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

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 × 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 × 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 β-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 × 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 × 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 × 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 × 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 × 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.

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. 4⇓A). 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 β-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 β-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. 4⇑B). 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. 4⇑B) 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. 4⇑C). 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 × 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. 6⇓A), inhibit LAAO by substrate competition (20, 28). Diacetyl and 2H3B, both with nonaromatic chemical structures (Fig. 6⇓A), 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. 6⇓B). 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. 7⇓A). Using an Eadie-Hofstee plot (Fig. 7⇓B), the apparent maximum velocity (V_max) of the overall enzyme reaction and the apparent Michaelis-Menten constant K_m were estimated to be 0.0099 U/mg and 6.5 mM, respectively. This apparent K_m 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 K_m 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 K_m (in millimolar concentration) and apparent V_max (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).