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

Abstract

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 inEscherichia coli and purified to homogeneity. The recombinant protein was found to have characteristics comparable with those of the native enzyme. 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 inEscherichia coli and purified to homogeneity. The recombinant protein was found to have characteristics comparable with those of the native enzyme. 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. intestinalishas recently been described as the “missing link” between prokaryotes and eukaryotes (1Kabnick K.S. Peattie D.A. Am. Sci. 1991; 79: 34-43Google Scholar). 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. InGiardia, the arginine dihydrolase (ADH) 1The 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-β-d-thiogalactoside; MALDI-TOF, matrix assisted laser desorption-time of flight; CHEF, contour-clamped homogeneous electric field. 1The 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-β-d-thiogalactoside; MALDI-TOF, matrix assisted laser desorption-time of flight; CHEF, contour-clamped homogeneous electric field. pathway is an important source of energy, with arginine the preferential fuel used in the early and most proliferative stages of in vitro growth (2Edwards M.R. Schofield P.J. O'Sullivan W.J. Costello M. Mol. Biochem. Parasitol. 1992; 53: 97-104Crossref PubMed Scopus (48) Google Scholar). This pathway has been reported in only two other eukaryotes,Trichomonas vaginalis (3Linstead D. Cranshaw M.A. Mol. Biochem. Parasitol. 1983; 8: 241-252Crossref PubMed Scopus (53) Google Scholar) and Tritrichomonas fetus (4Yarlett N. Lindmark D.G. Goldberg B. Ali Moharrami M. Bacchi C.J. J. Eukaryotic Microbiol. 1994; 41: 554-559Crossref PubMed Scopus (32) Google Scholar), both also placed on early branches of the eukaryotic evolutionary tree (5Sogin M.L. Gunderson J.H. Elwood H.J. Alonso R.A. Peattie D.A. Science. 1989; 243: 75-77Crossref PubMed Scopus (585) Google Scholar). The ADH pathway is normally confined to the prokaryotic kingdom, where it provides both a source of energy and nitrogen (6Cunin R. Glansdorff N. Pierard A. Stalon V. Microbiol. Rev. 1986; 50: 314-352Crossref PubMed Google Scholar). 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 → citrulline → ornithine + carbamyl phosphate → CO2 + NH4+ + ATP. The activities of all the arginine dihydrolase pathway enzymes have been demonstrated in Giardia (7Schofield P.J. Edwards M.R. Matthews J. Wilson J.R. Mol. Biochem. Parasitol. 1992; 51: 29-36Crossref PubMed Scopus (72) Google Scholar), 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 (7Schofield P.J. Edwards M.R. Matthews J. Wilson J.R. Mol. Biochem. Parasitol. 1992; 51: 29-36Crossref PubMed Scopus (72) Google Scholar, 8Schofield P.J. Edwards M.R. Kranz P. Mol. Biochem. Parasitol. 1991; 45: 39-48Crossref PubMed Scopus (44) Google Scholar). 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 (9Knodler L.A. Schofield P.J. Edwards M.R. Microbiology. 1995; 141: 2063-2070Crossref PubMed Scopus (27) Google Scholar). 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 (7Schofield P.J. Edwards M.R. Matthews J. Wilson J.R. Mol. Biochem. Parasitol. 1992; 51: 29-36Crossref PubMed Scopus (72) Google Scholar). 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 (10Knodler L.A. Schofield P.J. Gooley A.A. Edwards M.R. Exp. Parasitol. 1997; 85: 77-80Crossref PubMed Scopus (13) Google Scholar). 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. G. intestinalistrophozoites, Portland I strain, were cultured as described previously (11Knodler L.A. Edwards M.R. Schofield P.J. Exp. Parasitol. 1994; 79: 117-125Crossref PubMed Scopus (44) Google Scholar). Encystation of trophozoites was as described previously (12Que X. Svard S.G. Meng T.C. Hetsko M.L. Aley S.B. Gillin F.D. Mol. Biochem. Parasitol. 1996; 81: 101-110Crossref PubMed Scopus (37) Google Scholar). G. intestinalis genomic DNA was prepared by the method described by Yee and Dennis (13Yee J. Dennis P.P. J. Biol. Chem. 1992; 267: 7539-7544Abstract Full Text PDF PubMed Google Scholar), 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 (12Que X. Svard S.G. Meng T.C. Hetsko M.L. Aley S.B. Gillin F.D. Mol. Biochem. Parasitol. 1996; 81: 101-110Crossref PubMed Scopus (37) Google Scholar). Degenerate sense and antisense oligonucleotides were derived from tryptic peptides obtained from the purified giardial arginine deiminase (10Knodler L.A. Schofield P.J. Gooley A.A. Edwards M.R. Exp. Parasitol. 1997; 85: 77-80Crossref PubMed Scopus (13) Google Scholar) (Table I) and synthesized on a Beckman Oligo 1000 DNA Synthesizer. PCR conditions were as described in the manufacturer's protocol for Tthpolymerase (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) (12Que X. Svard S.G. Meng T.C. Hetsko M.L. Aley S.B. Gillin F.D. Mol. Biochem. Parasitol. 1996; 81: 101-110Crossref PubMed Scopus (37) Google Scholar). The resulting PCR product was cloned into the pGEM-T vector (Promega, Madison, WI) for sequencing.Table ISynthetic oligonucleotides used in PCR for amplification of a portion of the arginine deiminase gene and the tryptic peptides from which they were designedPeptideOligonucleotideNucleotide sequence1415′-GTI GA (A/G) GGI GCI CCN GCN TA-3′25′-GTA IGC IGG NGC NCC (T/C)TC-3′2335′-GCI TA(C/T) GGI (C/T)TI TA(C/T) CA(A/G) AA-3′45′-CTT (T/C)TG (A/G)TA IAG NCC (G/A)TA-3′2755′-GTI GA(C/T) GA(A/G) TGG AT(A/C/T) GA(C/T) GT-3′65′-AC (G/A) TC IAT CCA (T/C)TC (G/A)TC-3′ Open table in a new tab 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-lengthKpnI/BamHI ADI fragment. Giardia chromosome blots, prepared from chromosome-sized DNA molecules separated by contour-clamped homogeneous electric field (CHEF) gel electrophoresis (14Upcroft J.A. Healey A. Upcroft P. Int. J. Parasitol. 1993; 23: 609-616Crossref PubMed Scopus (26) Google Scholar), 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. 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 theEscherichia 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 withHindIII-restricted fragments of approximately 3.1 kb. The clones chosen from the PstI and HindIII “mini” libraries were subjected to to the DNA was with the (Amersham Corp.) or the The was used for analysis of sequence fragment representing the arginine deiminase sequence was amplified by PCR genomic DNA using polymerase The fragment was cloned into the expression vector The recombinant expression was into coli cells and in with 100 of expression with to the conditions were used for protein were at °C with for and by at 4 °C for at The cells were in 0.9% (w/v) and in phosphate phosphate 500 were prepared by on at a of of cells to of using a cycle, were at 4 °C for 40 at to Purification of the recombinant protein was performed using a with of phosphate The recombinant protein was onto the and was with and of containing and 50 The recombinant protein was with of phosphate 500 in recombinant and total protein were using the protein arginine deiminase activity was by the of citrulline (9Knodler L.A. Schofield P.J. Edwards M.R. Microbiology. 1995; 141: 2063-2070Crossref PubMed Scopus (27) Google Scholar). of recombinant arginine deiminase activity by arginine was and the were with previously reported on the native enzyme (9Knodler L.A. Schofield P.J. Edwards M.R. Microbiology. 1995; 141: 2063-2070Crossref PubMed Scopus (27) Google Scholar). were in the or of at with of in the was performed in on (w/v) with a (w/v) to the method of (12Que X. Svard S.G. Meng T.C. Hetsko M.L. Aley S.B. Gillin F.D. Mol. Biochem. Parasitol. 1996; 81: 101-110Crossref PubMed Scopus (37) Google Scholar). were transferred onto membrane at for were for with in (10 and the membrane was probed with to at a of The blot was using chloride in + phosphate in molecular mass of the active recombinant protein was by gel using a of purified recombinant protein was performed using was carried in a containing μg of purified recombinant μg of 50 at °C for The peptides were separated from using a The were first with 4 of acid in and then the peptides were in of B in The peptides were to 100 analysis was performed using onto a matrix which was then subjected to mass analysis using a mass analysis of data was with from from tryptic peptides of the purified arginine deiminase were used in PCR to amplify a portion of the target gene. the of the tryptic peptides in the giardial enzyme was it was to all of sense oligonucleotides from one with antisense oligonucleotides from the other two peptides in PCR, oligonucleotide in all (Table one oligonucleotide consistent in PCR 4 and a single PCR product of approximately The had oligonucleotides and 4 to the and of the an amino sequence that be a to tryptic (10Knodler L.A. Schofield P.J. Gooley A.A. Edwards M.R. Exp. Parasitol. 1997; 85: 77-80Crossref PubMed Scopus (13) Google Scholar) was present the of the PCR This 600-bp amplicon was used in Southern hybridization analysis of genomic with a fragment for The fragment were kb for kb 3.1 kb for HindIII, and 3.2 kb from fragments was and probed with the 600-bp PCR of two clones and representing the PstI fragment into pTZ18 in both were by overlapping DNA fragments by analysis an open reading frame from of and at the of the This open reading frame contained amino acids encoded in the amplified PCR fragment and an sequence to tryptic (10Knodler L.A. Schofield P.J. Gooley A.A. Edwards M.R. Exp. Parasitol. 1997; 85: 77-80Crossref PubMed Scopus (13) Google Scholar). obtain the of the arginine deiminase a HindIII was and two clones were identified and and sequence of the arginine deiminase gene was obtained by on both using oligonucleotides from An amino acid sequence consistent with tryptic (10Knodler L.A. Schofield P.J. Gooley A.A. Edwards M.R. Exp. Parasitol. 1997; 85: 77-80Crossref PubMed Scopus (13) Google Scholar) was in this all five tryptic peptides derived from the purified enzyme (10Knodler L.A. Schofield P.J. Gooley A.A. Edwards M.R. Exp. Parasitol. 1997; 85: 77-80Crossref PubMed Scopus (13) Google Scholar) are present in the amino acid sequence of the open reading of sequence was obtained from and of which were an open reading frame arginine The amino acid sequence is in for HindIII and PstI and the of oligonucleotides 4 and that the 600-bp PCR product are acid sequences consistent with the tryptic peptides from the purified enzyme (10Knodler L.A. Schofield P.J. Gooley A.A. Edwards M.R. Exp. Parasitol. 1997; 85: 77-80Crossref PubMed Scopus (13) Google Scholar) are in The open reading frame a of which is in with the reported molecular mass of by of the purified arginine deiminase from G. intestinalis (10Knodler L.A. Schofield P.J. Gooley A.A. Edwards M.R. Exp. Parasitol. 1997; 85: 77-80Crossref PubMed Scopus (13) Google Scholar). of in gene in G. intestinalis were in the and downstream of the arginine deiminase gene. These are in In most giardial there is a at the which has been to be a with RNA polymerase J. Dennis P.P. Microbiol. 1994; Scopus Google Scholar). The arginine deiminase gene has an to the a with the or has also been as a J. Dennis P.P. Microbiol. 1994; Scopus Google Scholar) and occurs a of in the of giardial including arginine deiminase The sequence is to as a polyadenylation in Giardia D.A. Alonso R.A. A. J. Biol. 1989; PubMed Scopus Google Scholar), and a sequence is in the of the arginine deiminase downstream from the using an oligonucleotide specific for the arginine deiminase gene and an oligo(dT) in PCR with trophozoite polyadenylation of the arginine deiminase transcript was found to downstream of this sequence Total RNA from trophozoites and encysting cells and in encysting was subjected to Northern blot hybridization with a 1.5-kb HindIII/PstI fragment of the arginine deiminase gene. of approximately kb was have been reported for giardial to this transcript is in with the of the gene sequence a polyadenylation The transcript for arginine deiminase were of comparable in trophozoites and in and encysting cells. of mRNA for an transcript A. P. J. and M. R. of RNA for were obtained when trophozoite mRNA was subjected to Northern blot DNA molecules were separated using gel electrophoresis and subjected to Southern hybridization with the fragment. The ADI gene was on chromosome (14Upcroft J.A. Healey A. Upcroft P. Int. J. Parasitol. 1993; 23: 609-616Crossref PubMed Scopus (26) Google Scholar) of Giardia for the arginine deiminase gene is available from to all prokaryotic J. Mol. PubMed Scopus Google Scholar, M. M. PubMed Google Scholar, M. M. J. 1994; PubMed Scopus Google Scholar), R. M. Microbiol. 1992; PubMed Scopus Google R. M. Microbiol. 1992; PubMed Scopus Google Scholar), Stalon V. A. D. J. Biochem. 1989; PubMed Scopus Google Scholar), and of these amino acid sequences with the giardial sequence is in The giardial protein is the reported to with the of the amino acid sequence as a extension with the other Arginine deiminase from G. intestinalis was most similar to the with and between the two The giardial arginine deiminase gene was cloned into the expression vector and in cells. containing the were and the recombinant protein was expressed with was to the cell. The active recombinant protein was purified in a single step using a This single step in a purification of with of enzyme activity being (Table of the recombinant arginine deiminase be purified from of enzyme activity for the was for phosphate coli containing the expression vector had arginine deiminase for recombinant arginine deiminase from G. Open table in a new tab analysis the purified recombinant protein had a molecular mass of The native molecular mass of the active recombinant enzyme was to be by gel that the active recombinant enzyme is a analysis with a that to the identified the purified recombinant protein and a single from an coli the peptides were by mass The mass corresponded to the mass for the amino acid sequence data of the recombinant arginine deiminase total of mass that were for than of the sequence of the recombinant of recombinant arginine deiminase activity by arginine at a of in the of arginine was The arginine for the recombinant enzyme (Table and the native enzyme (9Knodler L.A. Schofield P.J. Edwards M.R. Microbiology. 1995; 141: 2063-2070Crossref PubMed Scopus (27) Google Scholar) was of the of recombinant and native arginine deiminase by a of arginine were previously reported amino or or chain were previously reported (9Knodler L.A. Schofield P.J. Edwards M.R. Microbiology. 1995; 141: 2063-2070Crossref PubMed Scopus (27) Google Scholar). Open table in a new tab G. intestinalis as a of one of the branches of the eukaryotic tree (5Sogin M.L. Gunderson J.H. Elwood H.J. Alonso R.A. Peattie D.A. Science. 1989; 243: 75-77Crossref PubMed Scopus (585) Google Scholar) has prokaryotic We have previously demonstrated that the arginine in Giardia is of as a between prokaryotes and and the of arginine also the primitive nature of this parasite. we report the cloning and expression of the first enzyme in the catabolism of arginine the ADH pathway, arginine This is the first sequence available for this gene from a eukaryotic organism. The arginine deiminase gene to be This the of degenerate oligonucleotides based on sequences to obtain a fragment of the arginine deiminase gene. were from tryptic peptides obtained from purified arginine deiminase that we have recently reported (10Knodler L.A. Schofield P.J. Gooley A.A. Edwards M.R. Exp. Parasitol. 1997; 85: 77-80Crossref PubMed Scopus (13) Google Scholar). This is the first of the of a gene from G. intestinalis using this The amino acid sequence homology of the giardial arginine deiminase was with the deiminase The with P. Stalon V. A. D. J. Biochem. 1989; PubMed Scopus Google Scholar) and M. R. M. Microbiol. 1992; PubMed Scopus Google Scholar) was and that the sequences are well for there is between the M. J. Mol. PubMed Scopus Google Scholar) and M. R. M. Microbiol. 1992; PubMed Scopus Google Scholar) when sequences between the is with between the M. J. Mol. PubMed Scopus Google Scholar) and P. Stalon V. A. D. J. Biochem. 1989; PubMed Scopus Google Scholar) for There are three small but well to all arginine deiminase sequences as follows: at amino acid and these are important in enzyme and these were found in the amino acid sequences for other or including deiminase (EC (EC arginine (EC and (EC This that in the of arginine deiminase may be in the arginine site and arginine has been arginine deiminase gene was cloned into the expression vector the at the The recombinant protein was expressed by coli with was to the cells. is that both the expression and function of the native enzyme are as expression of the and active arginine deiminase protein and other We have previously that the arginine in Giardia trophozoites is (11Knodler L.A. Edwards M.R. Schofield P.J. Exp. Parasitol. 1994; 79: 117-125Crossref PubMed Scopus (44) Google Scholar). conditions expression at a that was by the cells. purification procedure in the of homogeneous recombinant arginine The activity of the recombinant protein was comparable with that of the purified native arginine deiminase (10Knodler L.A. Schofield P.J. Gooley A.A. Edwards M.R. Exp. Parasitol. 1997; 85: 77-80Crossref PubMed Scopus (13) Google Scholar), and the of by arginine were similar for both the recombinant and native major was that the recombinant enzyme was than the native which all activity of (10Knodler L.A. Schofield P.J. Gooley A.A. Edwards M.R. Exp. Parasitol. 1997; 85: 77-80Crossref PubMed Scopus (13) Google Scholar). by mass analysis of a tryptic of the purified recombinant arginine deiminase a mass of to of specific tryptic peptides of the recombinant arginine The only mass that were and be for by the tryptic of the recombinant protein were a small and the mass The of sequence that were as tryptic peptides may be for by by the of small peptides or by of peptides The provides a and method for the sequence of an expressed a containing the was the of the was demonstrated by analysis and by the of the purification it that the and properties of the recombinant and native enzymes are In addition to being specific for the it is that a new chemotherapeutic has on the host. For this metabolic and that are to a or to those of the are for future drug design. In this paper, we present a site for chemotherapeutic against Arginine deiminase is found in and is in an important energy pathway in of of recombinant enzyme the for future on the and of arginine deiminase in of the recombinant protein the of of enzyme that is for of this enzyme to giardial infection.