Cloning and Expression of a Prokaryotic Enzyme, Arginine Deiminase, from a Primitive Eukaryote Giardia intestinalis*

Leigh A. KnodlerDagger , Eric O. Sekyere, Thomas S. Stewart, Philip J. Schofield, and Michael R. Edwards§

From the School of Biochemistry and Molecular Genetics, University of New South Wales, Sydney 2052, Australia

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Arginine deiminase (EC 3.5.3.6) catalyzes the irreversible catabolism of arginine to citrulline in the arginine dihydrolase pathway. This pathway has been regarded as restricted to prokaryotic organisms but is an important source of energy to the primitive protozoan Giardia intestinalis. In this paper we report the cloning and expression of the arginine deiminase gene from this parasite. Degenerate oligonucleotides based on amino acid sequences of tryptic peptides from the purified protein were used to amplify a portion of the arginine deiminase gene. This was then used as a probe to screen HindIII and PstI "mini" libraries to obtain two overlapping clones that contained the arginine deiminase gene. The open reading frame encoded 581 amino acids including all of the tryptic peptides that were sequenced and corresponded to a molecular mass of 67 kDa. Northern blot analysis identified a single 1.8-kilobase transcript in both trophozoites and encysting cells. Arginine deiminase was successfully expressed in Escherichia coli and purified to homogeneity. The recombinant protein was found to have characteristics comparable with those of the native enzyme.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Giardia intestinalis is one of the most commonly transmitted intestinal pathogens in the world. There are two stages in the life cycle, both well adapted to two different, hostile environments. Infection is most often by ingestion of the cyst from a contaminated water or food supply. Excystation is induced by passage through the stomach, resulting in the emergence of the motile trophozoite form, which colonizes the upper small intestine and causes the symptoms of giardiasis, including vomiting and diarrhea, by an as yet unknown mechanism. Completion of the life cycle occurs when trophozoites are carried downstream by the intestinal fluid and then encyst, followed by emergence from the host. Current treatment against giardial infection is problematic, due to the increasing emergence of drug-resistant strains and lack of compliance. Hence there is a need for new and specific drugs against infection.

In addition to being a major health burden, G. intestinalis has recently been described as the "missing link" between prokaryotes and eukaryotes (1). Therefore Giardia is an important tool for the study of the evolution of the complex nature of the eukaryotic cell. Metabolism in Giardia, for example, is indicative of the primitive nature of this organism. In Giardia, the arginine dihydrolase (ADH)1 pathway is an important source of energy, with arginine the preferential fuel used in the early and most proliferative stages of in vitro growth (2). This pathway has been reported in only two other eukaryotes, Trichomonas vaginalis (3) and Tritrichomonas fetus (4), both also placed on early branches of the eukaryotic evolutionary tree (5).

The ADH pathway is normally confined to the prokaryotic kingdom, where it provides both a source of energy and nitrogen (6). The pathway consists of three enzymatic steps, involving arginine deiminase, ornithine transcarbamolyase, and carbamate kinase, with ATP production at the final enzymatic step as follows: arginine right-arrow citrulline right-arrow ornithine + carbamyl phosphate right-arrow CO2 + NH4+ + ATP.

The activities of all the arginine dihydrolase pathway enzymes have been demonstrated in Giardia (7), with carbamate kinase being the most active enzyme yet reported in cell-free extracts. The activity of ornithine transcarbamolyase, the least active enzyme in the pathway, is approximately 5-fold higher than the reported activities of some glycolytic enzymes (7, 8). An arginine-ornithine exchange transporter has been described in Giardia that has properties similar to arginine transporters found in some prokaryotes that utilize the ADH pathway (9). The function of the pathway in intact trophozoites has been demonstrated in metabolic flux experiments by measuring the release of radiolabeled CO2 from [guanidino-14C]arginine (7). These are all properties consistent with the ADH pathway representing a major metabolic capacity of the protozoan.

We are interested in characterizing the first enzyme in the pathway, arginine deiminase, which catalyzes the irreversible conversion of arginine to citrulline, primarily as a candidate for future drug design. Very few sequences of arginine deiminase are available from genome data bases, and there is very little homology between these published sequences, which prevents the cloning of the arginine deiminase gene from Giardia from degenerate oligonucleotides. We have recently reported the purification of arginine deiminase from Giardia and the sequence of five tryptic peptides (10). In this paper, we present the first report of the isolation, characterization, and overexpression of the arginine deiminase gene from a eukaryotic organism, and we suggest that it may be an attractive chemotherapeutic target against giardial infection.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Cell Culture Conditions-- G. intestinalis trophozoites, Portland I strain, were cultured as described previously (11). Encystation of trophozoites was as described previously (12).

Nucleic Acid Isolation-- G. intestinalis genomic DNA was prepared by the method described by Yee and Dennis (13), followed by cesium chloride equilibrium density centrifugation. Total RNA was isolated using RNAZol B (Tel-Test, Friendswood, TX), and cDNA was synthesized as described previously (12).

Oligonucleotides and Polymerase Chain Reaction-- Degenerate sense and antisense oligonucleotides were derived from tryptic peptides obtained from the purified giardial arginine deiminase (10) (Table I) and synthesized on a Beckman Oligo 1000 DNA Synthesizer. PCR conditions were as described in the manufacturer's protocol for Tth polymerase (Biotech International, Australia) with 500 ng of genomic DNA. Annealing was at either 45 or 50 °C and extension at 72 °C for 40 cycles. PCR products were treated with Klenow fragment and ligated into the SmaI site of the pTZ18 vector. For identification of the polyadenylation addition position, PCR of trophozoite cDNA was performed with a sense oligonucleotide ADI 1840 (nts 1840-1859 of the giardial arginine deiminase sequence) and an oligo(dT) primer SGS-10 (CGAGCTGCGTCGACAGGC(T)17) (12). The resulting PCR product was cloned into the pGEM-T vector (Promega, Madison, WI) for sequencing.

Nucleic Acid Hybridizations-- Trophozoite genomic DNA (10 µg) was digested to completion with either BamHI, EcoRI, HindIII, or PstI, and the resulting fragments were separated by electrophoresis on 1% (w/v) agarose gels. The DNA was transferred to Hybond N+ membrane (Amersham Corp.) and probed overnight at 55 °C with a randomly labeled (MegaPrime Kit, Amersham Corp.) 600-bp PCR product, amplified with oligonucleotides 4 and 5.

Total RNA (15 µg) from trophozoites and encysting cells was separated on a 1.3% (w/v) agarose gel containing 0.9% (v/v) formaldehyde. The RNA was transferred to Zeta-Probe blotting membrane (Bio-Rad) and hybridized overnight at 60 °C with a randomly labeled (MegaPrime, Amersham Corp.) 1.5-kb HindIII/PstI fragment from the arginine deiminase gene.

Poly(A+) mRNA was isolated from 100 µg of total trophozoite RNA using the Dynabeads oligo(dT) Purification Sytem (Dynal). Total RNA and mRNA were similarly separated on 1.0% (w/v) agarose containing 7.5% (v/v) formaldehyde. The total RNA and mRNA were transferred onto nylon membrane and probed with the full-length KpnI/BamHI ADI fragment.

Chromosomal Location-- Giardia chromosome blots, prepared from chromosome-sized DNA molecules separated by contour-clamped homogeneous electric field (CHEF) gel electrophoresis (14), were generously supplied by Dr. Jacqueline Upcroft (Queensland Institute of Medical Research). Southern hybridization was performed using the full-length BamHI/KpnI fragment of the ADI gene at 60 °C.

Colony Hybridizations-- PstI-restricted DNA fragments of approximately 3.2 kb were excised from an agarose gel, electroeluted, and ligated into pTZ19 vector that had been digested with PstI and dephosphorylated. Transformation was into the Escherichia coli strain JM101. Colonies were transferred to Hybond N+ membrane and hybridized overnight at 60 °C with the 600-bp amplicon that had been randomly labeled (MegaPrime, Amersham Corp.). This procedure was repeated with HindIII-restricted fragments of approximately 3.1 kb.

DNA Sequencing and Analysis-- The clones chosen from the PstI and HindIII "mini" libraries were subjected to exonuclease III digestion according to the Erase-A-Base kit instructions (Promega). DNA sequencing was with the Sequenase Version 2.0 sequencing kit (Amersham Corp.) or the Pharmacia T7 sequencing kit.

The ANGIS (Australian National Genomic Information Service) network was used for computer analysis of sequence data.

Expression of the Recombinant Protein-- A 1.8-kb KpnI/BamHI fragment representing the entire arginine deiminase coding sequence was amplified by PCR from Giardia genomic DNA using Pfu polymerase (Boehringer Mannheim). The KpnI/BamHI fragment was subsequently cloned into the pQE-30 expression vector (Qiagen). The recombinant expression shuttle (pQE-30-ADI) was transformed into E. coli M15(pREP 4) cells (Qiagen) and grown in LB media supplemented with 100 µg/ml ampicillin. Induction of expression with IPTG appeared toxic to the E. coli, thus non-inducing conditions were used for protein expression. Cells were grown at 37 °C with shaking for 5.5 h and harvested by centrifugation at 4 °C for 15 min at 2000 × g. The pelleted cells were washed once in phosphate-buffered saline (1.8 mM KH2PO4, 5 mM K2HPO4, pH 7.4, 0.9% (w/v) NaCl) and resuspended in phosphate buffer (20 mM phosphate buffer, pH 7.4, 500 mM NaCl, 10 mM imidazole, 0.1 mM leupeptin). Cell lysates were prepared by sonication on ice at a concentration of 1 g of cells to 5 ml of buffer using a Branson Sonifier 250 (40% duty cycle, output 2-3, 2-min bursts). Cell extracts were centrifuged at 4 °C for 40 min at 27,000 × g to remove particulate material.

Enzyme Purification-- Purification of the recombinant protein was performed using a His-trapR chelating column (Pharmacia Biotech Inc.) equilibrated with 10 ml of start buffer (20 mM phosphate buffer, pH 7.4, 0.5 M NaCl, 10 mM imidazole). The histidine-tagged recombinant protein was absorbed onto the column and was washed twice with 10 and 16 ml of start buffer containing 10 and 50 mM imidazole, respectively. The recombinant protein was eluted with 5 ml of elution buffer (20 mM phosphate buffer, pH 7.4, 0.5 M NaCl, 500 mM imidazole) in 1-ml fractions. Purified recombinant and total cellular protein were quantified using the Bio-Rad protein assay. Recombinant arginine deiminase activity was measured by the colorimetric determination of citrulline formation (9).

Inhibition of the Recombinant Enzyme-- Inhibition of recombinant arginine deiminase activity by various arginine analogues was performed, and the results were compared with their previously reported effects on the native enzyme (9). Inhibition studies were in the presence or absence of potential inhibitors at 10 mM concentration with 1 mM arginine, 3.5 µg/ml of protein, 20 mM Hepes, pH 7.0, in the assay.

SDS-Polyacrylamide Gel Electrophoresis and Western Blot Analysis-- SDS-PAGE was performed in Tris/glycine buffer, pH 8.3, on 10% (w/v) separating gels with a 4% (w/v) stacking according to the method of Laemmli (12). Proteins were transferred onto polyvinylidene difluoride membrane (Novex) at 25 V for 90 min. Transfer membranes were blocked for 1 h with 3% bovine serum albumin in Tris-buffered saline buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl), and the membrane was probed with Nickel-NTA conjugate coupled to alkaline phosphatase (Qiagen) at a dilution of 1:1000. The blot was developed using 5% nitro blue tetrazolium chloride in 70% dimethylformamide + 5% 5-bromo-4-chloro-3-indolyl phosphate in dimethylformamide. Native molecular mass of the active recombinant protein was determined by gel filtration using a Smart Chromatography System (Pharmacia).

Matrix Assisted Laser Desorption/Time of Flight (MALDI-TOF) Mass Spectrometry Analysis-- Tryptic digestion of purified recombinant protein was performed using modified sequencing grade trypsin (Boehringer Mannheim). Digestion was carried out in a 1-ml reaction volume containing 400 µg of purified recombinant enzyme, 10 µg of trypsin, 2 M urea, 0.1 M (NH4)2CO3, 50 mM glycine at 37 °C for 17.5 h. The peptides were separated from salts using a Sep-Pak C18 column (Waters, Millipore). The salts were first eluted with 4 ml of solvent A (0.1% trifluoroacetic acid in water), and then the peptides were eluted in 2 ml of solvent B (0.09% trifluoroacetic acid, 90% acetonitrile in water). The eluted peptides were concentrated to 100 pmol/µl. Mass spectrometric analysis was performed using 10-pmol fractions spotted onto a matrix (alpha -cyano-4-hydroxyceramide) which was then subjected to MALDI-TOF mass spectrometer analysis using a Laser MAT 2000 MALDI-TOF mass spectrometer (Finnigan). Computer analysis of data was with MacProMass from PSIEX.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Amplification of a Fragment of the Arginine Deiminase Gene-- Oligonucleotide primers designed from tryptic peptides of the purified arginine deiminase were used in PCR to amplify a portion of the target gene. Since the order of the tryptic peptides in the giardial enzyme was unknown, it was necessary to test all possible combinations of sense oligonucleotides from one peptide with antisense oligonucleotides from the other two peptides in PCR, i.e. six oligonucleotide combinations in all (Table I). Only one oligonucleotide combination gave consistent results in PCR (results not shown). Oligonucleotides 4 and 5 gave a single PCR product of approximately 600 bp. The clone had oligonucleotides 5 and 4 perfectly matched to the 5'- and 3'-ends of the amplicon, respectively. Additionally, an amino sequence that could be a possible match to tryptic peptide 6 (10) was present toward the N-terminal end of the translated PCR amplicon. This 600-bp amplicon was used in Southern hybridization analysis of genomic DNA, with a unique fragment detected for each restriction digest. The fragment sizes detected were 13.5 kb for BamHI, 6.1 kb for EcoRI, 3.1 kb for HindIII, and 3.2 kb for PstI (results not shown).

                              
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Table I
Synthetic oligonucleotides used in PCR for amplification of a portion of the arginine deiminase gene and the tryptic peptides from which they were designed
Letters in parentheses indicate wobble position nucleotides included in the probe mixtures. I indicates inosine residues. N indicates each of the four nucleotides. Tryptic peptide sequences from purified giardial arginine deiminase were previously reported (10). Sense oligonucleotides are odd-numbered and antisense oligonucleotides are even-numbered.

Cloning and Sequence Analysis-- A mini library from 3.2-kb PstI-restricted fragments was constructed and probed with the 600-bp PCR amplicon. Sequences of two positively hybridizing clones P6/19B and P7/19B, representing the PstI fragment inserted into pTZ18 in both orientations, were determined by overlapping DNA fragments generated by exonuclease III digestion. Sequence analysis revealed an open reading frame from nt 1680 of P6/19B and truncated at the 3'-end of the insert. This open reading frame contained amino acids encoded in the originally amplified PCR fragment and an additional sequence identical to tryptic peptide 17 (10). To obtain the 3'-end of the arginine deiminase gene, a HindIII mini library was screened, and two positively hybridizing clones were identified and designated HE1/19 and HE9/19. Additional sequence of the arginine deiminase gene was obtained by "primer walking" on both strands using oligonucleotides designed from known sequence. An amino acid sequence consistent with tryptic peptide 14 (10) was located in this additional sequence. Thus all five tryptic peptides derived from the purified enzyme (10) are present in the deduced amino acid sequence of the open reading frame.

Approximately 3700 bp of sequence was obtained from the PstI- and HindIII-derived clones, of which 1743 bp were an open reading frame encoding arginine deiminase. The deduced amino acid sequence is presented in Fig. 1. Restriction sites for HindIII and PstI and the location of oligonucleotides 4 and 5 that generated the original 600-bp PCR product are indicated. Amino acid sequences consistent with the tryptic peptides from the purified enzyme (10) are shown in bold. The 1743-nt open reading frame encodes a polypeptide of 64,090 Da, which is in close agreement with the reported molecular mass of 64,000 Da determined by SDS-PAGE of the purified arginine deiminase from G. intestinalis (10).


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Fig. 1.   Nucleotide sequence and the deduced amino acid sequence of the arginine deiminase gene from G. intestinalis. Restriction sites are underlined with dots, and elements proposed to be involved in gene transcription are underlined with solid lines. Oligonucleotides 4 and 5 used to generate the initial PCR product are also underlined. The first nucleotide of the initiation codon is numbered +1. The site of polyadenylation addition is marked by an asterisk. The positions of the five tryptic peptide sequences obtained from the purified arginine deiminase (10) are indicated with bold letters.

A number of motifs identifying possible regulatory elements involved in gene transcription in G. intestinalis were located in the upstream and downstream regions of the arginine deiminase gene. These regulatory elements are underlined in Fig. 1. In most giardial genes there is a short AT-rich region ending at the ATG translation start codon, which has been proposed to be a promoter element associated with RNA polymerase II transcription (16). The arginine deiminase gene has such an AT-rich element adjacent to the start codon (-16 AAAAAAATCCTAGTAC -1). Additionally, a second motif, with the consensus CAAAT, ATTTG, or abbreviated forms, has also been suggested as a regulatory element (16) and occurs a number of times in the region upstream of many giardial genes, including arginine deiminase (Fig. 1). The sequence AGTPuoAAPyd is proposed to serve as a polyadenylation signal in Giardia (17), and such a sequence (AGTGAAT) is located in the 3'-untranslated region of the arginine deiminase gene, 3 nts downstream from the TGA termination codon. By using an oligonucleotide specific for the arginine deiminase gene and an oligo(dT) in PCR with trophozoite cDNA, polyadenylation of the arginine deiminase transcript was found to occur 10 nts downstream of this heptanucleotide sequence (Fig. 1).

Northern Analysis-- Total RNA from trophozoites and encysting cells (6 and 24 h in encysting medium) was subjected to Northern blot hybridization with a 1.5-kb HindIII/PstI fragment of the arginine deiminase gene. A band of approximately 1.8 kb was detected (Fig. 2, panel A). Since no introns have been reported for giardial genes to date, this transcript size is in agreement with the predicted size of the transcribed gene sequence plus a polyadenylation tract. The transcript levels for arginine deiminase were of comparable abundance in trophozoites and in 6- and 24-h encysting cells. Levels of mRNA for an internal control whose transcript levels show no developmental variation2 ascertained equal loading of RNA for each condition. Similar results were obtained when trophozoite poly(A+) mRNA was subjected to Northern blot hybridizations (Fig. 2, panel B).


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Fig. 2.   Northern analysis of the arginine deiminase transcript. Panel A, total RNA from trophozoites (T), 6-h encysting cells (6h), and 24-h encysting cells (24h) was isolated from G. intestinalis, separated by electrophoresis, and transferred to nylon membrane. The transfer was probed with a 1.5-kb HindIII/PstI fragment of the arginine deiminase gene at 60 °C overnight. Panel B, lanes 1 and 2 are trophozoite poly(A+) RNA and total RNA, respectively, separated by formaldehyde agarose gel electrophoresis. The RNA was transferred to nylon membrane and probed with the full-length ADI gene, and the results are shown in lanes 3 (poly(A+)) and 4 (total).

Chromosomal Location-- Chromosome-sized DNA molecules were separated using CHEF gel electrophoresis and subjected to Southern hybridization with the 1.8-kb BamHI/KpnI ADI fragment. The ADI gene was located on chromosome 5 (14) of Giardia (Fig. 3).


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Fig. 3.   Location of the giardial arginine deiminase gene on chromosome 5. Panel A shows the fractionation of chromosome-sized DNA molecules of G. intestinalis by CHEF gel electrophoresis in lanes 1 and 2. Yeast size standards are separated in lane 3. Panel B shows a blot of lanes 1 and 2 of the gel in panel A hybridized with a full-length ADI probe.

Sequence Homology with Other Arginine Deiminases-- Sequence information for the arginine deiminase gene is available from seven sources to date, all prokaryotic organisms, Mycoplasma arginini (18-20), Mycoplasma hominis (21), Mycoplasma orale (21), Pseudomonas aeruginosa (22), Pseudomonas putida (GenBankTM accession number U07185), Halobacterium salinarium (GenBankTM accession number X80931), and Clostridium perfringens (GenBankTM accession number X97684). A ClustalW alignment of these amino acid sequences with the giardial sequence is shown in Fig. 4. The giardial protein is the largest reported to date, with the majority of the additional amino acid sequence occurring as a C-terminal extension compared with the other deiminases. Arginine deiminase from G. intestinalis was most similar to the H. salinarium enzyme with 26% identity and 50% similarity between the two sequences.


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Fig. 4.   Alignment of the deduced amino acid sequences of arginine deiminases from G. intestinalis (accession number U49236), M. arginini (accession number X54141), M. hominis (accession number D13314), P. aeruginosa (accession number X14694), P. putida (accession number U07185), H. salinarium (accession number X80931), and C. perfringens (accession number X97684) using the ClustalW program. Asterisks indicate identical amino acids, and a single dot indicates a conserved amino acid substitution.

Expression and Purification of Recombinant Arginine Deiminase-- The giardial arginine deiminase gene was cloned into the expression vector pQE 30 and transformed in E. coli M15[pREP 4] cells. Clones containing the pQE 30-ADI construct were isolated, and the recombinant protein was expressed under non-inducing conditions, since induction with IPTG was toxic to the host cell. The active 6x histidine-tagged recombinant protein was purified in a single step using a His-TrapR (Pharmacia) chelating column. This single step resulted in a purification of 33-fold with 67% of enzyme activity being recovered (Table II). Overall 2 mg of the recombinant arginine deiminase could be purified from 96 mg of starting material. To maintain enzyme activity for longer periods the elution buffer was exchanged for 20 mM phosphate buffer, pH 7.4, 0.1 mM EDTA, 10 mM beta -mercaptoethanol, 20% glycerol. E. coli containing the expression vector alone had no detectable arginine deiminase activity.

                              
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Table II
Purification scheme for recombinant arginine deiminase from G. intestinalis
Unit of enzyme activity = 1 µmol of citrulline formed (min)-1.

SDS-PAGE analysis indicated the purified recombinant protein had a molecular mass of 66 kDa (Fig. 5). The native molecular mass of the active recombinant enzyme was determined to be 140 kDa by gel filtration, indicating that the active recombinant enzyme is a dimer. Western analysis with a nickel-NTA/alkaline phosphatase conjugate that binds to the histidine tag identified the purified recombinant protein and a single band from an E. coli extract expressing the pQE-30-ADI construct (Fig. 5).


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Fig. 5.   SDS-PAGE and Western blot analysis of purified recombinant arginine deiminase and cell-free extracts. Panel A, lanes 1-3 are molecular mass standards, purified recombinant arginine deiminase, and a cell-free extract of E. coli expressing the pQE-3-ADI plasmid, respectively, and stained with Coomassie Brilliant Blue R250. Panel B, lanes 2 and 3 are the same as panel A and probed with nickel-NTA/alkaline phosphatase conjugate, directed specifically against the 6x histidine tag on the recombinant protein.

Tryptic peptides were analyzed by MALDI-TOF mass spectrometry. The experimental mass spectrum corresponded to the mass profiles predicted for the amino acid sequence data of the recombinant arginine deiminase (Fig. 6). A total of 19 mass peaks that were observed could account for more than 50% of the putative sequence of the recombinant protein.


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Fig. 6.   MALDI-TOF mass spectrometry analysis. Panel A, MALDI-TOF mass spectrum of tryptic peptides of purified recombinant arginine deiminase. The x axis represents the mass charge ratio (m/z), and the y axis represents the relative abundance for each peptide. Panel B shows the putative amino acid sequence of the recombinant protein and the tryptic peptides that were observed. Arrows indicate the tryptic peptides for which peaks with the correct mass (± 0.2%) were observed in the spectrum shown in panel A. Broken arrows indicate pairs of tryptic peptides with identical masses. Tryptic peptides that would not be observed in the mass spectrum shown in panel A because their mass is below 750 Da are underlined.

Inhibition of the Recombinant Enzyme-- Inhibition of recombinant arginine deiminase activity by various arginine analogues at a concentration of 10 mM in the presence of 1 mM arginine was assessed. The arginine analogue inhibition profile for the recombinant enzyme (Table III) and the native enzyme (9) was comparable.

                              
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Table III
Comparison of the inhibition of recombinant and native arginine deiminase by a number of arginine analogues
The influence of arginine analogues at a final concentration of 10 mM on the activity of recombinant arginine deiminase with 1 mM arginine at 37 °C over 10 min was studied.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

G. intestinalis as a representative of one of the earliest diverging branches of the eukaryotic tree (5) has retained many prokaryotic features. We have previously demonstrated that the arginine transport system in Giardia is supportive of its position as a transition between prokaryotes and eukaryotes, and the metabolism of arginine also reflects the primitive nature of this parasite. Here we report the cloning and expression of the first enzyme involved in the energy-producing catabolism of arginine via the ADH pathway, arginine deiminase. This is the first sequence available for this gene from a eukaryotic organism.

The arginine deiminase gene family appears to be poorly conserved. This prevented the use of degenerate oligonucleotides based on consensus sequences to obtain a fragment of the arginine deiminase gene. Instead primers were designed from tryptic peptides obtained from purified arginine deiminase that we have recently reported (10). This is the first example of the isolation of a gene from G. intestinalis using this approach. The amino acid sequence homology of the giardial arginine deiminase was highest with the deiminase from H. salinarium (accession number X80931) (50% similarity). The similarity with P. aeruginosa (22) and M. hominis (21) was 49 and 46%, respectively. It seems that within species the sequences are well conserved, for example there is 82% similarity between the M. arginini (18) and M. hominis (21) sequences. However, when comparing sequences between species, the similarity is markedly reduced, with 54% similarity between the M. arginini (18) and P. aeruginosa (22) deiminases for example.

There are three small but well conserved regions common to all arginine deiminase sequences (Fig. 4) as follows: at amino acid residues 173-176 (F(M/T/Q)R(D/E)), 226-231 ((I/L/V)EGGD(F/V)), and 280-284 (MHLD(C/T)). Presumably these conserved regions are important in enzyme structure and functioning. However, these conserved regions were not found in the amino acid sequences for other arginine-utilizing or arginine-producing enzymes, including peptidyl-arginine deiminase (EC 3.5.3.15), arginase (EC 3.5.3.1), argininosuccinate lyase (EC 4.3.2.1), arginine decarboxylase (EC 4.1.1.19), and nitric-oxide synthase (EC 1.14.23). This suggests that residues distantly located in the primary structure of arginine deiminase may be involved in the arginine binding site and catalysis. To date, no arginine binding motif has been identified.

Giardial arginine deiminase gene was cloned into the expression vector pQE-30 carrying the 6x histidine tag at the 5'-end. The recombinant protein was expressed by transformed E. coli under non-inducing conditions, since induction with IPTG was highly toxic to the expressing cells. It is likely that both the expression and function of the native enzyme are highly controlled, as uncontrolled expression of the highly soluble and active arginine deiminase would compromise protein synthesis and other biochemical processes requiring arginine. We have previously shown that the intracellular arginine pool in Giardia trophozoites is almost undetectably low (11). Non-inducing conditions allowed expression at a level that was tolerated by the host cells. A one-step purification procedure resulted in the isolation of homogeneous recombinant arginine deiminase. The activity of the recombinant protein was comparable with that of the purified native arginine deiminase (10), and the patterns of inhibition by various arginine analogues were similar for both the recombinant and native enzymes. A major advantage was that the recombinant enzyme was much more stable than the native enzyme, which loses all activity within 8 h of isolation (10). Further characterization by MALDI-TOF mass spectrometry analysis of a tryptic digest of the purified recombinant arginine deiminase showed a mass spectrum consisting of peaks corresponding to mass:charge ratios of specific tryptic peptides of the recombinant arginine deiminase. The only mass peaks that were observed and could not be accounted for by the tryptic hydrolysis of the recombinant protein were a small peak (m/z 1622.0) and the highest mass peak (m/z 3853.8). The regions of sequence that were not observed as tryptic peptides may be accounted for by incomplete hydrolysis, by the generation of small peptides (m/z <750) or by losses of polar peptides during desalting. The approach provides a quick and simple method for confirming the sequence of an expressed protein. Although a peptide containing the N-terminal 6x histidine tag was not observed, the presence of the tag was demonstrated by Western analysis (Fig. 5) and by the effectiveness of the one-step purification procedure. Thus it appears that the physical and kinetic properties of the recombinant and native enzymes are comparable.

In addition to being specific for the parasite, it is desirable that a new chemotherapeutic agent has minimal deleterious effects on the human host. For this reason, metabolic pathways and cell components that are unique to a parasite, or significantly different to those of the host, are desirable targets for future drug design. In this paper, we present a potential site for chemotherapeutic attack against giardiasis. Arginine deiminase is not found in humans and is involved in an important energy producing pathway in Giardia. Availability of large quantities of recombinant enzyme should pave the way for future studies on the regulation and mechanism of arginine deiminase action in Giardia. Furthermore, availability of the recombinant protein will facilitate the pursuit of detailed knowledge of enzyme structure that is required for designing synthetic competitors of this enzyme to combat giardial infection.

    FOOTNOTES

* This work was supported in part by a grant from the National Health and Medical Research Council of Australia.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U49236.

Dagger Recipient of a Dora Lush (Biomedical) Postgraduate Scholarship. Present address: Division of Infectious Diseases, Dept. of Pathology, University of California, San Diego, 214 Dickinson St., San Diego, CA 92103-8416.

§ To whom correspondence should be addressed. Tel.: 61 2 93852017; Fax: 61 2 93851483; E-mail: m.edwards{at}unsw.edu.au.

1 The abbreviations used are: ADH, arginine dihydrolase; PAGE, polyacrylamide gel electrophoresis; nt, nucleotide(s); bp, base pair(s); kb, kilobase(s); PCR, polymerase chain reaction; IPTG, isopropyl-beta -D-thiogalactoside; MALDI-TOF, matrix assisted laser desorption-time of flight; CHEF, contour-clamped homogeneous electric field.

2 L. A. Knodler, E. O. Sekyere, T. S. Stewart, P. J. Schofield, and M. R. Edwards, unpublished observations.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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