Characterization of an indoleamine 2,3-dioxygenase-like protein found in humans and mice
Introduction
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 synthesis (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 acute depletion of tryptophan has effects on mood and aggression 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 enzyme whose activity can be induced by dietary tryptophan and steroid hormones (Schimke et al., 1965, Knox, 1966). Indoleamine 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 an immunomodulatory role in inducing tolerance by suppressing T lymphocyte proliferation via tryptophan depletion (Munn et al., 1999).
The downstream metabolites of the kynurenine pathway can have physiological or patho-physiological activities, and it is not always clear whether the induction of this pathway exerts its effects through tryptophan depletion, by production of kynurenine 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). Kynurenic acid acts as an antagonist for NMDA receptors whereas QA is an agonist and may be neuroexcitotoxic at physiological concentrations (Perkins and Stone, 1982, Schwarcz et al., 1983). Elevated levels of QA have been found in the cerebrospinal fluid of people with dementia due to acquired immune deficiency syndrome and cerebral malaria as well as the lesions associated with Alzheimer's disease (Heyes et al., 1989, Medana et al., 2002, Medana et al., 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 catalysing the same reaction, albeit in different cell types. For example, kynurenine aminotransferase catalyzes the formation of kynurenic 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 evolutionary relationship among a sample of INDO and INDOL1 sequences.
Section snippets
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 observed (at positions 86 and 107 in relation to the transcription start site), so primers were designed to amplify the corresponding gene
Mouse and human indoleamine 2,3-dioxygenase-like proteins
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-coding codon 21 base pairs upstream of the predicted start codon 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 the second methionine
Discussion
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-tryptophan, the
Acknowledgment
We would like to thank Sonia Cattley from ANGIS for bioinformatics support, Ghassan Maghzal for technical assistance with the HPLC analysis and Jane Radford for assistance with immunohistochemistry.
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