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The Journal of Immunology, 2004, 173: 4561-4567.
Copyright © 2004 by The American Association of Immunologists

IL-4-Induced Gene-1 Is a Leukocyte L-Amino Acid Oxidase with an Unusual Acidic pH Preference and Lysosomal Localization1

James M. Mason*, Mamta D. Naidu{dagger}, Michele Barcia*, Debra Porti*, Sangeeta S. Chavan{dagger} and Charles C. Chu2,{dagger}

* Gene Therapy Vector Laboratory, and {dagger} Laboratory of Gene Activation, North Shore-Long Island Jewish Research Institute, and Departments of Medicine, North Shore University Hospital and New York University School of Medicine, Manhasset, NY 11030


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IL-4-induced gene-1 (Il4i1 or Fig1) initially isolated as a gene of unknown function from mouse B lymphocytes, is limited in expression to primarily immune tissues and genetically maps to a region of susceptibility to autoimmune disease. The predicted Il4i1 protein (IL4I1) sequence is most similar to apoptosis-inducing protein and Apoxin I, both L-amino acid oxidases (LAAO; Enzyme Commission 1.4.3.2). We demonstrate that IL4I1 has unique LAAO properties. IL4I1 has preference for aromatic amino acid substrates, having highest specific activity with phenylalanine. In support of this selectivity, IL4I1 is inhibited by aromatic competitors (benzoic acid and para-aminobenzoic acid), but not by nonaromatic LAAO inhibitors. Il4i1 protein and enzyme activity is found in the insoluble fraction of transient transfections, implying an association with cell membrane and possibly intracellular organelles. Indeed, IL4I1 has the unique property of being most active at acidic pH (pH 4), suggesting it may reside preferentially in lysosomes. IL4I1 is N-linked glycosylated, a requirement for lysosomal localization. Confocal microscopy of cells expressing IL4I1 translationally fused to red fluorescent protein demonstrated that IL4I1 colocalized with GFP targeted to lysosomes and with acriflavine, a green fluorescent dye that is taken up into lysosomes. Thus, IL4I1 is a unique mammalian LAAO targeted to lysosomes, an important subcellular compartment involved in Ag processing.



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  		FIGURE 1. IL4I1 is most similar to LAAO. Schematic of mouse IL4I1 is shown to scale with N-linked glycosylation sites (N-Gly) ({blacksquare}) and potential tyrosine (Y) phosphorylation sites ({circ}). Predicted FAD cofactor binding domains are indicated by white boxes in the schematic corresponding to the three black lines underneath. The signal peptide and short regions of similarity to many known FAD-binding proteins (MANY) and to specific proteins (phytoene desaturase (PDS), monoamine oxidase (MAO), and tryptophan 2-monooxygenase (TMO)) are shown by the next row of black lines underneath the schematic, corresponding to the gray-spotted boxes. Regions of similarity to bacterial (Bacillus cereus) and eukaryotic (fish (Scomber japonicus) and snake (Crotalus atrox, C. adamanteus)) LAAO are shown by the remaining black lines underneath the schematic of IL4I1.

 

   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interleukin-4-induced gene-1 (Il4i13 or Fig1) was initially isolated in a screen for IL-4-induced genes from mouse B cells using cDNA representational difference analysis (1, 2). Recently, Fig1 was renamed Il4i1 in keeping with recommendations from the Human Gene Nomenclature Committee (3). Il4i1 may play an important role in the immune system for several reasons. It is induced in an immediate-early fashion by IL-4, a key regulator of the immune response (4); its expression is strikingly limited to immune tissues, with clear expression in B cells (1, 5); and it is also found in professional APCs (dendritic cells (DC) and macrophages) (6, 48).

Mouse Il4i1 genetically maps to chromosome 7 between the Klk6 and Fut1 genes (23.1 cM; Mouse Genome Database, Mouse Genome Informatics; The Jackson Laboratory, Bar Harbor, ME; www.informatics.jax.org (July 2004)) (1, 7), a region where susceptibility to systemic lupus erythematosus (SLE) has been implicated by a locus or loci derived from the NZW mouse strain (8, 9, 10). Interestingly, the NZW allele for Il4i1 contains three amino acid substitutions (11), which could alter a function that results in SLE susceptibility. Furthermore, the Sle3 susceptibility locus from this region appears to be expressed primarily in APCs (12), where we have also detected Il4i1 expression. Finally, the human Il4i1 ortholog is located on chromosome 19q13.3–19q13.4 (11) in a region that is a hot spot for autoimmune disease susceptibility in general, including rheumatoid arthritis, multiple sclerosis, insulin-dependent diabetes mellitus, and SLE (13, 14, 15). Thus, an alteration in Il4i1 could contribute to autoimmune disease in both mouse and man.

One clue to the function of Il4i1 is its sequence similarity to L-amino acid oxidases (LAAO; Enzyme Commission (EC) 1.4.3.2) (Fig. 1). The cDNA sequences of both mouse and human Il4i1 predict a protein containing a putative signal peptide sequence for entry into the endoplasmic reticulum (ER), a region of similarity to LAAO, and a C-terminal region with no homology to known proteins (1, 11). The LAAO region of similarity most closely resembles apoptosis-inducing protein found in fish (43% identical over 485 aa) (16) and snake venom LAAO (37% identical over 484 aa) (17), also known as Apoxin I (18). The LAAO-similar portion conserves key domains and residues that bind the flavin adenine dinucleotide (FAD) cofactor required for its enzymatic activity, as well as residues in the active site of the LAAO crystal structure (11, 19).

Because of the similarity of IL4I1 to LAAO, we examined the LAAO enzyme reaction catalyzed by IL4I1. LAAO catalyzes the oxidation of L-amino acid to 2-oxo acid, via an L-imino acid intermediate (20) (Fig. 2). Water and oxygen are required in this reaction resulting in hydrogen peroxide (H2O2) and ammonia byproducts. We developed an enzyme assay demonstrating that IL4I1 is the first LAAO found in immune cells. Although IL4I1 has similar enzyme kinetics to other LAAO from other cells and organisms, it is most reactive with aromatic amino acids at acidic pH. Because acidic pH preference is a common feature of lysosomal enzymes, we investigated the subcellular localization of IL4I1 and found that it is the first known LAAO that is targeted to lysosomes. Thus, IL4I1 may have a fundamental role in lysosomal Ag processing and presentation.



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  		FIGURE 2. LAAO reaction. The chemical structure of L-phenylalanine is shown as a typical L-amino acid oxidized by LAAO. LAAO is indicated with the FAD cofactor and its oxidation state. The reaction is shown in two steps, with an L-imino acid intermediate.

 

   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Expression plasmid construction

Plasmid pIl4i1-iGFP was constructed by digesting vector pIRES2-EGFP (BD Clontech, Palo Alto, CA) with restriction enzymes Ecl136II and EcoRI, isolating the 5.3-kb linearized vector, and then ligating a 1.9-kb PvuII/EcoRI fragment from plasmid pIl4i1-IRES-dsRED (data not shown) containing the full-length mouse Il4i1 cDNA into the vector (Fig. 3). In pIl4i1-iGFP, both Il4i1 and enhanced GFP gene (EGFP) are expressed on a bicistronic mRNA with an internal ribosome entry site (IRES) between Il4i1 and EGFP that permits translation of both proteins. Plasmid pIl4i1-dsRED was constructed by restriction enzyme digesting vector pDs-Red1-N1 (BD Clontech) with SmaI and AgeI, isolating the 4.7-kb linearized vector, and ligating a similarly digested 1.9-kb DNA fragment containing the complete mouse Il4i1 coding sequence and optimized Kozak translational start site (21), but lacking a stop codon (Fig. 3). This DNA fragment was generated by PCR using primers NS217 (5'-AAA CCC GGG GCC GCC ACC ATG GCT GGG CTG GCC CTG CGT-3') and NS218 (5'-GGC GAC CGG TGA GTG GTC CCC CAC TCG GTG CAT-3') and plasmid template pLXSN-Il4i1 (data not shown) using a GeneAmp kit (PerkinElmer, Branchburg, NJ). After DNA sequencing of this PCR fragment cloned into pT7Blue-3 (Novagen, Madison, WI) confirmed that no errors were introduced, the 1.9-kb SmaI/AgeI DNA fragment was inserted into pDsRed1-N1. The resulting pIl4i1-dsRED plasmid expresses the complete Il4i1 coding sequence translationally fused to the Discosom red fluorescent protein (dsRED).



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  		FIGURE 3. Il4i1 expression plasmid constructs. Schematics of pIl4i1-iGFP and pIl4i1-dsRED plasmids are shown to scale. Restriction enzyme sites used in construction are indicated. Coding regions of Il4i1, neomycin resistance gene (neo), EGFP, and Il4i1-dsRED are indicated with solid arrows (except dsRED portion is gray spotted). The CMV immediate-early promoter (Pcmv), neo promoters, and IRES are shown in white.

 
Cells and transfections

NIH3T3 cells were cultured in D10 medium (high-glucose DMEM supplemented with 10% heat-inactivated FBS (Invitrogen Life Technologies, Carlsbad, CA)) to ~50% confluence (~1.5 x 106 cells) in 10-cm-diameter tissue culture dishes (BD Falcon, Bedford, MA) at 5% CO2 and 37°C. After incubating cells in fresh medium for 1 h at 37°C, cells were transfected with 40 µg of plasmid DNA using the Calcium Phosphate Transfection System (Invitrogen Life Technologies). The next day, cells were washed with PBS (pH 7.2; Invitrogen Life Technologies) to remove the Ca3(PO4)2/DNA precipitate, and fresh D10 medium was added. Transfection efficiency was determined by fluorescent light microscopy 1 or 2 days postwash. Transfections with >30% fluorescent cells were further analyzed.

Total RNA isolation and RT-PCR

Total RNA was isolated using the guanidine isothiocyanate protocol (22). In brief, 1 x 106 transfected cells were lysed in guanidine isothiocyanate solution, extracted with phenol/chloroform mixture, and then precipitated with isopropanol. The resulting RNA was resuspended in 20–50 µl of diethyl-pyrocarbonate-treated H2O and quantitated using UV absorption measurements at 260/280 nm.

For first-strand cDNA synthesis of RT-PCR, 7 µl of RNA (200–1000 ng) plus 2 µl of 10 µM oligo(dT) (5'-TTT TTT TTT TTT TTT V) was heated at 65°C for 5 min, cooled at room temperature, and then kept on ice. To this mixture, 4 µl of 2.5 mM dNTP, 1 µl of 100 mM DTT, 1 µl of 200 U/µl Moloney murine leukemia virus reverse transcriptase, 4 µl of 15 mM MgCl2, 375 mM KCl, 250 mM Tris (pH 8.3) buffer (Invitrogen Life Technologies), and 1 µl of 1 U/µl RNase inhibitor (5 Prime->3 Prime, Boulder, CO) were added and incubated at 42°C for 60 min, heated to 99°C for 5 min, and stored at 4°C. This cDNA (1 µl) was PCR amplified in a 20-µl reaction containing 10 mM Tris (pH 8.3), 2.0 mM MgCl2, 50 mM KCl, 0.2 mM each dNTP, 1.5 U of AmpliTaq Gold (Roche Molecular Biochemicals, Indianapolis, IN), and 0.5 mM each of Il4i1-specific primers (CCC52 (5'-CGG AAT TCG GTA CCG GAG AAG ATG CCA GAA AAG) and CCC54 (5'-CGG AAT TCG GTA CCG TAA GGC TTC TGC AAA)) or {beta}-actin-specific primers (M309-11 (5'-GAT GAC GAT ATC GCT GCG CTG) and M309-12 (5'-GTA CGA CCA GAG GCA TAC AGG)) under the following PCR conditions: 95°C for 5 min; (94°C for 5 s, 55°C for 15 s, and 72°C for 30 s) times 25–35 cycles; 72°C for 30 s and soak at 4°C. PCR products were electrophoresed on 1% agarose gels and visualized after ethidium bromide staining under UV illumination.

Immunoblot analysis

Supernatants were collected from transfected cell cultures 2 days postwash and concentrated ~100-fold to 100–150 µl at 4°C in 20-ml Centricon Plus-20 tubes (30-kDa molecular mass cutoff; Millipore, Bedford, MA). Cell fractions were prepared from ~4 x 106 transfected cells resuspended in 250 µl of PBS (pH 7.4) containing protease inhibitors (Sigma-Aldrich, St. Louis, MO): 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM PMSF, 10 µg/ml pepstatin A, and 10 µg/ml trypsin inhibitor. Cells were sonicated on ice for 5–10 s at 5 Hz in an Ultrasonic Processor, model XL2010 (Heat Systems, Farmingdale, NY). The sonicate was centrifuged at 13,000 x g for 10 min at 4°C. The soluble fraction was removed and the insoluble cell pellet was resuspended in 250 µl of PBS (pH 7.4) with protease inhibitors. Fractions were stored as 50-µl aliquots at –80°C. For deglycosylation, 50 µg of protein was treated with 2 µl (1000 U) of PNGase F (New England Biolabs, Beverly, MA) in manufacturer’s buffer for 1 h at 37°C.

Supernatants and soluble or insoluble cell fractions (15 µl) were mixed with an equal volume of 0.001% bromophenol blue (Bio-Rad, Hercules, CA), 4% SDS, 10% 2-ME, 20% glycerol, and 125 mM Tris (pH 6.8; Sigma-Aldrich), and denatured at 95°C for 5 min. This mixture was separated by SDS-PAGE (4% stacking, 7.5% resolving gel) in duplicate for 2–3 h at 40 mA on a MiniPROTEAN II Electrophoresis Cell (Bio-Rad). To detect proteins, one gel was stained with 0.05% Coomassie brilliant blue R-250 (Bio-Rad) in 40% methanol and 10% glacial acetic acid (Fisher Scientific, Fair Lawn, NJ) overnight and destained by washes in 40% methanol and 10% acetic acid.

Proteins on the duplicate gel were transferred onto a Trans-Blot nitrocellulose membrane (0.45 µm; Bio-Rad) overnight at 4°C at 15 mA in transfer buffer (20% methanol, 20 mM Tris (pH 8.3), and 153.6 mM glycine (Sigma-Aldrich)) using a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad). Membranes were probed using Living Colors Ds Peptide Ab (anti-dsRED; BD Clontech) at 1/1000 dilution or affinity-purified polyclonal rabbit anti-IL4I1 peptide Ab at 1/2000 dilution (generous gift from Dr. S.-K. Jung (Institute for Virus Research, Kyoto University, Kyoto, Japan)) in TTBS (0.1% Tween 20 (Fisher Scientific) in TBS (pH 7.5)). After washing with TTBS, the primary Ab was detected using the ECL Western blotting analysis system (Amersham Biosciences, Piscataway, NJ). Briefly, membranes were incubated with HRP-conjugated goat anti-rabbit polyclonal Ab (1/50,000 dilution), washed, and incubated with luminol plus H2O2. Chemiluminescence was detected with Hyperfilm ECL (Amersham Biosciences) after 1- to 2-min exposure.

Il4i1 enzyme assay

One day postwash, supernatants were collected and the cells were harvested by trypsinization (one 10-cm-diameter dish yields ~2 x 106 cells). Collected supernatants were filtered through a 0.2-µm filter before assay. Cell pellets were freeze-thawed three times in microfuge tubes alternately between –70 and 37°C. After centrifugation at 13,000 x g for 10 min at 4°C, the soluble fraction was removed to a separate tube. The remaining insoluble fraction was resuspended in PBS (pH 7.2; 120 µl per dish). All fractions were kept on ice before enzyme assay.

In a 96-well flat-bottom Nunc-Immuno Maxisorp plate (Nunc, Roskilde, Denmark), 100 µl of L-amino acid or amine substrate (all from Sigma-Aldrich, except for dopamine (American Reagent Labs, Shirley, NY)) at indicated concentrations or dilution of known concentrations of H2O2 (from 1.47 to 1470 µM; Sigma-Aldrich) was added to individual wells in duplicate. A fresh premix of 10 µl of 200 U/ml HRP type VI-A, 10 µl of 10 mg/ml o-phenylenediamine, and 20 µl of 500 mM phosphate citrate buffer (pH 5.0) (Sigma-Aldrich) was prepared per well. To the premix was added 60 µl of supernatant, cell extract, or H2O2 control. The complete 100-µl premix was added to the wells, and the 200-µl reaction mix was incubated for 2 h at 37°C with atmospheric oxygen in a humidified 5% CO2 incubator. The reaction was stopped by addition of 11 µl of 36 N H2SO4. Samples were centrifuged at 13,000 x g for 10 min to pellet any insoluble material. Soluble material was transferred to a fresh 96-well plate, and A490 was measured on a SPECTRAmax 340 microplate spectrophotometer (Molecular Devices, Sunnyvale, CA). The OD490 background reading from pIRES2-EGFP transfectants was subtracted from that of pIl4i1-iGFP transfectants. From the H2O2 standard curve, the OD490 was converted to micromolar concentration of H2O2 generated, equivalent to micromolar concentration of amino acid oxidized based on the enzyme reaction (Fig. 2), and used to calculate Il4i1 or LAAO enzyme units (nanomoles of amino acid oxidized per minute at 37°C). As a positive control, 10 µl of 0.05 mg/ml crude snake venom (Crotalus adamanteus) LAAO extract (Sigma-Aldrich) was also tested. To determine specific LAAO activity (units per milligram of total protein), total protein was measured by the method of Bradford using the Bio-Rad Protein Assay. On average, resuspended insoluble extracts from pIl4i1-iGFP transfectants had 234 ± 89 mg/ml total protein.

To vary the pH, the o-phenylenediamine and premix solutions were prepared with alternate buffers (acetic acid/sodium acetate (pH 4.0); 2-morpholinoethanesulfonic acid/NaOH (pH 6.0); or 3-morpholino-2-hydroxypropanesulfonic acid/NaOH (pH 7.0) (Sigma-Aldrich)) to result in a 50 mM final concentration in the assay. Benzoic acid (BA), para-aminobenzoic acid (PABA), 2,3-butanedione (diacetyl; Sigma-Aldrich), and 2-hydroxy-3-butynoic acid (2H3B; TCI America, Portland, OR) were added at the indicated inhibitory concentrations.

Subcellular colocalization

In 35-mm glass-bottom, coated microwell dishes (MatTek Corporation, Ashland, MA), NIH3T3 cells were cotransfected with pIl4i1-dsRED and a vector expressing green fluorescent fusion protein targeted to a particular subcellular localization (pEGFP-Peroxi, pEYFP-ER, pEGFP-Endo (BD Clontech), pHYAL2-EGFP (23), PTS2-EGFP (24), or YFP-GL-GPI (25)). After 24 h, the cells were washed and cultured for another 48 h. Alternatively, 48 h postwash pIl4i1-dsRED-transfected cells were incubated with 0.5 µg/ml acriflavine (Sigma-Aldrich) for 20 min, washed, and cultured in fresh medium (26). Cells were examined by confocal laser-scanning microscopy (Fluoview 300-IX; Olympus, Melville, NY) to determine subcellular localization by overlapping fluorescence.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
High-efficiency transient transfection overexpresses detectable Il4i1 protein

To test whether Il4i1 protein had the predicted LAAO activity, we attempted to generate stable cell lines that overexpressed Il4i1 protein. However, stable lines expressing Il4i1 could not be easily obtained, due to toxic effects of the protein (data not shown). To examine Il4i1 enzyme activity despite its toxicity, we overexpressed Il4i1 protein in high-efficiency transient transfections of NIH3T3 cells. To monitor transfection efficiency, Il4i1 was expressed in the pIRES2-EGFP vector (pIl4i1-iGFP) or as an Il4i1-dsRED fusion protein in the pDsRed1-N1 vector (pIl4i1-dsRED). Transfection efficiency was monitored by observing the percentage of fluorescent cells. Cells transiently transfected at >30% efficiency expressed Il4i1 mRNA (Fig. 4A). Cells transfected with pIl4i1-iGFP or pIl4i1-dsRED expressed the expected 267-bp Il4i1 RT-PCR fragment, whereas cells transiently transfected with the parental control plasmids pIRES2-EGFP and pDsRed1-N1 did not. A 440-bp {beta}-actin RT-PCR fragment demonstrated that RNA from cells transiently transfected with the plasmids is functional for successful reverse transcription.



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  		FIGURE 4. IL4I1 is expressed in transient transfectants and glycosylated. A, RNA prepared from NIH3T3 cells transiently transfected (2 days postwash) with pIl4i1-iGFP, pIl4i1-dsRED, or vector alone (either pIRES2-EGFP (iGFP) or pDsRed1-N1 (dsRED)) was tested for Il4i1 and {beta}-actin expression by RT-PCR. B, Protein from supernatants (Sup.) or soluble (Sol.) and insoluble (Insol.) cell fractions of these transfectants were analyzed by immunoblot using anti-IL4I1 peptide antisera. Molecular masses (in kilodaltons) of protein standards run in parallel are indicated. Anti-IL4I1-specific proteins are observed in the Il4i1 expression plasmid transfectants. Samples from dsRED transfectants had similar background as iGFP transfectants (data not shown). Transfection efficiency assessed by fluorescent microscopy was 40–60% for all samples in this representative experiment. Fluorescent microscopy inspection generally agrees with efficiencies observed by flow cytometry in subsequent experiments. From many experiments, pIl4i1-iGFP typically produces less anti-IL4I1-specific protein than pIl4i1-dsRED. This may be due to variation in transfection efficiency or protein stability. Small amount of observed soluble IL4I1 may be genuine or may be due to incomplete fractionation. Cell viability is difficult to assess in transient transfections, but does not appear to be affected by Il4i1 expression plasmid transfectants in measurements from subsequent experiments (not shown). C, Protein from insoluble cell fraction of pIl4i1-dsRED transfected were untreated or treated with PNGase F buffer with or without PNGase F enzyme and analyzed by immunoblot with anti-dsRED Ab. N-Linked glycosylation sites are not found in dsRED. Thus, all PNGase F deglycosylation is found in IL4I1.

 
The predicted Il4i1 protein sequence has a typical N-terminal signal peptide and no hydrophobic transmembrane domain (Fig. 1). This suggests that Il4i1 traffics into and through the ER, ultimately targeted for secretion or into the lumen of an intracellular organelle. Supernatants, and the soluble and insoluble cell fractions were collected from transfected cells and separated by SDS-PAGE. Staining by Coomassie blue showed that equal amounts of protein were loaded for each fraction (data not shown). Immunoblot analysis of pIl4i1-iGFP-transfected cells demonstrated that Il4i1 protein (~90 kDa) is located mainly in the insoluble fraction of cell lysates (Fig. 4B). Similarly, in pIl4i1-dsRED fusion-transfected cells, the Il4i1-dsRED fusion protein (~120 kDa) was localized mainly to the insoluble fraction as detected by both anti-IL4I1 (Fig. 4B) and anti-dsRED Ab (data not shown).

The expected sizes for IL4I1 and IL4I1-dsRED are 70–68 and 97–95 kDa based on the predicted amino acid sequence and cleavage of the predicted signal peptide. The larger observed protein sizes may be due to posttranslational modifications, such as N-linked glycosylation at three putative sites conserved between human and mouse IL4I1 (11). Indeed, like snake venom LAAO (27), IL4I1 is N-linked glycosylated, because PNGase F deglycosylation yielded a protein of smaller size (Fig. 4C). Furthermore, transfectants treated with tunicamycin, which prevents glycosylation, yielded protein of similar small size (data not shown).

Il4i1 enzyme preferentially oxidizes aromatic amino acids

Supernatant and cell fractions from transfections overexpressing Il4i1 were tested for LAAO enzyme activity using a panel of L-amino acid and amine substrates (Fig. 5). The supernatant and soluble cell fraction had little or no LAAO activity, consistent with the amount of Il4i1 protein. The insoluble cell fraction, which contained the most Il4i1 protein, exhibited easily detectable LAAO activity, with highest enzymatic activity using common aromatic L-amino acid substrates: phenylalanine, tyrosine, and tryptophan. The remaining common protein encoding L-amino acids, as well as some uncommon L-amino acids (cystine, L-DOPA, trans-4-hydroxy-proline) and amines (dopamine, putrescine) did not exhibit significant activity. Thus, IL4I1 is a newly reported LAAO (EC 1.4.3.2) with a preference for common aromatic L-amino acids.



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  		FIGURE 5. IL4I1 LAAO activity prefers aromatic L-amino acids. Insoluble cell extracts from NIH3T3 cells transiently transfected with pIl4i1-iGFP or pIRES2-EGFP vector were assayed for specific LAAO activity (units per milligram) at pH 5 with various L-amino acid or amine substrates (common amino acids (standard single letter code), cystine (CC), trans-4-hydroxy-proline (H-Pro), dopamine, L-DOPA, putrescine) at 10 mM final concentration under atmospheric oxygen. Background activity of extracts from pIRES2-EGFP transfections was subtracted. The mean of duplicate measurements of a representative of three experiments is shown. Supernatants from these cell cultures had no detectable activity (<1 x 10–5 U/mg). With the common aromatic L-amino acid substrates, soluble cell extracts containing IL4I1 occasionally had barely detectable activity that was at least 10-fold less than the insoluble cell extracts.

 
Il4i1 is inhibited by aromatic competitors

LAAO inhibitors have very different chemical structures. PABA and BA, both with aromatic chemical structures (Fig. 6A), inhibit LAAO by substrate competition (20, 28). Diacetyl and 2H3B, both with nonaromatic chemical structures (Fig. 6A), bind the active site and inactivate arginine residues in the active site (29) or modify the FAD cofactor of LAAO (30, 31), respectively. In contrast to other LAAO, IL4I1 prefers aromatic L-amino acid substrates, suggesting that PABA and BA would inhibit Il4i1 enzyme activity better than diacetyl and 2H3B. At concentrations known to inhibit other LAAO, PABA and BA inhibit Il4i1 enzyme activity in a concentration-dependent manner, with >50% inhibition at the highest inhibitor concentration (Fig. 6B). For comparison, at 1.0, 5.0, and 10.0 mM PABA and BA, snake venom LAAO activity was inhibited 26, 80, 100%, and 19, 65, and 100%, respectively (28). In comparison, diacetyl (data not shown) or 2H3B did not inhibit Il4i1 enzyme activity at any concentration, including that known to inhibit other LAAO (29, 30, 31). Thus, these inhibitor studies confirm the preference of IL4I1 for aromatic substrates.



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  		FIGURE 6. IL4I1 is inhibited by aromatic competitors. A, Chemical structures of amino acid substrates (Phe and Leu) and LAAO inhibitors (BA, PABA, 2H3B, and diacetyl). B, Il4i1 enzyme activity was measured as in Fig. 5, except inhibitors were added at the indicated concentration, and 25 mM phenylalanine was used. Percent inhibition of Il4i1 enzyme activity was calculated from samples with no inhibitor added. Representative of two experiments is shown.

 
Il4i1 enzyme kinetics

Under atmospheric oxygen conditions, saturation of the enzyme reaction was obtained with increasing substrate (phenylalanine) concentrations, a behavior typical of an apparent first-order enzyme reaction that obeys Michaelis-Menten kinetics (Fig. 7A). Using an Eadie-Hofstee plot (Fig. 7B), the apparent maximum velocity (Vmax) of the overall enzyme reaction and the apparent Michaelis-Menten constant Km were estimated to be 0.0099 U/mg and 6.5 mM, respectively. This apparent Km falls within the range observed for all substrates of LAAO (0.011–100 mM) (32, 33). Using phenylalanine as a substrate under atmospheric oxygen conditions, the apparent Km has been reported to vary from 0.011 to 3.5 mM in LAAO from microbial, reptilian, and avian organisms (33, 34, 35, 36).



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  		FIGURE 7. Il4i1 enzyme kinetics. A, Insoluble cell extracts from NIH3T3 cells transiently transfected with pIl4i1-iGFP were assayed for specific LAAO activity at various concentrations of phenylalanine. These values are minus background activity from insoluble extracts from vector (pIRES2-EGFP) transfectants. The means and SDs are shown. Logarithmic trend line fitted by least-squares calculation is shown. B, An Eadie-Hofstee plot was used to calculate the apparent Km (in millimolar concentration) and apparent Vmax (in units per milligram) from the best-fit line calculated by linear regression.

 
IL4I1 prefers acidic pH

The pH preference of Il4i1 enzyme activity was evaluated to help provide insight into its cellular localization (Fig. 8). IL4I1 activity was highest at pH 4.0, suggesting that IL4I1 is most active in an acidic cellular compartment (lysosome, late endosome) and less active at neutral pH normally found in the cytoplasm (37). For comparison, we confirmed that LAAO from C. adamanteus, which has an apparent pH optimum of 7.2–7.5 (38, 39), is less active at acidic pH (40) (Fig. 8).



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  		FIGURE 8. Il4i1 enzyme activity is highest at acidic pH. Insoluble cell extracts were measured as in Fig. 7, except that the highest possible concentration of phenylalanine (50 mM) was used to detect Il4i1 or snake venom (C. adamanteus) LAAO activity at varying pH. Phenylalanine was insoluble at higher concentrations. The mean of duplicate measurements from a representative of eight experiments is shown. Different pH buffers did not significantly affect the H2O2 standard curves (data not shown), which were used to calculate LAAO activity.

 
IL4I1 is targeted to lysosomes

To determine whether IL4I1 is targeted to an acidic subcellular compartment, we analyzed cells cotransfected with pIl4i1-dsRED and a vector expressing a green fluorescent fusion protein targeted to a known subcellular compartment. Visualization by laser-scanning confocal microscopy demonstrated that IL4I1-dsRED colocalized with protein expressed in the ER as expected by the presence of a signal peptide sequence in IL4I1 (Fig. 9). Furthermore, IL4I1-dsRED colocalized with protein or dye targeted to lysosomes. However, it did not colocalize with protein targeted to endosomes, peroxisomes, and plasma membrane. Thus, IL4I1 is a LAAO targeted to lysosomes.



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  		FIGURE 9. IL4I1 localized to lysosomes. Representative NIH3T3 cells transfected with pIl4i1-dsRED and cotransfected with a vector expressing a green fluorescent fusion protein or costained with acriflavine are shown as separate red, green, and merged fluorescence images. IL4I1-dsRED colocalizes with ER (pEYFP-ER) and lysosomal markers (pHYAL2-EGFP, acriflavine). IL4I1-dsRED does not colocalize with endosome (pEGFP-Endo), peroxisome (PTS2-EGFP, pEGFP-Peroxi (not shown)), and plasma membrane (YFP-GL-GPI (not shown)) fluorescent fusion proteins. pDsRED1-N1 vector transfectants had broad red fluorescence throughout the cell that did not exhibit this punctate pattern and also was not excluded from the nucleus (not shown).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To characterize Il4i1 enzyme activity despite its cellular toxicity, the gene was transiently overexpressed in NIH3T3 cells at high efficiency. IL4I1 has a prototypic signal peptide sequence directing it to the lumen of the ER and lacks a hydrophobic transmembrane domain for retention in the cell membrane. Mouse IL4I1 contains three putative N-linked glycosylation sites that are conserved in human (11). These sites may contain mannose-6-phosphate, which targets proteins to lysosomes (41). Indeed, IL4I1 behaves like a typical lysosomal enzyme with a preference for acidic pH (Fig. 8). This is unique for a LAAO. Previously reported pH optima of other LAAO vary between pH 6.6 and 9.5 (32). Decreased IL4I1 activity at normal physiological pH may serve as a safety feature, ensuring that IL4I1 does not generate highly toxic H2O2 until the enzyme is sequestered into an acidic intracellular compartment (lysosomes/endosomes). Indeed, IL4I1 was found in lysosomes as determined by colocalization studies (Fig. 9).

Substrate specificities of LAAO measured at atmospheric oxygen vary from species to species. Snake venom LAAO from Calloselasma rhodostoma, Naja naja kaouthia, and Ophiophagus hannah, like IL4I1, had highest activity with phenylalanine and tryptophan (33). However, unlike IL4I1, these LAAO had high activity with additional L-amino acids (methionine and leucine for C. rhodostoma and N. kaouthia, or lysine, leucine, arginine, and methionine for O. hannah). Snake venom LAAO from Trimeresurus mucrosquamatus had highest activity with a nonaromatic L-amino acid, leucine, but also had activity with phenylalanine, methionine, and tyrosine as substrates (28). Finally, apoptosis-inducing protein from fish had LAAO activity only with lysine as a substrate (16). In comparison, IL4I1 efficiently oxidized only the aromatic L-amino acid substrates: phenylalanine, tyrosine, and tryptophan (Fig. 5). This preference for aromatic substrates was supported by the observation that only LAAO inhibitors containing aromatic rings were able to inhibit IL4I1 (Fig. 6). Although the biological significance of these substrate preferences is not known, they may be accounted for by sequence differences in the enzymatic active site (11, 33).

IL4I1 is the first characterized mammalian LAAO enzyme that is similar to the snake venom LAAO family. Previous studies have identified mammalian LAAO activity in peroxisomes and mitochondria of rat kidney or liver (42, 43). This LAAO activity was due to the B form of L-{alpha}-hydroxy acid oxidase (EC 1.1.3.15) (30), which has no sequence similarity with IL4I1 or LAAO from snake venom or fish (44, 45). Thus, we have characterized IL4I1 as a novel mammalian LAAO with unique properties.

Recently, the murine minor histocomptibility locus H46 on chromosome 7 was identified as a polymorphic peptide derived from endogenous Il4i1 protein prepared by the MHC class II processing pathway within APC and subsequently presented by MHC class II (6). Our demonstration that IL4I1 localizes to lysosomes is consistent with the notion that endogenous IL4I1 may be naturally presented by MHC class II by virtue of its targeting to this processing compartment. In addition, processing of endogenous IL4I1 required the N terminus (6), which contains the N-linked glycosylation sites essential for lysosomal targeting. Interestingly, the IL4I1 polymorphism that is distinguished as minor H46 is identical with a polymorphism that distinguishes wild type (BALB/c, C57BL/6, and NZB) from the NZW allele.

The Sle3 susceptibility locus, which is derived from the NZW strain, maps to the same region of mouse chromosome 7 as Il4i1 (1, 9). The role of Sle3 in SLE susceptibility appears to involve APC (12). Il4i1 expression is limited to APC, such as B cells stimulated with IL-4, macrophages, and DC (6, 48). IL-4 may also regulate expression of Il4i1 in DC, because human DC were prepared from peripheral blood by culture with GM-CSF and IL-4 (48). The Sle3 susceptibility locus could involve an Il4i1-mediated function in Ag processing. Indeed, a transfected B cell line overexpressing Il4i1 was more efficient in processing and presenting protein Ag (48). Variations in the amount of Il4i1 protein may affect Ag presentation by occupation of MHC class II peptide binding sites with Il4i1 peptides. Alternatively, changes in Il4i1 LAAO activity could affect Ag presentation by several possible mechanisms: 1) directly modifying antigenic peptides by oxidation, 2) additionally cleaving peptides affecting the repertoire of antigenic peptides, and/or 3) controlling the rate of amino acid removal, which would indirectly control the rate of upstream peptidases. Abnormal lysosomal peptidase activity and modified amino acids are known to alter immunogenicity and possibly autoimmunity (46, 47).


   Acknowledgments
 
This article is dedicated to the memory of Dr. Vincent Massey. We thank Dr. Vincent Massey, Dr. Sandro Ghisla, Dr. Peter Macheroux, Diana Chang, Dr. Yih-Pai Chu, Dr. Hsin Chu, Dr. Raul Wapnir, Dr. Saul Teichberg, and Dr. Nicholas Chiorazzi for critical review, advice, support, and encouragement; and Dr. Leslie Godwin, Dr. Dorothy Guzowski, and Craig Gawel for help with confocal microscopy, oligonucleotide synthesis, and DNA sequencing.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by the North Shore-Long Island Jewish Research Institute, the Muriel Fusfeld Foundation, the Leonard Wagner Autoimmunity Fund, the SLE Foundation, and U.S. Public Health Service Grant AI44837 awarded by the National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Charles C. Chu, Laboratory of Gene Activation, North Shore-Long Island Jewish Research Institute, and Departments of Medicine, North Shore University Hospital and New York University School of Medicine, 350 Community Drive, Manhasset, NY 11030. E-mail address: cchu{at}nshs.edu Back

3 Abbreviations used in this paper: Il4i1, IL-4-induced gene-1; IL4I1, Il4i1 protein; DC, dendritic cell; SLE, systemic lupus erythematosus; LAAO, L-amino acid oxidase; ER, endoplasmic reticulum; FAD, flavin adenine dinucleotide; EGFP, enhanced GFP gene; IRES, internal ribosome entry site; dsRED, Discosom red fluorescent protein; BA, benzoic acid; PABA, para-aminobenzoic acid; diacetyl, 2,3-butanedione; 2H3B, 2-hydroxy-3-butynoic acid. Back

Received for publication June 18, 2003. Accepted for publication July 23, 2004.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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