Characterization of an indoleamine 2,3-dioxygenase-like protein found in humans and mice.
ABSTRACT Indoleamine 2,3-dioxygenase (INDO) and tryptophan 2,3-dioxygenase (TDO) each catalyze the first step in the kynurenine pathway of tryptophan metabolism. We describe the discovery of another enzyme with this activity, indoleamine 2,3-dioxygenase-like protein (INDOL1), which is closely related to INDO and is expressed in mice and humans. The corresponding genes have a similar genomic structure and are situated adjacent to each other on human and mouse chromosome 8. They are likely to have arisen by gene duplication before the origin of the tetrapods. The expression of INDOL1 is highest in the mouse kidney, followed by epididymis, and liver. Expression of mouse INDOL1 was further localized to the tubular cells in the kidney and the spermatozoa. INDOL1 was assigned its name because of its structural similarity to INDO. We demonstrate that INDOL1 catalyses the conversion of tryptophan to kynurenine therefore a more appropriate nomenclature for the enzymes might be INDO-1 and INDO-2, or the more commonly-used abbreviations, IDO-1 and IDO-2. Although the two proteins have similar enzymatic activities, their different expression patterns within tissues and during malaria infection, suggests a distinct role for each protein. This identification of INDOL1 may help to explain the regulation of the diversity of physiological and patho-physiological processes in which the kynurenine pathway is involved.
- SourceAvailable from: Richard A Metz[Show abstract] [Hide abstract]
ABSTRACT: IDO2 is a relative of IDO1 implicated in tryptophan catabolism and immune modulation but its specific contributions to normal physiology and pathophysiology are not known. Evolutionary genetic studies suggest that IDO2 has a unique function ancestral to IDO1. In mice, IDO2 gene deletion does not appreciably affect embryonic development or hematopoiesis, but it leads to defects in allergic or autoimmune responses and in the ability of IDO1 to influence the generation of T regulatory cells. Gene expression studies indicate that IDO2 is a basally and more narrowly expressed gene than IDO1 and that IDO2 is uniquely regulated by AhR, which serves as a physiological receptor for the tryptophan catabolite kynurenine. In the established KRN transgenic mouse model of rheumatoid arthritis, where IDO1 gene deletion has no effect, IDO2 deletion selectively blunts responses to autoantigen but has no effect on responses to neoantigen challenge. In human populations, natural variations in IDO2 gene sequence that attenuate enzymatic activity have been reported to influence brain cancer control and adaptive immune responses to the IDO2 protein itself, consistent with the concept that IDO2 is involved in shaping immune tolerance in human beings. Biochemical and pharmacological studies provide further evidence of differences in IDO2 enzymology and function relative to IDO1. We suggest that IDO2 may act in a distinct manner from IDO1 as a set-point for tolerance to "altered-self" antigens along the self-non-self continuum where immune challenges from cancer and autoimmunity may arise.Frontiers in Immunology 11/2014; 5:585.
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ABSTRACT: Indoleamine 2,3-dioxygenases (IDOs) are tryptophan-catabolizing enzymes with immunomodulatory functions. However, the biological role of IDO2 and its relationship with IDO1 are unknown. To assess the relationship between IDO2 and IDO1, we investigated the effects of co-expression of human (h) IDO2 on hIDO1 activity. Cells co-expressing hIDO1 and hIDO2 showed reduced tryptophan metabolic activity compared with those expressing hIDO1 only. In a proteomic analysis, hIDO1-expressing cells exhibited enhanced expression of proteins related to the cell cycle and amino acid metabolism, and decreased expression of proteins related to cell survival. However, cells co-expressing hIDO1 and hIDO2 showed enhanced expression of negative regulators of cell apoptosis compared with those expressing hIDO1 only. Co-expression of hIDO1 and hIDO2 rescued the cell death induced by tryptophan-depletion through hIDO1 activity. Cells expressing only hIDO2 exhibited no marked differences in proteome profiles or cell growth compared with mock-transfectants. Cellular tryptophan metabolic activity and cell death were restored by co-expressing the hIDO2 mutant substituting the histidine 360 residue for alanine. These results demonstrate that hIDO2 plays a novel role as a negative regulator of hIDO1 by competing for heme-binding with hIDO1, and provide information useful for development of therapeutic strategies to control cancer and immunological disorders that target IDO molecules.Experimental and Molecular Medicine 11/2014; 46:e121. · 2.46 Impact Factor
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ABSTRACT: The kynurenine pathway (KP) is the principal route of L-tryptophan (TRP) catabolism leading to the production of kynurenine (KYN), the neuroprotectants, kynurenic acid (KYNA) and picolinic acid (PIC), the excitotoxin, quinolinic acid (QUIN) and the essential pyridine nucleotide, nicotinamide adenine dinucleotide (NAD+). The enzymes indoleamine 2,3-dioxygenase-1 (IDO-1), indoleamine 2,3-dioxygenase-2 (IDO-2) and tryptophan 2,3-dioxygenase (TDO-2) initiate the first step of the KP. IDO-1 and TDO-2 induction in tumors are crucial mechanisms implicated to play pivotal roles in suppressing anti-tumor immunity. Here, we report the first comprehensive characterisation of the KP in 1) cultured human glioma cells and 2) plasma from patients with glioblastoma (GBM). Our data revealed that interferon-gamma (IFN-γ) stimulation significantly potentiated the expression of the KP enzymes, IDO-1 IDO-2, kynureninase (KYNU), kynurenine hydroxylase (KMO) and significantly down-regulated 2-amino-3-carboxymuconate semialdehyde decarboxylase (ACMSD) and kynurenine aminotransferase-I (KAT-I) expression in cultured human glioma cells. This significantly increased KP activity but significantly lowered the KYNA/KYN neuroprotective ratio in human cultured glioma cells. KP activation (KYN/TRP) was significantly higher, whereas the concentrations of the neuroreactive KP metabolites TRP, KYNA, QUIN and PIC and the KYNA/KYN ratio were significantly lower in GBM patient plasma (n = 18) compared to controls. These results provide further evidence for the involvement of the KP in glioma pathophysiology and highlight a potential role of KP products as novel and highly attractive therapeutic targets to evaluate for the treatment of brain tumors, aimed at restoring anti-tumor immunity and reducing the capacity for malignant cells to produce NAD+, which is necessary for energy production and DNA repair.PLoS ONE 11/2014; 9(11):e112945. · 3.53 Impact Factor
Characterization of an indoleamine 2,3-dioxygenase-like protein
found in humans and mice
Helen J. Balla,⁎, Angeles Sanchez-Pereza, Silvia Weisera, Christopher J.D. Austina,
Florian Astelbauera, Jenny Miua, James A. McQuillana, Roland Stockera,
Lars S. Jermiinb,c,d, Nicholas H. Hunta
aDiscipline of Pathology and Bosch Institute, University of Sydney, NSW 2006, Australia
bSchool of Biological Sciences, University of Sydney, NSW 2006, Australia
cSydney Bioinformatics, University of Sydney, NSW 2006, Australia
dCentre for Mathematical Biology, University of Sydney, NSW 2006, Australia
Received 1 February 2007; received in revised form 27 March 2007; accepted 4 April 2007
Available online 18 April 2007
Received by A. Bernardi
Indoleamine 2,3-dioxygenase (INDO) and tryptophan 2,3-dioxygenase (TDO) each catalyze the first step in the kynurenine pathway of
tryptophan metabolism. We describe the discovery of another enzyme with this activity, indoleamine 2,3-dioxygenase-like protein (INDOL1),
which is closely related to INDO and is expressed in mice and humans. The corresponding genes have a similar genomic structure and are situated
adjacent to each other on human and mouse chromosome 8. They are likely to have arisen by gene duplication before the origin of the tetrapods.
The expression of INDOL1 is highest in the mouse kidney, followed by epididymis, and liver. Expression of mouse INDOL1 was further localized
to the tubular cells in the kidney and the spermatozoa. INDOL1 was assigned its name because of its structural similarity to INDO. We
demonstrate that INDOL1 catalyses the conversion of tryptophan to kynurenine therefore a more appropriate nomenclature for the enzymes might
be INDO-1 and INDO-2, or the more commonly-used abbreviations, IDO-1 and IDO-2. Although the two proteins have similar enzymatic
activities, their different expression patterns within tissues and during malaria infection, suggests a distinct role for each protein. This identification
of INDOL1 may help to explain the regulation of the diversity of physiological and patho-physiological processes in which the kynurenine
pathway is involved.
© 2007 Elsevier B.V. All rights reserved.
Keywords: Tryptophan catabolism; Gene duplication; Kynurenine pathway; Spermatozoa; Kidney tubule
Tryptophan is an essential amino acid that is required in
several physiological processes in addition to protein synthesis.
Dietary tryptophan enters the liver through the hepatic portal
system, where it has been demonstrated to induce protein syn-
thesis (Sidransky, 1976). Excess tryptophan can be delivered to
the bloodstream, where it can be taken up by tissues and used in
protein synthesis or in the synthesis of serotonin and melatonin.
The dietary intake of tryptophan correlates with the ratio of
tryptophan to large neutral amino acids in the plasma and with
brain serotonin levels (Fernstrom and Wurtman, 1972). Thus
through causing low serotonin levels (Young and Leyton, 2002).
An alternative fate for l-tryptophan is catabolism through the
kynurenine pathway. The first and rate-limiting step in the
pathway converts tryptophan to N-formylkynurenine, which is
quickly catabolized to kynurenine. In the liver this step is
catalyzed primarily by tryptophan 2,3-dioxygenase (TDO), an
Gene 396 (2007) 203–213
Abbreviations: Indoleamine 2,3-dioxygenase, INDO; indoleamine 2,3-
dioxygenase-like protein, INDOL1; tryptophan 2,3-dioxygenase, TDO; inter-
feron gamma, IFNγ; kynurenic acid, KA; quinolinic acid, QA; N-methyl-d-
aspartate, NMDA; heme oxygenase, Hmox.
⁎Corresponding author. Molecular Immunopathology Unit, Medical Foun-
dation Building, K25, 92-94 Parramatta Rd, University of Sydney, Camper-
down, 2006, Australia. Tel.: +61 2 90363238; fax: +61 2 90363286.
E-mail address: email@example.com (H.J. Ball).
0378-1119/$ - see front matter © 2007 Elsevier B.V. All rights reserved.
steroid hormones (Schimke et al., 1965; Knox, 1966). Indo-
leamine 2,3-dioxygenase (INDO) also performs this reaction
and is found throughout the body. It is highly expressed in
inflammatory states with interferon-gamma (IFNγ) playing a
key mediatory role in its expression (Takikawa et al., 1999).
IFNγ has anti-proliferative effects on mammalian and microbial
cells through induction of INDO expression and subsequent
tryptophan depletion in the microenvironment (Pfefferkorn,
1984; Takikawa et al., 1988). The expression of INDO also has
T lymphocyte proliferation via tryptophan depletion (Munn
et al., 1999).
The downstream metabolites of the kynurenine pathway can
havephysiologicalorpatho-physiological activities, anditisnot
always clear whether the induction of this pathway exerts its
effects through tryptophan depletion, by production of kynur-
enine and/or kynurenine-derived metabolites or a combination
of these two processes. For example, the proliferation of T
lymphocytes also is inhibited by downstream catabolites of the
kynurenine pathway (Frumento et al., 2002). Two of the major
metabolites formed by the pathway are kynurenic acid (KA) and
quinolinic acid (QA), both of which can bind to glutamate
receptors activated by N-methyl-d-aspartate (NMDA). Kynure-
nic acid acts as an antagonist for NMDA receptors whereas QA
is an agonist and may be neuroexcitotoxic at physiological
concentrations (PerkinsandStone,1982;Schwarcz etal.,1983).
Elevated levels of QA have been found in the cerebrospinal
fluid of people with dementia due to acquired immune defi-
ciency syndrome and cerebral malaria as well as the lesions
associated with Alzheimer's disease (Heyes et al., 1989; Me-
dana et al., 2002, 2003; Guillemin et al., 2005).
Although TDO and INDO have evolved to be functionally
similar, there is little structural similarity to suggest a common
ancestral gene. Interestingly, the ancestral gene of mammalian
INDO has a different role in molluscs, where it has myoglobin-
like activity (Suzuki and Takagi, 1992). Other steps of the
kynurenine pathway have structurally-similar isozymes catalys-
ing the same reaction, albeit in different cell types. For example,
kynurenine aminotransferase catalyzes the formation of kynure-
nic acid from kynurenine and three phylogenetically-conserved
genes encoding enzymes with this activity have been identified
(Yu et al., 2006). Here we report the existence of a paralog of the
INDO gene, called INDOL1 for indoleamine 2,3-dioxygenase-
like protein 1. We describe its sequence in mice and humans, the
distribution and activity of the mouse protein, and the evolu-
tionary relationship among a sample of INDO and INDOL1
2. Materials and methods
2.1. Mouse INDOL1 expression construct
A cDNA (accession number BC026393) predicted to encode
an INDO-like protein was cloned in a large-scale cDNA cloning
project (Strausberg et al., 2002). We obtained the clone from the
IMAGE consortium and used it as a template to amplify the
predicted amino acid sequence with Accuprime Pfx polymerase
(Invitrogen, CA, USA). Two potential start codons were ob-
served (at positions 86 and 107 in relation to the transcription
start site), so primers were designed to amplify the corres-
ponding gene products (mINDOL1a for ATG at position 86 and
mINDOL1bfor the ATG at position 107 in combination with the
reverse primer mINDOL1c). The coding sequences were ligated
into the pENTR vector (Invitrogen) using blunt-end ligation.
The cDNA clone obtained from the IMAGE consortium was
found to contain a 1-basepair (bp) mismatch at position 852
when compared with the genomic sequence and sequences we
amplified from mouse liver. This would result in an amino acid
substitution, so we substituted the 3’ end of the coding sequence
using a restriction enzyme site and a partial INDOL1 clone
derived from mouse liver. The inserts were transferred to the
pDEST-17 and pDEST 26 vectors using the LR recombinase kit
(Invitrogen). This allowed expression of the mouse INDOL1
protein in bacterial (pDEST-17) or mammalian (pDEST-26)
cells with an N-terminal 6 × Histidine tag. A cDNA encoding
the INDO protein was also amplified from mouse tissue
(mINDOa and mINDOb) and cloned into the same vectors to
compare INDOL1 expression and activity.
2.2. Cloning of human INDOL1 cDNA
RNA was extracted from human liver using Tri reagent
was primed with a gene-specific primer (hINDLO1d), and the
mouse INDOL1 amino acid sequence was aligned against a
translation in all six frames of human genomic DNA located
upstream of the human INDO gene. Primers were designed
based on the human sequence in areas appearing to be homo-
logous with the mouse INDOL1 sequence. Two overlapping
fragments of the human INDOL1 coding sequence were amp-
lified from human liver cDNA using Accuprime Pfx polymerase
and the primer combinations hINDOL1a/hINDOL1b and
hINDOL1c/hINDOL1d. Forty PCR cycles were performed at
95 °C for 15 s, 60 °C for 30 s and 72 °C for 90 s. PCR products
were phosphorylated with T4 polynucleotide kinase (Fermentas,
Canada), purified with a Qiaquick PCR purification kit (Qiagen,
Australia) and blunt-end ligated using the CloneSmart LCAmp
Elite Blunt Cloning Kit (Lucigen, WI, USA).
Primers used to amplify mRNA and cDNA from the human
and mouse genomes: mINDOa, CACCATGGCACTCAG-
TAAAATATCTC; mINDOb, CACTAAGGCCAACTCAGAA-
GAGCTT; mINDOc, AGATGAAGATGTGGGCTTTGCT;
mINDOd, GGCAGATTTCTAGCCACAAGGA; mINDOL1a,
CACCATGACGCTGGAGGTGC; mINDOL1c, GCTAAGCA-
CCAGGACACAGGAG; mINDOL1d, CAAAGTCAGAGCA-
hINDOL1a, CACCATGGAGCCCCACAGACC; hINDOL1b,
CCACGTGGGTGAAG; mRPL13aF, CTTAGGCACTGCTC-
204 H.J. Ball et al. / Gene 396 (2007) 203–213
The chromosomal location and genomic structure of the
INDOL1 genes from human and mouse were determined by
aligning the two cDNAs to their respective genomes in Gen-
2.3. Sequences used in phylogenetic study
The inferred INDOL1 amino acid sequences of human and
mouse were compared to 26 homologous amino acid sequences
(see table in supplemental online material) from human (Homo
sapiens), chimpanzee (Pan troglodytes), macaque (Macaca
mulatta), dog (Canis familiaris), cow (Bos taurus), mouse (Mus
musculus), rat (Rattus norvegicus), toad (Xenopus tropicalis),
chicken (Gallus gallus), sea urchin (Strongolycentrotus purpur-
and seven yeasts (Aspergillus fumigatus Af293, A. terreus
NIH2624, Candida albicans SC5314, Debaryomyces hansenii
CBS767, Yarrowia lipolytica CLIB122, Eremothecium gossypii
and Saccharomyces cerevisiae). Homologous sequences en-
coded by other relevant genomes (e.g., zebrafish and pufferfish)
were found, but they were incomplete or appeared to be an-
notated incorrectly (based on the alignment of mammalian
sequences, it was easy to see whether an annotation was plau-
sible). Of the sequences analysed, four were incomplete: the
INDOL1 gene products from chimpanzee, cow and chicken
lack the first ∼60 amino acids, and the INDOL1 gene product
from macaque is lacking ∼47 amino acids in a region cor-
responding to Exon 11 (the current annotation encodes a peptide
with little similarity with a highly conserved region of the
alignment, so the data from this exon were excluded).
A multiple sequence alignment at the amino acid level was
produced using MUSCLE (Edgar, 2004) and refined using
Seaview (Galtier et al., 1996) to remove perceived alignment
errors. The alignment is available as online material from http://
www.bio.usyd.edu.au/jermiin/Ball_2007.mase. A subset of
sites from the alignment was used in the phylogenetic analysis.
Sites with many gaps in the alignment were ignored because the
amino acids at such sites may be due to insertions and because
the gaps contribute both ambiguity and a computational burden.
Sites used for the phylogenetic analysis are underlined in the
2.4. Choosing an appropriate substitution model
Most phylogenetic methods are model-based and rely on
substitution models that assume that the sites evolved under
stationary, reversible and homogeneous conditions and that the
evolutionary processes are independent and identical across
sites (Jermiin et al., in press). We used two methods to deter-
mine under what conditions the sequences may have evolved.
Following Ababneh et al. (2006), we used a matched-pairs test
of symmetry to detect whether the sequences had evolved under
stationary, reversible and homogeneous conditions. Given the
results of these tests, we also used ProtTest (Abascal et al.,
2005) to select an appropriate substitution model for the phy-
logenetic analysis. The model was chosen using the Akaike
2.5. Phylogenetic analysis
Given the results from the previous section, we used PHYML
Version 2.4.4 (Guindon and Gascuel, 2003) to identify the most
trees. The analysis was done using the WAG substitution model
(Whelan and Goldman, 2001) with rate-heterogeneity across sites
modelled using a discrete Γ distribution with eight rate categories
and invariant sites (parameter values estimated from the data).
To account for topological model uncertainty during the
phylogenetic analysis (see Wolf et al., 2000), we used another
strategy. First, we used Tree-puzzle version 5.2 (Schmidt et al.,
2002) to obtain a set of optimal and near optimal trees — the set
j-option. Second, we estimated the likelihood of each of the 3273
trees thus obtained. Third, based on the Shimodaira–Hasegawa
test (Shimodaira and Hasegawa, 1999), we concluded that topo-
logical model uncertainty is present, so we obtained the expected
likelihood weight (Strimmer and Rambaut, 2002) for each tree
(provided by Tree-puzzle). Fourth, a likelihood-weighted consen-
sus tree was inferred on the basis of the 3273 trees, each with its
version 3.66 (Felsenstein, 2006). Fifth, the consensus tree was
compared using the Shimodaira–Hasegawa test to (i) the most
likely tree found, and (ii) a plausible tree in which the origin of
Strongylocentrotus purpuratus was changed to match the
perceived monophyletic origin of Deuterostomata. Finally, we
used the same test to compare two hypotheses on the origin of
INDO and INDOL1: an ancient gene duplication (a single gene
duplication,predating the divergenceoftetrapods,gaveriseto the
INDO and INDOL1 sequences); or recent gene duplications
(many gene duplications, postdating the divergence of tetrapods,
gave rise to the INDO and INDOL1 sequences). Trees were
illustrated using NJPLOT (Perriere and Gouy, 1996).
2.6. Expression of INDO proteins
and INDO−/−mice (Baban et al., 2004) (courtesy of A. Mellor,
Medical College of Georgia, USA) were housed at the Uni-
versity of Sydney with free access to food and water. All
procedures were performed in accordance with the guidelines of
the University of Sydney Animal Care and Ethics Committee.
Mice were infected with malaria by intraperitoneal (i.p.)
inoculation with 100 μl phosphate buffered saline containing
106parasitized red blood cells from a Plasmodium berghei
ANKA (PbA)-infected mouse (courtesy of G. Grau, University
of Sydney, Australia). Tissues were removed for RNA and
protein extraction and immunohistochemistry.
RNAwas extracted frommouse tissues using Tri reagent. One
INDOL1 mRNA (mINDOL1d and mINDOL1e primers) and a
reference gene RPL13a (mRPL13aF and mRPL13aR primers)
were measured by real-time PCR. This was performed in a two-
step PCR of 40 cycles with 95 °C for 15 s and 60 °C for 45 s
using a Rotorgene PCR machine (Corbett Research, Australia)
205H.J. Ball et al. / Gene 396 (2007) 203–213
and Platinum SYBR Green qPCR Supermix-UDG (Invitrogen).
Expression levels were measured via a standard curve.
Polyclonal antibodies directed against peptides derived from
the mouse INDO (peptide sequence CKP SKK KPT DGD) and
INDOL1 (CTR ARS RGL TNP SPH) were generated by im-
munizing rabbits, then collecting and affinity purifying the
antisera (performed by Genscript, NJ, USA). For protein ex-
pression studies, tissues were homogenised in lysis buffer of
50 mM Tris HCl (pH 7.5), 150 mM NaCl, 10% Glycerol, 1%
Triton X-100, 10 mM EDTA, complete protease inhibitor
(Roche, Switzerland) and, after centrifugation, the supernatants
were collected. The bicinchoninic acid protein assay (Pierce, IL,
USA) was used to determine protein concentration. 25 μg of
total cell lysate were separated by 10% SDS-PAGE and trans-
ferred to PVDF membrane (GE Healthcare, UK). All incuba-
tions were followed by three washes of PBS with 0.1% Tween
20. Non-specific binding was blocked with PBS, 5% Skim milk
and 0.1% Tween 20 for 1 h. The primary INDOL1 antibody
(0.5 ng/μl) was added overnight at 4°C. The secondary anti-
rabbit antibody linked to horseradish peroxidase was incubated
for 1 h, followed by detection with enhanced chemilumines-
cence (GE Healthcare) and expression measured by densitom-
HEK293Tcells, a similar method was used with the substitution
of a mouse monoclonal antibody against the Histidine tag (Cell
Signalling Technology, USA) and a secondary anti-mouse
antibody (GE Healthcare).
OCT medium (Tissue-Tek, CA, USA) and frozen in cooled
hexane. Sections were cut onto charged slides and fixed in
acetonefor 10min.After air-drying, thesections were incubated
in TNT (0.1 M Tris pH 7.5, 0.15 M NaCl, 0.01% Tween 20) for
10 min before blocking endogenous peroxidase activity by
rinsed in TNT, after which endogenous biotin expression was
blocked using a Biotin blocking kit (Dako, Denmark) according
to the manufacturer's instructions. Peptide blocking was per-
MA, USA). After 10 min peptide blocking, the solution was
with 0.3% blocking agent). Sections were incubated for 30 min
at room temperature then washed in TNT (three washes of 3 min
in TNT with 0.5% blocking agent and incubated with the
sections for 30 min. After washing, the sections were incubated
with streptavidin-conjugated horseradish peroxidase (ABC
peroxidase kit, Vectastain) 30 min and, following more washes,
staining was visualised by a four minute incubation with 3,3′-
diaminobenzidine (Dako, Denmark). The tissue was counter-
stained with hematoxylin, dehydrated through graded ethanols
and xylene, and coverslipped.
2.7. Activity of indoleamine 2,3-dioxygenase proteins
Flasks (75 cm2) of HEK293T cells were plated with 7.5 ×
106cells and the medium replaced the next day with 10 ml of
Opti-MEM I medium (Invitrogen) supplemented with 10% fetal
bovine serum (FBS). A suspension containing 12 μl of
Lipofectamine 2000 (Invitrogen) with the 10 μg of pDEST26
expression plasmid (Invitrogen) containing mouse INDO,
mouse INDOL1 or as a control, the Arabidopsis thaliana gene
for β-glucuronidase (GUS) (Invitrogen) in 1 ml Opti-MEM I
medium was added to each flask. The medium was replaced the
next day with DMEM medium supplemented with 10% FBS
and 200 μM L-tryptophan (final concentration estimated to be
280 μM). The cells were incubated for 7 h and the amount of
tryptophan and kynurenine present in the medium was mea-
sured using high-performance liquid chromatography (HPLC)
(Christen et al., 1994). Briefly, the cell culture supernatant was
deproteinized with the addition of trichloroacetic acid (4%) and
the resulting supernatant separated on an Agilent 1100 HPLC
system (Agilent, USA) equipped with a Hypersil 3u ODS C18
column (Phenomenex, USA) eluted with 100 mM chloroacetic
acid / 9% acetonitrile pH 2.4. Tryptophan and kynurenine were
detected by UVabsorbance at 280 and 364 nm respectively. The
enzyme activity was estimated according to the amount of
kynurenine detected adjusted for total protein concentration in
the cellular fraction and time.
3.1. Mouse and human indoleamine 2,3-dioxygenase-like
A cDNA (GenBank accession number BC026393), cloned
from mouse liver, was predicted to encode a protein (GenBank
accession number NP_666061) with high similarity to mouse
INDO. Our analysis of the cDNA suggested that a methionine-
was more likely to be the actual start of translation. It has a
Kozak consensus sequence and the first few amino acids are
similar to the human INDOL1 sequence, which does not have
in replacement of valine with alanine (when compared to the
from liver tissue: GenBank accession number EF137182). As
the INDOL1 clone was obtained from the FVB/N mouse strain
while the mouse genome and other INDOL1 cDNAs were
obtained from the C57Bl/6 strain, it is possible that this dif-
ference simply represents a single nucleotide polymorphism.
However, two partial cDNAs from the FVB/N strain deposited
in the GenBank database (accession numbers BI218956 and
BI221365) also match the C57Bl/6-derived sequence, suggest-
ing that the full-length clone contains a mutation. The valine at
this position is also well conserved in INDO and INDOL1
proteins from other species (see http://www.bio.usyd.edu.au/
jermiin/Ball_2007.mase for alignment). During the preparation
of this manuscript a predicted protein sequence (accession
number XP_988036) was deposited in GenBank that matches
exactly the mouse INDOL1 sequence we have determined.
The human ortholog of the mouse INDOL1 was cloned by
PCR amplification using primers based on human genomic
206 H.J. Ball et al. / Gene 396 (2007) 203–213
DNA that was similar to the mouse cDNA. Human INDOL1
contains 407 aminoacids, 73% of which are identical to those of
mouse INDOL1. The cDNA of human INDOL1 and its corres-
ponding amino acid sequence have been deposited in GenBank
(accession number EF052681). Another human cDNA with
some similarity to the mouse gene exists (GenBank accession
number NM_194294), but the sequence contains a retained
intron resulting in a predicted protein that does not closely
match mouse INDOL1, so the sequence was ignored in this
study. Several human INDOL1 sequences cloned as part of this
study contain a 1-base pair mismatch at position 969 when
compared to the human genomic sequence. There is a single
nucleotide polymorphism (A/C) at this location (GenBank ac-
cession number rs2981161) and it does not alter the gene pro-
duct. There is ∼43% similarity between both mouse and human
INDOL1 in relation to their respective INDO proteins whereas
the similarity between the two proteins and TDO is very low.
An alignment of the mouse and human INDO and INDOL1
proteins is shown in Fig. 1.
The genomic structure of human INDO has been determined
(Kadoya et al., 1992). We aligned the cDNA sequences of the
mouse INDO, human INDOL1 and mouse INDOL1 to the
corresponding genomic sequences to determine the genomic
structure. In both species the genes are less than 20 kilobases
apart. The lengths of the first and last exons differ somewhat but
the structure is otherwise identical (Fig. 2A). The fifth exon is
unusually small, encoding only five amino acids.
3.2. Phylogenetic analysis of INDO and INDO-like proteins
INDOhas beenclonedfroma varietyofspecies,rangingfrom
mammals to fungi (see table in supplemental online material for
GenBank accession numbers). Based on gene predictions and
evidence from expressed sequence tags (EST), it is clear that
INDO or INDOL1 also are present in other species. For example,
like gene product that acts as myoglobin rather than being
involved in tryptophan catabolism. In some species there is also
understand how these sequences are related, we conducted a
phylogenetic study on the basis of the aligned peptides.
The matched-pairs test of symmetry was used to determine
whether it could be assumed that the sequences had evolved
under stationary, reversible and homogeneous conditions. A
total of 10 tests (2.65% of the tests conducted) produced a
probability less than 5%, implying that there is little evidence
that these data have evolved under more complex conditions.
This, in turn, implied that the use of time-reversible Markov
models would be appropriate during the phylogenetic analysis.
A suitable substitution model was found using ProtTest.
Using the phylogenetic approach described above, at least
2382 optimal and near optimal trees (i.e., trees that do not differ
significantly at a 5% level of significance from the most likely
tree) were identified, implying that it would be necessary to
consider topological model uncertainty. Fig. 2B shows the
Fig. 1. An alignment of the mouse and human INDO and INDOL1 proteins. Identical residues are shaded in black and functionally similar residues are shaded in grey.
For clarity, when residues are identical in the mouse and human homologs but functionally different in the paralog amino acid sequences, the INDO identical residues
are shaded in black and the INDOL1 identical residues are underlined. The immunizing peptides used to make the antibodies for mouse INDO and INDOL1 are
marked with arrows.
207 H.J. Ball et al. / Gene 396 (2007) 203–213
consensus tree that resulted from using procedures that accounts
for topological model uncertainty. The tree is less likely than the
most likely tree (▵lnL = 4.1) but the difference is not significant
(p = 0.787). A similar tree with the lineage leading to the sea
urchin arising from the last common ancestor with chordates
alsodidnotdiffersignificantlyfromthemost likelytree (▵lnL=
5.0; p = 0.766), implying that a monophyletic origin of deutero-
stome species (here represented by the echinoderm and chordate
sequences) is within reason, given the data, and that the phy-
logeny of these sequences concurs with current thought on the
animal evolution. The yeast sequences form a distinct group that
is well separated from the animal sequences. Ignoring the
unexpected origin of the sea urchin in Fig. 2B, which is justified
given the statistical result (the unexpected origin of the sea
urchin may be an artefact caused by the limited number of sites
in the alignment), the animal sequences are separated into two
groups. The division of the animal sequences concurs with the
protostome–deutostome division of animals that is based on
early embryonic development. The phylogeny also shows that
the INDO and INDOL1 sequences of animals have evolved in a
similar manner following an ancient gene duplication that
(∼360 million years ago). An alternative scenario with gene
duplications occurring independently after the divergence of
mammalian lineages, which was found to be the most likely
cause of an abundance of type I interferon a genes in mouse and
humans (Hardy et al., 2004), was found tobe extremelyunlikely
(▵lnL = 2643.8; p = 0.000).
Fig. 2. A. A schematic representation of the gene structure of the mouse and human INDO and INDOL1 genes. The coding region is shown in the clear boxes and the
darker shaded regions represents the 3′ or 5′ untranslated regions (UTR). The extent of the UTR has not been determined for human INDOL1 and the 3′UTR is not
shown in its entirety for the other cDNA sequences. B. A phylogenetic tree illustrating the inferred evolutionary relationship of the INDO/INDOL1 family of proteins.
Different colours are used to emphasize different subsets of the family. The edges of the tree are drawn to scale (corresponding to the inferred amount of time over
which the evolution occurred — the scale bar corresponds to 0.2 substitutions per site). Relative support for each edge was obtained using differential weighting of the
optimal and near optimal trees — a value of 1.0 is good and a value of 0.0 is bad (for details, see Materials and methods).
208 H.J. Ball et al. / Gene 396 (2007) 203–213
3.3. Expression of mouse INDOL1
Expression of mouse INDOL1 was demonstrated by Western
Blot analysis on proteins extracted from mouse tissue.
Specificity of the polyclonal sera for INDOL1 was determined
by Western Blot analysis of lysates from E. coli transformed
with the pDEST17-mINDO or pDEST17-mINDOL1 (data not
shown). A specific band corresponding to the predicted size for
mouse INDOL1 (approximately 41 kDa) was observed in the
lysate of pDEST17-mINDOL1 transformed E. coli (Fig. 3A,
lane 3) and this band disappeared by competition with the
immunizing peptide (Fig. 3A, lane 6). A specific band, which
disappeared after competition with the immunizing peptide, was
also observed in protein extracted from kidney tissue Fig. 3A,
lanes 1 and 4). This protein had a slightly higher molecular
weight (approximately 45 kDa) compared to the bacterially-
expressed protein suggesting that INDOL1 may undergo post
translational modification in mammalian cells. The highest
levels of INDOL1 protein expression were observed in the
kidney, then epididymis, followed by the liver (Fig. 3A and B).
in the liver followed by kidney then testis and epididymis (data
not shown). The expression of INDOL1 protein and mRNAwas
not shown). Although expression levels of the INDOL1 tran-
(INDO−/−) mouse (Table 1), the levels of INDOL1 were main-
tained (Fig. 3B).
We previously have shown that INDO is induced in malaria-
infected mice (Sanni et al., 1998; Hansen et al., 2004), a disease
with high levels of circulating cytokines, including IFNγ, a key
regulator of INDO expression. In contrast, an analysis of
INDOL1 mRNA and protein expression during malaria in-
fection by quantitative PCR and Western Blotting shows no
changes in expression or tissue-specific down-regulation of
expression (Table 1; Fig. 3B). Although there is an apparent
increase in INDOL1 expression during Plasmodium berghei
ANKA (PbA) infection in the kidney (Fig. 3B), an analysis of a
larger number of mice found this induction of INDOL1 ex-
pression was variable and did not attain statistical significance
(data not shown). There is also a down-regulation of INDOL1
mRNA and protein levels during PbA infection that is specific
to the liver (Table 1; Fig. 3B). In addition, INDOL1 expression
is not dependent on the presence of IFNγ as IFNγ−/−mice
maintained expression of the INDOL1 protein (Fig. 3B).
Immunohistochemistry was performed on mouse tissues
(Fig. 4). The epididymis is known to contain high levels of
INDO expression and activity. Localization of INDO was in the
principal and apical cells of the caput epididymidis (Fig. 4B) in
accordance a previous study (Britan et al., 2006). In compa-
rison, INDOL1 was not detected in the cells of the epididymis
but in the spermatozoa, specifically in the tails (Fig. 4C). TDO
expression previously had been observed only in the heads of
spermatozoa (Britan et al., 2006). As might be expected, ex-
pression of INDOL1 is also observed in the spermatozoa in the
testis; however, INDO expression was undetectable in this
tissue (Fig. 4F and E, respectively). In the kidney, INDO ex-
pression was primarily localized to the blood vessels (Fig. 4H)
whilst INDOL1 was expressed only in the tubules (Fig. 4I).
3.4. Activity of mouse INDOL1
Preliminary studies on a bacterially-expressed INDOL1 and
INDO protein and lysates from transiently-transfected
HEK293T (human embryonic kidney) cells demonstrated that
INDOL1 had only 3–5% (average 3.9%) of the enzymatic
activity of INDO in the in vitro assay conditions (Takikawa
et al., 1988) developed for INDO (data not shown). Much
higher activity levels were observed when enzyme activity was
measured by supplying transiently-transfected HEK193T cells
with L-tryptophan and, after time had elapsed, measuring L-
tryptophan and kynurenine in the medium by HPLC (Table 2).
Fig. 3. Expression of mouse INDOL1 protein in mouse tissues. A. Polyclonal
serum generated against a peptide from the mouse INDOL1 sequence was use to
detect INDOL1 expression in mouse tissues. Lanes 1 and 4 contain protein from
the kidney. Lanes 2 and 5 have a molecular weight marker and lanes 3 and 6
contain lysate from E. coli transformed with the INDOL1 expression construct.
In lanes 4 and 6, the antibody was incubated with an excess of the immunizing
peptide before being added to the membrane. The amount of INDOL1 in the
epididymis (lane 8) is shown relative to the kidney expression (lane 7). B.
INDOL1 expression in C57BL/6, INDO−/−and PbA-infected C57BL/6 mice is
shown in the kidney (lanes 1, 2 and 3, respectively) and liver (lanes 4, 5 and 6,
respectively). The expression of INDOL1 in IFNg−/−mice was detected in the
kidney, epididymis and liver (lanes 7, 8 and 9) respectively. Representative
results are shown from a total of 3–6 mice/strain or PbA infection. C.
Expression of INDOL1 (lanes 2 and 3) and INDO (lanes 4 and 5) proteins in
lysates of HEK293T cells transfected with expression plasmids encoding
6×histidine-tagged INDOL1 and INDO proteins. Before lysis, medium was
removed for measurement of kynurenine to determine enzyme activity (see
Table 2). The Western Blot analysis was performed with an antibody specific for
the Histidine tag and a molecular weight marker is in lane 1.
Expression of INDO and INDOL1 mRNA
Expression of mRNA relative to uninfected C57BL/6 mice
Expression of INDO and INDOL1 mRNA (mean±SEM) in INDO−/−mice or
PbA-infected C57BL/6 mice relative to uninfected C57BL/6 mice was
determined by real-time reverse transcriptase polymerase chain reaction
(normalized to reference gene expression). Statistical analysis was performed
using the Mann–Whitney test.
⁎Denotes expression significantly different to uninfected mice (pb0.05) and
ND indicates values not determined.
209 H.J. Ball et al. / Gene 396 (2007) 203–213
Western Blot analysis of cell lysates with an antibody directed
against the N-terminal histidine tag of both proteins indicated
the enzymes were expressed at comparable levels (Fig. 3C). To
accurately measure enzyme activity requires quantifying the
amount of enzyme added to an in vitro assay. Whilst we were
able to purify bacterially-expressed INDOL1 protein, we were
unable to determine appropriate in vitro assay conditions for
enzymatic activity. Therefore, although INDOL1 can convert L-
tryptophan to kynurenine, its exact activity relative to INDO is
difficult to determine at present. We also tested the activity of
the alternative INDOL1 expression construct that utilises the
start codon further downstream and found it had reduced
activity when transfected into HEK293T cells compared with
the longer protein, suggesting the longer protein is the correct
sequence (data not shown).
The identification of a third enzyme with the capacity to
metabolise tryptophan along the kynurenine pathway adds a
new dimension to the understanding of how this pathway is
regulated under normal and abnormal physiological conditions.
INDO and TDO are two phylogenetically-unrelated genes that
have evolved to have a similar function (i.e. catalysing the first
step in tryptophan catabolism). Although there are differences
in enzyme activity in regard to substrates other than L-trypto-
phan, the major difference between INDO and TDO appears to
be the expression patterns of the two enzymes. We report the
existence of an INDO-like protein that is encoded by a gene
adjacent to the INDO gene. The two genes most likely arose
through gene duplication that occurred before the origin of
tetrapods. Preliminary results demonstrate that the INDOL1
protein has INDO activity but it is currently unclear whether it
has additional functions (e.g. whether it can act as an oxygen
carrier similar to myoglobin or utilize other substrates).
This finding suggests that INDO is similar to many other
heme enzymes in that there is more than one isozyme catalysing
Fig. 4. CellularlocalizationofINDOandINDOL1proteins.ImmunohistochemistrywithantiseraraisedagainstINDO(B,EandH)andINDOL1(C,FandI)peptideswas
used to localize the proteins in the mouse epididymis (A, B and C), testis (D, E and F) and kidney (G, H and I). Rabbit IgG was used as an isotype control (A, D and G).
Enzymatic activity of INDO and INDOL1
Enzyme activity nmoles kynurenine/h/mg protein
Amount of kynurenine, adjusted for total cellular protein, detected in the
medium of HEK293T cells transfected with either the INDO or INDOL1
expression plasmid (mean±SEM, n=4). In cells transfected with the control
GUS expression plasmid, the amount of kynurenine was less than 1% of that
detected in either the INDO or INDOL1-transfected cells.
210H.J. Ball et al. / Gene 396 (2007) 203–213
the same reaction. For example, there are multiple isozymes of
heme oxygenase (HMOX), cyclooxygenase and nitric oxide
synthase. There is a high degree of similarity among amino acid
sequences from these enzyme families (e.g. greater than 50%
similarity between the nitric oxide synthase proteins), suggest-
ing that they each had a common ancestral gene. Unlike the
INDO and INDOL1 genes, however, the genes encoding other
heme isozymes are on different chromosomes (e.g., Hmox1 on
22q13.1 and Hmox2 on 16p13.3). The proximity of the INDO
and INDOL1 genes potentially could result in their co-ordinated
regulation. Consistent with this notion, in an INDO gene
knockout mouse strain (Baban et al., 2004) we found significant
down-regulation of the INDOL1 transcript in some tissues. This
could potentially be due to a deletion of part of the INDO gene
disrupting regulatory elements of the adjacent INDOL1 gene.
A duplication of an enzyme-encoding gene may lead to
situations where: (i) a particular reaction can be completed
faster (because the enzyme is present at a higher concentration),
(ii) the two gene products evolve towards being expressed
differently (e.g., expressed in different cells or at different
times), or (iii) one of the duplicated genes may evolve towards
another function. The phylogeny presented in Fig. 2B provides
the framework for examining how the structures and functions
of the INDO/INDOL1 gene family have changed. Given that
the INDO/INDOL1 gene duplication took place ∼360 million
years ago, the INDO and INDOL1 genes have had ample of
time to evolve towards encoding different functions and/or to be
differentially expressed. One of the first mechanisms that may
play a role during the generation of functional diversity is the
emergence of different expression patterns of the proteins. TDO
is seen as the enzyme responsible for tryptophan metabolism in
the liver, but it is also expressed in the brain and epididymis
(Haber et al., 1993; Britan et al., 2006). INDO is expressed in
the intestines, kidney and reproductive system, including the
epididymis, and is also induced in many tissues in response to
stimuli such as inflammatory mediators. The overlapping ex-
pression of INDOL1 with the other two enzymes in the kidney,
epididymis and liver might be taken to indicate some redun-
dancy, but the different cellular localisation of the enzymes also
suggests a distinct role for each of the enzymes.
In the epididymis INDO protein was localized to the prin-
cipal and apical cells of the caput epididymidis whereas the
TDO protein was detected in smooth muscle cells of the epi-
didymal duct and on the head of spermatozoa (Britan et al.,
2006). In this study, we observed INDOL1 immunoreactivity in
the tail of the spermatozoa. The different patterns of expression
suggest that there is no functional redundancy amongst the three
tryptophan-catabolizing enzymes in the epididymis, but the role
of each enzyme remains to be defined. Tryptophan metabolism
is possibly involved in several processes in this tissue. The
epididymis has a local serotonin synthesis that regulates sexual
maturation (Jimenez-Trejo et al., 2007). Tryptophan depletion is
involved in immune regulation, allowing implantation of the
embryo but also may be involved earlier in conception and
fertilisation, and it is possible that the TDO and INDOL1
expression are involved in this process (Gutierrez et al., 2003).
Interestingly, L-tryptophan functions as a chemo-attractant for
spermatozoa in some species of abalone (Riffell et al., 2002).
The epididymis is also suggested to be a tissue where high
levels of reactive oxygen species might contribute to DNA
damage. Therefore, indoleamine 2,3-dioxygenase activity may
offer protection, by recycling superoxide as its cofactor (Britan
et al., 2006).
INDO enzyme activity has been measured in the kidney in a
number of animals (Allegri et al., 2003), although its locali-
zation within the normal kidney is unclear. We found murine
INDO protein to be localized primarily to the renal vasculature.
This is in accordance with a previous study performed on
tissues from mouse infected with malaria (Hansen et al., 2004).
This study showed a large and systemic induction of INDO in
the vasculature during malaria infection. The kidney differs
from most other tissues in that it also has detectable INDO
expression in the microvasculature of uninfected mice. In con-
trast, we observed that INDOL1 is localized to the kidney
tubules. The kidney tubules express transporters for neutral
amino acids and rapidly reabsorb L-tryptophan (Chan and
Huang, 1971). One of these transporters is mutated in Hartnup
disease, a disorder of amino acid absorption in the intestine and
reabsorption in the kidney tubules resulting the loss of tryp-
tophan and other amino acids in the urine (Broer et al., 2004).
The role that INDOL1 activity might play in the kidney is
unclear. It is possible that it protects the kidney tubules from
damage caused by oxidative stress via recycling superoxide
anion. Alternatively, along with TDO expression in the liver,
INDOL1 activity may regulate plasma L-tryptophan levels by
catabolizing excess L-tryptophan reabsorbed by the tubules. The
concentration of amino acids within kidney tubules and L-tryp-
tophan in the epididymis is higher than in the plasma (Hinton,
1990; Silbernagl, 1988) so it is possible that INDOL1 ex-
pression, like TDO expression, can be regulated by substrate
INDO, in most tissues, has low basal levels of expression but
expression can be induced, with IFNγ being a key regulator
(Takikawa et al., 1999). In malaria infection, a disease with high
circulating levels of IFNγ, INDO expression is induced in the
endothelium of every tissue studied (Hansen et al., 2004). In
contrast, we see little induction of INDOL1 mRNA expression
in malaria infection and, even, down-regulation in the liver. The
levels of INDOL1 are maintained in IFNγ−/−mice further
suggesting the cytokine is not a major regulator of INDOL1
expression. This suggests that INDO may be the enzyme more
likely to be involved catalysing tryptophan catabolism during
inflammatory processes. Similarities exist with other heme-
containing enzymes where multiple isozymes are expressed.
The expression of some of the isozymes, such as heme oxy-
genase 1, is highly induced in pathological processes, whereas
another isozyme that catalyzes the same reaction, heme oxy-
genase 2, has a more stable pattern of expression and is thought
to play a role in homeostasis (Ewing and Maines, 1991).
We show that expression of INDOL1 increases the con-
version of L-tryptophan to kynurenine in transfected HEK293T
cells. The HUGO committee on gene nomenclature assigned
the name INDOL1 (for indoleamine 2,3-dioxygenase-like 1
protein) to the gene adjacent to INDO based on its sequence
211H.J. Ball et al. / Gene 396 (2007) 203–213
similarity to INDO with no functional information available. In
light of the INDOL1 protein having tryptophan-catabolizing
activity, a more appropriate nomenclature might be INDO1 for
INDO and INDO2 for INDOL1 (or with the abbreviation more
commonly used in the literature, IDO1 and IDO2). We have
used the current HUGO nomenclature for this report but re-
commend adoption of the name change in the future by the
Gene duplication and subsequent accumulation of substitu-
tions can lead to different functions of the gene products. In
molluscs, INDO-like genes have evolved to produce a protein
with a myoglobin-like activity rather than tryptophan-catabo-
lizing activity. We have not investigated whether INDOL1 has
functions that are additional to or alternative to tryptophan
metabolism. The kynurenine pathway is important in numerous
physiological and patho-physiological processes, and the way
that this diversity might be achieved and regulated has not been
clear to date. The discovery of a second indoleamine 2,3-
dioxygenase isozyme may explain this diversity and reveal new
roles of indoleamine 2,3-dioxygenase enzyme activity.
We would like to thank Sonia Cattley from ANGIS for
bioinformatics support, Ghassan Maghzal for technical assis-
tance with the HPLC analysis and Jane Radford for assistance
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