The mouse Gm853 gene encodes a novel enzyme: Leucine decarboxylase

Accepted Manuscript The mouse Gm853 gene encodes a novel enzyme: Leucine decarboxylase

Ana Lambertos, Bruno Ramos-Molina, David Cerezo, Andrés J. López-Contreras, Rafael Peñafiel PII: DOI: Reference:

S0304-4165(17)30358-6 doi:10.1016/j.bbagen.2017.11.007 BBAGEN 28983

To appear in: Received date: Revised date: Accepted date:

21 September 2017 31 October 2017 2 November 2017

Please cite this article as: Ana Lambertos, Bruno Ramos-Molina, David Cerezo, Andrés J. López-Contreras, Rafael Peñafiel , The mouse Gm853 gene encodes a novel enzyme: Leucine decarboxylase. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Bbagen(2017), doi:10.1016/ j.bbagen.2017.11.007

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ACCEPTED MANUSCRIPT Gm853 is a leucine decarboxylase

The Mouse Gm853 Gene Encodes a Novel Enzyme: Leucine Decarboxylase

Ana Lambertos1,2, Bruno Ramos-Molina1,3, David Cerezo1, Andrés J. López-Contreras1,4,

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and Rafael Peñafiel1,2*

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Department of Biochemistry and Molecular Biology B and Immunology. Faculty of Medicine.

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University of Murcia. 2Instituto Murciano de Investigación Biosanitaria (IMIB). Murcia, Spain. 3

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Present address: Department of Endocrinology and Nutrition, Virgen de la Victoria University

Hospital, Institute of Biomedical Research in Malaga (IBIMA) and University of Malaga,

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Malaga, Spain; CIBER Physiopathology of Obesity and Nutrition (CIBERObn), Institute of Health Carlos III, Spain. 4Present address: Center for Chromosome Stability, Department of

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Cellular and Molecular Medicine, Panum Institute, University of Copenhagen, Copenhagen,

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Denmark.

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(*) Corresponding author: Rafael Peñafiel; [email protected]

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Running title: Novel leucine decarboxylase encoded by Gm853 Keywords: polyamine; isopentylamine; amino acid decarboxylases; ornithine decarboxylase antizyme inhibitor; AZIN2 paralogue; Gm853; enzyme catalysis; protein evolution; testosterone Abbreviations: AZ, antizyme; AZIN, antizyme inhibitor; DFMO, α-difluoromethylornithine; DMEM, Dulbecco's Modified Eagle Medium; HEK, human embryonic kidney; LDC, leucine decarboxylase; ODC, ornithine decarboxylase; PLP, pyridoxal phosphate.

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ACCEPTED MANUSCRIPT Gm853 is a leucine decarboxylase ABSTRACT Ornithine decarboxylase (ODC) is a key enzyme in the biosynthesis of polyamines. ODCantizyme inhibitors (AZINs) are homologous proteins of ODC, devoid of enzymatic activity but acting as regulators of polyamine levels. The last paralogue gene recently incorporated into the ODC/AZINs family is the murine Gm853, which is located in the same chromosome as AZIN2,

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and whose biochemical function is still unknown. By means of transfection assays of HEK293T cells with a plasmid containing the coding region of Gm853, we show here that unlike ODC,

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GM853 was a stable protein that was not able to decarboxylate L-ornithine or L-lysine and that

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did not act as an antizyme inhibitor. However, GM853 showed leucine decarboxylase activity, an enzymatic activity never described in animal cells, and by acting on L-leucine (Km=

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7.03x10-3 M) it produced isopentylamine, an aliphatic monoamine with unknown function. The other physiological branched-chain amino acids, L-valine and L-isoleucine were poor substrates

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of the enzyme. Gm853 expression was mainly detected in the kidney, and as Odc, it was stimulated by testosterone. The conservation of Gm853 orthologues in different mammalian

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species, including primates, underlines the possible biological significance of this new enzyme.

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In this study, we describe for the first time a mammalian enzyme with leucine decarboxylase activity, therefore proposing that the gene Gm853 and its protein product should be named as

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leucine decarboxylase (Ldc, LDC).

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ACCEPTED MANUSCRIPT Gm853 is a leucine decarboxylase 1. Introduction Amino acid decarboxylases are a group of relevant enzymes that participate in the generation of biogenic amines or neurotransmitters, such as gamma-aminobutyric acid (GABA). According to their amino acid sequences, these pyridoxal-5'-phosphate (PLP)-dependent enzymes have been divided in four different groups (1). Eukaryotic ornithine decarboxylase (ODC) (EC 4.1.1.17),

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the enzyme that catalyzes the formation of putrescine from L-ornithine belongs to group IV. ODC has also been included in the alanine racemase structural family, together with prokaryotic

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arginine decarboxylase and diaminopimelate decarboxylase (2).

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In vertebrates, ODC is a key enzyme for the synthesis of polyamines, organic polycations that are essential for different cellular functions, such as those associated with cell

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growth, proliferation, differentiation, apoptosis and aging (3-5). ODC is regulated at post-

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translational level by a complex circuit of proteins involving ornithine decarboxylase antizymes (AZs) and antizyme inhibitors (AZINs). AZ synthesis is stimulated by increasing intracellular polyamine concentrations, through a singular mechanism involving a +1 ribosomal

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frameshifting at the premature stop codon of the antizyme mRNA (6,7). Mammalian cells

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express several AZ genes, AZ1 being the first AZ identified (8).

AZ1 stimulates ODC

degradation by the 26S proteasome by an exceptional mechanism independent of ubiquitin

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conjugation, and also inhibits polyamine uptake by a mechanism still unidentified (9,10). AZ2 is expressed at lower levels than AZ1, whereas and AZ3 is specific of testis (11). AZINs are

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considered as positive regulators of polyamine metabolism, since they tightly bind to AZs and prevent the negative effects that AZs exert on ODC and polyamine uptake (12, 13). Two antizyme inhibitors (named AZIN1 and AZIN2) have been characterized, so far, as the products of genes localized in different chromosomes. In contrast to ODC, both AZINs are degraded after ubiquitination, and their binding with AZs markedly stabilize them (14-16). AZIN1 and AZIN2 have a high sequence homology with ODC, but they lack the specific residues that have been shown to be essential for the decarboxylating activity of the enzyme (2,17). Although both of them act as AZ antagonists, they have different expression patterns. Whereas AZIN1 is mainly

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ACCEPTED MANUSCRIPT Gm853 is a leucine decarboxylase expressed in normal and malignant proliferating cells (12,18-20), AZIN2 is more abundant in differentiated cells (21, 22). Even though a high expression of AZIN2 mRNA was initially observed in testis and brain (23), more recent histological studies, using human and mouse tissues, have revealed that the protein is expressed not only in differentiated testicular and neural cells but it also is abundant in secretory cells of the adrenal medulla, pancreas, digestive

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tract, lung, kidney and sweat gland (21,22,24-26). In addition, it was shown that AZIN2 participates in the regulation of vesicular transport in breast cancer cells (27). All these studies

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suggest that AZIN2 may have a role in the secretory or vesicular activity of cells.

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Genomic studies have revealed the existence of many homologous genes of ODC in different species. In the mouse genome, four paralogues are included in the Odc family.

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Functional studies have demonstrated that Azin1 and Azin2 are paralogous of Odc that, although expressed as inactive forms of the enzyme, have acquired regulatory functions (15,16,28). The

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last member, named Gm853, is a predicted gene whose putative function, if any, is still unknown. Since it is localized in the same chromosome as Azin2, it is conceivable that it might

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act as an antizyme inhibitor. Conversely, the conservation of all the essential catalytic residues

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of mouse ODC, suggests that it might have enzymatic activity. To test these possibilities, we studied the activity of the Gm853 protein by means of transient transfection experiments in

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HEK293T cells with an expression plasmid containing the ORF corresponding to Gm853. Our results indicate that this protein does not act as an AZIN, neither have ODC activity, but,

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interestingly, it is able to decarboxylate L-leucine, showing therefore a novel decarboxylase activity, never described before in animal cells.

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ACCEPTED MANUSCRIPT Gm853 is a leucine decarboxylase 2. Experimental prodecures 2.1. Materials. L-[1-14C] ornithine was purchased from American Radiolabeled Chemicals Inc. (St. Louis, MO, USA). L-[U-14C] leucine was purchased from Amersham Biosciences (Buckinghamshire, UK). Anti-FLAG M2 monoclonal antibody peroxidase conjugate, testosterone propionate, EDTA,

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Igepal CA-630, aminooxi acetic acid, cycloheximide, L-ornithine monohydrochloride, L-lysine, L-leucine, D-leucine, DL-norleucine L-valine, L-isoleucine, L-cysteine, L-methionine, L-

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phenylalanine, L-glutamine, L-glycine, L-arginine hydrochloride, L-histidine, L-serine, L-

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isopentylamine hydrochloride, putrescine dyhydrochloride, spermidine trihydrochloride, spermine tetrahydrochloride, 1,6-hexanodiamine, 1,7-diaminoheptane, dansyl chloride, protease

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inhibitor cocktail (4-(2-aminoethyl)benzenesulfonyl fluoride, EDTA, bestatin, E-64, leupeptin, aprotinin), and suberic acid bis (3-sulfo-N-hydroxysuccinimide ester) sodium salt (BS3) were

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obtained from Sigma Aldrich (St. Louis, MO). Lipofectamine 2000 transfection reagent, Dulbecco's Modified Eagle Medium (DMEM GlutaMAX), RPMI 1640 medium, foetal bovine

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serum (FBS) and penicillin/streptomycin were purchased from Invitrogen (Carlsbad, CA).

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Pierce ECL Plus Western Blotting Substrate was from Thermo Scientific (IL, USA). Rabbit anti-ERK2 antibody (SC-154) was purchased from Santa Cruz Biotechnology (Texas, USA). D,L-alpha-difluoromethylornithine (DFMO) was provided by Dr. Patrick Woster (Medical

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2.2. Animals.

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University of South Carolina, Charleston, SC).

Mice were maintained in standard conditions at the Service of Laboratory Animals (University of Murcia). All animal procedures were compliant with the international guidelines of animal welfare and approved by the Bioethics Committee 548/2011 (University of Murcia). C57BL/6 mixed genetic background male and female mice, approximately 3 month old, were used. Testosterone propionate (100mg/kg, dissolved in vegetal oil) was administered by subcutaneous injection, 48 h apart for 8 days. Animals were euthanized, after light anesthesia. Tissues were

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ACCEPTED MANUSCRIPT Gm853 is a leucine decarboxylase dissected, and used for RNA extraction or enzymatic assays. Urine was collected from the urinary bladder after euthanasia. 2.3. Cell culture and transient transfections. Human embryonic kidney (HEK) 293T cells, obtained from ATCC, were cultured in DMEM, containing 10% FBS, 100 units/ml penicillin, and 100 µg/ml streptomycin, in a humidified

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incubator containing 5% CO2 at 37°C. Cells were grown to ~80% confluence and then were

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transiently transfected with Lipofectamine 2000 using 1.5 µl of reagent and 0.3 µg of plasmid per well (12-well plates). In co-transfection experiments, the mixtures contained equimolecular

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amounts of each construct. The plasmid pcDNA3 without gene insertion was used as negative control. After 6 h of incubation the transfection medium was removed, fresh complete medium

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was added, and cells were grown for 16 hours. In most experiments, the cells were collected and

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homogenized for enzymatic assays and Western blot, whereas the culture media was dansylated and analyzed by HPLC. In other experiments, the transfected cells were cultured in RPMI media and incubated with 1 mM of different amino acid solutions, to determine intracellular and

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extracellular polyamine content. All the constructs used in transient transfections were cloned

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into the expression vector pcDNA3.1. The FLAG epitope DYKDDDDK was included to the N terminus of GM853, ODC and AZIN2, and to the C terminus of functional isoforms of AZ1,

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AZ2 and AZ3. Except FLAG-AZIN2 construct (15), the rest of the clones were generated and purchased from GenScript.

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2.4. Real-time and conventional PCR analysis. Total RNA was extracted with PureLink RNA Mini Kit (Life Technologies) and cDNA was generated from 5 g of total RNA using MMLV-Reverse Transcriptase (Sigma) according to manufacturer instructions. The primers used in the analysis of Gm853 transcripts by semiquantitative PCR were: Gm853 E2 Forward (5’-TGCTGGGAGTTGCAGAACAC-3’), Gm853 E9 Reverse (5’-AACTCATGGA GTACTGTAGGCAC-3’) and Gm853 E10 Reverse (5’-GCCAAATGAGCACCACACTG-3’). For real-time PCR the primers were: Gm853 (forward, 5’-TGCTGGGAGTTGCAGAACAC-3’; reverse, 5’-GCGGCCCGAGATGGA-3’, 6

ACCEPTED MANUSCRIPT Gm853 is a leucine decarboxylase Odc

(forward,

5´-ATGGGTTCCAGAGGCCAAA-3’;

reverse

5’-

CTGCTTCATGAGTTGCCACATT-3’), Ddc (forward 5’-GGACTA AAGTTATCCGCCAG3’; reverse 5’-TTCTACAGAGGAATGCGCCT-3’). The values were normalized against the housekeeping gene β-actin (forward, 5’-GATT ACTGCTCTGGCTCCTAGCA-3’; reverse, 5’GCTCAGGAGGAGCAATGATCTT-3’).

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2.5. Western blot analysis.

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Transfected HEK 293T cells were collected in PBS, pelleted, and lysed in solubilization buffer (50 mM Tris–HCl pH 8, 1% Igepal and 1 mM EDTA) with protease inhibitor cocktail (Sigma

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Aldrich). The cell lysate was centrifuged at 14,000 ×g for 20 min. Equal amounts of protein were separated in 10% SDS-PAGE. The resolved proteins were electroblotted to PVDF

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membranes, and the blots were blocked with 5% non-fat dry milk in PBS-T (Tween 0.1%) and

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incubated overnight at 4 °C with the anti-FLAG antibody peroxidase labelled (1:10000). Immunoreactive bands were detected by using ECL Plus Western Blotting Substrate. ERK2, detected by a rabbit anti ERK2 antibody (Santa Cruz, USA), was used as loading control.

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Densitometric analysis was achieved with ImageJ software. 2.6. Cross-linking analysis.

Cell transfected with ODC-FLAG and GM853-FLAG constructs were lysed in solubilization

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buffer, and the lysates were incubated for 1 h at 25°C in 1 mM Tris-HCl pH 7.5, either alone or

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with 1 mM BS3 as crosslinking agent. The cross-linking reaction was terminated by the addition of 1 M Tris–HCl pH 7.5, and the cross-linked material was analyzed by Western blotting and incubation with anti-FLAG antibody. 2.7. Enzymatic measurements. Enzyme activity was measured in both cells transfected with Gm853 (in vivo assays), and in cell homogenates from transfected cells (in vitro assays). Higher activity was observed under in vivo conditions (about 4-fold, Fig. S2B).

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ACCEPTED MANUSCRIPT Gm853 is a leucine decarboxylase a) Decarboxylating activity. Transfected HEK 293T cells were collected in phosphate buffered saline (PBS), pelleted and lysed in solubilization buffer (50 mM Tris-HCl, 1% Igepal and 1 mM EDTA) or buffer B (25 mM phosphate buffer, 0.1 mM PLP, 0.2 mM EDTA and 1mM dithiothreitol) for ODC and LeuDC activities, respectively. For ODC activity determination, the extract was centrifuged at 14,000 ×g for 20 min, and a fraction of the supernatant was taken to a

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final volume of 50µl with buffer A (10 mM Tris-HCl, 0.25 M sacarose, 0.1 mM pyridoxal phosphate, 0.2 mM EDTA and 1 mM dithiothreitol), whereas the cell lysated in buffer B was

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directly used to determine LeuDC activity. Decarboxylating activity was determined in the

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supernatant by measuring 14CO2 released from L-[1-14C] ornithine or L-[U-14C] leucine. The reaction was performed in glass tubes with tightly closed rubber stopper, hanging from the

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stoppers two disks of filter paper wetted in 0.5 M benzethonium hydroxide dissolved in methanol. The samples were incubated at 37°C from 15 to 120 minutes, and the reaction was

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stopped by adding 0.5 ml of 2 M citric acid. The filter paper disks were transferred to scintillation vials and counted by liquid scintillation.

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b) Polyamine content by HPLC. Intracellular polyamines were extracted with 0.4 M perchloric

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acid from pelleted cells, and the supernatant obtained after centrifugation at 10,000g for 10 min was used for polyamine determination by HPLC. For extracellular polyamine determination, a

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fraction of the cell culture media was concentrated with a Speedvac Concentrator (Savant Instruments Inc. Farmingdale, NY, USA) and the resulting residue was resuspended in 0.4 M

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perchloric acid as described previously. In brief, polyamines underwent dansylation according to the method described by Seiler (29) with some modifications, and the dansylated polyamines were separated by HPLC using a BondaPak C18 column (4.6 x 300 mm; Waters) and acetonitrile/water mixtures (running from 70:30 to 96:4 during 30 min of analysis) as mobile phase and at a flow rate of 1 ml/min. 1,6-Hexanediamine and 1,7-diaminoheptane were used as internal standards, and standard solutions of putrescine, spermidine, spermine and isopentylamine were used to calibrate the column. Detection of the derivatives was achieved

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ACCEPTED MANUSCRIPT Gm853 is a leucine decarboxylase using a Waters 420-AC fluorescence detector (Millipore), with a 340-nm excitation filter and a 435-nm emission filter. 2.8. Mass spectrometry/ HPLC-MS/MS analysis. The analysis were carried out on a HPLC-MS system consisting of an Agilent 1100 Series HPLC (Agilent Technologies, Santa Clara, CA, USA) equipped with a thermostated µ-wellplate

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autosampler and a quaternary pump, and connected to an Agilent Ion Trap XCT Plus Mass

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Spectrometer (Agilent Technologies, Santa Clara, CA, USA) using an electrospray (ESI) interface. Samples and standards (40 µl) were injected into a Waters XBridge C18 HPLC

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column (2.1  150 mm, 5 µm, Agilent Technologies), thermostated at 40C, and eluted at a flow

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rate of 200 µl/min during the whole separation. Standards were prepared in MilliQ water. Samples were filtered through Amicon 3K centrifugal units to eliminate proteins and the clean

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filtrates were used for the analysis. Mobile phase A, consisting of 0.1% formic acid (w/v) in MilliQ water, and mobile phase B, consisting of 0.1% formic acid (w/v) in acetonitrile, were used for the chromatographic separation. The initial HPLC running conditions were solvent A:B

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90:10 (v/v). The gradient elution program was 10% solvent B for 10 min; a linear gradient from

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10 to 100% solvent B in 20 min; 10 min at constant 100% solvent B. The column was equilibrated with the starting composition of the mobile phase for 15 min before each analytical

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run. The mass spectrometer was operated in the positive mode with a capillary spray voltage of 3500 V, and a scan speed of 26000 (m/z)/sec (Ultrascan mode) from 50-500 m/z. The nebulizer

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gas pressure, drying gas flow rate, and drying gas temperature were set at 30 psi, 8 l/min, and 350C. Other instrument parameters were optimized for generating the highest signal intensities. Data were obtained in the MS and MS/MS mode using MRM and processed using the DataAnalysis program for LC/MSD Trap Version 3.2 (Bruker Daltonik, GmbH, Germany) provided by the manufacturer. Authentic isopentylamine and putrescine were detected as the [M+H]+ ions at 88.3 and 89.2 m/z, respectively, and they were confirmed with the transitions 88.3>71.4 and 89.2>72.4 m/z, respectively. The ion chromatograms of these compounds from both standards and samples were extracted and the peak area was quantified using the 9

ACCEPTED MANUSCRIPT Gm853 is a leucine decarboxylase DataAnalysis program for LC/MSD Trap Version 3.2 (Bruker Daltonik, GmbH, Germany). The peak area data of standards were used for the calculation of the calibration curve, from which the concentration of compounds in samples was obtained. 2.9. Confocal microscopy. Cells grown on coverslips were transfected with Gm853. Twenty-four hours after transfection,

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cells were fixed with 4% paraformaldehyde in PBS and permeabilized with 0.5% Igepal in PBS.

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For detection of FLAG labeled GM853, cells were incubated with an anti-FLAG M2 monoclonal antibody (1:7.000), followed by an Alexa 488-conjugated secondary antibody

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(1:400). For the staining of nucleus cells were loaded with DAPI (1:10000) during 5 minutes. Finally, samples were mounted by standard procedures using a mounting medium from Dako

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(Carpinteria) and examined with a Leica True Confocal Scanner TCS-SP2 microscope.

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2.10. Statistical analysis.

The data were analyzed by Student's t-test for differences between means. P<0.05 was

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considered statistically significant.

3. Results

paralogues.

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3.1. Comparative study of the genetic and protein structure of murine Gm853 with those of its

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According to the Ensembl genome browser, the murine Gm853 predicted gene is annotated as ENSMUSG00000023120. It is located, as its paralogue mouse Azin 2 (mAzin2), in chromosome 4 and, like this gene, it has 9 coding exons, whereas the other 2 paralogues, mOdc and mAzin1, contain 10 coding exons (Fig. S1). In this regard, it appears that the gene structure of Gm853 is closer to that of Azin2 than to those of Odc or Azin1. Protein sequence comparison of GM853 with its paralogues is shown in Fig. 1. GM853 is the shortest protein among the four homologues, with a length of 425 amino acid residues. Sequence homology, calculated by the Clustal Omega multiple sequence alignment program, showed a 46.1% and 45.7% identity with respect to ODC and AZIN2, respectively, and 41% with AZIN1. Higher dissimilarities were 10

ACCEPTED MANUSCRIPT Gm853 is a leucine decarboxylase found in the N- and C-terminal regions, showing GM853 a C-terminal truncation relative to the other paralogues. Conversely to AZIN1 and AZIN2, GM853 conserves all the 22 amino acid residues of ODC related to the catalytic activity (2,17, 30-34), suggesting that GM853 may have some enzymatic activity. 3.2. Functional aspects of GM853 in transfected HEK 293T cells.

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To determine whether GM853 is a new catalytic form of ODC or a third antizyme inhibitor, we

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used a clone containing the ORF of Gm853 (NCBI Reference Sequence: NM_001034872) in the expression vector pcDNA3.1, fused with the sequence corresponding to the FLAG epitope

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in the N-terminal extreme, to facilitate the detection of the expressed protein using an antiFLAG antibody. To test the possible function of GM853, HEK 293T cells were transiently

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transfected with that clone, and different assays were carried out. First, the capacity of GM853

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to decarboxylate L-ornithine was measured in homogenates of cells transfected with Gm853, by means of a radiometric method. In parallel assays, this activity was also determined in extracts from cells transfected with the empty vector or with clones of mouse Odc or Azin2. Fig. 2A

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shows that ODC activity in ODC-transfected cells, as expected, was remarkably higher than the

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activity of mock-transfected cells. On the contrary, ODC activity in the cells transfected with Gm853 was indistinguishable from that of mock transfected cells. In addition, the ODC activity

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in cells transfected with Gm853 was also significantly much lower than that of the cells transfected with Azin2. These results indicate that GM853 is not able to decarboxylate L-

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ornithine, at least as efficiently as ODC. On the other hand, they also suggest that GM853 does not appear to act as an AZIN. To corroborate that GM853 was unable to abrogate the action of AZs on ODC, HEK 293T cells were co-transfected with ODC and different combinations of plasmids, and ODC activity and protein levels of ODC, AZIN2 and GM853 were analyzed. Whereas AZIN2 was able to rescue ODC activity and ODC protein completely from the action of AZ1, GM853 did not affect the AZ1-mediated inhibition of ODC activity and ODC degradation (Fig. 2B, 2C). This again suggested that it is unlikely that GM853 could act as an antizyme inhibitor. To get further information on any hypothetical interaction between GM853

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ACCEPTED MANUSCRIPT Gm853 is a leucine decarboxylase and AZs, cells were co-transfected with Gm853 and each of the three different Azs. Fig. 3A shows that neither AZ1, nor AZ2 or AZ3 affected the levels of GM853, in contrast to the marked effect of AZ1 on the stimulation of ODC degradation (35) or the protection elicited by the AZs on the degradation of AZIN1 or AZIN2 (36,37). This lack of effect of the AZs on GM853 stability was also observed when protein synthesis was inhibited by treatment of the co-

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transfected cells with cycloheximide for 240 min (Fig. 3B). Importantly, immunoprecipitation experiments were unable to demonstrate binding of GM853 with AZs (results not shown),

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confirming that GM853 lacks antizyme inhibitor activity.

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We next tested whether GM853 may somehow affect polyamine metabolism. For this purpose, we analyzed polyamine levels in transfected cells by HPLC both intracellularly and in

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the extracellular media. Major changes were found in the chromatograms derived from cells transfected with Gm853, in comparison to those obtained from mock-transfected cells (Fig. 4A

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and 4B). In particular, we detected a dansylable metabolite (X) in the culture media of HEK 293T cells transfected with Gm853, which was not present in the media of mock- transfected

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cells (Fig. 4A). This product was also detected, although at lower levels, in the homogenates of

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Gm853 transfected cells (Fig. 4B), and was different from putrescine, as shown in Fig. 4C. These results substantiate the hypothesis that GM853 could transform some component present

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in the DMEM culture media, presumably an amino acid, to generate the dansylable product. When DMEM media was substituted by RPMI, a culture media with lower concentration of

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amino acids (Table S1), the unknown product was still formed, but in significantly lower amounts (Fig. 4D), supporting the contention that some amino acid of the media could be the substrate of GM853. To corroborate this hypothesis, Gm853 transfected cells, grown in RPMI media, were supplemented with 1mM mixtures of different amino acids present in the culture media, and the products formed were analyzed by HPLC. Among all mixtures assayed, only the one formed by the branched-side chain amino acids (leucine, isoleucine and valine) led to a significant increase of the unknown compound (Fig. 4E).

When the RPMI media was

supplemented separately with each amino acid of this group, this product only increased in

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ACCEPTED MANUSCRIPT Gm853 is a leucine decarboxylase presence of leucine (Fig. 4F and Fig. S2A). These results suggested that the dansylated metabolite could be monodansyl isopentylamine and, therefore, that GM853 could act as a decarboxylase of leucine, generating the monoamine 3-methyl butylamine (isopentylamine or isoamylamine) (Fig. 4G). To corroborate this possibility, several additional experiments were performed.

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First, to verify the nature of the product, the HPLC eluted fraction corresponding to the retention time of the dansylated product, presumable dansyl isopentylamine, was analyzed by

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HPLC-mass spectrometry. Fig. 5A shows the mass spectra obtained, in which the major

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compound with a m/z of 320.7 is compatible with the molecular weight of monodansyl isopentylamine. In addition, the HPLC chromatograms of the dansylation of authentic

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isopentylamine hydrochloride and the dansylated products of Gm853 transfected cells were compared. As illustrated in Fig. 5B a perfect match between the peaks corresponding to the

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products in both chromatograms was evident. Moreover, the direct analysis of the media of Gm853 transfected cells by mass spectrometry identified a compound with m/z of 88.3,

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corresponding with the spectra given by authentic isopentylamine (Fig. 5C). Finally, to confirm

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that isopentylamine was formed through the decarboxylation of L-leucine by GM853, the CO2 from

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C-labelled L-leucine, incubated with homogenates of Gm853

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transfected cells, was measured. As shown in Fig. 5D, the lysates from cells transfected with Gm853 were able to decarboxylate L-leucine and that this activity was markedly decreased by

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aminooxy acetic acid (AOA), a general inhibitor of PLP-containing enzymes. From all these results, it can be concluded that the protein encoded by Gm853 is an enzyme with leucine decarboxylase activity (LDC) and that it requires PLP for its catalytic activity. From now on we will call it GM853/LDC or just LDC. 3.3. Properties of leucine decarboxylase encoded by Gm853. Enzyme activity was measured in both cells transfected with Gm853 (in vivo assays), and in cell homogenates from transfected cells (in vitro assays). Higher activity was observed under in vivo conditions (about 4-fold, Fig. S2B). To verify whether this enzyme followed Michaelis-Menten 13

ACCEPTED MANUSCRIPT Gm853 is a leucine decarboxylase kinetics, the rate of isopentylamine formation was measured at different L-leucine concentrations, and the results plotted in Fig. 6A. From the hyperbolic graph obtained, an apparent Km= 7.03±0.73 x 10-3 M was calculated. Table 1 shows the effect of different leucine isomers, on the formation of isopentylamine from L-leucine. Whereas the supplementation of the media with 1mM L-leucine increased about 4-fold the rate of amine synthesis, D-leucine

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inhibited the process more than 50%. The addition of 1mM of L-norleucine, the linear isomer of L-leucine, did not appear to inhibit the enzyme. On the contrary, a marked increase in the

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formation of a product, with a retention time very close to the dansylated form of

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isopentylamine, was observed. These results suggest that the linear isomer of L-leucine could be decarboxylated by the enzyme to generate pentylamine, a compound not distinguishable from

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isopentylamine by HPLC. Table 1 also shows that none of the basic amino acids assayed, either the ODC substrates (L-ornithine and L-lysine) or the substrates of other decarboxylases (L-

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arginine and L-histidine), did inhibit the enzyme. In addition, difluoromethylornithine (DFMO), a potent irreversible inhibitor of mammalian ODC, was unable to decrease the rate of

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isopentylamine formation. In cells co-transfected with Gm853/Ldc and Az1, the enzymatic

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activity was similar to those transfected with Gm85/Ldc3 alone (results not shown), reinforcing the notion that AZ1 does not interact with GM853/LDC.

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Since it is known that the active form of ODC is a homodimer, and that the increase in salt concentration inhibited the enzyme by favoring the dissociation of the dimer into inactive

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monomers (38), the effect of the ionic strength on LDC activity was assayed, by incubating cell homogenates of Gm853/Ldc-transfected cells with increasing amount of NaCl. Fig. 6B clearly shows that leucine decarboxylase activity markedly decreased with the rise in salt concentration, similarly to previous findings in ODC, suggesting that LDC may have an oligomeric structure in solution. Western blot analysis of the LDC-flag, before and after cross-linking experiments with bis(sulfosuccinimidyl) suberate, suggested that the native enzyme may be present within the cell as dimeric and tetrameric forms (Fig. 6C). In order to exclude that LDC could affect the activity of ODC, through the formation of heterodimers, cells were co-transfected with

14

ACCEPTED MANUSCRIPT Gm853 is a leucine decarboxylase Gm853/Ldc and Odc. Neither ODC activity nor ODC protein resulted affected by LDC (Fig. 6 D), excluding that possibility. Finally, the subcellular localization of the enzyme and its metabolic turnover were studied. For the first purpose, HEK 293T cells were transfected with a construct of Gm853/Ldc tagged with the FLAG epitope, and analyzed by confocal microscopy. In addition, cells were

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also transfected with constructs of mouse ODC and AZIN2, as controls. Fig. 6E shows that GM853/LDC, like ODC, is located predominantly in the cytosol, differing from AZIN2 that

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localizes at the ER-Golgi Intermediate Compartment (ERGIC), as shown earlier (39). Cell

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fractionation experiments, using differential centrifugation, also showed that LDC was mainly present in the soluble fraction (results not shown). Further, we studied the half-life of LDC, by

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treating the transfected cells with cycloheximide, a protein synthesis inhibitor, and measuring the levels of the protein at different time after cycloheximide addition. As shown in Fig. 7, LDC

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remained stable for at least 8h, in clear contrast to ODC, which decayed rapidly, with a half-life of approximately 144 min. This marked effect in the rate of protein degradation, could be

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related to the differences in the C-terminal sequence between both proteins.

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3.4. Expression of Gm853/Ldc in murine tissues. The expression of Gm853/Ldc in mice was studied by semiquantitative RT-PCR of RNA

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samples obtained from different male mouse tissues, in which our previous studies had shown that its paralogue Azin2 is expressed (19,21). For this amplification, a forward primer,

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corresponding to exon 2, and two reverse primers corresponding to exon 9 (coding region) and exon 10 (3’UTR) were used. Fig. 8A shows that among all tissues analyzed from male mice, the kidney showed a marked expression of the gene. By contrast, no expression was detected in other tissues such as testis, brain, heart, adrenal gland, epididymis or lung, tissues where Azin2 is significantly expressed (19,21). Of the two bands detected, the lower one corresponded to the expected size, whereas the size of the upper one was slightly smaller than expected. This discrepancy can be due to sequence differences in the 3’UTR between the isolated mRNA and those annotated in Ensembl and NCBI gene banks (40). Since it is well known the existence of 15

ACCEPTED MANUSCRIPT Gm853 is a leucine decarboxylase a marked sexual renal dimorphism in mice, affecting to Odc (41,42) and Dopa decarboxylase (Ddc) (43) expression levels, we decided to analyze the influence of sex on the expression of Gm853/Ldc in the mouse kidney. Fig. 8B shows that the gene expression of Gm853/Ldc was higher in males than in females. In addition, the treatment of female mice with testosterone propionate increased the levels of Gm853/Ldc mRNA in the kidney (Fig. 8C), indicating that its

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expression is regulated by androgens. However, the sex influence on the expression of this gene was less intense than that found for Odc or Ddc (Fig. 8C). Finally, the analysis of the urine of

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female mice detected the presence of isopentylamine, and that its concentration increased after

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testosterone treatment (Fig. 8D).

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4. Discussion

In the present work, we have studied the possible expression and function of the

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predicted gene Gm853, located in chromosome 4 of mouse genome. Gm853 is a paralogous gene in the ODC family included in group IV of amino acid decarboxylases. Genomic studies

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have revealed that most eukaryotic enzyme families include inactive homologues considered as

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“dead” enzymes (44). In the ODC family, it is well established that the protein homologues of ODC denominated as AZINs are devoid of catalytic activity but, interestingly, they exert regulatory functions in polyamine metabolism (5,8,13). The analysis of the gene structure of

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Gm853, together to its localization in the same chromosome as Azin2, initially suggested that

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the product of Gm853 could function as a novel antizyme inhibitor. However, the alignment of its protein sequence with those of other mouse paralogues revealed that it contains all the amino acid residues that are critical for the enzymatic activity of ODC (30-34). As suggested by others, the maintenance of all these critical residues is a good predictor for assigning a decarboxylating activity to new members of the family (2,17). Our results, based on the functional studies of the protein synthesized in the cells transfected with the murine gene Gm853, have clearly shown that this new homologue of ODC possesses decarboxylating activity, and that it does not act as an antizyme inhibitor. More

16

ACCEPTED MANUSCRIPT Gm853 is a leucine decarboxylase interestingly, this enzyme is not able to decarboxylate the basic amino acids L-ornithine or Llysine as ODC does, but instead it can specifically decarboxylate L-leucine to generate isopentylamine, an alkylmonoamine never described to be formed by any metazoan enzyme. In fact, in mammalian cells the first steps in the catabolism of the branched-chain amino acids are catalyzed by branched-side amino acid transaminase and branched-chain α-ketoacid

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dehydrogenase (45). To our knowledge, leucine decarboxylating activity has only been reported in some bacteria, such as Proteus vulgaris (46) or Bacillus sphaericus (47), with the name of

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valine decarboxylase (EC 4.1.1.14). Whereas the bacterial enzyme can decarboxylate the three

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branched-side amino acids (valine, isoleucine and leucine), the murine GM853 cannot decarboxylate valine or isoleucine, suggesting that the branching at the beta-carbon of the side

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chain of the amino acid interferes with the catalytic process. The catalytic capacity of GM853/LDC is not surprising, since it contains, as above mentioned, the 22 amino acids

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required to sustain the enzymatic activity of ODC. It is known that in ODC there are two identical active sites, each made up by catalytic residues contributed from both subunits

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(31,32,48). Sequence homology between GM853/LDC and ODC, together with the similarities

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in the responses to salt concentration and crosslinking, suggest that the generation of the active sites in GM853/LDC would require of interactions between subunits. It appears evident that the shift in the substrate specificity in GM853/LDC· must be related to subtle changes in the

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structure of the active center. One may speculate that the gap of ten amino acid residues in the

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internal segment of GM853/LDC, in comparison to the sequence K293-Y317 in mODC, could be related to some conformational changes affecting the accommodation of the aliphatic chain of the substrate. However, it is clear that X-ray structure determination of GM853 is required to provide additional information on this issue. Two related properties of the new protein described here that differ from those of ODC deserve some consideration. The first is the marked stability of GM853/LDC, which may be related to the lack of the sequences found in the carboxy terminal domain of mouse ODC, required for the rapid degradation of the latter (49-51). The second is its lack of interaction with

17

ACCEPTED MANUSCRIPT Gm853 is a leucine decarboxylase antizymes, because co-transfections with Azs did not affect either the enzymatic activity or the protein stability of GM853/LDC, in clear contrast with ODC or AZINs. The reason for this inability cannot be ascribed to the absence in the GM853/LDC sequence of the segment equivalent to residues 117 to 140 of mouse ODC, which is essential for the interaction with antizymes (52), since a sequence very similar to that of ODC and AZINs is present in

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GM853/LDC. In fact, in GM853/LDC, this so called antizyme-binding region contains the five residues that are conserved in all ODC orthologues and paralogues (34,36). Interestingly, these

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two properties of GM853/LDC are shared with ODC from Trypanosoma brucei, since the

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trypanosomal enzyme also lacks sequences found in the carboxyl terminus of murine ODC (49), and does not interact with antizyme (52). In addition, in both GM853/LDC and T. brucei ODC

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the comparison of the antizyme-binding regions revealed the substitution of the glutamine 120 of mouse ODC and AZINs by a histidine (Fig. S3). Finally, the incapacity of GM853 to form

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heterodimers with ODC is similar to that reported for trypanosomal ODC (53). All these results indicate that the similarity of GM853/LDC with the parasitic ODC is higher than that with the

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mammalian ODC. Although the reasons for this circumstance remain to be elucidated, this

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finding is in agreement with the postulated hypothesis on the horizontal gene transfer of the Trypanosoma brucei Odc gene from a vertebrate source (54).

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Orthologue genes of the murine Gm853 are present in the genomes of several mammals, including some primates (See Table S2). In all the cases where the chromosomal locations are

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known, the orthologue is located at the same chromosome as Azin2, suggesting that both genes are the result of the duplication and divergent evolution of a common ancestral gene with ODC activity. This is a new interesting example of the evolutionary plasticity of the ODC family, in which the new group of paralogues retained the catalytic activity but with changed substrate specificity. This group would correspond with the ODC2/AZID subgroup, proposed by Ivanov and coworkers (17). According to the HUGO Gene Nomenclature Committee (HGNC), the human orthologue of Gm853 is the pseudogene LINC01225 (long intergenic non-protein coding RNA 1225) located, as human AZIN2, in chromosome 1. Recent studies have verified that

18

ACCEPTED MANUSCRIPT Gm853 is a leucine decarboxylase LINC01225 is upregulated in hepatocellular carcinoma, having been suggested that it can act as a biomarker for the diagnosis and prognosis of this type of cancer (55). The relevance of GM853/LDC in mammalian physiology remains to be determined, although its expression profile suggests that it may have some function in the kidney. At present, there are no available data on the possible biological effects of isopentylamine. The

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results presented here reveal that the product formed from leucine by GM853/LDC is excreted in the urine. This finding is in agreement with earlier studies that showed that, in mice, most of

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the putrescine formed in the kidney from ornithine by ODC was eliminated in the urine (49, 56).

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At present, still there are not definitive answers to this behavior. The renal profile of expression of Gm853 in the mouse kidney shown in this work is in agreement with a recent publication that

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showed that the renal protein named (ODCrp, gm853) is responsive to androgens (57).

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In conclusion, our results demonstrate that the predicted gene Gm853 is expressed in mice, and most importantly, that it encodes a protein having leucine decarboxylase activity, catalyzing the formation of a novel biological amine, isopentylamine. Such enzymatic activity,

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never described before in animal cells, presented by GM853, the most recently identified

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homologous protein of ODC, is an interesting example of molecular evolution that derived into a new enzymatic activity. According to the present results, we propose the name of leucine

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descarboxylase (Ldc) for Gm853.

Acknowledgments

We thank Dr. Alex Torrecillas (SAI) for his help in the Mass spectrometry analysis.

Funding This work was supported by the Spanish Ministry of Economy and Competitiveness, grant SAF2011-29051 (with European Community FEDER support) and Seneca Foundation

19

ACCEPTED MANUSCRIPT Gm853 is a leucine decarboxylase (Autonomous Community of Murcia), contract 19875/GERM/15. AL was recipient of a FPU pre-doctoral fellowship.

Conflict of interest

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The authors declare that they have no conflicts of interest with the contents of this article.

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Author contributions

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A.L. and R.P contributed to conception and design of the study; A.L. and D.C. performed the experiments; A.L., B.R.M, A.J.L.C. contributed to the analysis of data and manuscript

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preparation; R.P. directed the project and wrote the manuscript.

20

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ACCEPTED MANUSCRIPT Gm853 is a leucine decarboxylase FIGURE LEGENDS

FIGURE 1. Comparison of the amino acid sequences of mouse GM853, ODC, AZIN1 and AZIN2 using Clustal Omega. Asterisks represent amino acid identity; colon and dots represent amino acid similarity between the proteins. Black background indicates important residues for ODC activity conserved in GM853 and grey background indicates non-conserved residues in AZINs.

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FIGURE 2. Analysis of the putative ODC and AZIN activities of GM853. (A) Cell lysates from HEK293T cells transfected with plasmids containing the coding region of Odc, Azin2, Gm853 or empty vector (pcDNA3.1) were used. After 16 h of transfection, the cells were harvested and homogenized as described in “Experimental procedures”. Top: Western blot of the different proteins detected with anti-Flag antibody. Bottom: Relative ODC activity of the different extracts with respect to those from Odc-transfected cells (100%). (B) Relative ODC activity in cell homogenates from HEK293T cells transfected with single, double or triple combinations of cDNAs. In these experiments, each well contained equimolecular amounts of each construct. ODC activity is expressed as the percentage of the single ODC transfectant in each experiment. Results are the mean±SD of three experiments; (*) P<0.05 vs ODC transfectant; (***) P<0.001 vs ODC +AZ1 double transfectant. (C) Western blot analysis of F-ODC protein in the different transfection experiments. The material was resolved by 10% SDS-PAGE, the bands were detected using anti-Flag and antiERK2 antibodies, and the intensity of each band in the blots was quantified with ImageJ. In the double and triple transfectants, F-ODC protein is expressed as percentage of single ODC-transfectant (ODC %).

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FIGURE 3. Influence of the mouse antizymes (AZ1, AZ2 and AZ3) on the intracellular stability of GM853 protein. (A) Western blot analysis of GM853 in homogenates from cells transfected with Gm853 for 16 h, alone or in combination with each of the three antizymes Az1, Az2 or Az3. F-GM853 was detected with anti-FLAG antibody and ERK2, used as loading control, was determined by means of polyclonal ERK2 antibody. The intensity of each band in the blots was quantified with ImageJ, and the ratio between F-GM853/ERK2 in the single transfectant was considered as 100%. (B) Influence of antizymes on the rate of GM853 degradation in transfected HEK293T. Cycloheximide (CHX) 200 µM was added to the growth media 16 h post-transfection and the cells were harvested after 4 h of incubation. The levels of GM853 were determined by Western blot analysis using anti FLAG antibodies. Each of the above experiments was repeated twice, with similar results.

FIGURE 4. Analysis of the products formed by HEK293T cells transfected with Gm853. After 16 h of transfection, the culture media was aspirated and the cells collected. An aliquot of the media was concentrated to dryness and resuspended in perchloric acid 0.4M, whereas the cells were homogeneized in the same acid (200 µl per well). After centrifugation at 12.000xg for 15 min, the supernatants were dansylated and analyzed by HPLC as described in the Experimental section. The pellets were dissolved in 2M NaOH, and used for protein determination. (A) Overlapped HPLC chromatograms of the dansylated media from cells transfected with Gm853 (red line) or with the empty vector pcDNA 3.1 (green line). Hexanediamine (Hxd) and heptanediamine (Hpd) were used as internal standards. Note the abundance of a dansylated compound (X) that is likely formed from a product present in the media of cells transfected with Gm853, which is absent in the media from the mock transfected cells. (B) Overlapped HPLC chromatograms of homogenates from cells transfected with Gm853 (red line) or with the empty vector pcDNA 3.1 (green line). The same compound (X) was detected, but in lesser amount than in the media. (C) Comparison of the chromatograms of the unknown product (X in the red line) 24

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and dansylated putrescine (green line). (D) Overlapped chromatograms of culture media 16 h after transfection HEK293T cells with Gm853; DMEM Media (red line), RPMI Media (green line). (E) HPLC chromatograms of RPMI Media from cells transfected with Gm853: RPMI alone (control) or supplemented with 1 mM mixtures of different amino acids that are components of the culture media. Note that among all mixtures, only the supplementation with branched-chain amino acids markedly stimulated the unknown compound. (F) HPLC chromatograms of RPMI Media from cells transfected with Gm853: RPMI alone (control) or supplemented with 1 mM solution of each branched-side chain amino acid. Note that only Lleucine markedly increased the formation of X. (G) Panel showing the theoretical decarboxylation reactions of the branched-side amino acids. According to the results shown in panel F, only isopentylamine is the monoamine that is likely to be formed in the media of the Gm853-transfected cells from the decarboxylation of L-leucine.

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FIGURE 5. Analysis of the products formed by the action of GM853. (A) Mass spectra of the dansylated product (X in Fig 4A) corresponding to the 1 ml fraction collected between 8.5-9.5 min after injection to the HPLC column. The m/z=320.7 value of the main compound matched with the calculated theoretical value of monodansyl isopentylamine. (B) HPLC chromatograms showing the correspondence between the retention times of the dansylated product X (Fig. 4A) and authentic dansylated isopentylamine. (C) MS spectra of authentic isopentylamine showing a peak located at 88.3 m/z. MS/MS showing the fragmentation of the compound, giving a peak at 71.4 m/z. (D) Leucine decarboxylase activity determined by the radiometric method. 14CO2 release from cell homogenates from Gm853 transfected cells incubated with 14C(U)-L-leucine. ND, non-detectable; AOA, aminooxy acetic acid 2mM.

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FIGURE 6. Kinetics and other properties of GM853/LDC. (A) Influence of substrate concentration on the rate of formation of isopentylamine in homogenates from by HEK 293T cells transfected with Gm853. After 15 h of transfection, cells were lysed in 25 mM phosphate buffer (pH 7.2) containing 0.1 mM pyridoxal phosphate, 0.2 mM EDTA, 1 mM dithiothreitol, and the homogenates were incubated with different L-leucine concentrations for a period of 3h. Isopentylamine was analyzed by HPLC as described in the experimental section. Duplicated values for each concentration were plotted, and the apparent Michaelis-Menten constant was calculated by using a nonlinear regression analysis (GraphPad software). Km= 7.03±0.73 x 10-3 M, Vm= 32.1±1.6 nmol/h (r2=0.995). (B) Influence of salt concentration on the enzymatic activity of GM853/LDC. Cell homogenates of Gm853 transfected cells, obtained as in A, were incubated with different concentration of NaCl, and the amount of isopentylamine formed was analyzed by HPLC. Results are means of duplicate determinations. (C) Western blot analysis of GM853/LDC before and after incubation with bis(sulfosuccinimidyl) suberate (BS3) as crosslinking agent. Cell homogenates from Gm853 transfected cells were incubated for 1h with 1mM BS3. Proteins were analyzed by SDS/PAGE/immunobloting using anti-FLAG antibody. (D) Left. Influence of GM853 on the enzymatic activity of ODC. Cell homogenates from single and double transfectants of Gm853 and Odc were analyzed for ODC activity, using the radiometric method. GM853 did not significantly affect the decarboxylating activity of ODC. Results are expressed as percentage of ODC single transfectants (mean±SD from triplicate determinations). Right. Influence of GM853 on ODC protein levels. Proteins were analyzed by SDS/PAGE/immunoblotting using anti-FLAG antibody. No significant variation on protein levels was detected. (E) Subcellular location of GM853/LDC in transfected cells. Laser scanning confocal micrographs of HEK 293T cells transfected with GM853 fused to the FLAG epitope. After transfection, cells were fixed, permeabilized and stained with anti-FLAG antibody and ALEXA anti-mouse, and then examined in a confocal microscope. Nuclei were stained with DAPI.

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ACCEPTED MANUSCRIPT Gm853 is a leucine decarboxylase FIGURE 7. Protein stability of GM853/LDC in transfected cells. Left panel: after 16 h of transfection, either with Gm853 or Odc, cells were incubated with 200 µM cycloheximide (CHX), and harvested at the indicated times, lysed in buffer containing a protease inhibitor cocktail, and GM853 and ODC protein levels determined by Western blot analysis and incubation with anti-FLAG antibody. The indicated percentages on values at time 0 represent a mean of two experiments. Right panel: Half-lives of GM853 and ODC in transfected cells calculated by linear regression analysis (GraphPad software); t1/2 ODC= 144 min.; t1/2 GM853> 8h. (*) Statistical significance of slope comparison: P<0.001

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FIGURE 8. Expression of Gm853/Ldc in mice. (A) Total RNA was extracted from tissues of C57BL/6J adult male mice and analyzed by semiquantitative RT-PCR using specific primers as described under “Experimental procedures”. (B) Comparison of Gm853 mRNA levels in the kidneys of male and female mice. Results from quantitative RT-PCR were normalized with respect to β-actin and expressed as percentage of male values.* P<0.05 (n=4). (C) Influence of testosterone treatment (100 mg/kg, 8 days) on the expression of Gm853, Odc and Ddc (DOPA decarboxylase) in the kidney of female mice. Results from quantitative RT-PCR were normalized with respect to β-actin. * P<0.05 (n=3 animals per group). (D) Influence of testosterone on the excretion of isopentylamine and putrescine in the urine of female mice. Urine from control and testosterone treated females were analyzed by HPLC/MS. ** P<0.01; *** P<0.001.

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Figure 2

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Figure 4

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Figure 5

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ACCEPTED MANUSCRIPT Gm853 is a leucine decarboxylase TABLE 1. Influence of different amino acids on the leucine decarboxylase activity of GM853/LDC.

Relative enzymatic activity

100 425 42 402* 113 107 103 101 99

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Control (RPMI Media) L-leucine D-leucine L-norleucine L-ornithine L-lysine L-arginine L-histidine Difluoromethylornithine

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Amino acid

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Enzyme activity was assayed in HEK293T cells transfected with Gm853 grown in RPMI media (containing 0.38 mM L-leucine). After 15h of transfection, the initial media was changed by fresh RPMI media, without added amino acids (control) or supplemented with 1mM of different amino acids, and the formation of isopentylamine was analyzed by HPLC.

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(*) Isopentylamine+Pentylamine.

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ACCEPTED MANUSCRIPT Gm853 is a leucine decarboxylase Highlights

The mouse gene Gm853 encodes a protein with a novel leucine decarboxylating activity.

Contrary to its paralogue ODC, GM853 does not decarboxylate basic amino acids.

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GM853 is a stable protein that does not act as an antizyme inhibitor.

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Gm853 is expressed in the kidney, generating isopentylamine excreted in the urine.

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