3-Hydroxykynurenine oxidizes alpha-crystallin: potential role in cataractogenesis

The alpha-, beta-, and gamma-crystallins are the major structural proteins of mammalian lenses. The human lens also contains tryptophan-derived UV filters, which are known to spontaneously deaminate at physiological pH and covalently attach to lens proteins. 3-Hydroxykynurenine (3OHKyn) is the third most abundant of the kynurenine UV filters in the lens, and previous studies have shown this compound to be unstable and to be oxidized under physiological conditions, producing H2O2. In this study, we show that methionine and tryptophan amino acid residues are oxidized when bovine alpha-crystallin is incubated with 3-hydroxykynurenine. We observed almost complete oxidation of methionines 1 and 138 in alphaA-crystallin and a similar extent of oxidation of methionines 1 and 68 in alphaB-crystallin after 48 h. Tryptophans 9 and 60 in alphaB-crystallin were oxidized to a lesser extent. AlphaA-crystallin was also found to have 3OHKyn bound to its single cysteine residue. Examination of normal aged human lenses revealed no evidence of oxidation of alpha-crystallin; however, oxidation was detected at methionine 1 in both alphaA- and alphaB-crystallin from human cataractous lenses. Age-related nuclear cataract is associated with coloration and insolubilization of lens proteins and extensive oxidation of cysteine and methionine residues. Our findings demonstrate that 3-hydroxykynurenine can readily catalyze the oxidation of methionine residues in both alphaB- and alphaA-crystallin, and it has been reported that alpha-crystallin modified in this way is a poorer chaperone. Thus, 3-hydroxykynurenine promotes the oxidation and modification of crystallins and may contribute to oxidative stress in the human lens.     

Protein-bound kynurenine is a photosensitizer of oxidative damage

Human lens proteins become progressively modified by tryptophan-derived UV filter compounds in an age-dependent manner. One of these compounds, kynurenine, undergoes deamination at physiological pH, and the product binds covalently to nucleophilic residues in proteins via a Michael addition. Here we demonstrate that after covalent attachment of kynurenine, lens proteins become susceptible to photo-oxidation by wavelengths of light that penetrate the cornea. H2O2 and protein-bound peroxides were found to accumulate in a time-dependent manner after exposure to UV light (lambda > 305-385 nm), with shorter-wavelength light giving more peroxides. Peroxide formation was accompanied by increases in the levels of the protein-bound tyrosine oxidation products dityrosine and 3,4-dihydroxyphenylalanine, species known to be elevated in human cataract lens proteins. Experiments using D2O, which enhances the lifetime of singlet oxygen, and azide, a potent scavenger of this species, are consistent with oxidation being mediated by singlet oxygen. These findings provide a mechanistic explanation for UV light-mediated protein oxidation in cataract lenses, and also rationalize the occurrence of age-related cataract in the nuclear region of the lens, as modification of lens proteins by UV filters occurs primarily in this region.     

Tryptophan-derived ultraviolet filter compounds covalently bound to lens proteins are photosensitizers of oxidative damage

The human eye is chronically exposed to light of wavelengths >300 nm. In the young human lens, light of wavelength 300-400 nm is predominantly absorbed by the free Trp derivatives kynurenine (Kyn), 3-hydroxykynurenine (3OHKyn), and 3-hydroxykynurenine-O-beta-D-glucoside (3OHKynG). These ultraviolet (UV) filter compounds are poor photosensitizers. With age, the levels of the free UV filters in the lens decreases and those of protein-bound UV filters increases. The photochemical behavior of these protein-bound UV filters and their role in UV damage are poorly elucidated and are examined here. UVA illumination of protein-bound UV filters generated peroxides (principally H2O2) in a metabolite-, photolysis-time-, and wavelength-dependent manner. Unmodified proteins, free Trp metabolites, and Trp metabolites that do not bind to lens proteins gave low peroxide yields. Protein-bound 3OHKyn (principally at Cys residues) yielded more peroxide than comparable Kyn and 3OHKynG adducts. Studies using D2O and sodium azide implicated 1O2 as a key intermediate. Illumination of the protein-bound adducts also yielded protein-bound Tyr oxidation products (DOPA, di-tyrosine) and protein cross-links via alternative mechanisms. These data indicate that the covalent modification of lens proteins by Kyn derivatives yields photosensitizers that may enhance oxidation in older lenses and contribute to age-related nuclear cataract.     

Identifying sites of attachment of UV filters to proteins in older human lenses

Recent results indicate that covalent modification of proteins by tryptophan-derived UV filters may explain the age-dependent coloration of human lenses, and play a role in age-related cataract. The sites of attachment of the UV filters to the lens crystallins, however, have not been determined. This study utilized a database of predicted masses of UV filter-modified tryptic peptides to target sites of UV filter attachment. Proteins were isolated from old normal lenses and digested with trypsin at pH 6, in order to preserve the integrity of the sites of modification. Peptides were separated by high-performance liquid chromatography and characterized by mass spectrometry. Major colored and fluorescent peaks in the digest were found to correspond to cysteine-containing peptides in which the sulfur atom of the sidechain was linked to the major UV filter compound, 3-hydroxykynurenine glucoside. Three of the peptides originated from gammaS-crystallin and one from betaB1-crystallin. These results show that a predicted mass database can be used to facilitate the identification of sites of UV filter modification in human lens crystallins. Furthermore, this work represents the first evidence that UV filters bind to specific residues on lens proteins in vivo, and suggests that sulfhydryl groups may be important sites for the attachment of UV filters.     

3-hydroxykynurenine-mediated modification of human lens proteins: structure determination of a major modification using a monoclonal antibody

Tryptophan can be oxidized in the eye lens by both enzymatic and non-enzymatic mechanisms. Oxidation products, such as kynurenines, react with proteins to form yellow-brown pigments and cause covalent cross-linking. We generated a monoclonal antibody against 3-hydroxykynurenine (3OHKYN)-modified keyhole limpet hemocyanin and characterized it using 3OHKYN-modified amino acids and proteins. This monoclonal antibody reacted with 3OHKYN-modified N(alpha)-acetyl lysine, N(alpha)-acetyl histidine, N(alpha)-acetyl arginine, and N(alpha)-acetyl cysteine. Among the several tryptophan oxidation products tested, 3OHKYN produced the highest concentration of antigen when reacted with human lens proteins. A major antigen from the reaction of 3OHKYN and N(alpha)-acetyl lysine was purified by reversed phase high pressure liquid chromatography, which was characterized by spectroscopy and identified as 2-amino-3-hydroxyl-alpha-((5S)-5-acetamino-5-carboxypentyl amino)-gamma-oxo-benzene butanoic acid. Enzyme-digested cataractous lens proteins displayed 3OHKYN-derived modifications. Immunohistochemistry revealed 3OHKYN modifications in proteins associated with the lens fiber cell plasma membrane. The low molecular products (<10,000 Da) isolated from normal lenses after reaction with glucosidase followed by incubation with proteins generated 3OHKYN-derived products. Human lens epithelial cells incubated with 3OHKYN showed intense immunoreactivity. We also investigated the effect of glycation on tryptophan oxidation and kynurenine-mediated modification of lens proteins. The results showed that glycation products failed to oxidize tryptophan or generate kynurenine modifications in proteins. Our studies indicate that 3OHKYN modifies lens proteins independent of glycation to form products that may contribute to protein aggregation and browning during cataract formation.     

Non-oxidative modification of lens crystallins by kynurenine: a novel post-translational protein modification with possible relevance to ageing and cataract

In humans, the crystallin proteins of the ocular lens become yellow-coloured and fluorescent with ageing. With the development of senile nuclear cataract, the crystallins become brown and additional fluorophores are formed. The mechanism underlying crystallin colouration is not known but may involve interaction with kynurenine-derived UV filter compounds. We have recently identified a sulphur-linked glutathionyl-3-hydroxykynurenine glucoside adduct in the lens and speculated that kynurenine may also form adducts with GSH and possibly with nucleophilic amino acids of the crystallins (e.g. Cys). Here we show that kynurenine modifies calf lens crystallins non-oxidatively to yield coloured (365 nm absorbing), fluorescent (Ex 380 nm/Em 450-490 nm) protein adducts. Carboxymethylation and succinylation of crystallins inhibited kynurenine-mediated modification by approx. 90%, suggesting that Cys, Lys and possibly His residues may be involved. This was confirmed by showing that kynurenine formed adducts with GSH as well as with poly-His and poly-Lys. NMR studies revealed that the novel poly-Lys-kynurenine covalent linkage was via the epsilon-amino group of the Lys side chain and the betaC of the kynurenine side chain. Analysis of tryptic peptides of kynurenine-modified crystallins revealed that all of the coloured peptides contained either His, Cys or an internal Lys residue. We propose a novel mechanism of kynurenine-mediated crystallin modification which does not require UV light or oxidative conditions as catalysts. Rather, we suggest that the side chain of kynurenine-derived lens UV filters becomes deaminated to yield an alpha,beta-unsaturated carbonyl which is highly susceptible to attack by nucleophilic amino acid residues of the crystallins. The inability of the lens fibre cells to metabolise their constituent proteins results in the accumulation of coloured/fluorescent crystallins with age.     

Mechanism of insolubilization by a single-point mutation in alphaA-crystallin linked with hereditary human cataracts

AlphaA-crystallin is a small heat shock protein that functions as a molecular chaperone and a lens structural protein. The R49C single-point mutation in alphaA-crystallin causes hereditary human cataracts. We have previously investigated the in vivo properties of this mutant in a gene knock-in mouse model. Remarkably, homozygous mice carrying the alphaA-R49C mutant exhibit nearly complete lens opacity concurrent with small lenses and small eyes. Here we have investigated the 90 degrees light scattering, viscosity, refractive index, and bis-ANS fluorescence of lens proteins isolated from the alphaA-R49C mouse lenses and found that the concentration of total water-soluble proteins showed a pronounced decrease in alphaA-R49C homozygous lenses. Light scattering measurements on proteins separated by gel permeation chromatography showed a small amount of high-molecular mass aggregated material in the void volume which still remains soluble in alphaA-R49C homozygous lens homogenates. An increased level of binding of beta- and gamma-crystallin to the alpha-crystallin fraction was observed in alphaA-R49C heterozygous and homozygous lenses but not in wild-type lenses. Quantitative analysis with the hydrophobic fluorescence probe bis-ANS showed a pronounced increase in fluorescence yield upon binding to alpha-crystallin from mutant as compared with the wild-type lenses. These results suggest that the decrease in the solubility of the alphaA-R49C mutant protein was due to an increase in its hydrophobicity and supra-aggregation of alphaA-crystallin that leads to cataract formation. Our study further shows that analysis of mutant proteins from the mouse model is an effective way to understand the mechanism of protein insolubilization in hereditary cataracts.     

UVA Light-excited Kynurenines Oxidize Ascorbate and Modify Lens Proteins through the Formation of Advanced Glycation End Products: IMPLICATIONS FOR HUMAN LENS AGING AND CATARACT FORMATION*

Advanced glycation end products (AGEs) contribute to lens protein pigmentation and cross-linking during aging and cataract formation. In vitro experiments have shown that ascorbate (ASC) oxidation products can form AGEs in proteins. However, the mechanisms of ASC oxidation and AGE formation in the human lens are poorly understood. Kynurenines are tryptophan oxidation products produced from the indoleamine 2,3-dioxygenase (IDO)-mediated kynurenine pathway and are present in the human lens. This study investigated the ability of UVA light-excited kynurenines to photooxidize ASC and to form AGEs in lens proteins. UVA light-excited kynurenines in both free and protein-bound forms rapidly oxidized ASC, and such oxidation occurred even in the absence of oxygen. High levels of GSH inhibited but did not completely block ASC oxidation. Upon UVA irradiation, pigmented proteins from human cataractous lenses also oxidized ASC. When exposed to UVA light (320–400 nm, 100 milliwatts/cm², 45 min to 2 h), young human lenses (20–36 years), which contain high levels of free kynurenines, lost a significant portion of their ASC content and accumulated AGEs. A similar formation of AGEs was observed in UVA-irradiated lenses from human IDO/human sodium-dependent vitamin C transporter-2 mice, which contain high levels of kynurenines and ASC. Our data suggest that kynurenine-mediated ASC oxidation followed by AGE formation may be an important mechanism for lens aging and the development of senile cataracts in humans.     

Identification of the new UV filter compound cysteine-L-3-hydroxykynurenine O-beta-d-glucoside in human lenses

UV filters protect the human lens and retina from UV light-induced damage. Here, we report the identification of a new UV filter, cysteine-l-3-hydroxykynurenine O-beta-d-glucoside, which is present in older normal human lenses. Its structure was confirmed by independent synthesis. It is likely this novel UV filter is formed in the lens by nucleophilic attack of cysteine on the unsaturated ketone derived from deamination of 3-hydroxykynurenine O-beta-d-glucoside. Quantitation studies revealed considerable variation in normal lens levels that may be traced to the marked instability of the cysteine adduct. The novel UV filter was not detected in advanced nuclear cataract lenses.     

Rate of formation of AGEs during ascorbate glycation and during aging in human lens tissue

The similarity of the yellow chromophores isolated from human cataracts with those from ascorbic acid modified calf lens proteins was recently published [Biochim. Biophys. Acta 1537 (2001) 14]. The data presented here additionally quantify age-dependent increases in individual yellow chromophores and fluorophores in the water-insoluble fraction of normal human lens. The water-insoluble fraction of individual normal human lens was isolated, solubilized by sonication and digested with a battery of proteolytic enzymes under argon to prevent oxidation. The level of A(330)-absorbing yellow chromophores, 350/450 nm fluorophores and total water-insoluble (WI) protein were quantified in each lens. The total yellow chromophores and fluorophores accumulated in parallel with the increase in the water-insoluble protein fraction during aging. The digest from each single human lens was then subjected to Bio-Gel P-2 size-exclusion chromatography. The fractions obtained were further separated by a semi-preparative prodigy C-18 high-performance liquid chromatography (RP-HPLC). Bio-Gel P-2 chromatography showed four major fractions, each of which increased with age. RP-HPLC of the amino acid peak resolved five major A(330)-absorbing peaks and eight fluorescent peaks, and each peak increased coordinately with age. A late-eluting peak, which contained hydrophobic amino acids increased significantly after age 60.Aliquots from an in vitro glycation of calf lens proteins by ascorbic acid were removed and subjected to the same enzymatic digestion. Ascorbic acid-modified calf lens protein digests showed an almost identical profile of chromophores, which also increased in a time-dependent manner. The late-eluting peak, however, did not increase with the time of glycation and may not be an advanced glycation endproduct (AGE) product. The data indicate that the total water-insoluble proteins, individual yellow chromophores and fluorophores increased equally both with aging in normal human lens and during ascorbate glycation in vitro. The major protein modifications, which accumulate during aging, therefore, appear to be AGEs. Whereas the late-eluting peak, which showed poor correlation to ascorbylation, may represent UV filter compounds bound to lens proteins.     

Oxygen radical induced fluorescence in proteins; identification of the fluorescent tryptophan metabolite,N-formyl kynurenine,as a biological index of radical damage

Summary The effect of oxygen derived free radicals (OFR) on aromatic and sulphur containing amino acids has been investigated, both in their free form and within protein backbones. Aerated amino acids and proteins in solution were exposed to three discrete OFR generating systems; (1) gamma radiation in the presence or absence of formate (2) photolysis by UV light at 254 and 366 nm, and (3) site specific modification by H₂O₂ in the presence of CuII ions.A sensitive reverse-phase HPLC technique with dual detection systems (UV absorbance and fluorescence monitoring) was developed to analyse the products of amino acid oxidation. OFR denatured amino acids were chromatographed by this procedure, and all radical species generated, with the exception of the superoxide anion, resulted in the formation of identifiable fluorescent metabolites of tryptophan, kynurenines. The identity of peaks was confimed by spiking with authentic material and scanning absorption spectroscopy. After complete proteolytic hydrolysis, OFR treated proteins were also analysed by this technique; again the dose dependent production of kynurenines was detected in IgG, lens crystallins and albumin. Bityrosine was not detected in any of the proteins studied using this procedure, however, several novel unidentified fluorophores were detected in proteolytic hydrolysates, possibly the product of two different amino acid radicals.Immunoglobulin G isolated from the sera of normals and rheumatoid arthritis (RA) patients was examined for the presence of one specific tryptophan metabolite, N-formyl kynurenine. Significantly elevated levels of this metabolite were detected in rheumatoid sera, suggesting increased OFR activity in RA.These results have demonstrated firstly, that specific oxidised products of amino acids are retained in the protein backbone after exposure to OFR generating systems. Secondly, in aerated solution, oxidised tryptophan residues confer the major new visible fluorescence in non-haem proteins, not tyrosine products. In addition, this work has demonstrated that the measurement of a specific product of an oxidised amino acid can be applied to biological macromolecules, and may be important in implicating free radical reactions in certain disease processes.     

A general mechanism of polypeptide cross-linking by 3-hydroxykynurenine

Abstract

The human lens contains a group of fluorescent compounds, derived from tryptophan, which act to absorb UV light in the 300–400 nm region of the spectrum.¹ The major component is the glucoside of 3-hydroxykynurenine (3HK), 3-hydroxykynurenine glucoside (3HKG).²In the lens, 3HKG represents a unique pathway of tryptophan metabolism. Smaller amounts of kynurenine and 3HK have been detected in human lens extracts.^3,4 . More recently, a new UV-filter compound derived from tryptophan, 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid O-glucoside (AHBG), was identified, and constitutes the second most abundant UV-filter in the human lens.⁵     

Kynurenine binds to the peptide binding region of the chaperone alphaB-crystallin

UV filters, such as kynurenine, are present in the human lens. They are spontaneously unstable at neutral pH and deaminate to form reactive alpha, beta unsaturated ketones. This process becomes more prominent after the lens barrier develops in middle age. Here we show that deaminated kynurenine reacts primarily with histidine residues in alphaB-crystallin: a major lens protein that lacks cysteine. Five of the nine histidines in alphaB-crystallin were found to be conjugated with kynurenine. Furthermore, a major site of covalent modification was at histidine 83, which is found in the putative peptide binding region of alphaB-crystallin; a site crucial for its role as a chaperone. We propose that modification of alphaB-crystallin by UV filters may compromise the chaperone action of this protein.     

Glutathionylation of lens proteins through the formation of thioether bond

Formation of lanthionine, a dehydroalanine crosslink, is associated with aging of the human lens and cataractogenesis. In this study we investigated whether modification of lens proteins by glutathione could proceed through an alternative pathway: that is, by the formation of a nonreducible thioether bond between protein and glutathione. Direct ELISA of the reduced water-soluble and water-insoluble lens proteins from human cataractous, aged and bovine lenses showed a concentration-dependent immunoreactivity toward human nonreducible glutathionyl-lens proteins only. The reduced water-insoluble cataractous lens proteins showed the highest immunoreactivity, while bovine lens protein exhibited no reaction. These data were confirmed by dot-blot analysis. The level of this modification ranged from 0.7 to 1.6 nmol/mg protein in water-insoluble proteins from aged and cataractous lenses. N-terminal amino acid determination in the reduced and alkylated lens proteins, performed by derivatization of these preparations with dansyl chloride followed by an exhaustive dialysis, acid hydrolysis and fluorescence detection of dansylated amino acids by RP-HPLC, showed that N-terminal glutamic acid was present in concentration of approximately 0.2 nmol/mg of lens protein. This evidence points out that at least some of the N-terminal amino groups of nonreducible glutathione in the reduced human lens proteins are not involved in a covalent bond formation. Since disulfides were not detected in the reduced and alkylated human lens proteins, GSH is most likely attached to lens proteins through thioether bonds. These results provide, for the first time, evidence that glutathiolation of human lens proteins can occur through the formation of nonreducible thioether bonds.     

Changes in proteins of the human lens in development and aging.

A number of proteins have been isolated from the human lens at different stages of development, from before birth to old age. These proteins have been characterized and compared with each other and with corresponding proteins from bovine lens. Many similarities were found between human and bovine crystallins, but alpha-crystallin isolated from old human lenses using DEAE-cellulose, unlike bovine alpha-crystallin similarly isolated, is not found as large soluble aggregates. The amide contents of various lens protein fractions were determined. No extensive changes were found during adult life, but there was evidence that significant deamidation of alpha-crystallin had occurred before birth and possibly during infancy. The results are related to the unique development and aging of the lens.     

Photoreactions of human lens monomeric crystallins

Human lens beta s- and gamma A-crystallins exhibit very similar tryptophan fluorescence emission maxima (329 nm). gamma A isolated from infant human lenses is photo-oxidized by 300 nm irradiation and forms water-insoluble aggregates; beta s or gamma A from young human lenses form a small amount of water-soluble crosslinked species. At least part of the mechanism of photodamage by 300 nm irradiation is photogeneration of the oxidant H2O2 via the generation of O2- radical, this reaction occurs via photosensitization by the tryptophan photo-oxidation product N-formylkynurenine (N-FK) or related species. These results indicate that even though the tryptophan residues of beta s- and gamma A-crystallins are in hydrophobic (buried) microenvironments as compared to those of the alpha- and beta-crystallins, the photogeneration of N-FK is sufficient to produce O2- and H2O2.     

Polypeptide modification and cross-linking by oxidized 3-hydroxykynurenine

3-Hydroxykynurenine (3OHKyn) is present in the mammalian lens as a UV filter and is formed from kynurenine in the tryptophan metabolic pathway. 3OHKyn is a readily autoxidized o-aminophenol which binds to proteins in vitro. The lens, particularly its central region, the nucleus, becomes increasingly oxidized with age. Under such conditions, the oxidation products of 3OHKyn may bind to lens proteins and contribute to nuclear cataract formation. The purpose of this study was to determine the structures of in vitro reaction products of 3OHKyn with model peptides as a general model for 3OHKyn modification of proteins. 3OHKyn was incubated with the dipeptide glycylglycine (GG) and the tetrapeptide tuftsin (sequence TKPR) under oxidizing conditions, and the reaction products were characterized by a variety of spectroscopic techniques. The major 3OHKyn-GG reaction product involves formation of a benzimidazole moiety between the GG N-terminus and the oxidized amino and/or phenol groups of 3OHKyn. In contrast, tuftsin, which has an N-terminal threonine, forms predominantly a cross-linked dimer with oxidized 3OHKyn. This product is analogous in structure to the dimeric reaction product, quinilinobenzoxamine, formed between oxidized 3OHKyn and glycyllysine [Aquilina, J. A., et al. (1999) Biochemistry 38, 11455-11464], which contains a benzoxazole moiety. The identification of a tuftsin dimer suggests that 3OHKyn can react with any peptide having a free alpha-amino group, via a general side chain elimination mechanism. The identification of both benzimidazole and benzoxazole adducts in peptides with a free N-terminus suggests that peptide amino groups can react initially at either the aromatic amino or hydroxyl group of oxidized 3OHKyn. The proportion of each adduct may change, however, depending on the amino acid sequence at the N-terminus.     

Human lens coloration and aging. Evidence for crystallin modification by the major ultraviolet filter, 3-hydroxy-kynurenine O-beta-D-glucoside.

The human lens becomes increasingly yellow with age and thereby reduces our perception of blue light. This coloration is associated with lens proteins (crystallins), but its molecular basis was unknown. Here we show that the coloration occurs because of the interaction of crystallins with a UV filter compound, 3-hydroxykynurenine glucoside (3-OHKG). Crystallin modification results from deamination of the 3-OHKG amino acid side chain, yielding an unsaturated ketone that is susceptible to nucleophilic attack by cysteine, histidine, and lysine residues. This novel protein modification contributes to age-related lens coloration and may play a role in human nuclear cataractogenesis.     

Nanosensor detection of an immunoregulatory tryptophan influx/kynurenine efflux cycle

Mammalian cells rely on cellular uptake of the essential amino acid tryptophan. Tryptophan sequestration by up-regulation of the key enzyme for tryptophan degradation, indoleamine 2,3-dioxygenase (IDO), e.g., in cancer and inflammation, is thought to suppress the immune response via T cell starvation. Additionally, the excreted tryptophan catabolites (kynurenines) induce apoptosis of lymphocytes. Whereas tryptophan transport systems have been identified, the molecular nature of kynurenine export remains unknown. To measure cytosolic tryptophan steady-state levels and flux in real time, we developed genetically encoded fluorescence resonance energy transfer nanosensors (FLIPW). The transport properties detected by FLIPW in KB cells, a human oral cancer cell line, and COS-7 cells implicate LAT1, a transporter that is present in proliferative tissues like cancer, in tryptophan uptake. Importantly, we found that this transport system mediates tryptophan/kynurenine exchange. The tryptophan influx/kynurenine efflux cycle couples tryptophan starvation to elevation of kynurenine serum levels, providing a two-pronged induction of apoptosis in neighboring cells. The strict coupling protects cells that overproduce IDO from kynurenine accumulation. Consequently, this mechanism may contribute to immunosuppression involved in autoimmunity and tumor immune escape.     

Double mechanism for apical tryptophan depletion in polarized human bronchial epithelium

Indoleamine 2,3-dioxygenase is an enzyme that catabolizes tryptophan to kynurenine. We investigated the consequences of IDO induction by IFN-gamma in polarized human bronchial epithelium. IDO mRNA expression was undetectable in resting conditions, but strongly induced by IFN-gamma. We determined the concentration of tryptophan and kynurenine in the extracellular medium, and we found that apical tryptophan concentration was lower than the basolateral in resting cells. IFN-gamma caused a decrease in tryptophan concentration on both sides of the epithelium. Kynurenine was absent in control conditions, but increased in the basolateral medium after IFN-gamma treatment. The asymmetric distribution of tryptophan and kynurenine suggested the presence of a transepithelial amino acid transport. Uptake experiments with radiolabeled amino acids demonstrated the presence of a Na(+)-dependent amino acid transporter with broad specificity that was responsible for the tryptophan/kynurenine transport. We confirmed these data by measuring the short-circuit currents elicited by direct application of tryptophan or kynurenine to the apical surface. The rate of amino acid transport was dependent on the transepithelial potential, and we established that in cystic fibrosis epithelia, in which the transepithelial potential is significantly more negative than in noncystic fibrosis epithelia, amino acid uptake was reduced. This work suggests that human airway epithelial cells maintain low apical tryptophan concentrations by two mechanisms, a removal through a Na(+)-dependent amino acid transporter and an IFN-gamma-inducible degradation by IDO.