Molecular Cloning and Expression of a Purine-specific N-Ribohydrolase from Trypanosoma brucei brucei
SEQUENCE, EXPRESSION, AND MOLECULAR ANALYSIS*

Roger PelléDagger , Vern L. Schramm§, and David W. ParkinDagger

From the Dagger  International Livestock Research Institute, Nairobi, Kenya and the § Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

N-Ribohydrolases, including the inosine-adenosine-guanosine-preferring (IAG) nucleoside hydrolase, have been proposed to be involved in the nucleoside salvage pathway of protozoan parasites and may constitute rational therapeutic targets for the treatment of these diseases. Reported is the complete sequence of the Trypanosoma brucei brucei iagnh gene, which encodes IAG-nucleoside hydrolase. The 1.4-kilobase iagnh cDNA contains an open reading frame of 981 base pairs, corresponding to 327 amino acids. The iagnh gene is present as one copy/haploid genome and is located on the size-polymorphic pair of chromosome III or IV in the genome of T. b. brucei. In Southern blot analysis, the iagnh probe hybridized strongly with Trypanosoma brucei gambiense, Trypanosoma brucei rhodesiense, Trypanosoma evansi, Trypanosoma congolense, and Trypanosoma vivax and, to a lesser extent, with Trypanosoma cruzi genomic DNA. The iagnh gene is expressed in bloodstream forms and procyclic (insect) life-cycle stages of T. b. brucei. There are no close amino acid homologues of IAG-nucleoside hydrolase outside bacterial, yeast, or parasitic organisms. Low amino acid sequence similarity is seen with the inosine-uridine-preferring nucleoside hydrolase isozyme from Crithidia fasciculata. The T. b. brucei iagnh open reading frame was cloned into Escherichia coli BL21(DE3), and a soluble recombinant IAG-nucleoside hydrolase was expressed and purified to >97% homogeneity. The molecular weights of the recombinant IAG-nucleoside hydrolase, based on the amino acid sequence and observed mass, were 35,735 and 35,737, respectively. The kinetic parameters of the recombinant IAG-nucleoside hydrolase are experimentally identical to the native IAG-nucleoside hydrolase.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Protozoan parasites rely on preformed purine nucleosides or bases for the biosynthesis of purine ribonucleotides, since they do not contain de novo purine biosynthetic pathways (1-3). Parasites and host cells have several common steps in purine salvage pathways, but unique intermediate steps are determined by the response of the parasite to the purine composition of the host's cells or bloodstream. Protozoan parasites have evolved pathways to utilize any purine nucleoside or base in the environment (1). Intracellular hemoparasites, exemplified by the American trypanosomes, have different purine nucleosides or bases available than the extracellular hemoprotozoan parasites, such as the African trypanosomes. In addition, the various life-cycle stages of the African trypanosomes are exposed to diverse environments, which vary from the mouth and midgut of tsetse flies to the extracellular bloodstream of livestock, wildlife, and humans (4, 5).

The purine salvage pathway can be divided into stages: 1) the biosynthesis of IMP from free nucleosides or bases, and 2) the conversion of IMP to adenylate and guanylate nucleotides (1). The biosynthetic enzymes of the purine salvage pathway are purine nucleoside kinases, hypoxanthine-guanine-xanthine phosphoribosyl-1-pyrophosphate transferases, and the purine N-ribohydrolases and/or phosphorylases.

N-Ribohydrolases found in both African and American trypanosomes are potential chemotherapeutic targets, since no N-ribohydrolase activity or encoding genes have been identified in mammals (6). N-Ribohydrolases catalyze the hydrolysis of the N-ribosidic bond between N-9 of the purine base and C-1' of the (deoxy-) ribose, as shown by Reaction 1. 
(<UP>Deoxy-</UP>)<UP>nucleoside + H<SUB>2</SUB>O → base + </UP>(<UP>deoxy-</UP>)<UP>ribose</UP>
<UP><SC>Reaction</SC> 1</UP>
These enzymes have been identified and characterized, to a greater or lesser extent, from Trypanosoma brucei brucei (7), Trypanosoma brucei gambiense (8), Trypanosoma cruzi (9), Leishmania donovani (10), Leishmania mexicana (11), and Crithidia fasciculata (12-14). In C. fasciculata, over 90% of nucleoside salvage occurs through the inosine-uridine-preferring (IU-)1 nucleoside hydrolase and guanosine-inosine-preferring nucleoside hydrolases, establishing a role for these isozymes in the purine nucleoside salvage pathway (14).

The IU- (13) and guanosine-inosine (14) nucleoside hydrolase from C. fasciculata and the inosine-adenosine-guanosine-preferring (IAG-) nucleoside hydrolase from T. b. brucei (7) have been extensively characterized. The IU-nucleoside hydrolase has been purified, the chemical and kinetic mechanisms determined (13), the transition state determined from kinetic isotope effects (15), the cDNA cloned and overexpressed in Escherichia coli (6), and the recombinant enzyme has been characterized by x-ray crystallography (16).

Recent research has focused on the IAG-nucleoside hydrolase from T. b. brucei, a hemoprotozoan parasite of livestock (7). Studies of the T. b. brucei enzyme have established that IAG-nucleoside hydrolase has different substrate and inhibitor specificity, kinetic mechanism, and transition state structure than IU-nucleoside hydrolase from C. fasciculata (7). Reported in this paper is the nucleotide sequence of the iagnh open reading frame, the predicted amino acid sequence, genetic distribution among several parasitic organisms, the relative level of expression of the iagnh mRNA in different life-cycle stages of T. b. brucei, and a comparison of the kinetic parameters of the purine nucleoside substrates for the native and recombinant IAG-nucleoside hydrolase. The overexpression of IAG-nucleoside hydrolase in E. coli and the purification of the recombinant enzyme provide sufficient protein for structural and transition state studies.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Parasites-- The different bloodstream forms of T. b. brucei, ILTat1.1, a pleomorphic strain (17); T. b. gambiense, IL3250; Trypanosoma brucei rhodesiense, IL3953; Trypanosoma congolense, IL1180; Trypanosoma vivax, IL2160; were grown and isolated as described (18). Procyclic T. b. brucei, ILTat1.1 (tsetse fly midgut stage), were cultured in vitro and isolated as described (19). The trypanosomes were used immediately, or stored frozen as cell pellets at -70 °C. Theileria parva (piroplasm form) DNA was a generous gift from Dr. ole-MoiYoi, International Livestock Research Institute, Nairobi, Kenya (20).

Spectrophotometric Assay-- A continuous spectrophotometric assay for IAG-nucleoside hydrolase was performed, at the appropriate wavelength, with inosine, guanosine, adenosine, or p-nitrophenylriboside as substrate, in 50 mM phosphate, pH 7.2 (7).

Amino Acid Sequence of the N-terminal and CNBr Fragments-- Native IAG-nucleoside hydrolase was isolated from the long slender bloodstream form of T. b. brucei and purified to near homogeneity (7). The IAG-nucleoside hydrolase was dialyzed extensively against 1 mM phosphate, pH 7.2, lyophilized, treated with CNBr and the fragments isolated using reverse-phase high performance liquid chromatography, eluting with a gradient of acetonitrile in 0.1% trifluoroacetic acid. The amino acid sequences were obtained for the N-terminal region and CNBr fragments using automated Edman gas-phase sequencing. The protein fragmentation, sequencing, and mass analysis were provided by the Laboratory of Macromolecular Analysis, Albert Einstein College of Medicine, Bronx, NY.

RNA and DNA Preparation-- Total parasitic RNA was isolated by the modified guanidinium thiocyanate-phenol-chloroform extraction procedure (21, 22). Poly(A)+ RNA was purified from total RNA by oligo(dT) chromatography and genomic DNA was isolated by the SDS-proteinase K-phenol method (23). Phage DNA was isolated by the LambdasorbTM phage adsorbent (Promega) protocol. Plasmid DNA was purified from overnight bacterial culture, by the WizardTM miniprep (Promega) DNA purification system.

DNA Library Construction-- A genomic DNA library from T. b. brucei clone ILTat 1.1 (long slender actively dividing bloodstream form) was constructed in the cloning vector lambda gt11. Genomic DNA was sheared by sonication to 2-8-kilobase pair DNA fragments, which were purified on an agarose gel and converted to blunt ends with T4 DNA polymerase. The fragments were ligated both to EcoRI adaptors and to lambda gt11 vector dephosphorylated arms, followed by packaging into phage heads and plating on E. coli Y1090 cells (24). Clones and subclones were sequenced using the fmolTM DNA sequencing system (Promega).

Cloning of the IAG-Nucleoside Hydrolase Open Reading Frame-- Two cDNA fragments encoding the 5' and 3' halves of the full-length cDNA of the iagnh gene were generated by PCR amplification of total single-stranded cDNA from actively dividing long slender bloodstream form of ILTat 1.1 (25). The 5'-region was cloned by PCR amplification using the synthetic sense primer (5'-TAGAACAGTTTCTGTACTATATTG-3') for the mini-exon-derived sequence (common to all African trypanosome mRNAs) and the codon-based antisense primer (5'-TC(ACGT)AC(AGT)AT(AG)TT(ACGT)A(AG)(AG)AA(ACGT)GG-3') for the internal CNBr amino acid fragment, PFLNIVE, of IAG-nucleoside hydrolase. The 3' region was cloned using the degenerate sense primer (5'-CC(ACGT)TT(CT)(CT)T(ACGT)AA(CT)AT(ACT)GT(ACGT)GA-3') designed from the CNBr fragment and the antisense degenerate primer based on the oligo(dT) primer 5'-CCT20-3'.

The above PCR products generated (550 and 850 bp, respectively) were used as 32P-labeled probes to isolate a 3.5-kilobase pair lambda gt11 clone (24) containing the iagnh open reading frame (ORF) with the ATG initiation codon located 300 bp downstream of the 5' end. This clone was used as the template in PCR amplification to generate the full-length iagnh sequence. Primers 5'-GGGCCATGGCGAAAACAGTGATCCTCG-3' and 5'-CCCCTGCAGTCAGTAAACGCGTGTGGAAGC-3', based on the nucleotide sequence of the 5'- and 3'-regions, were designed to incorporate the underlined unique NcoI and PstI sites and to create the ATG initiation and the TCA termination codons (in bold), respectively. The double-stranded DNA was purified from agarose gel using the GenecleanTM system (BIO101, Inc.) and cloned into the pMOS-T vector and used to transform E. coli JM109. A modified pET-28a(+) expression vector was constructed by excising the BamHI-HindIII cloning site sequence and replacing it with a linker containing 5'-BamHI-PstI-HindIII-3' sites, thus introducing a unique PstI site in the expression vector. The iagnh ORF was excised from the pMOS-T expression vector with NcoI and PstI and subcloned into the modified pET-28a expression plasmid vector. Both DNA strands were sequenced using overlapping primers. The modified pET-28a expression vector, containing the iagnh ORF, was used to transform E. coli BL21(DE3) cells made competent using the CaCl2 method (26).

Chromosomal Localization of the iagnh Gene-- A filter blot containing pulsed field gel electrophoresis (PFGE)-separated chromosomes from T. b. brucei isolate TREU927/4 was a generous gift of Dr. Sara Melville and Vanessa Leech (Cambridge University, Cambridge, United Kingdom). Briefly, chromosomes were separated on an agarose gel by PFGE as described (27). Following staining of the gel with ethidium bromide to visualize the chromosome bands under UV light, chromosomal DNA was transferred onto a nylon filter using standard methods (23), after nicking the DNA by soaking the gel in 0.25 M HCl for 15 min. The filter was prehybridized, hybridized with 32P-labeled iagnh probe, and washed under the conditions described for Southern blots.

Expression of T. b. brucei IAG-Nucleoside Hydrolase-- An overnight culture was generated by inoculating 50 ml of 2 × YT (tryptic soy broth 16 g/liter; yeast extract, 10 g/liter; NaCl, 5.0 g/liter) containing 30 µg/ml kanamycin monosulfate (kanamycin A), with a loop of liquid culture E. coli BL21(DE3) cells, containing the iagnh ORF in modified pET-28a, and incubated overnight at 37 °C in a shaking incubator. The next morning, 5 ml of this overnight culture was added to 300 ml of Terrific Broth (tryptic soy broth 12 g/liter; yeast extract, 24 g/liter; glycerol, 4.0 ml/liter, containing 50 mM Hepes, pH 7.2, containing 30 µg/ml kanamycin A) and incubated at 37 °C with shaking until the cells reached an A600 between 1 and 2. The cells were then transferred to 15 liters of Terrific Broth, containing 30 µg/ml of kanamycin A, grown in a 16-liter New Brunswick Fermentor, with agitation at 37 °C. Upon reaching A600 ~ 4, the medium was cooled to 20 °C and isopropyl-1-thio-beta -D-galactopyranoside, dissolved in 50% ethanol, was added to the medium to give a final concentration of 0.2 mM isopropyl-1-thio-beta -D-galactopyranoside. The cells were incubated with stirring and aeration overnight at 20 °C. The following morning, the cells were harvested by centrifugation, washed once with 10 mM phosphate buffer, pH 7.5, containing 140 mM NaCl, 3 mM KCl, 1 mM CaCl2, 0.4 mM MgCl2 (Dulbecco's phosphate-buffered saline), and stored at -20 °C.

Purification of Recombinant IAG-Nucleoside Hydrolase-- Purification of the recombinant IAG-nucleoside hydrolase (rIAG-nucleoside hydrolase) was based on the previous published scheme (7) with two additional chromatography steps to obtain highly purified protein. Superdex 75 molecular sieve, 50 mM HEPES, pH 7.2, with 0.3 M KCl, as elution buffer, and/or a "mixed dye" binding column consisting of 2.5 ml each of Reactive Brown (Sigma R-2757), Green 5 (R-2257), Green 19 (R-2882), Red 120 (R-0503); Yellow 3 (R-3757), and Yellow 86 (R-2382), using 10 mM MES, pH 6.5 as elution buffer, was utilized. The rIAG nucleoside hydrolase does not bind to the dyes, and contaminating proteins are retained. The purity of the protein was estimated by electrophoresis in polyacrylamide gels containing SDS. The enzyme was flash-frozen in dry ice/ethanol and stored at -70 °C.

Northern and Southern Blot Hybridizations-- Northern blot analysis of poly(A)+ RNA (~1 µg) was performed as described (28). Southern blot analysis of purified genomic DNA (2 µg) digested with a variety of restriction endonucleases was performed as described (23). Two 32P-labeled probes from either the iagnh ORF (iagnh probe) or the 663-bp 5' fragment of the ORF for the iunh gene (iunh probe) were generated by random priming (29). The 32P-labeled probe based on the beta -tubulin gene (beta -tubulin probe) was used as an internal control to determine the amount of mRNA transferred to the nylon filters (30).

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Genomic Analysis of the iagnh Locus in T. b. brucei-- Genomic DNA from T. b. brucei (ILTat 1.1) was digested with restriction endonucleases, which cleave internally and/or externally to the iagnh ORF, and hybridized with the iagnh probe (Fig. 1, panel B). Endonucleases with one cleavage site within the iagnh ORF yielded two unequal size hybridization bands. A time-dependent cleavage using RsaI did not produce a pattern expected for tandemly repeated genes (data not shown). All endonucleases (except NcoI) that have no restriction sites in the iagnh locus produced a single hybridizing fragment. NcoI, which does not contain a restriction site in the iagnh locus, cleaved the genomic DNA to generate two distinct hybridization bands on a highly resolving long range separation gel (data not shown).


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Fig. 1.   Southern blot and chromosomal analysis of the iagnh locus. Panel A, Southern blot hybridization of PFGE gel with iagnh probe was as described under "Materials and Methods." Lane 1 shows the EtBr-stained pattern of chromosomal bands after separation by PFGE. The legends and Roman numerals on the left to the chromosomal bands indicate the corresponding chromosome equivalents of each band. The chromosomal nomenclature has been defined by the T. b. brucei Genome Project Initiative, with the chromosomes labeled according to increasing size in PFGE using Roman numerals (27). A filter containing PFGE-separated chromosomes was hybridized with the following T. b. brucei ILTat 1.1 DNA probes: cysteine protease (lane 2), cyclophilin A (lane 3), glyceraldehyde-3-phosphate dehydrogenase (lane 4), and iagnh (lane 5). CZ indicates the compression zone. Panel B, genomic DNA (2 µg) from T. b. brucei ILTat1.1 was digested with the indicated restriction enzymes, fractionated on a 0.8% agarose gel, transferred to a Nytran filter, and probed with the iagnh probe. Post-hybridization washes in 1 × SSC, 0.1% SDS at 65 °C were followed by autoradiography. The ORF of the T. b. brucei iagnh contains a single restriction site each for HaeII (lane 5), KpnI (lane 6), PvuII (lane 7), RsaI (lane 8), and SalI (lane 9), while BamHI (lane 1), EcoRI (lane 3), HindIII (lane 2), and NcoI (lane 4) do not cleave within the iagnh ORF. Panel C, Southern blot analysis of various parasitic genomic DNA. Total genomic DNA (~2 µg) from T. b. brucei (lane 1), T. b. gambiense (lane 2), T. b. rhodesiense (lane 3), T. evansi (lane 4), T. vivax (lane 5), T. congolense (lane 6), T. cruzi (lane 7), Th. parva (lane 8), and C. fasciculata (lane 9) were digested to completion with PstI and then analyzed by Southern blot hybridization with the iagnh probe at 55 °C. Post-hybridization washes was in 2 × SSC, 0.1% SDS at 55 °C, followed by autoradiography at -70 °C for 3 days (lanes 1-5) and 14 days (lanes 6-9). Numbers on left are DNA size markers in kilobases (kbp).

The chromosomal localization of the iagnh gene was determined by Southern blot hybridization of PFGE-separated chromosomes of T. b. brucei isolate TREU927/4 (27) using the iagnh probe. The ethidium bromide-stained gel showed the chromosomal band profile (Fig. 1, panel A, lane 1). The autoradiograph showed that the iagnh probe hybridized to two bands with the larger-sized band containing chromosomes III, IV, V, and VI and the smaller-sized band containing chromosomes III and IV. The iagnh probe hybridization signal to the III, IV chromosome band was of the same intensity as that to the III, IV, V, VI chromosome band.Three control probes were used: a cDNA probe containing the T. b. brucei cyclophilin A homologue (cyclophilin probe),2 which hybridized to the compression zone (lane 3), and a cysteine protease probe (lane 2) (31) and glyceraldehyde-3-phosphate dehydrogenase probe (lane 4) (32), which hybridized to the band containing chromosome VI and the band containing chromosome III, IV, V, and VI, as expected (27).

The PstI-digested genomic DNA from several protozoan parasites was probed with the iagnh probe at 55 °C and washed under medium stringency conditions (2 × SSC, 0.1% SDS at 55 °C) and showed strong hybridization signals with T. brucei gambiense and rhodesiense, T. evansi, T. vivax, T. congolense, and T. cruzi DNA and a faint signal with Th. parva and C. fasciculata DNA (Fig. 1 panel C). There were only weak hybridization bands observed against genomic DNA from T. vivax, T. congolense, or T. b. brucei using the IU-nucleoside hydrolase probe (data not shown).

Primary Gene Sequence of IAG-Nucleoside Hydrolase-- The full-length cDNA for IAG-nucleoside hydrolase contains an ORF of 981 bases, a 5' mini-exon 186 bases upstream to the ATG start codon, and a poly(A) tail extending 90 bases downstream from the TGA stop codon (Fig. 2). The coding region is 53% GC, which is consistent with the average coding region GC content of 51.6% from 31 previously reported T. brucei genes (33).


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Fig. 2.   cDNA and predicted amino acid sequence of the IAG-nucleoside hydrolase from T. b. brucei. The nucleotide sequence shown in italics and not numbered is from the genomic DNA clone. The 5' mini-exon spliced leader on the cDNA is underlined. Nucleotides within the open reading frame start with number 1. Nucleotides 5' to the ATG start codon are negatively numbered. The 5' end of the nucleotide sequence that is trans-spliced onto the mini-exon is shown in bold letters. The amino acids in bold represent those residues that correspond to the amino acids determined from the N-terminal sequence and an internal CNBr sequence.

Sequence Alignment-- A BlastX search of the amino acid sequence, deduced from the iagnh ORF (Fig. 3), revealed homology to only two ORF, one that encodes the IU-nucleoside hydrolase from C. fasciculata (32% amino acid identity) and one that encodes the YEIK E. coli hypothetical 33.7-kDa protein in the nfo-fruA intergenic region (33% amino acid identity) (34, 35). Regions of significant amino acid sequence similarity include the N-terminal 20 amino acids and the region from amino acids 158-196. The N-terminal motif XKXXXILDXDXXXDD of the IAG- and IU nucleoside hydrolases are conserved, as is a P residue at amino acid 26. Another conserved motif (amino acid 182-196) is AEXNIXXDPXAAXXV. The N-terminally processed IAG-nucleoside hydrolase contains 326 amino acids compared with 314 amino acids from the N-terminally processed IU-nucleoside hydrolase of C. fasciculata (6). The additional 12 amino acids in the IAG-nucleoside hydrolase are located around positions 5, 170, and 260 relative to the similar proteins.


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Fig. 3.   Sequence analysis of IAG-nucleoside hydrolase from T. b. brucei and similar deduced protein sequences. The data were collected from the On-Line National Center for Biotechnology Information, (http://www.ncbi.nlm.nih.gov). The search was conducted using the default parameters of the BLAST X algorithm. Only the deduced amino acid sequences from the IU-nucleoside hydrolase and the intergenic reading frame of YIEK E. coli (YIEK_Ecoli) were obtained using these search parameters. The remaining sequences were obtained from Ref. 6 and aligned manually. Only the regions showing substantial similarity to the IAG-nucleoside hydrolase are shown. Abbreviations are defined at bottom of figure.

Expression during Development of mRNA of One iagnh Gene-- The mRNAs from the actively dividing long slender and intermediate bloodstream forms, non-dividing short stumpy bloodstream form, and the procyclic tsetse midgut form life-cycle stages of T. b. brucei were hybridized with the iagnh probe (Fig. 4, panel A). The relative amount of mRNA loaded in each lane was determined by hybridization to the same blot of a beta -tubulin (Fig. 4, panel B). The iagnh mRNA was expressed equally in the actively dividing long slender and intermediate bloodstream forms and was slightly less expressed in the procyclic culture form. The non-dividing short stumpy bloodstream form showed a decrease in the levels of expression of the iagnh mRNA. However, the levels of stable mRNA expressed between the life-cycle stages is similar to that observed for beta -tubulin.


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Fig. 4.   Northern blot analysis of the iagnh transcripts. Approximately 1 µg of poly(A)+ RNA isolated from long slender bloodstream form (lanes 1 and 6), intermediate bloodstream form (lanes 2 and 5), short stumpy bloodstream form (lane 3), and procyclic insect form (lane 4) was denatured and electrophoresed on a 1.4% agarose gel blotted onto a Nytran filter and hybridized with iagnh probe (A) or with a beta -tubulin cDNA probe (B) as described under "Materials and Methods." Post-hybridization washes in 0.1 × SSC, 0.1% SDS at 65 °C were followed by autoradiography at -70 °C. RNA size markers (Life Technologies, Inc.) are indicated in kilobases (kb).

Overexpression, Purification, and Kinetic Analysis of IAG-Nucleoside Hydrolase-- E. coli (BL21(DE3)) transformed with the modified pET-28a-IAG-nucleoside hydrolase construct (Fig. 5) expressed over 10% of the soluble protein as IAG-nucleoside hydrolase (Fig. 6, panels A and B). About 200 mg of >97% homogeneous protein was obtained from 150 g (wet weight) of E. coli (Table I and Fig. 6, panel C). The protein eluted at a Ve/Vo corresponding to a Mr of 72,000 by Superdex 200 Gel chromatography, while SDS-PAGE showed a single band of a Mr of 35,000, consistent with the native protein being a dimer of Mr 35,000 subunits. The calculated molecular weight, based on the predicted amino acid sequence from the cDNA, was 35,735 for the rIAG-nucleoside hydrolase. This is within experimental error of the observed molecular weight for the rIAG-nucleoside hydrolase determined by mass spectrometry, Mr = 35,737. The observed molecular weight of the native IAG-nucleoside hydrolase, as determined by mass spectrometry, is 35,759, which is 22 atomic mass units different from the rIAG-nucleoside hydrolase. A single codon change introduced by the PCR cloning could account for the difference, so kinetic analysis was conducted to ensure that the recombinant and native IAG-nucleoside hydrolase have identical kinetic properties.


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Fig. 5.   Plasmid for expression of IAG-nucleoside hydrolase from T. b. brucei. The 981-bp ORF of the iagnh gene is contained within the NcoI-PstI restriction fragments and placed in the homologous sites in a modified pET-28a(+) vector. The ORF of the cDNA iagnh gene is shown in Fig. 2. A PstI restriction site was cloned next to the existing HindIII restriction site to obtain the modified pET-28a(+) expression vector.


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Fig. 6.   SDS-PAGE of IAG-nucleoside hydrolase. Panel A: 12.5% SDS-PAGE stained with Coomassie Blue R250. Approximately 10 µg of protein from total cell lysate of uninduced (in triplicate, lanes 1-3) and isopropyl-1-thio-beta -D-galactopyranoside-induced (in triplicate, lanes 4-6) bacterial cultures were loaded in each lane. Protein standards with their respective sizes in kilodaltons (kDa) are indicated on the left. Panel B, 12.5% SDS-PAGE stained with Coomassie Blue R250 showing purification of recombinant IAG-nucleoside hydrolase. Lane M contains molecular weight standards; lane 1 contains 1 µg of native purified IAG-nucleoside hydrolase; lanes 2-4 contain approximately 20 µg of protein from Superdex 75/Red A, Superdex 200, Q-Sepharose, (NH4)2SO4, and initial extract steps of the purification, respectively. Panel C, 12.5% SDS-PAGE stained with Coomassie Blue R250 to determine the purity of IAG-nucleoside hydrolase. Lane 1, native IAG-nucleoside hydrolase; lane M, molecular weight standards; lanes 2-4, 1, 10, and 100 µg of IAG-nucleoside hydrolase following purification using Superdex 75/Red A chromatography. Comparison of the minor bands that appear in lane 4 to that of lanes 2 and 3 indicate that the purified protein is 97% pure.

                              
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Table I
Purification of recombinant IAG-nucleoside hydrolase from E. coli
The activity is based on the Delta A280 using 250 µM inosine in 50 mM phosphate buffer, pH 7.2. 

The kinetic parameters for the recombinant IAG-nucleoside hydrolase are compared with the published data of the native IAG-nucleoside hydrolase in Table II. The Km and kcat for all the naturally occurring purine nucleoside substrates and the synthetic substrate, p-nitrophenylriboside, were experimentally identical.

                              
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Table II
Comparison of kinetic constants and subunit molecular weight for native and recombinant IAG-nucleoside hydrolase from T. b. brucei

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Genomic DNA Analysis and Sequence of the iagnh Gene-- The cDNA for IAG-nucleoside hydrolase, a purine salvage enzyme from the African trypanosome T. b. brucei, contains both the mini-exon found in the mRNA of all trypanosomes and the poly(A) tail. Both native and recombinant IAG-nucleoside hydrolases have a processed N-terminal Met as established by N-terminal sequencing data. The calculated molecular weight of the rIAG-nucleoside hydrolase, based on the translated amino acid sequence of the cDNA, and the observed molecular weight, determined by mass spectroscopy, was 35,735 and 35,737, respectively. Thus, the ORF and the DNA sequence assignment are established.

The iagnh locus in T. b. brucei (ILTat 1.1) was analyzed for both copy number and tandem repeats using restriction enzyme analysis and Southern blotting. A number of genes in T. b. brucei, including tubulins (36), calmodulin (37), and glyceraldehyde-3-phosphate dehydrogenase (32), are arranged as tandem repeat sequences, while others have one gene/haploid genome. These include hypoxanthine-guanine phosphoribosyltransferase (38) and glucosephosphate isomerase (39). Restriction endonuclease digestion analysis and Southern blotting with partial and complete digestion indicate the existence of one iagnh gene/haploid genome in this T. b. brucei clone. In-contrast, T. vivax DNA digested with PstI produces at least five hybridization bands greater than 2 kilobase pairs, suggesting that the "nh gene" in T. vivax is present in at least two copies per haploid genome (Fig. 1, panel C).

Chromosome-sized molecules of T. b. brucei DNA were separated using pulse-field gradient electrophoresis, blotted, and hybridized with the iagnh probe. The two chromosome-sized bands that hybridized with the iagnh probe consist of the band containing chromosomes III, IV, V, and VI and that containing only chromosomes III and IV. Because the iagnh probe hybridizes with equal intensity to both chromosome bands, the data support the location of the iagnh gene on chromosome III or IV. Although the mechanism underlying chromosome size polymorphism is not yet known, telomere lengthening and collapse or genetic exchange during cyclical transmission in the vector could generate size variability in T. b. brucei (40, 41).

Strong hybridization signals against the iagnh probe establishes the presence of an N-ribohydrolase gene in all African trypanosomes tested. However, the absence of a strong hybridization signal using the iagnh probe does not preclude the existence of N-ribohydrolase isozymes. N-Ribohydrolase isozymes have been identified in T. cruzi (9) and a cDNA-derived N-terminal amino acid sequence, which shows a high level of homology with the IU-nucleoside hydrolase, has been identified in Leishmania major (6). In contrast, there was only a weak hybridization signal with T. b. brucei, T. congolense, or T. vivax genomic DNA, when using the iunh probe from C. fasciculata (data not shown). Therefore, the above data suggest that the N-ribohydrolases are ubiquitous enzymes for purine salvage in protozoan parasites.

Analysis of iagnh mRNA Expression-- The life-cycle stages of T. b. brucei in the bloodstream differentiate from actively dividing long slender and intermediate life-cycle stages, to the non-dividing, short stumpy life-cycle stage (4). The short stumpy forms are more capable of surviving in the tsetse fly's blood meal and are proposed to be the life-cycle stage that can reinitiate an infection in the tsetse fly (5). Northern blots show that the iagnh gene is expressed in all developmental stages of the parasite that were tested. Although the iagnh mRNA is produced at higher levels in the actively dividing bloodstream forms and the in vitro cultured procyclic form when compared with the non-dividing short stumpy form, expression of iagnh mRNA is substantial in all actively dividing forms. In comparison, another gene that encodes a purine salvage enzyme from T. b. brucei, the hgprt gene, which encodes hypoxanthine-guanine phosphoribosyltransferase, is not developmentally regulated. This enzyme is also encoded in one gene/haploid genome (38). In contrast, the cysteine proteases from T. b. brucei are expressed at higher levels in the non-dividing short stumpy bloodstream form and exist as multiple tandemly repeated arrays (31, 42, 43).

Biological Role of IAG-Nucleoside Hydrolase-- Berens et al. (1) have proposed that the salvage pathways utilized by parasitic organisms respond to the purine composition of the parasite's environment. The equal expression of the iagnh and hgprt genes, which encode two vital purine salvage enzymes, in the actively dividing life-cycle stages of T. b. brucei support this hypothesis. Fig. 7 summarizes the processes by which a purine nucleoside or base is converted to IMP in T. b. brucei. An important component of purine salvage is the transport mechanisms for purine nucleosides and bases. T. b. brucei has efficient adenosine and inosine transporter systems with Km values in the submicromolar range (44). Adenosine, released during tissue damage, has been proposed as the most important purine source for T. b. brucei (45). Another proposal is that the constitutive expression of the hypoxanthine-guanine phosphoribosyltransferase enzymes is due to hypoxanthine as the major source of purines (38, 46).


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Fig. 7.   Conversion of adenosine, inosine, and hypoxanthine to AMP or IMP in the bloodstream form of T. b. brucei. The reported enzyme activities and transporters involved in the adenosine-inosine-hypoxanthine purine salvage pathway of the bloodstream form of T. b. brucei. Abbreviations are as follows: IAG-NH, IAG-nucleoside hydrolase (7); IG-NH, inosine-guanosine nucleoside hydrolase (7); P1 and P2, adenosine and inosine transporters; ?, a proposed hypoxanthine transporter (45); PRPP, phosphoribosylpyrophosphate; HGPRTase, hypoxanthine-guanine phosphoribosyltransferase (38); APRTase, adenosine phosphoribosyltransferase (53); A-Ado-Lase, adenylosuccinate lyase; A-Ado-Sase, adenylosuccinate synthetase (A-Ado-Sase) (54). Paths with a question mark (?) indicate that the enzyme or transporter has not been reported or characterized. Enzymatic activities of the poorly characterized purine nucleoside kinases, and deaminases are not shown (1).

The concentration of available purine nucleotide precursors in the bloodstream of both humans and domestic animals is still in question. Estimates of the concentration of free hypoxanthine in the blood of humans varies over 250-fold from 0.092 µM to 24 µM (47-50). The concentrations of hypoxanthine vary by as much as 20-fold depending on the method of collection and storage (51). The levels of adenosine, inosine, and hypoxanthine are reported to be 2, 0.2, and 0.7 µM, respectively, when plasma was prepared in the presence of the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (50). Free concentrations of adenosine, inosine, and hypoxanthine in the bloodstream of livestock have not been well documented. The continuous expression of both the iagnh and hgprt genes in African trypanosomes suggests that both the hypoxanthine-guanine phosphoribosyltransferase and N-ribohydrolases are required for the biosynthesis of IMP, with adenosine and/or inosine being the major source of purines in the bloodstream of the mammalian host.

Amino Acid Sequence Comparison-- The IU- and IAG-nucleoside N-ribohydrolase isozymes have distinct amino acid sequences with only two relatively conserved regions. The x-ray crystal structure of the IU-nucleoside hydrolase from C. fasciculata reveals that the N-terminal Asp residues are clustered near the catalytic site (16). The site contains a tightly bound metal ion, and the ion is held in place by ionic interactions with amino acids Asp-10 and Asp-15 (16). The conserved Asp-8 and Asp-14 residues are in the second sphere of amino acids, which stabilize the metal ion-binding amino acids. Recent results establish that the ion is a tightly bound Ca2+ present in both the IU- and IAG-nucleoside hydrolase.3 The second group of conserved amino acids from 158 to 196 contains the AEXNI motif. From the crystal structure of the IU-nucleoside hydrolase, amino acids corresponding to Glu-183 and Asn-185 in the IAG-nucleoside hydrolase are also located in the proposed catalytic site pocket (16). Thus, the conserved regions are associated with the catalytic site elements, despite the substantial differences in substrate and inhibitor specificity for the IU- and IAG- N-ribohydrolase isozymes (7, 52). The conserved region 249-267 in IAG-nucleoside hydrolase contains Asp-258, which corresponds to Asp-242 in IU-nucleoside hydrolase, a residue that is in contact with the catalytic site Ca2+ (16).3 Amino acid His-241 of the IU-nucleoside hydrolases has been demonstrated to be the proton donor involved in leaving group activation (6). However, no corresponding histidine is located near the corresponding region in IAG-nucleoside hydrolase. IAG- and IU-nucleoside hydrolases have different substrate and inhibitor specificities and pH profiles (7, 52), indicating that different amino acids are involved in aglycone activation. The pH profiles for IAG-nucleoside hydrolase show that two basic amino acids are essential for catalysis, pKa 8.8 (7), making amino acids of the corresponding region 254 YYAWD 258 of IAG-nucleoside hydrolase the most likely candidates for involvement in leaving group activation.

Purification and Characterization of the Recombinant IAG-Nucleoside Hydrolase-- The recombinant enzyme has experimentally indistinguishable kinetic and chromatographic characteristics relative to the native IAG-nucleoside hydrolase. The kinetic parameters of the naturally occurring purines and the synthetic substrate p-nitrophenylriboside are identical. The observed molecular weight of 72,000 for the rIAG nucleoside hydrolase corresponds to a homodimer of 35,000 Mr. The similarity of the kinetic parameters, alignment of both the N-terminal amino acids and CNBr-generated fragments, and the comparison of calculated and observed molecular weights establish that the cDNA encodes a protein with the same kinetic and structural properties as the native IAG-nucleoside hydrolase. The coding region for the rIAG-nucleoside hydrolase provides access to structural, mechanistic, and metabolic studies of the purine salvage pathway in African trypanosomes.

Conclusion-- Protozoan parasites are known to use nucleoside hydrolase enzymes in the essential pathways of purine salvage. The IAG-nucleoside hydrolase is the first purine-specific N-ribohydrolase to be characterized by molecular analysis, and is shown to be a common enzyme of purine salvage in African trypanosomes. Sequence data banks reveal no corresponding enzymes in the mammalian hosts. The IAG-isozyme differs substantially from the nonspecific IU-nucleoside hydrolase in amino acid sequences, subunit structure, and kinetic and chemical mechanisms. Amino acid sequences that are involved in the binding of Ca2+ to the catalytic site and those proposed for binding the ribose are part of the conserved sequences. Amino acids involved in the purine base-binding region are different, reflecting the leaving-group activation mechanism for IAG-nucleoside hydrolase. The unique chemical, structural, and kinetic properties of N-ribohydrolase isozymes support their use as targets for chemotherapeutic agents and as diagnostic probes.

    ACKNOWLEDGEMENTS

We thank Dr. Ruth Angeletti, Edward Nieves, and staff of the Laboratory of Macromolecular Analysis, Albert Einstein College of Medicine, for providing the protein fragmentation, sequencing, and mass analysis. We also thank Drs. Onesmo ole-MoiYoi and Noel Murphy for their support and critical evaluation of the manuscript.

    FOOTNOTES

* This work was supported by an African Regional Research Fulbright Award (to D. W. P. for 1994-1995), by National Institutes of Health Research Grant GM41916 (to V. L. S.), and by the International Livestock Research Institute (ILRI), Nairobi, Kenya. This is ILRI Publication 97065.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) AF017231.

To whom correspondence should be addressed: International Livestock Research Institute, P. O. Box 30709, Nairobi, Kenya. Tel.: 254-2-630-743; Fax: 254-2-631-499; E-mail: d.parkin{at}cgnet.com.

1 The abbreviations used are: IU-, inosine-uridine-preferring; IAG-, inosine-adenosine-guanosine-preferring; bp, base pair(s); PCR, polymerase chain reaction; ORF, open reading frame; PFGE, pulsed field gel electrophoresis; MES, 4-morpholineethanesulfonic acid.

2 R. Pelle and N. B. Murphy, manuscript in preparation.

3 V. L. Schramm, D. W. Parkin, M. Degano, and O. Varlamova, manuscript in preparation.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
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