Identification and characterization of guanosine 5′-monophosphate reductase of Trypanosoma congolense as a drug target

Article abstract of DOI:10.1016/j.parint.2017.03.006

Trypanosoma congolense is one of the most prevalent pathogens which causes trypanosomosis in African animals, resulting in a significant economic loss. In its life cycle, T. congolense is incapable of synthesizing purine nucleotides via a de novo pathway, and thus relies on a salvage pathway to survive. In this study, we identified a gene from T. congolense, TcIL3000_5_1940, as a guanosine 5′-monophosphate reductase (GMPR), an enzyme that modulates the concentration of intracellular guanosine in the pathogen. The recombinant protein was expressed in Escherichia coli, and the gene product was enzymatically confirmed as a unique GMPR, designated as rTcGMPR. This enzyme was constitutively expressed in glycosomes at all of the parasite's developmental stages similar to other purine nucleotide metabolic enzymes. Mycophenolic acid (MPA) was found to inhibit rTcGMPR activity. Hence, it is a potential lead compound for the design of trypanocidal agents, specifically GMPR inhibitor.

Full text of DOI:10.1016/j.parint.2017.03.006

Accepted Manuscript
Identification and characterization of guanosine 5′-
monophosphate reductase of Trypanosoma congolense as a drug
target
Albertus Eka Yudistira Sarwono, Keisuke Suganuma, Shinya
Mitsuhashi, Tadashi Okada, Simon Peter Musinguzi, Kengo
Shigetomi, Noboru Inoue, Makoto Ubukata
PII:
DOI:
Reference:
S1383-5769(16)30496-2
doi: 10.1016/j.parint.2017.03.006
PARINT 1660
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Parasitology International
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Revised date:
Accepted date:
18 November 2016
17 February 2017
27 March 2017
Please cite this article as: Albertus Eka Yudistira Sarwono, Keisuke Suganuma, Shinya
Mitsuhashi, Tadashi Okada, Simon Peter Musinguzi, Kengo Shigetomi, Noboru Inoue,
Makoto Ubukata , Identification and characterization of guanosine 5′-monophosphate
reductase of Trypanosoma congolense as a drug target. The address for the corresponding
author was captured as affiliation for all authors. Please check if appropriate. Parint(2017),
doi: 10.1016/j.parint.2017.03.006
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ACCEPTED MANUSCRIPT
Identification and characterization of guanosine 5’-monophosphate reductase of Trypanosoma
congolense as a drug target
Running title:GMP reductase of Trypanosoma congolense
a
b, c
a
Albertus Eka Yudistira SARWONO  Keisuke SUGANUMA
 Shinya MITSUHASHI Tadashi
b, d
b
a
e
OKADA
UBUKATA
Simon Peter MUSINGUZI Kengo SHIGETOMI Noboru INOUE Makoto
a
a Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Kita-ku,
Sapporo, Hokkaido 060-8589, Japan
b National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary
Medicine, Inada, Obihiro, Hokkaido 080-8555, Japan
c Research Center for Global Agromedicine, Obihiro University ofAgriculture and Veterinary
Medicine, Inada, Obihiro, Hokkaido 080-8555, Japan
d Division of Neurology, Resoirology, Endocrinology and Metabolism, Department of Internal
Medicine, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki, 889-1692,
Japan
e Obihiro University ofAgriculture and Veterinary Medicine, Inada, Obihiro, Hokkaido 080-8555,
Japan
Albertus Eka Yudistira Sarwono and Keisuke Suganuma contributed equally to this work
# Address correspondence to: Prof. Makoto Ubukata, Ph.D.
Research Faculty ofAgriculture, Hokkaido University, Kita-9, Nishi-9, Kita-ku, Sapporo 060-8589,
Japan
E-mail: m-ub@for.agr.hokudai.ac.jp
Tel/FAX: 011-706-3638

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Conflict of interest statement
The authors declare that there is no conflict of interest
Acknowledgments
This study was financially supported by the Institute for the Fermentation, Osaka (IFO) Japan, Japan
Society for the Promotion of Science (JSPS) (25660051); Grants-in-Aid for Scientific Research from
the Ministry of Education, Culture, Sports, Science and Technology (MEXT) (25292167); and the
Japan Agency for Medical Research and Development (AMED) /Japan International Cooperation
Agency (JICA) and Science and Technology Research Partnership for Sustainable Development
(SATREPS).
We thank Ms.Yoko Matsushita forher excellent technical assistance.We thankMs. Ma Min for her
helpful discussion on the enzyme kinetics study.
ABSTRACT
Trypanosoma congolense is one of the most prevalent pathogens which causes trypanosomosis
in African animals, resulting in a significant economic loss.In its life cycle, T. congolense is incapable
of synthesizing purine nucleotides via a de novo pathway, and thus relies on a salvage pathway to
survive. In this study,we identified a gene from T. congolense,TcIL3000_5_1940, as a guanosine 5’-
monophosphate reductase (GMPR), an enzyme that modulates the concentration of intracellular
guanosine in the pathogen.The recombinant protein was expressed in Escherichia coli, and the gene
product was enzymatically confirmed as a unique GMPR, designated as rTcGMPR. This enzyme was
constitutively expressed in glycosomes at all of the parasite’s developmental stages similar to other
purine nucleotide metabolic enzymes. Mycophenolic acid (MPA) was found to inhibit rTcGMPR
activity. Hence, it is a potential lead compound for the design of trypanocidal agents, specifically
GMPR inhibitor.
KEYWORDS: African trypanosomosis; GMP reductase; purine metabolic pathway; Trypanosoma
congolense

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1. Introduction
The purine nucleotide is an essentialbuilding block of genetic materials in virtually all
organisms. Guanosine 5’-monophosphate dehydrogenase (GMPR) (EC 1.7.1.7) is a regulatory enzyme
for purine biosynthesis,which acts as a deamination catalyst of guanosine 5’-monophosphate (GMP)
conversion to inosine 5’-monophosphate (IMP) (Fig. 1). This enzyme complements two other purine
regulatory enzymes: inosine 5’-monophosphate dehydrogenase (IMPDH) (EC 1.1.1.205) and GMP
synthase (GMPS) (EC 6.3.5.2), with each catalyzing conversion of IMP to xanthosine 5’-
monophosphate (XMP) and XMP to GMP, respectively. Consequently, these enzymes cooperatively
maintain the intracellular balance of adenine and guanine nucleotides.However, due to the similarity
between GMPR and IMPDH at the primary structure level, misannotation of these two enzymes is
common [1]. The similarities between these enzymes were even more pronounced in reported
Kinetoplastida GMPR, which apparently featured a cystathione β-synthase (CBS) pair domain;
previously it was only found in IMPDHs [2,3].
(Fig. 1)
Due to the lack of metabolic machinery for synthesis via de novo pathway, some protozoa rely
on salvage pathway to produce purine nucleotides.On that account, the purine biosynthesis pathway in
protozoan studies has been considered as an interesting target for drugs [4–6] . Purine nucleotide
synthesis in Kinetoplastida parasites is also dependent on a salvage pathway, and most of the reactions
catalyzed by the above enzymes are proceeded in the glycosomes [7]. Pathogenic protozoa T.
congolense also possess this unique feature,which could be exploited as a novel chemotherapeutic
target.
T. congolense itself is a pathogenic protozoan that infects a broad range ofAfrican animals,
inflicting fatal animal trypanosomosis,which is also known as nagana disease. Up until recent time,
the infection of domesticated animals in central Africa has resulted in severe economical loss to the
society [8]. Currently, chemotherapeutic strategies against T. congolense infection consist of outdated
and ineffective therapeutic compounds, resulting in the emergence of chemo-resistant T. congolense
strains [9–11]. Generally, humans are resistant to T. congolense infection due to the trypanosome lytic
factor in the serum. Several research groups,however, have reported some cases of viable T.
congolense strains in human plasma, posing a future threat to human health [12–14].

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This study provides the first enzymatic and cytological identification of a unique GMPR-
encoding gene product in all life cycle stages ofT. congolense. Firstly, by analyzing the primary
structure of TcGMPR, we proposed a hypothesis on Kinetoplastida GMPR evolution lineage and
distinctive amino acids for simple GMPR identification. Subsequently, the recombinant TcGMPR
(rTcGMPR), expressed in Escherichia coli,was found to exhibit significantly different enzymatic
parameters from those of mammalian GMPRs. In the protozoan cell, TcGMPR was continuously
expressed in the glycosome throughout its life cycle. We also found that mycophenolic acid, a selective
IMPDH inhibitor, was an inhibitor for rTcGMPR at micromolar levels.

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2. Materials and Methods
2.1. Trypanosome in vitro culture
T. congolense IL3000, a Savannah subgroup strain isolated near the national border between
Kenya and Tanzania in 1966, was used in this study.The blood stream form (BSF) of T. congolense
IL3000 was propagated at 33ºC in 5% CO2 with Hirumi’s modified Iscove’s medium (HMI)-9 [15].
Procyclic trypomastigotes (PCF) and epimastigotes (EMF) were propagated at 27ºC using T. vivax
medium [16]. Metacycleic trypomastigote (MCF) were separated from the supernatant ofconfluently
cultured EMF culture by a DE52 anion exchange column [17]. Trypanosome cultures were maintained
by replacing the entire culture supernatant with fresh medium every other day.
2.2. Cloning of T. congolense GMP reductase
T. congolense GMP reductase (TcGMPR) (accession number TcIL3000_5_1940) was
identified at TriTrypDB (http://tritrypdb.org/tritrypdb/).The full-length TcGMPR gene was PCR-
amplified from T. congolense IL3000 strain genomic DNA using TcGMPR-F and TcGMPR-R primer
sets (Table S1). A full-length TcGMPR gene was digested by Bam HI and Eco RI, and inserted into a
pGEX 6P-2 plasmid (GE Healthcare Bio-Science Corp., UK).
2.3. Homology analysisand phylogenic analysisof GMPRs and IMPDHs
A homology analysis was performed using ClustalW ver. 2.1 (http://clustalw.ddbj.nig.ac.jp).
A phylogenetic tree was constructed by neighbor-joining method using MEGA 5.2 software.
2.4. Expression and purification of recombinant TcGMPR protein
The E. coli cells harboring TcGMPR-pGEX 6P-2 plasmid were grown at 37ºC until the optical
density at 600 nm reached 0.4. A recombinant TcGMPR fused with glutathione S-transferase (GST)
was induced with 0.1 mM isopropyl thio-β-D-galactoside (IPTG) for 12 h at 18ºC. rTcGMPR was
purified and cleaved using glutathione Sepharose 4B beads with PreScission protease (GE Healthcare
Bio-Science Corp., UK) according to the manufacturer’s protocol. rTcGMPR concentrations were
determined using a BCA Protein Assay Kit (Thermo Fisher Scientific Japan, Yokohama, Japan) in
accordance with the manufacturer’s instructions.The protein was stored at -80ºC as a 50% glycerol
mixture until use.

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2.5. Effect of pH to rTcGMPR activity
Method 2.5, 2.6, and 2.8 were carried out according to a previous report, with some
modifications [2]. Briefly, the activity of the recombinant protein was measured at 35ºC in 75 mM Tris-
HCl buffer under various pH (7.0-8.7) conditions.The reaction solution contained 200 μM NADPH
(Sigma Aldrich, St. Louis, MO, USA), 1 mM GMP (Sigma Aldrich Japan, Tokyo, Japan), 100 mM
KCl, 3 mM EDTA, 0.1 mg/ml bovine serum albumin (BSA), and 1 mM dithiothreitol. The ionic
strength of the solution was set to 0.2 with NaCl. The enzyme assay was started by the addition of
rTcGMPR to the reaction solution.Activity of rTcGMPR was monitored at 340 nm using
Biophotometer plus spectrophotometer(Eppendorf, Germany) to measure the conversion of NADPH to
NADP+. Activity for each pH value was expressed as percent difference of initial velocity (V ) value
0
from V0 at pH 7.0.
2.6. Kinetic parameter determination of rTcGMPR
Enzymatic activity was measured with an assay solution containing 75 mM Tris-HCl pH 7.0, 100
mM KCl, 3 mM EDTA, 1 mM DTT, and 0.1 mg/ml BSA at 35ºC. The ionic strength ofthe solution
was set to 0.2 with NaCl. Various concentrations ofGMP were used with fixed dose of NADPH at 200
µM for measurement of steady-state kinetics for GMP. By contrast,various concentrations of NADPH
were used with fixed dose of GMP at 1 mM for measurement of steady-state kinetics for NADPH.
Measurements of Vmax and Km were conducted by fitting the acquired initial velocity data from three
independent experiments to the Michaelis-Menten equation using GraFit 7 software (Erithacus
software, U.K.).
2.7. Inhibition of rTcGMPR by MPA
MPA inhibition to rTcGMPR activity was measured with an assay solution containing 75 mM
Tris-HCl pH 7.0, 100 mM KCl, 3 mM EDTA, 1 mM DTT, 0.1 mg/ml BSA, 100 μM NADPH, 100 μM
GMP, and various concentrations of MPA at 30ºC. DMSO was used as vehicle, with a final
concentration of 1%. The reaction was started by adding 50 nM of rTcGMPR. Reaction was carried out
for 1 h.Activity of rTcGMPR was monitored as a decrease of absorbance at 340 nm. Each point was
expressed as a percentage of consumed NADPH in each treatment sample relative to the vehicle

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control. The IC50 value was calculated using GraFit 7 software (Erithacus software, U.K.)
2.8. Activation of rTcGMPR by monovalent cation
Enzyme activation by monovalent cation was conducted with an assay solution containing 75 mM
Tris-HCl pH 7.0, 3 mM EDTA, 1 mM DTT, 0.1 mg/ml BSA, 200 µM NADPH, 1 mM GMP, and 100
mM monovalent chloride (either LiCl, NaCl, KCl, CsCl, or NH4Cl) at 35ºC. Activity for each
monovalent cation was expressed as a percent difference of the initial velocity (V0) value from V0 in
the assay with NaCl.
2.9. Polyclonal antibody production
Two 8-weeks-old imprinting control region mice were immunized with 50 µg (100 µL)
emulsion of equal volume of rTcGMPR and TiterMax® Gold (TiterMax USA Inc., Norcross,GA).
Primary immunization and two boosters (at two-week intervals) were administered subcutaneously.
One week after the second boosterinjection, blood was collected by cardiac puncture at terminal
anesthesia.Sera were prepared by centrifugation at 5,000×g for 10 min at 4ºC. The animal experiments
were carried out in accordance with the Standards Relating to the Care and Management of
Experimental Animals of Obihiro University of Agriculture and Veterinary Medicine (No. 25-134).
2.10. Western blot analysis
To prepare the parasite cell extracts, each stage of trypanosome in four life cycles was
suspended in lysis buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 10%
glycerol, and 1.0% Triton X-100) supplemented with a protease inhibitor cocktail (Complete Mini,
Roche Diagnostics K.K. Tokyo, Japan). After 30 min incubation on ice, the extract was centrifuged at
21,500×g, 4ºC for 10 min. Five-µg of each extract was separated by sodiumdodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE), and transferred into a polyvinylidene difluoride
membrane (Hybond-P, GE Healthcare Co.). The membrane was blocked by 5% skim milk in Tris-
buffered saline containing 0.05% Tween 20 (TBS-T), which was then incubated with a primary
antibody (anti-TcGMPR serum) and further incubated with secondary antibody ECLTM anti-mouse
IgG, horseradish peroxidase linked whole antibody from sheep (GE Healthcare Co., UK). The result
was visualized using an ECL Western Blotting Detection System (GE Healthcare Co., UK) according

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to the manufacturer’s instructions.The relative quantity of TcGMPR was evaluated by measuring the
signal densities of TcGMPR normalized by T. congolense α-tubulin signal densities using Quantity One
software (Bio-Rad, Berkeley, CA) [18].
2.11. Immunofluorescence microscopy
T. congolense specimens corresponding to each developmental stage were suspended in PBS
with 1% glucose.The cell suspensionswere spread over glass slides printed with highly water-repellent
mark (Matsunami Glass Ind., Ltd., Tokyo, Japan), air-dried, and fixed with 100% methanol for 10 min
at room temperature. For blocking, the specimens were incubated with 5% skim milk in TBS-T for 1 h
at room temperature. The specimens were incubated with a primary antibody (anti-TcGMPR serum),
subsequently incubated with a secondary antibody (Alexa Fluor 488 goat anti-mouse IgG [H+L],
Invitrogen, Life Technologies Japan, Tokyo, Japan) and Hoechest 33342 (Dojundo, Co. Ltd.,
Kumamoto, Japan). The specimens were observed using a confocal laser scanning microscope (Leica
TCS SP5, Leica Microsystems,Wetzlar, Germany).

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3. Results
3.1. Identification,bioinformaticsanalysis,and expression of TcGMPR
By utilizing a BLAST search, a GMPR/IMPDH family gene, TcIL3000_5_1940, was
identified in T. congolense as the homolog with the highest similarity to our previously discovered T.
congolense IMPDH [18]. The similarity of amino acid sequence between GMPRs and IMPDHs,
however, often results in misleading differentiation between enzymes if it is carried out only by
computational gene prediction methods [1]. Phylogenetic analysis revealed that IMPDHs and GMPRs
were classified into different clades. However, while GMPR from L. donovani and T. brucei clearly
exhibited a GMPR enzyme activity [2,19], they were not classified into the GMPR clade, but to in the
IMPDH related clade instead. TcIL3000_5_1940 itself was classified in the same clade as L. donovani
and T. brucei GMPR as shown in Fig. 2. Amino acid sequences ofGMPR homologs were aligned to
examine the conservation of its sequence in Fig. 3.
(Fig. 2)
(Fig. 3)
The predicted secondary structure of the TcIL3000_5_1940 protein consisted ofthe
TIM_phosphate_binding superfamily and two tandem repeats of CBS pair domains. To date,it has
been generally accepted that the CBS pair domain was contained in IMPDH but not in GMPR [1],
whereas this domain was found in L. donovani GMPR, T. brucei GMPR, and TcIL3000_5_1940 (Fig.
3). TcIL3000_5_1940 itself shares 72% of amino acid similarity with L. donovani GMPR and 80% of
amino acid similarity with T. brucei GMPR. However, TcIL3000_5_1940 shares only 32-35% gene
similarity to Homo sapiens GMPR1 (HsGMPR1), HsGMPR2, and Bos taurus GMPR1 (BtGMPR), and
BtGMPR2 as indicated in Table 1 [2,20–23]. In similar order, we observed up to 44% of shared identity
in the primary structures between TcIL3000_5_1940 and the IMPDHs in this study (Table S2).
(Table 1)
A full length TcIL3000_5_1940 candidate was cloned and a recombinant protein of
TcIL3000_5_1940 was expressed in E. coli. The purified protein was detected as a single band with a
molecular weight of 52 kDa on SDS-PAGE (Fig. 4). The protein did not show any IMPDH activity
(Fig. S1), while the activity of GMPR was confirmed as described in the following sections.
(Fig. 4)
3.2. Effect of pH to rTcGMPR activity

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To determine the optimum assay condition for steady-state kinetics measurement, activity of
the recombinant protein was evaluated at several pH values under neutral to basic conditions. In
agreement with a previous report on T. brucei GMPR, activity of rTcGMPR was found to be highest at
pH 7.0, and gradually decreased as the pH of the assay solution increased (Fig. 5) [2]. Acidic
conditions are generally avoided for GMPR studies due to the instability of co-factor NADPH under
such conditions.
(Fig. 5)
3.3. Enzymatic analysisof rTcGMPR
The apparent steady-state kinetics parameters of rTcGMPR were characterized with varying
either GMP or NADPH, and the acquired initial velocity was charted into a Lineweaver-Burk plot (Fig.
S2). The values of Km GMP, KmNADPH,and kcat were determined to be 91.6 ± 4.7 μM, 11.3 ± 2.3 μM, and
0.499 ± 0.014 s-1,respectively. These values were quite similar to the reported values for TbGMPR, but
the enzymatic properties were significantly different from those of HsGMPR1, HsGMPR2, BtGMPR1,
and BtGMPR2 as indicated in Table 2 [2]. The values of these parameters indicated that rTcGMPR has
at least 4-fold lower affinity to GMP, but up to 5-fold strongeraffinity to NADPH, compared to those
of mammalian GMPRs. Moreover, the kcat value of rTcGMPR indicated that the Trypanosomatidae
enzyme is up to 1.7-fold more efficient compared to its mammalian counterparts for catalyzing GMP
conversion to IMP.
(Table 2)
3.4. Inhibition of rTcGMPR by MPA
MPA is a known inhibitor of IMPDH. The structuralsimilarities at the primary level between
IMPDH and GMPR make MPA an intriguing candidate as a multi-target inhibitor. The rTcGMPR
inhibition assay of MPA showed a clear activity with IC50 of 239.6 ± 26.7 μM (Fig. 6). MPA also
showed potent inhibition activity in the T. congolense culture, where the presence of 1 μM inhibitor
resulted in a 99.6% decrease in free-living protozoa [18].
(Fig. 6)
3.5. Activation of rTcGMPR by monovalent cation
The results suggested that the cation effect for rTcGMPR is correlated with the radius size of
the corresponding cation. In the presence of Li+ (0.60 Å), the enzyme activity was lowest at 3.8 ± 0.2
nM/s. Subsequently,the addition of larger radius cations: Na+ (0.95 Å), K+ (1.33 Å), Cs+ (1.69 Å), and

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+
NH4 (1.48 Å) enhanced the enzyme activity to 5.8 ± 0.2 nM/s,28.5 ± 1.0 nM/s,9.7 ± 0.8, and 19.5 ±
2.5 nM/s,respectively (Fig. 7).
(Fig. 7)
3.6. Expression pattern and cellularlocalization ofTcGMPR
Previous studies have revealed that purine metabolic enzymes of Kinetoplastida were mostly
located in glycosomes [7,24]. In addition, further amino acid sequence analysis of TcGMPR indicated a
C-terminal Ser-Lys-Leu peroxisomal (glycosomal) targeting signal similar to that of L. donovani
GMPR (Fig. 3) and other glycosomal proteins [7,24,25]. TcGMPR was expressed in all stages ofthe
life cycle of T. congolense as shown in Fig. 8A and 8C. By Western blot analysis, TcGMPR was found
to be expressed up to 2-3 fold higher in EMF than in BSF, PCF, and MCF as shown in Fig. 8B. This
result was consistent with the previous report concerning the differential protein expression analysis of
all stages ofthe T. congolense lifecycle [26]. TcGMPR was expressed in the cytosolas a granular
localization, which was consistent with the glycosomes localization of T. brucei GMPR and T.
congolense IMPDH (Fig. 8C) [2,7,18,24].
(Fig. 8 – Colored Figure)
4. Discussion
Due to the lack of de novo purine synthesis pathway in trypanosomes,the protozoa is entirely
dependent on a salvage pathway, thus making this pathway an interesting drug target against any
pathogen of this genus [2,5,27–29]. GMPR is one of the enzymes in the pathway that maintains
intracellular adenine and guanine balance, along with IMPDH and GMPS [1]. More recent reports have
confirmed that an inhibition to one of the enzymes in the streamlined pathway effectively inhibits the
growth and proliferation of pathogens having a similar purine synthesis pathway,thus validating the
pathway as a promising drug target for anti-infection chemotherapy [3,27,28,30–34]. Therefore,
holistic understanding ofthis pathway is essential for designing novel therapeutic strategies against
such pathogens.
In gene data banks,an IMPDH/GMPR gene TcIL3000_5_1940 of T. congolense was annotated
as either GMPR or IMPDH, allegedly as a result of identity determination based on genomic sequence
analysis.This sort of discrepancy is common in the IMPDH/GMPR family gene, and has caused
confusions [1]. We have recently discovered a protein in the IMPDH/GMPR family from T.
congolense,which was later identified as an IMPDH [18]. Therefore, to fully understand the purine
biosynthesis pathway in T. congolense,further analysis was conducted to accurately identify the

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TcIL3000_5_1940 gene product as a possible GMPR candidate.
To date, it was assumed that IMPDHs and GMPRs were classified into different clades, with
only IMPDHs having CBS pair domain. However, GMPRs of Kinetoplastida contain a CBS pair
domain despite clearly exhibiting GMPR activity. These results suggest that GMPR is a polyphyletic
group, and Kinetoplastida GMPR evolved independently, separated from otherGMPRs in the earlier
evolutionary process.While it is understood that the CBS pair domain is not a core enzyme domain, it
may act as a regulatory domain to bind ATP, AMP,AdoMet,and so on [35]. Moreover, in a report on
Leishmania GMPR, CBS has been linked to biological responses ofGMPR at the intracellular level of
GTP and ATP, which subsequently controls the state of the enzyme complex to modulate its catalytic
activity [3].
Specific amino acid residues have actually been proposed as distinctive points between GMPR
and IMPDH at the primary structure level. One proposal was the consensus sequence Tyr303-Arg304
and Glu307 (Y-R-X-X-E) of GMPR (human GMPR2 numbering) [1]. This proposalwas derived from
the non-conserved region of IMPDHs at position 303-304 and a Glu to Gln substitution at position
307.. However, this might not be the case because Tyr303-Arg304 dyad is also non-conserved in the
reported L. donovani and T. brucei GMPR, while Glu431 (Tritrichomonas foetus IMPDH numbering)
is also present in several IMPDHs instead of Gln. Surprisingly, all GMPRs that were included in this
study are were conserved in the catalytic loop region in residue Leu217-Ser218 (Fig. S3). Due to the
fact that TcIL3000_5_1940 was also conserved in this position, we were confident that the gene was in
fact a GMPR-encoded gene in T. congolense (TcGMPR). The role of this conserved region for GMPR
activity might be necessary to investigate in further studies.
The kinetics parameters of rTcGMPR were found to be similar to those of TbGMPR. In
contrast, these values were significantly different from those of Bos taurus GMPR (BtGMPR), one of
the susceptible animals to T. congolense infection (Table 2) [2]. Calculated Km values against GMP and
NADPH suggested that rTcGMPR exerted up to 6-fold higher affinity for GMP and conversely 5-fold
lower affinity for NADPH, compared to BtGMPR. These differences implied two key takeaways: that
the development of a TcGMPR-targeting drug might also be useful as a TbGMPR-targeting drug, and
that an anti-trypanosomal drug for infected cattle or humans might be developed by targeting
trypanosome GMPRs, such as TbGMPR and TcGMPR.

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Currently, the discovery of novel chemotherapeutic agents against trypanosomosis has been
thwarted by limited attention and funding from major pharmaceutical companies [36,37].
Consequently, infections were treated with outdated drugs, most of which have been tolerated by some
protozoan strains.While GMPRs could be an attractive drug target, at present no potent GMPR
inhibitor has been proposed as having a broad therapeutic window and minimal adverse effects. Bessho
et al. suggested ribavirin as a possible chemotherapeutic compound or a lead compound for the
development of new GMPR inhibitors targeting the GMP-binding site of TbGMPR [2]. L. donovani
GMPR was also reported to be inhibited by IMP analogs [38,39]. Our current investigation revealed
that mycophenolic acid, a selective nicotinamide-binding site inhibitor of IMPDH, showed a moderate
inhibitory activity against rTcGMPR. Similar finding on GMPR inhibition by MPA was also reported
with Leishmania major GMPR, which exerted a Ki value of 20 μM [3]. However, MPA is also known
as a potent IMPDH inhibitor, with higher affinity to mammalian enzymes than to protozoan enzymes
[31]. In our previous publication, we showed that MPA has relatively potent inhibitory activity against
rTcIMPDH [17]. Therefore, it is expected that MPA potently inhibited growth of T. congolense in
cultures but showed less potent activity against rTcGMPR in vitro. By considering these facts,
appropriate derivatization of MPA and further drug designs are necessary for the development of anti-
infective agents for mammalian hosts [18,32,40]. Nevertheless, our findings re-highlight the possibility
of a new class of Trypanosomatidae GMPR inhibitor.
T. congolense GMPR was found to exhibit higher activity in the presence of larger radius
+
cations,especially K+ and NH4 . A similar activation pattern was also reported in IMPDHs, where the
presence of smaller ions such as Li+ and Na+ could cause a negative (inhibition), positive (activation),
+
or neutral effect (no effect) on enzyme activity, but larger ions (K+, Rb+, Cs+, Tl+, and NH4 ) always
activate the reaction [32,41,42]. Mammalian GMPRs on the other hand, showed no activation in the
+
presence of K+ and NH4 in comparison with Na+ [2]. This is the first report on a similar response
between GMPR and IMPDH to a broad range of cations.
GMPR could also catalyze a reverse reaction, the direct conversion of IMP to GMP. This
activity is directly dependent on the concentration of ammonium ion in the reaction [43]. The
activation of the deamination reaction, however, could be observed in a much lower ammonium
concentration than the progression of the aforementioned reverse reaction. While K+ has been
described as a molecular “lubricant” that facilitates the conformational changes in IMPDH reaction, the

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role of ammonium ion may be more complex, since it may play a very different role in the forward and
reverse reaction. Nevertheless, the ability of GMPR to catalyze the conversion between GMP and IMP
in both directions suggeststhat it might have an important role in the early development of
IMPDH/GMPR family, since the earliest forms of life are believed to have emerged in an ammonia rich
environment [44]. The opposite reaction by rTcGMPR was observed in an enzyme-concentration-
dependent manner. However, the reaction progressed very slowly even at high concentrations of
enzyme and substrates (Fig. S4). Trypanosome itself has been known as an ammonotelic organism, and
is able to tolerate relatively high ammonium concentrations [45–47]. Therefore, the protozoa might be
able to survive when there is a high concentration of ammonium ion in the blood and under repressed
activity of other enzymes, provided that TcGMPR can produce sufficient GMP for its survival [43].
TcGMPR was constitutively expressed and localized in cytosolas a granular localization during
the developmental stages.In addition to our experimental data, previous reports also suggested that
purine metabolic enzymes was localized in glycosomes of Kinetoplastida [7,48]. These results implied
that TcGMPR was also expressed in glycosomes, similar to otherpurine metabolic enzymes.
In conclusion, the present study revealed that TcIL3000_5_1940 is the first enzymatically
analyzed GMPR-encoded gene in T. congolense. Although rTcGMPR showed remarkably similar
kinetic parameters to TbGMPR, it differs significantly to mammalian GMPRs. In addition, Leu217-
Ser218 dyad was identified by the amino acid alignment as a practical distinction point to identify
GMPRs from IMPDHs. Lastly, a selective IMPDH inhibitor, mycophenolic acid, was found to inhibit
rTcGMPR activity, demonstrating its potential as a lead compound for the development of effective
chemotherapeutic agents against Trypanosoma infection.
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Fig. 1. Deamination reaction of GMP catalyzed by GMPR.
Fig. 2. Phylogenetic tree based on the GMPRs and IMPDHs amino acid sequence. The phylogenetic
tree was generated using the neighbor-joining method incorporated into the MEGA 5.2 software.

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Fig. 3. Amino acid alignment of TcGMPR and other GMPRs. The square indicates the predicted CBS
pair domain amino acid residues.The dashed square indicates the catalytic loop. The bold characters
indicate the C-terminal Ser-Lys-Leu peroxisomal (glycosomal) targeting signal. Star symbol indicates
the catalytic Cys204 residue (HsGMPR2 numbering). Diamond symbols indicate Leu217-Ser218, the
conserved dyad that differentiates a GMPR from IMPDHs. Protein sequence ofTcGMPR (TriTrypDB:
TcIL3000_5_1940); T. brucei GMPR [(TbGMPR) TriTrypdDB: Tb927.5.2080]; L. donovani GMPR
[(LdGMPR) Uniprot: E9BDA8]; B. taurus GMPR1 and 2 [(BtGMPR 1) Uniprot: AAI23864.1;
(BtGMPR2) NCBI: NP_001033208]; H. sapiens GMPR1 and 2 [(HsGMPR1) NCBI: NP_006868.3;
(HsGMPR2) NCBI: NP_057660.2] were aligned using ClustalW (http://clustalw.ddbj.nig.ac.jp).
Fig. 4. SDS-PAGE of recombinant TcGMPR. Protein expression in the E. coli culture was monitored
from the induction until purification steps. Lane M: protein marker, 1: E. coli culture before IPTG
addition, 2: E. coli culture 12 h after IPTG addition, 3: purified rTcGMPR fused with GST, and 4:
purified rTcGMPR with cleaved GST.
Fig. 5. Determination of optimum pH for rTcGMPR. Each rTcGMPR activity was plotted against the
corresponding value of experimental pH. The activity at pH 7.0 was set to 100% relative activity.
Acidic conditions were excluded from the experiment due to NADPH instability. Each data point
represents mean ± standard deviation from three independent experiments.
Fig. 6. Inhibitory effect of MPA against rTcGMPR. Each data point represents mean ± standard
deviation from three independent experiments.
Fig. 7. Effect of monovalent cation on rTcGMPR activity. rTcGMPR activity was evaluated in the
presence of each monovalent cation in the form of a chloride salt. The activity in the presence of NaCl
was set as 100%. Each bar represents mean ± standard deviation from three independent experiments .
Fig. 8. Expression profile and localization of TcGMPR. (A) Western blot analysis was performed with
five µg of total cell protein extracted from BSF, PCF, EMF and MCF using anti-TcGMPR and anti-
Tcα-tubulin antibody.(B) The graph showed the relative quantity (each stage/BSF) of TcGMPR in each

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stage. Each bar represents mean ± standard deviation from three independent experiments . (C) Indirect
immunofluorescence staining using anti-TcGMPR antibody was observed by confocal laser scanning
microscopy. Nucleolus and kinetoplast DNA were stained by Hoechest 33342 and are shown in red.
For the microscopy, each of the images was captured using the same photomultiplier tube gain and
voltage.
Fig. S1. IMPDH activity assay ofTcGMPR. Unlike human IMPDH (closed circles), TcIL3000_5_1940
(open circles) did not show any IMPDH activity. IMPDH activity assay was conducted with assay
solution containing 50 mM Tris-HCl pH 8.0, 100 mM KCl, 3 mM EDTA, 1 mM DTT, 0.1 mg/ml BSA,
800 μM NAD+, and 250 μM IMP. Reaction was initiated by adding enzyme solutions to the assay
solution.The reaction was carried out at 30ºC for 1 h. IMPDH activity was monitored as an increase of
absorbance in 340 nm, indicating formation of NADH. Each point was expressed as the amount of
NADH formed during reaction.
Fig. S2. Steady-state kinetic analysis of TcGMPR. (a) The Km NADPH was determined at a fixed GMP
concentration of 1.0 mM and varying concentration of NADPH. (b) The Km GMP was determined at a
fixed NADPH concentration of 200 μM and varying concentration of GMP. The initial velocity values
of the reactions were fitted to the Michaelis-Menten equation using GraFit 7 software (Erithacus
software, U.K.). Each point represents mean ± standard deviation from three independent experiments.
Fig. S3. Leu217-Ser218 as a distinctive residue dyad of GMPR. Fraction of active loops in GMPR and
IMPDH were aligned. Diamond symbols indicate Leu217-Ser218 (HsGMPR2 numbering), the residue
that differentiates GMPRs from IMPDHs. Star symbol indicates Cys204, an important catalytic residue
for GMPR and IMPDH.
Protein sequence was taken from NCBI database unless stated otherwise.Protein sequences for
H. sapiens (accession number of HsIMPDH1: NP_000874.2, HsIMPDH2: NP_000875.2, HsGMPR1:
NP_006868.3, HsGMPR2: NP_057660.2), B. taurus (BtGMPR1: AAI23864.1 [Uniprot], BtGMPR2:
NP_001033208), L. donovani (LdIMPDH: AAA29253.1 [Uniprot], LdGMPR: E9BDA8 [Uniprot]), T.
brucei (TbIMPDH: P50098.1 [Uniprot], TbGMPR: Tb927.5.2080 [TriTrypDB]), T. congolense
(TcIMPDH: BAT33662.1 [Uniprot], TcGMPR: TcIL3000_5_1940 [TriTrypDB]), B. gibsoni

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(BgIMPDH: AFL46337.1), P. falciparum (PfIMPDH: XP_001352079.1), T. gondii (TgIMPDH:
XP_002368170.1), T. foetus (TfIMPDH: AAB01581.1 [Uniprot]), C. parvum (CpIMPDH:
XP_625342.1), E. coli (EcIMPDH: NP_417003.1, EcGMPR: NP_414646.1), and S. pyrogenes
(SpIMPDH: NP_270113.1) were aligned using ClustalW program.
Fig. S4. Reverse reaction of TcGMPR. TcGMPR was able to convert IMP to GMP in the presence of
+
NH4 . The assay of GMPR reverse reaction activity was conducted with solution containing 75 mM
Tris-HCl pH 8.0, 100 mM KCl, 3 mM EDTA, 1 mM DTT, 1 mM NADP+, 50 mM NH4Cl, and various
IMP concentrations.Reaction was initiated by adding enzyme to the assay solution.The reaction was
carried out at 25ºC for 20 h. The reverse GMPR activity was monitored as an increase of absorbance in
340 nm, indicating formation of NADPH. Each point was expressed as the mean amount of NADPH
formed during reaction ± standard deviation from three independent experiments .

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Figure 7