.armigeracarboxypeptidase A
Identification of cDNAs encoding carboxypeptidases
inH armigera larval gut library The characterization of three similar cDNAs encoding carboxypeptidase A-like digestive proteases as a result of screening a cDNA library prepared from RNA extracted from gut tissue of corn earworm larvae has been described previously [10] In order to isolate cDNA clones encoding other digestive carboxypeptidases, degenerate oligonucleo-tide primers were designed using the N-terminal sequence data obtained from gut polypeptides, as described above Using these specific primers, and primers directed against vector sequences, PCR was carried out on the larval gut cDNA library Both N-terminal primers in combination with a generic 3¢ primer gave products of 1.0 kb PCR products were individually excised from gel, purifed, and cloned At least three independent clones for each product were characterized by a preliminary DNA sequencing run
Fig 1 Purification of native and recombinant carboxypeptidases (A)
Affinity chromatography of gut extract from larval H.armigera on
immobilized PCI Fractions eluted under conditions as shown were
analysed by SDS/PAGE Bands A–F refer to polypeptides subjected to
N-terminal sequence analysis (Table 1) (B) Purification of
recombin-ant HaCA42 carboxypeptidase from culture medium after expression
in P.pastoris The purified protein was analysed by SDS/PAGE.
Trang 5The PCR reactions using the two separate
carboxypepti-dase-specific primers each gave essentially a single product
with similarity to carboxypeptidases (although minor
het-erogeneity, potentially resulting from amplification errors,
was present) These sequences were then used as probes to
screen the cDNA library cDNAs detected by each of the
two PCR products were isolated and sequenced
Characterization ofH armigera
carboxypeptidase-encoding cDNAs
cDNAs encoding the 35 kDa H.armigera
carboxypepti-dase (band C, Fig 1A) are exemplified by a clone
designa-ted HaCA42 This cDNA (accession no AJ626862) was
fully sequenced on both strands; it is truncated at the 5¢ end,
and starts at nucleotide 8 of the coding sequence The
sequence at the 5¢ end of the mRNA was completed
by 5¢ RACE, from which two independent clones gave
identical sequences at the same starting point for the
mRNA A poly(A) tail is present The corresponding mRNA thus contains an 11 base 5¢ untranslated region (UTR), a coding sequence of 1275 bases (including stop codon), and an 89 base 3¢ UTR excluding the poly(A) sequence A cDNA clone with 98% identity with HaCA42
at the nucleotide level over the coding sequence and 99% identity with HaCA42 in the deduced amino acid sequence was also sequenced, and represents a second member of the subfamily of carboxypeptidase genes exemplified by HaCA42 The deduced amino acid sequence of HaCA42 (Fig 2) predicts that this is a secreted protein, the first 18 residues constituting a typical signal peptide (SignalP prediction, vs 2.0) The predicted proprotein is therefore
is 406 amino acids in length, with a predicted MW of 46.0 kDa When this sequence was used to query the protein sequence databases, the closest similarity (38–40% identity, based on identity of corresponding amino acid residues) was to the carboxypeptidase A sequences encoded
by the cDNAs previously isolated from H.armigera
Fig 2 Predicted protein sequence from cDNA HaCA42 The predicted signal peptide is indicated; propeptide and mature protein are designated from N-terminal sequence determined for carboxypeptidase purified from H.armigera larval gut extract (shaded) Sequence features of clan MC carboxypeptidases are denoted as follows (numbering from human carboxypeptidase A sequence): *, catalytically active residues (Arg127, Glu270);
d, zinc ligand residues (His69, Glu72, His196); b, substrate binding residues (Arg71, Asn144, Arg145, Tyr198, Tyr248); s, S1¢ site residue (Arg255) Potential N-glycosylation sequences are boxed.
Table 1 N-terminal sequences of polypeptides eluted from affinity column containing immobilized PCI at pH 12 and with 6 M guanidine hydrochloride Partial amino acid sequences predicted by specified cDNAs (accession numbers in brackets) are given in italic type.
Band
(kDa)
N-terminal sequence determined/sequence predicted
PCI
AYSSSSPA RIEDYPSTVQLETGIGRV Similar to serine protease (Y12274)
ALAY KNPHY AS GR T TMVHLFE a-amylase (AAA17751)
ALAYKNPHYASGRTTMVHLFE a-amylase (AAA17751)
6 M guanidine hydrochloride
RSRLSFDKIHSYEEVDAYLQELAKEFPNVVTVVEGG carboxypeptidase (AJ005176)
ASRLD S LPFDQIYTYHQVDTFLDMLA carboxypeptidase
C (35) SITWDTYYRHDEINDYLDELAEQNSD(L / I)XTV Ha cDNA CA42
SGKSITWDTYYRHDEINDYLDELAEQNSD L VTVINA carboxypeptidase
Trang 6(accession numbers AJ005176–8) Similar levels of similarity
were found to sequences of ORFs found in the genomes
of Drosophila (34–37% identity, NM139861-3), Anopheles
(42% identity, AAAB01008960) and Caenorhabditis elegans
(40% identity, NM074283) A carboxypeptidase B enzyme
from crayfish also lies within the group of sequences
showing the high levels of similarity to HaCA42 (37%
identity, P04069)
The N-terminal sequence determined for the 35 kDa
carboxypeptidase from H.armigera larval guts (band C;
SITWDTY…; Table 1) is located 98 amino acids from the
predicted N-terminus of the pro-region (Fig 2) The amino
acid sequence predicted by the cDNA is identical to the
sequence determined (29 amino acid residues) Removal of
the pro-region results in a predicted protein of 308 amino
acids, MW 34.8 kDa, in close agreement with that
deter-mined by SDS/PAGE Other features of the predicted
protein sequence are consistent with the conserved residues
in metallocarboxypeptidases of clan MC [2] The mature
sequence contains amino acid residues His69, Glu72 and
His196 (numbering based on human carboxypeptidase A)
which ligate the catalytic zinc ion in
metallocarboxypeptid-ases; Arg127 and Glu270 also involved in catalysis; and
Arg71, Asn144, Arg145, Tyr198 and Tyr248, which
parti-cipate in substrate binding (Fig 2) A distinguishing feature
of this predicted protein sequence is the amino acid residue
at position 255, which determines substrate specificity by
interacting with the side chain of the P1¢ residue In the
protein predicted by HaCA42 this residue is arginine There
are also two consensus N-glycosylation sites within the
amino acid sequence predicted by HaCA42, both of which
lie within the mature protein, with one near the C-terminus
The cDNAs encoding the polypeptide present in the
upper band of the 30 kDa doublet of H.armigera
carb-oxypeptidases (band D, Fig 1A) are exemplified by a clone
designated HaCB6 (accession no AJ626863) This cDNA
also encodes a clan MC metallocarboxypeptidase enzyme,
and will be described elsewhere
Expression and purification of recombinant
procarboxypeptidase HaCA42
A construct to allow the protein encoded by HaCA42 to
be expressed in the yeast P.pastoris was assembled by
amplifying the coding sequence of the cDNA by PCR
Primers were designed to allow the PCR product to be
inserted into the Pichia expression vector pGAPZaB with
the N-terminus of the proprotein in-frame and adjacent to
the cleavage point of the yeast a-mating factor secretion
signal encoded by the vector In addition, a (His)6-tag
encoded by the vector was added to the C-terminus of the
protein before the stop codon The construct was verified by
DNA sequencing after assembly, and linearized plasmid
DNA was used to transform competent P.pastoris After
selection for transformation on zeocin plates, colonies were
screened by PCR for the presence of the HaCA42 sequence
Positive colonies were individually grown in small-scale
cultures, and samples of culture medium were assayed for
expression of His-tagged protein by immunodot blot The
transformant that showed the highest level of expression
was chosen for protein production, and was grown up under
optimized conditions in a 2 L laboratory fermentor
The recombinant protein (referred to subsequently as HaCA42 procarboxypeptidase or carboxypeptidase) was purified from culture medium by hydrophobic interaction chromatography followed by affinity chromatography on immobilized nickel ions The purified protein ran as a single band when analysed by SDS/PAGE, with an estimated
MW of 50 kDa (Fig 1B) The yield of purified protein was 5 mgÆL)1of fermenter culture
Activation of recombinant procarboxypeptidase HaCA42 The recombinant HaCA42 procarboxypeptidase enzyme had no detectable activity when assayed against synthetic-furylacryloyl (FA)–peptide substrates for carboxypeptidases Three substrates were assayed: FAPP, with a C-terminal phenylalanine residue (hydrolysed by carboxypeptidase A); FAAK, with a C-terminal lysine residue (hydrolysed by carboxypeptidase B) and FAEE, with a C-terminal gluta-mate residue (hydrolysed by glutagluta-mate carboxypeptidase) However, after the protein was treated with substoichio-metric amounts of bovine trypsin (procarboxypeptidase/ trypsin molar ratio > 5 : 1) carboxypeptidase activity against FAEE could be detected Trypsin gave no activity against any of these substrates in the absence of the recombinant carboxypeptidase Because the activation of mammalian digestive procarboxypeptidases in vivo is known to be caused by cleavage of the propeptide by trypsin [19], the results suggested that a similar activation process was necessary for the HaCA42 procarboxypepti-dase, and that endogenous yeast proteases in the protease-deficient Pichia strain used were not sufficient to cause activation
To further investigate the activation process, recombinant HaCA42 procarboxypeptidase was incubated with trypsin (9.4 : 1 molar ratio) at 37C At various timepoints samples were withdrawn and trypsin activity was quenched; the carboxypeptidase activity against FAEE was then assayed, and the polypeptides present were analysed by SDS/PAGE Results are shown in Fig 3A A control sample of HaCA42 procarboxypeptidase (track C) contained no detectable polypeptides of < 48 kDa, but even after a nominal zero incubation time (track 0), corresponding to sampling the mixture of procarboxypeptidase and trypsin immediately after addition of trypsin, polypeptides of 36 kDa and
13 kDa are present in the sample Neither of these polypeptides was present in trypsin when this enzyme was analysed by SDS/PAGE (data not shown) By analogy with the activation of mammalian carboxypeptidases, these polypeptides correspond to the active HaCa42 carboxy-peptidase (36 kDa polypeptide) and the pro-region (13 kDa polypeptide) After 5 min the majority, and by 20 min all, of the original 50 kDa protein had been digested, with an increase in staining of the bands of 36 kDa and
13 kDa On further incubation with trypsin the 13 kDa polypeptide is itself digested by trypsin, and decreases in amount until it is no longer detectable after 80 min of digestion, but the amount of 36 kDa polypeptide remains constant up to 2 h digestion under these conditions When the carboxypeptidase activity (FAEE substrate) of the mixture was assayed, there was a qualitative correlation between the appearance of the putative 36 kDa activated carboxypeptidase polypeptide, and the level of activity
Trang 7detected Thus, the control procarboxypeptidase sample
had no detectable activity, but the zero time sample
contained detectable activity (5% of maximum activity)
which increased with time (Fig 3B) However, when
quantitative estimates of activity were compared to results
of the gel analysis, it was apparent that cleavage alone was
not sufficient for activation After 20 min digestion by
trypsin, all of the procarboxypeptidase band at 50 kDa
had been cleaved to 36 kDa and 13 kDa bands, but the
carboxypeptidase activity was only 55% of the maximum
activity (Fig 3A,B) The carboxypeptidase activity only
reaches a maximum after 60–80 min incubation with
trypsin, and further incubation with trypsin to 120 min does
not affect the level of activity against this substrate
Attainment of maximum carboxypeptidase activity in this
assay corresponds to the disappearance of the 13 kDa
pro-region polypeptide (Fig 3A,B); once this polypeptide
has been completely digested by trypsin, the
carboxypepti-dase activity is maximal
Characterization of recombinant HaCA42 carboxypeptidase activity
The pH optimum for hydrolysis of FAEE by the activated HaCA42 carboxypeptidase was determined over the range 2.2–10.5 using a variety of buffer systems There was a marked optimum activity at pH 8.5 in borate buffer with activity declining to 50% of maximum at pH 7.5 and 10.0 (data not shown) Various diagnostic inhibitors were used to characterize the activity of the recombinant enzyme No inhibition (< 10% reduction in activity compared to enzyme preincubated without inhibitor) was observed after preincubation with: the cysteine protease inhibitor E-64 (10)5Mfinal concentration); the aspartic protease inhibitor pepstatin (10)5M); the serine protease inhibitors phenyl methylsulphonyl fluoride, (2· 10)5M) and soybean kunitz trypsin inhibitor (5· 10)7M); chymostatin (10)5M) an inhibitor of chymotrypsin; and benzamidine (10)2M) an inhibitor of trypsin
The metalloprotease inhibitors phenanthroline (5·
10)3M) and EDTA (10)2M) both had marked effects on activity (82% inhibition and 96% inhibition, respectively)
as did the protein carboxypeptidase inhibitor PCI (94% inhibition at 2.5· 10)6M) Interestingly, preincubation with zinc, used by many authors in activating carboxy-peptidase, has a deleterious effect on activity; 10)5MZnCl2 inhibits activity by 68% and 10)6MZnCl2inhibits activity
by 21% The reducing agent dithiothreitol also inhibits activity of the recombinant HaCA42 carboxypeptidase at concentrations above 10)5M, resulting in 45% inhibition at
10)4Mand 86% inhibition at 10)3M The kinetic parameters for hydrolysis of FAEE by the recombinant HaCA42 carboxypeptidase were determined
by a standard Michaelis–Menten analysis using varying substrate concentrations Kmwas estimated as 6· 10)5M
(mean of three determinations), and Vmaxwas estimated as 7.3· 10)7moles FAEE hydrolysedÆs)1Æmg protein)1 Assu-ming a MW of 36 kDa for the active recombinant enzyme, and that all the proenzyme has been activated and remains active in the assay, these figures give values of 26 s)1for
iatand 4.3· 105s)1ÆM )1for kcat/Km
Substrate specificity of recombinant HaCA42 carboxypeptidase
The activated recombinant HaCA42 carboxypeptidase hydrolysed FAEE (Glu C-terminal residue), but gave no detectable hydrolysis of synthetic substrates for peptidase A (FAPP, Phe C-terminal residue) or carboxy-peptidase B (FAAK, Lys C-terminal residue) even when used in large amount for extended digestion periods The specificity of the activated enzyme was investigated in more detail by incubation with a selection of peptides of known sequence in the presence of trypsin inhibitors to prevent digestion by the activating enzyme The presence or absence
of digestion was assayed by MS over a mass range which included the peptide substrates Results are presented in Table 2, which defines the peptide sequences and their abbreviations
When hydrolysing peptide substrates at enzyme/substrate ratios of 1 : 200, the HaCA42 carboxypeptidase had a similar specificity to that observed when synthetic dipeptide
Fig 3 Activation of HaCA42 carboxypeptidase by trypsin (A) SDS/
PAGE of cleavage of procarboxypeptidase, sampled after stated times
of digestion with bovine trypsin (9.4 : 1 molar ratio procarboxypept
idase/trypsin) Pro-, procarboxypeptidase; Mature, mature
carboxy-peptidase; Activn peptide, pro-region The faint band at 25 kDa is
from the trypsin used for activation (B) Carboxypeptidase activity
(digestion of FAEE substrate) after stated times of digestion with
bovine trypsin.
Trang 8substrates were used Angiotensin 1, a peptide with a
C-terminal neutral, hydrophobic amino acid (Leu) was
not hydrolysed, like the synthetic substrate FAPP (Phe
C-terminal residue) Similarly, fibrinopeptide B, with a
C-terminal basic residue (Arg), like FAAK (Lys C-terminal
residue), was not hydrolysed The neutral hydrophilic
C-terminal serine of angiotensin (1–14), and the C-terminal
proline of ACTH 1–24 were also not hydrolysed On the
other hand, a peptide with a C-terminal glutamate residue
(b-endorphin amino acids 61–91) was readily cleaved by the
HaCA42 carboxypeptidase, like the synthetic substrate
FAEE (C-terminal Glu residue) The specificity was further
explored by using peptide substrates with the C-terminal
side-chain amide residues, asparagine (PDI substrate) and
glutamine (Cys-CD36) Neither peptide was hydrolysed
by the HaCA42 carboxypeptidase, suggesting that the
C-terminal amino acid must carry a negative charge on the
side chain Finally, an angiotensin 1 converting enzyme
(ACE) inhibitor peptide with a C-terminal aspartate residue
was assayed; this was hydrolysed by the carboxypeptidase,
but very slowly Under conditions sufficient to completely
cleave the b-endorphin substrate, < 5% of the
ACE-inhibitor peptide was cleaved, as estimated by the
appearance of a new peptide with lower molecular mass
(data not shown)
The specificity of the HaCA42 carboxypeptidase was also
investigated by carrying out a time-course experiment for
digestion of the b-endorphin substrate Results are presented
in Fig 4 At an enzyme/peptide ratio of 1 : 5000,
appear-ance of a peptide product of correct mass for cleavage of the
C-terminal glutamate from the b-endorphin peptide was
observed after 1 min The amount of this product relative to
the undigested peptide increased with time, until digestion
was essentially complete after 90 min After removal of the
C-terminal glutamate, the next residue is a glycine, but there
was no evidence for removal of this residue from the initial
product of HaCA42 carboxypeptidase digestion in the
timescale of this experiment (up to 120 min), or in
experi-ments where HaCA42 carboxypeptidase was present at
ratios up to 1 : 200 with respect to substrate
The HaCA42 carboxypeptidase was also assayed for its
ability to hydrolyse the folate analogue methotrexate, which
contains a glutamate residue linked via an amide bond to
pteroic acid No activity against this substrate could be
detected in a spectrophotometric assay in the presence of
excess enzyme
Glutamate carboxypeptidase activity inH armigera larvae
Activity towards synthetic substrates for carboxypeptidase
A and B (FAPP and FAAK) has previously been charac-terized in gut extracts from H.armigera larvae [10] However, crude extracts of H.armigera larval gut contents showed little detectable activity towards the glutamate carboxypeptidase substrate FAEE, although carboxypep-tidase A activity, and low levels of carboxypepcarboxypep-tidase B activity could be detected in the same material To confirm that the digestive carboxypeptidases in this insect did include enzymes with activity towards substrates with C-terminal glutamate residues, two approaches were taken When insects were induced to regurgitate gut contents, and the regurgitant was collected and analysed, carboxypepti-dase activity towards the FAEE substrate could be readily detected The activity was shown to be present in bulk gut contents by partial purification of total gut content proteins
by ammonium sulphate precipitation The redissolved ammonium sulphate pellet was assayed for carboxypepti-dase activity, and hydrolysis of both FAPP (carboxypepti-dase A activity) and FAEE (glutamate carboxypepti(carboxypepti-dase activity) were detected, although more activity towards the former substrate was present Quantitative analysis gave values of 4.3· 10)8 moles FAPP hydrolysedÆmin)1Ægut equivalent)1 and 7.3· 10)9 moles FAEE hydro-lysedÆmin)1Ægut equivalent)1 under the conditions of this assay, suggesting that approximately six times as much carboxypeptidase A activity as glutamate carboxypeptidase activity is present in bulk gut contents
A Northern blot of RNA extracted from gut tissue of larval H.armigera was probed with the HaCA42 cDNA A single band of estimated size 1.45 kb was observed after autoradiography (Fig 5), consistent with the estimated size
of the mRNA, and its assumed abundance in gut tissue
Discussion
Carboxypeptidases specific for glutamate have been char-acterized from a number of bacterial species; they are referred to as carboxypeptidase G (various subtypes), or more correctly, glutamate carboxypeptidases (NC-IUBMB preferred [25]), and have been given the EC classification 3.4.17.11 These enzymes are able to cleave C-terminal glutamate residues in peptides, and also the glutamate
Table 2 Digestion of peptide substrates by activated recombinant HaCA42 carboxypeptidase Digestion was detected by MS after varying times of digestion up to 2 h 0, no digestion detectable; ±, slight digestion detectable; +++, digestion readily detectable ACTH, adrenocorticotropic hormone.
b-endorphin (aa 61–91) Glutamate YGGFMTSEKSQTPLVTLFKNAIIKNAYKKGE +++
Trang 9residue linked via its a-amino group to pteroic acid in folic
acid and folate analogues, such as the drug methotrexate
(4-amino-N10-methylpteroylglutamate) A distinct enzyme,
known as glutamate carboxypeptidase II (EC 3.4.17.21),
which is active towards acidic dipeptides with C-terminal glutamate, and folate analogues, is present in mammalian nervous tissue and prostate [26] These enzymes all belong to clan MH of metalloproteinases, and have little sequence similarity or structural similarity to clan MC carboxy-peptidases The enzyme described in the present paper is different from these previously described glutamate carboxypeptidases in belonging to the clan MC of metallo-carboxypeptidases It is also more specific than the clan
MH glutamate carboxypeptidases, as it has no detectable activity towards glutamate residues linked to folic acid No other carboxypeptidase in clan MC has a similar specificity
to the HaCA42 enzyme, and to date the only eukaryotic digestive carboxypeptidase activity demonstrated has been
of the -A or -B type [2,27] The HaCA42 enzyme is therefore the first example of a new peptidase The best nomenclature for this enzyme would be carboxypeptidase C (which would emphasize its similarity to carboxypeptidases A and B), but this name is already used for a subclass of serine carboxy-peptidases, although not for any specific enzyme in this class Glutamate carboxypeptidase MC is a possible alternative
It seems unlikely that this type of carboxypeptidase is unique to H.armigera, and it would be reasonable to expect similar enzymes to be present in other lepidopteran
Fig 4 Time-course for digestion of b-endorphin peptide substrate by
activated HaCA42 carboxypeptidase Traces show mass spectra from
peptide sampled after varying times of digestion Mass ion at m/e
3465.0 corresponds to uncleaved peptide; mass ion at 3335.9
corres-ponds to removal of a glutamate residue from the C-terminus; this
product is then stable to further C-terminal exopeptidase action The
small peak at m/e 3150.7 visible after extended digestion results from
cleavage between lysine residues in the peptide C-terminal sequence
(…YKKGE) caused by residual trypsin activity from the activating
enzyme.
Fig 5 Expression of HaCA42 in gut mRNA RNA extracted from midgut tissue of H.armigera larvae fed on control diet (C) and diet supplemented with SKTI (S) was separated by formaldehyde/agarose gel electrophoresis and blotted onto nitrocellulose The blot was pro-bed with the HaCA42 cDNA (coding sequence and 3¢ UTR) and washed to a final stringency of 0.1 · NaCl/Cit, 0.1% SDS at 50 C The size of the hybridizing band was estimated from markers run on separate tracks of the same gel, which were excised and stained.
Trang 10herbivores, and possibly in a wider range of arthropods The
Drosophila melanogaster(fruit fly) genome contains 19 genes
encoding proteins with sequence similarity to the HaCA42
carboxypeptidase (BLASTcomparison, E < 10)30), plus two
genes encoding proteins with a low level of similarity
(CG4122, 4678; E¼ 7 · 10)6, 2· 10)6, respectively) A
phylogenetic tree based on sequence comparison between
HaCA42 and similar proteins predicted by the Drosophila
genome is shown in Fig 6A The HaCA42
carboxypepti-dase maps within the phylogenetic tree of similar Drosophila
predicted proteins Although not all the Drosophila genes
encode active carboxypeptidase enzymes, the majority
contain the residues necessary for activity, and have
sufficient similarity over the region corresponding to
residues 248–270 in human carboxypeptidase A to allow
the equivalent residue to amino acid 255, the specificity
determining residue, to be identified Three genes, CG4408,
CG12374 and CG14820, predict proteins with lysine
residues at position 255 (Fig 5B), where a positively
charged basic side chain should give these proteins a similar
specificity to HaCA42 All these proteins are predicted to
have metallocarboxypeptidase activity; the CG12374 product is annotated in FlyBase as having carboxy-peptidase A activity, but this assignment is based only
on overall sequence similarity and, we suggest, is probably incorrect
In contrast with the situation in Drosophila, the Anopheles gambiae(mosquito) genome does not contain genes enco-ding carboxypeptidases with similar predicted specificity to HaCA42 There are 22 genes predicting proteins with sequence similarity to HaCA42 (E < 10)29), but none of the genes predicting active enzymes have a basic residue at positions equivalent to Ile255 in human carboxypeptidase
A, all being carboxypeptidase A- or B-like in predicted specificity In support of a wider distribution of glutamate-specific carboxypeptidases beyond H.armigera, examina-tion of the global sequence databases suggests that a further enzyme similar in sequence to HaCA42, and with a similar predicted cleavage specificity, is present in one other insect species An incomplete cDNA from tsetse fly (Glossinia morsitans morsitans), designated GmZcp (accession number AAK07479; amino acid sequence given is not complete),
Fig 6 Sequence comparisons for carboxypeptidases (A) Phylogenetic tree for predicted carboxypeptidases of clan MC, family M14, from D.melanogaster (designated by CG-gene identifier) compared to HaCA42 carboxypeptidase (shaded branch) The S 1 ¢ site residue (AA255) and the predicted carboxypeptidase activity (A-like, B-like or glutamate-) based on this residue are as indicated Sequence similarity over the region corresponding to amino acids 248–270 (human carboxypeptidase A numbering) is not present for the predicted products of CG32379 and CG8945; CG15679 lacks both E270 and Y248, and CG3097 and CG8564 lack Y248 These genes are predicted to encode proteins inactive as carboxy-peptidases (B) Sequence alignment over the region including amino acids 246–272 (human carboxypeptidase A numbering) for human carb-oxypeptidase A, and enzymes of clan MC, family M14 predicted to show glutamate carbcarb-oxypeptidase activity H.armigera (shaded) and D.melanogaster genes are designated as above; GmZcp cDNA, protein predicted by G.morsitans (tsetse fly) gut cDNA clone.