Mouse spermine oxidase gene splice variants Nuclear subcellular localization of a novel active isoform Manuela Cervelli 1, *, Alessandro Bellini 1, *, Marzia Bianchi 1 , Lucia Marcocci 2 , Stefania Nocera 2 , Fabio Polticelli 1 , Rodolfo Federico 1 , Roberto Amendola 3 and Paolo Mariottini 1 1 Dipartimento di Biologia, Universita ` ‘Roma Tre’, Rome, Italy; 2 Dipartimento di Scienze Biochimiche ‘A. Rossi Fanelli’, Universita ` ‘La Sapienza’, Rome, Italy; 3 Istituto per la Radioprotezione, ENEA, CR Casaccia, Roma, Italy Spermine oxidase (SMO) is a flavoenzyme involved in polyamine homeostasis in animal cells. The mouse spermine oxidase gene (mSMO) codes for splice variants, including the previously reported major active isoform, herein named alfa (a). In the present work, eight additional gene splicing variants were characterized. The heterologous expression and biochemical characterization of three recombinant iso- forms (namely mSMOl,-c and -d) revealed that only the recombinant protein mSMOl displays biochemical charac- teristics similar to those of mSMOa; the other two recom- binant proteins contained no detectable SMO activity. In order to investigate in greater detail, the SMO enzyme activity associated with their subcellular localization, mSMOa and -l V5-tagged proteins were transiently and stably transfected in the murine neuroblastoma cell line, N18TG2. Very interestingly, the novel active mSMOl isoform was found to be present in both nuclear and cyto- plasmic compartments, thus providing the first evidence of SMO activity in the nucleus, while a cytoplasmic localization was confirmed for the mSMOa isoform. In addition, the relative transcription levels of the gene splicing variants were evaluated by RT-PCR analysis to verify a relationship with the SMO enzyme activity in various murine organs. Keywords: enzyme isoform; mouse; spermine oxidase; subcellular localization. In mammalian cells the maintenance of polyamine homeo- stasis is accomplished by the concerted action of three kinds of enzymes, namely spermidine/spermine N 1 -acetyltrans- ferase (SSAT), polyamine oxidase (PAO) and spermine oxidase (SMO). SSAT is responsible for the addition of N 1 -acetyl groups to both spermine (Spm) and spermidine (Spd) [1] and does not play a major role in the polyamine catabolic pathway in mouse embryonic stem cells, as dem- onstrated by Niiranen et al. [2]. A large number of studies have suggested that the depletion of polyamines and growth inhibition by polyamine analogues are involved in SSAT induction [3–8]. Very recently, Chen et al. [9] have identified and characterized a second human SSAT enzyme, named SSAT-2, which is putatively involved in basal or perturbed polyamine metabolism. The acetylated polyamines N 1 -acetylSpm (N 1 -AcSpm) and N 1 -acetylSpd (N 1 -AcSpd) are oxidized by the FAD-containing enzyme, PAO, to yield stoichiometric amounts of Spd and putrescine, respectively, plus 3-acetamidopropanal and H 2 O 2 [10–13]. PAO has been found in all vertebrate tissues [11], and purification by chromatographic methods has shown that it is localized subcellularly in both cytoplasm and peroxisomes [14–16]. Vujcic et al. [17], using a functional genomics approach, identified murine and human PAOs and demonstrated that PAO expression is inducible by polyamine analogues. Recently, the murine PAO was expressed in Escherichia coli cells and further characterized in its biochemical, spectro- scopic, redox and steady-state kinetic properties by Wu et al. [13], who named this enzyme N 1 -acetylated polyamine oxidase. As PAO plays a crucial role in polyamine catabolism, the mammalian enzyme has been considered as an important drug target and, in fact, it has been shown that a number of polyamine analogues have an antitumor effect in different cell lines [18–21]. The flavoprotein SMO is the most recently characterized enzyme found to be involved in mammalian polyamine homeostasis, being able to preferentially oxidize Spm and producing Spd, 3-amino- propanal and H 2 O 2 as reaction products [22–25]. On the other hand, Wang et al. [23] have recently reported that the human recombinant PAOh1/SMO enzyme is also able to oxidize N 1 -AcSpm, and shows a K m value for Spm that is 100 times lower than that found for the mouse SMO (mSMO). In particular, the mammalian enzyme was expressed, using an in vitro transcription/translation system [22], in transiently transfected human kidney 293 cells [24] and in E. coli BL21 DE3 cells [23,25]. The discovery of this novel enzymatic activity has enriched the complexity of the traditional polyamine interconversion pathway in which Spm is first acetylated by SSAT and then oxidized by PAO, because mammalian cells contain an enzyme capable of Correspondence to P. Mariottini, Dipartimento di Biologia, Universita` degli Studi ÔRoma TreÕ, Viale Guglielmo Marconi 446, 00146 Roma, Italy. Fax: + 39 06 55176321, Tel.: + 39 06 55176359, E-mail: [email protected]Abbreviations: HT, His-Tag; mPAO, mouse PAO; mSMO, mouse SMO; N 1 -acetylSpd, N 1 -acetyl derivative of spermidine; N 1 -acetyl- Spm, N 1 -acetyl derivative of spermine; PAO, polyamine oxidase; PAOh1/SMO, human SMO; SMO, spermine oxidase; Spd, spermidine; Spm, spermine; SSAT, spermidine/spermine N 1 -acetyltransferase. Enzyme: vertebrate polyamine oxidase (EC 1.5.3.11). *These authors provided equal contribution to this work. (Received 23 October 2003, revised 5 December 2003, accepted 5 January 2004) Eur. J. Biochem. 271, 760–770 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.03979.x
Transcript
Mouse spermine oxidase gene splice variantsNuclear subcellular localization of a novel active isoform
Manuela Cervelli1,*, Alessandro Bellini1,*, Marzia Bianchi1, Lucia Marcocci2, Stefania Nocera2,Fabio Polticelli1, Rodolfo Federico1, Roberto Amendola3 and Paolo Mariottini1
1Dipartimento di Biologia, Universita ‘Roma Tre’, Rome, Italy; 2Dipartimento di Scienze Biochimiche ‘A. Rossi Fanelli’,
Universita ‘La Sapienza’, Rome, Italy; 3Istituto per la Radioprotezione, ENEA, CR Casaccia, Roma, Italy
Spermine oxidase (SMO) is a flavoenzyme involved inpolyamine homeostasis in animal cells. The mouse spermineoxidase gene (mSMO) codes for splice variants, including thepreviously reported major active isoform, herein named alfa(a). In the present work, eight additional gene splicingvariants were characterized. The heterologous expressionand biochemical characterization of three recombinant iso-forms (namely mSMOl, -c and -d) revealed that only therecombinant protein mSMOl displays biochemical charac-teristics similar to those of mSMOa; the other two recom-binant proteins contained no detectable SMO activity. Inorder to investigate in greater detail, the SMO enzymeactivity associated with their subcellular localization,
mSMOa and -l V5-tagged proteins were transiently andstably transfected in the murine neuroblastoma cell line,N18TG2. Very interestingly, the novel active mSMOlisoform was found to be present in both nuclear and cyto-plasmic compartments, thus providing the first evidence ofSMOactivity in the nucleus, while a cytoplasmic localizationwas confirmed for the mSMOa isoform. In addition, therelative transcription levels of the gene splicing variants wereevaluated by RT-PCR analysis to verify a relationship withthe SMO enzyme activity in various murine organs.
In mammalian cells the maintenance of polyamine homeo-stasis is accomplished by the concerted action of three kindsof enzymes, namely spermidine/spermine N1-acetyltrans-ferase (SSAT), polyamine oxidase (PAO) and spermineoxidase (SMO). SSAT is responsible for the addition ofN1-acetyl groups to both spermine (Spm) and spermidine(Spd) [1] and does not play a major role in the polyaminecatabolic pathway in mouse embryonic stem cells, as dem-onstrated by Niiranen et al. [2]. A large number of studieshave suggested that the depletion of polyamines and growthinhibition by polyamine analogues are involved in SSATinduction [3–8]. Very recently, Chen et al. [9] have identifiedand characterized a second human SSAT enzyme, namedSSAT-2, which is putatively involved in basal or perturbedpolyamine metabolism. The acetylated polyaminesN1-acetylSpm (N1-AcSpm) and N1-acetylSpd (N1-AcSpd)are oxidized by the FAD-containing enzyme, PAO, to yield
stoichiometric amounts of Spd and putrescine, respectively,plus 3-acetamidopropanal and H2O2 [10–13]. PAO has beenfound in all vertebrate tissues [11], and purification bychromatographic methods has shown that it is localizedsubcellularly in both cytoplasm and peroxisomes [14–16].Vujcic et al. [17], using a functional genomics approach,identified murine and human PAOs and demonstrated thatPAO expression is inducible by polyamine analogues.Recently, the murine PAO was expressed in Escherichia colicells and further characterized in its biochemical, spectro-scopic, redox and steady-state kinetic properties by Wuet al. [13], who named this enzyme N1-acetylated polyamineoxidase. As PAO plays a crucial role in polyaminecatabolism, the mammalian enzyme has been consideredas an important drug target and, in fact, it has been shownthat a number of polyamine analogues have an antitumoreffect in different cell lines [18–21]. The flavoprotein SMO isthe most recently characterized enzyme found to be involvedin mammalian polyamine homeostasis, being able topreferentially oxidize Spm and producing Spd, 3-amino-propanal and H2O2 as reaction products [22–25]. On theother hand, Wang et al. [23] have recently reported that thehuman recombinant PAOh1/SMO enzyme is also able tooxidize N1-AcSpm, and shows a Km value for Spm that is100 times lower than that found for the mouse SMO(mSMO). In particular, the mammalian enzyme wasexpressed, using an in vitro transcription/translation system[22], in transiently transfected human kidney 293 cells [24]and in E. coli BL21 DE3 cells [23,25]. The discovery of thisnovel enzymatic activity has enriched the complexity of thetraditional polyamine interconversion pathway in whichSpm is first acetylated by SSAT and then oxidized by PAO,because mammalian cells contain an enzyme capable of
Correspondence to P. Mariottini, Dipartimento di Biologia, Universita
degli Studi �Roma Tre�, Viale Guglielmo Marconi 446, 00146 Roma,
*These authors provided equal contribution to this work.
(Received 23 October 2003, revised 5 December 2003,
accepted 5 January 2004)
Eur. J. Biochem. 271, 760–770 (2004) � FEBS 2004 doi:10.1111/j.1432-1033.2004.03979.x
directly oxidizing Spm to Spd. In this article we report that,similarly to the human counterpart, the mSMO gene ispresent as a single copy which codes for at least nine splicevariants, including that previously reported and character-ized as the major active isoform, named mSMOa herein.Cloning, heterologous expression and characterization ofthree additional SMO splice variants (namely mSMOl, -cand -d) revealed that the splicing variant coding for thenovel isoform, mSMOl, shows identical biochemical char-acteristics to those reported for mSMOa [8,17,22,25]. Theother two isoforms examined do not display any detectableSMO activity. RT-PCR analysis revealed different tran-scription levels of the gene splicing variants (correspondingto mSMOa, -l, -c and -d) in various mature murine tissues,which have been also examined for SMO enzyme activity.Furthermore, the subcellular localization of mSMOa, -l, -cand -d V5-tagged proteins, in the transiently and stablytransfected murine neuroblastoma cell line N18TG2, wasalso investigated. Very interestingly, the enzymatically activemSMOa isoform and the nonactive isoforms mSMOc andmSMOd are localized in the cytoplasm, while the activemSMOl isoform is localized in the nucleus. This is the firstindication of an SMO activity present in the nucleus andcontrasts with the cytoplasmic localization of the canonicalmSMOa enzyme.
Materials and methods
Reagents and other materials
Spd, Spm, N1-AcSpd and N1-AcSpm were purchased fromSigma-Aldrich-Fluka. Restriction and DNA-modifyingenzymes were purchased from MBI Fermetas. Otherchemicals were obtained from Sigma, Bio-Rad andJ. T. Baker.
Animals and organ sampling
Adult male DBA2 mice (Charles River, Italy) weremaintained under a standardized 12-h light:12-h dark cycleand had access to a standard diet and water at libitum.Mice were deeply anesthesised [Avertin, 240 lg per g bodyweight (i.p.)] and then killed by decapitation and thevarious organs were removed immediately afterwards,frozen in liquid nitrogen and stored at )80 �C for furtherhomogenate preparation. Mice were handled in accordancewith the guidelines of the European Communities CouncilDirective of 24 November 1986 (86/609/EEC).
Preparation of tissue or cell homogenates
Thawed samples of different organs and cells were homo-genized in 0.1 M borate buffer containing 1 mM EDTA,pH 8.5, then sonicated and dialyzed overnight against 1000volumes of the same buffer.
Amine oxidase activity of tissue and cell homogenates
The enzyme activity of organ and cell homogenates wasdetermined fluorometrically, according to Seiler et al. [26],with slight modifications, by measuring the production ofH2O2 upon substrate oxidation. Briefly, dialyzed samples
were preincubated at a concentration of 0.8 mgÆmL)1
protein at 37 �C for 45 min in 0.1 M borate buffer, 1 mM
EDTA (pH 8.5), in the presence of 3 mM sodium azide,0.2 mM 2-bromoethylamine, 0.15 lM clorgyline and 15 lM
deprenyl, to inhibit catalase, copper-containing amineoxidase and mitochondrial oxidase isoforms A and B,respectively. Then, 9 UÆmL)1 horseradish peroxidase,0.9 mM homovanillic acid and 1 mM polyamines wereadded and the samples incubated for a further 2 h. Spmand N1-AcSpm or N1-AcSpd, were used as the substrates ofmSMO and mPAO, respectively. Control samples wereincubated in the absence of polyamines. At 20 minintervals, aliquots were withdrawn from each sample anddiluted in 0.1 M NaOH. The fluorescence intensity was thenmeasured at 425 nm upon excitation at 315 nm; for eachsample a linear, time-dependent increase of fluorescencewas obtained and compared with standard curves preparedusing a serial dilution range of commercial H2O2. Theenzyme activity was then calculated after subtraction ofblank values and expressed as pmol H2O2 produced permin)1Æmg)1 of protein. Protein content was estimated bythe method of Markwell et al. [27] with bovine albumin asa standard.
DNA methodology and construction of mSMOexpression plasmids
The methods described by Sambrook et al. [28] were usedfor the extraction and manipulation of RNA and plasmidDNA, and general in vitro DNA manipulations. Nucleotidesequencing was carried out on both strands using theautomated fluorescent dye terminator technique (PerkinElmer ABI model 373A).
mSMO splice variant cloning
SMO isoforms were isolated using RT-PCR on total RNAfrom mouse brain. PCR products were ligated into thepGEMT-easy vector (Promega), sequenced and analyzedby comparison with the murine genomic SMO sequencelocated in GenBank (accession number AF498364). Novelsequences corresponding to alternative splicing were furthercloned into pET25b (Novagen) and pcDNA3-V5-TAG(Invitrogen) vectors, resequenced to check the accuracy ofthe nucleotide sequences and then utilized to transformE. coli BL21 DE3 cells (Novagen) and transfect theneuroblastoma N18TG2 cells [29]. Accession numbers(EMBL) for the mSMO splice variant cDNA sequencesare as follows: mSMOl, AJ567473; mSMOc, AJ567474;mSMOd, AJ567475; mSMOb, AJ567476; mSMOe,AJ567477; mSMO/, AJ567478; mSMOx, AJ567479; andmSMOg, AJ567480.
RT-PCR analysis of mSMO gene splice variantsin different murine tissues
The relative levels of mSMO gene splice variants weremeasured by RT-PCR using a set of primers based on thenucleotide sequence of the cloned alternative splicingproducts and designed to be specific for each splice variant.The design of the primers followed two criteria (a) primerpairs designed to anneal to noncontiguous exons, to check
� FEBS 2004 Expression of mouse spermine oxidase gene variants (Eur. J. Biochem. 271) 761
for the presence of genomic DNA and (b) primers designedto cover portions of contiguous exons, to ensure splicevariant specificity (Fig. 1). Total RNA was isolated fromdifferent tissues using the TRIZOL reagent (Gibco BRL),according to the manufacturer’s instructions. cDNA syn-thesis from the RNA of different mouse organs wasperformed by using primer random examers in a 20 lLreaction volume containing 1 lg of total RNA, according tothe manufacturer’s instructions (SuperScript First-StrandSynthesis System for RT-PCR; Invitrogen). The mSMOaspecific primer-pairs were: SMOa1 forward, 5¢-GTACCTGAAGGTGGAGAGC-3¢ and SMOa2 reverse, 5¢-TGCATGGGCGCTGTCTTGG-3¢; the mSMOl specific pri-mer-pairs were: SMOl1 forward, 5¢-CAGAGCAGCAGCCTGGTCACC-3¢ and SMOl2 reverse, 5¢-GGGCCCCTGCTGGAAGAGGTCTCG-3¢; the mSMOc specific pri-mer-pairs were: mSMOc1 forward, 5¢-AGTTCACAGCCCATGCAG-3¢ and SMOc2 reverse 5¢-AGGAACACATTTGGCAGTGAG-3¢; the mSMOd specific primer wasSMOd1 forward, 5¢-TTATACAACGAGCCCATGCAG-3¢,used together with SMOl2. Aliquots of reverse-transcribedRNA were amplified within the linear range by 20, 25 or 30PCR cycles (denaturation at 94 �C for 1 min, annealing at60 �C for 1 min and extension at 72 �C for 1 min). Inparallel, aliquots of reverse-transcribed RNA were ampli-fied using Taq DNA polymerase (Pharmacia), undersaturating experimental conditions, by 35 or 40 PCR cyclesto detect small quantities of cDNA (results not shown). TheRT-PCRs were normalized by comparison with the fourcontrols described below. The control primer-pairs formPAO, mSSAT and murine rpS7 and b-actin cDNAswere, respectively (a) mPAO-forward 5¢-GAGCCACCACTGCCTGCC-3¢ and mPAO-reverse 5¢-CCATGTGTGGCTTCCCC-3¢, corresponding to the regionbetween nucleotides 933 and 950 and nucleotides 1460and 1443, respectively, of the mPAO cDNA (GenBankaccession number XM_133921), (b) mSSAT-forward
5¢-CGTCCAGCCACTGCCTCTG-3¢ and mSSAT-reverse5¢-GCAAGTACTCTTTGTCAATCTTG-3¢, correspond-ing to the region between nucleotides 108 and 126 andnucleotides 582 and 560, respectively, of the mSSAT cDNA(GenBank accession number NM_009121), (c) rpS7-forward 5¢-CGAAGTTGGTCGG-3¢ and rpS7-reverse5¢-GGGAATTCAAAATTAACATCC-3¢, correspondingto the region between nucleotides 232 and 250 andnucleotides 648 and 629, respectively, of the rpS7 cDNA(GenBank accession number NM_011300), and (d) b-actin-forward 5¢-TGTTACCAACTGGGACGACA-3¢ andb-actin-reverse 5¢-AAGGAAGGCTGGAAAAGAGC-3¢,corresponding to the region between nucleotides 305 and320 and nucleotides 877 and 859, respectively, of the b-actincDNA (GenBank accession number X03765). Furthercontrol reaction mixtures, either without template (Figs 4and 5) or RT enzyme (results not shown), were prepared inparallel. These samples were uniformly negative.
Expression of mSMO gene splice variants in E. coli cells
E. coli BL21DE3 cells, transformed with pmSMOa,pmSMOl, pmSMOc and pmSMOd plasmids, were cul-tured at 30 �C, in LB (Luria–Bertani) medium containing50 lgÆmL)1 ampicillin, to an attenuance (D), at 660 nm,of 0.6, and then induced with isopropyl thio-b-D-galacto-pyranoside (0.4 mM final concentration), followed byfurther growth for 5 h at 30 �C. The E. coli BL21 DE3cells were harvested by centrifugation (4 �C, 10 min,10 000 g), washed with 0.4 culture volumes of 30 mM
Tris/HCl (pH 8.0), containing 20% sucrose and 1 mM
EDTA, and incubated for 5–10 min at room temperature.The suspension was then centrifuged (10 000 g, 10 min,4 �C) and the cell pellet resuspended in 0.05 culturevolumes of ice-cold 5 mM MgSO4 with vigorous shakingfor 10 min on ice. The cell resuspension wasthen centrifuged at 10 000 g for 10 min at 4 �C. The
Fig. 1. Exon structures of mouse spermine
oxidase (mSMO) splice variants. The sche-
matic representation is not in scale. Light gray
and dark gray boxes represent noncoding and
coding sequences, respectively. Black boxes
represent different ORFs. Exon VIa is marked
by a striped box. Positioning of the oligo-
nucleotides used as primers for RT-PCR
analysis are indicated.
762 M. Cervelli et al. (Eur. J. Biochem. 271) � FEBS 2004
supernatant, corresponding to the periplasmic fraction,was collected.
Rapid affinity purification of mSMO isoforms withpET His-Tag (HT) systems
The supernatant from E. coli BL21DE3 cells, trans-formed with the plasmids pmSMOa-HT, pmSMOl-HT,pmSMOc-HT and pmSMOd-HT, respectively, was ap-plied to a column (3 mL) with Ni2+ cations immobilizedon the His-Bind resin (Novagen), equilibrated with Bind-ing Buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris/HCl,pH 7.9). The column was washed with 60 mM imidazole,0.5 M NaCl, 20 mM Tris/HCl (pH 7.9), followed by elutionwith 750 mM imidazole, 0.5 M NaCl, 20 mM Tris/HCl(pH 7.9).
Determination of recombinant mSMO enzyme activityand kinetic constants
Enzyme activity was measured spectrophotometrically byfollowing the formation of a pink adduct (e515 ¼ 2.6 · 104
M)1Æcm)1) as a result of the oxidation and following
condensation of aminoantipyrine and 3,5-dichloro-2-hydroxybenzenesulfonic acid catalyzed by horseradish per-oxidase [30]. The measurements were performed in 0.1 M
sodium phosphate buffer, pH 8.5, with different substratesat various concentrations. Km and kcat values were deter-mined, using Spm as a substrate at concentrations rangingfrom 50 to 500 lM, while the concentration of mSMOisoforms was 2.0 · 10)8
M. Enzyme activities wereexpressed in International Units (IU; the enzyme concen-tration catalyzing the oxidation of 1 lmol of substrate perminute) per L of culture broth. Protein content wasestimated by using the method of Bradford [31] with BSAas a standard. SDS/PAGE was performed according to themethod of Laemmli [32].
Expression of mSMO gene splice variants in murineneuroblastoma N18TG2 cells
mSMOa, -l, -c and -d cDNA coding sequences were clonedinto the directional pcDNA3-V5-TAG plasmid (Invitrogen),following the manufacturer’s instructions, to producerecombinantV5-taggedproteins.ThemurineneuroblastomaN18TG2 cell line was cultured in DMEM (Dulbecco’smodified Eagle’s medium) containing 10% FBS, at 37 �C,5% CO2, in a humid incubator. The day before transfec-tion, three six-well chambers were seeded with 105 cells/well.Two micrograms of each recombinant pcDNA3-V5-TAGplasmid for the mSMOa, -l, -c and -d isoforms, plus acontrol plasmid, were chemically transfected into the hostcells using the Superfect reagent (Qiagen), according to themanufacturer’s instructions. Transfections were performedin triplicate, and the parental N18TG2 cell line wascontemporary grown in each six-well chamber. Forty-eighthours after transfection, cells were divided into T75 cultureflasks and a selective medium (300 lgÆmL)1 G418) wasadded to the transfected cells to produce three separatepools of stable transfected cell lines for each mSMOisoform, plus a control plasmid. After 2 weeks under G418selection, aliquots of each transfected N18TG2 cell pool
were seeded on chamber slides and, 24 h later, fixed (for15 min at 4 �C) with freshly prepared NaCl/Pi containing3.7% paraformaldehyde to determine the subcellular local-ization. The production of transfected variants in each poolof cells was analysed by RT-PCR, utilizing the same primer-pairs described above (see �RT-PCR analysis of mSMOgene splice variants in different murine tissues�). Threealiquots of 2 · 107 cells for each pcDNA3/mSMOa, -l, -cand -d/V5-tagged proteins stably transfected were analyzedfor mSMO activity. The subcellular localization ofmSMOa, -l, -c and -d/V5-tagged proteins was analysedby indirect immunofluorescence, using the mouse anti-V5mAb (Sigma) (1 lgÆmL)1, 1% BSA in NaCl/Pi) followed bya secondary reaction with goat polyclonal anti-mouse IgGconjugated to FITC (Sigma) (1: 60 dilution, 1% BSA inNaCl/Pi). Nuclei were counterstained by 4¢,6¢-diamidino-2-phenylindole (DAPI) and microphotographies were takenusing Zeiss Axioskop microscopy (Carl Zeiss, Milano,Italy), assisted by the digital Polaroid camera DMC II (oilimmersion lens, 63·). Confocal images of pcDNA3/mSMOl/V5 were taken by confocal microscopy(LSM510; Carl Zeiss, Milano, Italy) using propidium iodideto counterstain the nuclei.
Results
Cloning, sequencing of mSMO splice variants andsequence analysis
As it has been previously reported that mouse and humanSMO genes are single copy genes with the ability to code forsplice variants [8,17,22], as an initial step to characterize themSMO gene we carried out a Southern blot analysis (resultsnot shown), which confirmed the presence of a single copygene in the mouse genome. cDNAs from mouse brain wereisolated by RT-PCR, cloned and sequenced. Sequencecomparison of the cloned cDNAs with the mSMO gene(GenBank accession number AF498364) revealed that thereare at least nine splice variants, named in this work as a, b, c,d, l, /, e, g and x, whose exon structures are schematized inFig. 1. The deduced amino acid sequences are shown inFig. 2. All the isoforms share a common N-terminal portionconsisting of the same ORF spanning exon II to exon IV,with the exception of the mSMOd variant. The isoforms canbe divided in two groups according to their length, resultingin long (a,l,candb) andshort (d, e,g,xand/) translationalproducts. Among the long isoforms, besides the major splicevariant, mSMOa, only mSMOl retains the capability tobind the prosthetic group (Fig. 2), as all the protein regionsknown to interact with the FAD cofactor are present inthis splice variant [33,34]. On the contrary, mSMOc andmSMOb proteins are missing the exon VI or a portion of it,respectively, andhave anORFshifting at theC-terminus.Asa consequence, the resulting protein sequences lack theC-terminal region that interacts with the FAD cofactor.The mSMOd protein does not appear to be able to bind theprosthetic group, because it shows a more dramatic rear-rangement, lacking exons IV,VandVI, anddoesnot containthe FAD-binding regions (Fig. 2). All the remaining shortisoforms, which range from one-third to one-half of thelength of mSMOa, show rearrangements at the C-terminusowing to exon skipping and/or ORF shifting (Fig. 2).
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Fig. 2. Amino acid sequence alignment of the mouse spermine oxidase (mSMO) isoforms. Underlined amino acid sequences represent different
ORFs. Asterisks label the flavin-binding domain sequences [33,34]. The VIa exon is represented by a black box with white letters. Symbols used:
cb, major splicing site; ., minor splicing site.
764 M. Cervelli et al. (Eur. J. Biochem. 271) � FEBS 2004
Expression and purification of mSMO isoformsin E. coli cells
Four recombinant cDNA constructs (pmSMOa-HT,pmSMOl-HT, pmSMOc-HT and pmSMOd-HT), corres-ponding to the four secreted-tagged isoforms, were used totransform E. coli BL21 DE3 cells. After induction and over-expression, the proteins were purified by using a His-Bindchromatography kit (Novagen). SDS/PAGE analysis wasperformed on transformed E. coli extracts (results notshown) and purified recombinant mSMO isoforms (Fig. 3).The enzyme activity for the active forms (mSMOa andmSMOl) was measured spectrophotometrically and thecatalytically active proteins were expressed at a level of�5–10 IUÆL)1 of culture broth.
Kinetic properties of the mSMOl isoform
The biochemical properties of mSMOa have been repor-ted previously [22–25] and this splice variant can beconsidered as the major active isoform in mammaliancells. Interestingly, among the splice variants analyzed inthe present work, the mSMOl isoform also showscatalytic activity. The substrate specificity of mSMOlfor Spm, Spd and N1-acetylpolyamines has been investi-gated under standard conditions at pH 8.5. The purifiedenzyme oxidizes specifically Spm and is not active on Spd,N1-AcSpd and N1-AcSpm. Values of Km, Vmax and theoptimum pH were determined using Spm as substrate.The purified mSMOl exhibited a pH optimum of 8.5 inNa/Pi buffer; the values of Km and kcat were 150 lM and2.5 s)1, respectively.
Accumulation of mSMO splice variant mRNAsand amino oxidase activity in murine organs
The transcription level of four mSMO gene splice variants(a, l, c and d) was examined in various murine organs byRT-PCR analysis (Fig. 4A). PCR-amplified mRNA wasprobed with primer-pairs specific for each splice variant(Fig. 1), in order to determine the accumulation pattern ofeach isoform transcript. Under saturating PCR amplifica-
Sp, spleen; In, intenstine, C, control (no template). (B) Determination
of amino oxidase activity. Homogenates from different organs were
incubated with 1 mM spermine (Spm) or the N1-acetyl derivative of
spermidine (N1-AcSpd) as substrates. Measurements were carried out
as described in the Materials and methods. Results shown represent the
mean ± SD values of measurements obtained from three mouse
samples.
� FEBS 2004 Expression of mouse spermine oxidase gene variants (Eur. J. Biochem. 271) 765
tion conditions, the four mSMO mRNAs were detected inall the murine organs tested (results not shown), while,within the linear range of PCR amplification conditions, theisoform transcripts showed different accumulation patterns.The mSMOa and the mSMOl mRNAs have a similarprofile of expression, with both transcripts being accumu-lated at the highest levels in brain and skeletal muscle. ThemSMOc isoform mRNA was found to accumulate mainlyin brain and spleen, whereas the mSMOd transcriptaccumulated at a similar level in all organs examined, butto a lower extent in heart, kidney, liver and lungs. It isnoteworthy that, in spite of the fact that the mSMOaprimer-pair is able to amplify the mSMOb transcript,during the cDNA cloning of these two isoforms fromdifferent organs, a much higher number of cDNA clonescoding for the mSMOa isoform have been sequenced (19,38 and 21 mSMOa clones per each mSMOb clone fromcerebellum, brain and skeletal muscle samples, respectively).To further confirm the specificity of the primer-pair utilizedin this analysis, the amplified PCR product obtained fromcerebellum, brain and skeletal muscle samples were directlysequenced, showing that indeed the sequence correspondedto the mSMOa splice variant. On this basis, we considerthat the primer-pair used for mSMOa transcript detectionby RT-PCR is fairly specific. The transcription levels ofmurine PAO (mPAO), SSAT (mSSAT), ribosomal proteinS7 (rp-S7) and b-actin mRNAs have been analyzed in thesame organs, to compare the expression of the mSMO genesplice variants with that of two other genes related to thepolyamine homeostasis and of two typical control house-keeping genes. The mPAO mRNA level is in goodagreement with that reported by Wu et al. [13], beingexpressed in all tissue analyzed. Within the linear range ofPCR amplification conditions, the only differences observedin our experiments are the relatively higher level of themPAO transcript in brain and testis and the lower level inthe intestine. The rp-S7 and b-actin mRNAs levels arecomparable in all the organs analyzed within the linearrange of PCR amplification conditions, indicating theirhousekeeping nature. Noteworthy of mention is that themSSAT mRNA level was found to be constant in all theorgans examined.
mSMO and mPAO enzyme activities were measured onhomogenates from the same organ samples examined byRT-PCR, using Spm and N1-AcSpd as substrates, respect-ively (Fig. 4B). mSMO activity was detected at variouslevels in different organs at the following relative abun-dance: cerebellum > testis > heart > kidney > skeletalmuscles > brain. Little mSMO activity was detected inliver, lungs, spleen and intestine. mPAO activity showedthe following relative abundance: cerebellum > testis >kidney > brain > lungs > heart > spleen > liver >skeletal muscles > intestine.
The four mSMO splice variants studied in the presentwork were expressed transiently and ectopically in theneuroblastoma cell line, N18TG2, to gain insight on theirsubcellular localization and enzyme activity. Augmentedtranscript levels for each transfected isoform have been
Fig. 5. RT-PCR analysis of the mRNA level and amine oxidase enzyme
activity of mouse spermine oxidase (mSMO) gene splice variants, in
sentation of mSMO cDNA splice variants (see the legend to Fig. 1 for
explanation). (B) RT-PCR analysis. Total RNA extracted from dif-
ferent homogenates was analyzed by RT-PCR within the linear range.
A representative RT-PCR experiment from three independent
experiments is shown. Abbreviations: NT, nontransfected cells; P, cells
transfected with pcDNA3-V5-TAG; Ta-d, cells transfected with
pcDNA3/mSMOa, l, c and d/V5-TAG plasmids; C, control (no
template). (C) Determination of amino oxidase activity. Homogenates
from stably transfected cells were incubated with 1 mM spermine
(Spm) or the N1-acetyl derivative of spermine (N1-AcSpm) as sub-
strates. Measurements were carried out as described in the Materials
and methods. Results shown represent the mean ± SD values of
measurements obtained from three mouse samples.
766 M. Cervelli et al. (Eur. J. Biochem. 271) � FEBS 2004
Fig. 6. Subcellular localization of mouse spermine oxidase (mSMO) isoforms in the murine NB cell line, N18TG2. (A) Stably transfected cells are
indicated on the left side of the panel. Anti-V5 and 4¢,6¢-diamidino-2-phenylindole (DAPI) columns indicate the secondary immunofluorescence
detection and nuclei visualization, respectively. Merge column is the result of overlapping images. (B) Transiently transfected N18TG2/pcDNA3/
mSMOl-V5 have been treated with anti-V5 as in (A), nuclei were counterstained with propidium iodide dye. The merge column is the result of
overlapping images.
� FEBS 2004 Expression of mouse spermine oxidase gene variants (Eur. J. Biochem. 271) 767
detected in transiently (results not shown) and stablytransfected neuroblastoma cells (Fig. 5A,B), using b-actinas a control housekeeping gene for RNA stability and thequantity processed for each sample. In parallel, the aminooxidase activity was also measured in stably transfectedneuroblastoma cells to evaluate the concentration of activeprotein (Fig. 5C). A first experiment of subcellular local-ization was performed by fluorescence microscopy, usingthe V5-TAG as epitope to direct primary mAb. As depictedin Fig. 6A, in N18TG2/pcDNA3/mSMOa-V5, N18TG2/pcDNA3/mSMOl-V5, N18TG2/pcDNA3/mSMOc-V5and N18TG2/pcDNA3/mSMOd-V5 transiently transfectedcells, a cytoplasmic localization of the tagged isoforms isvisible. In fact, the microphotographies show that mSMOa,mSMOd, and mSMOc are localized in the cytoplasm. Onthe contrary, the mSMOl protein is clearly localized in boththe nucleus and cytoplasm. To better define the localizationof each isoform, a further confocal microscopy investigationwas carried out on both transiently and stably transfectedcells. This approach confirmed the cytoplasmic localizationof mSMOa, -c and -d (data not shown) and the nuclearlocalization of mSMOl (Fig. 6B). Taken together, theseresults consistently substantiate that mSMOa, mSMOd andmSMOc are cytoplasmic proteins. Very interestingly, thenovel mSMOl isoform is present in the nucleus and itsenzymatic activity is comparable to that of the cytoplasmicmSMOa isoform (Fig. 5C). In fact, mSMO enzyme activitywas measured on sonicated cultured cells using Spm as asubstrate, resulting in stably transfected cells with theisoforms mSMOa and mSMOl being present at levels ofabout five times higher than those detected in pcDNA3,mSMOc and mSMOd transfected and control cells. Inparallel, the mPAO activity measured using N1-AcSpm assubstrate was constant in all the samples analyzed (Fig. 5C).
The only difference observed between mSMOa andmSMOl isoforms consists of the insertion in the latterprotein of 30 amino acid residues, corresponding to the VIaexon, localized at the C-terminus (Figs 1 and 2). A MAXHOM
[34] search using, as a bait, a 49 residue amino acid sequencefrom mSMOl, including exon VIa (to minimize theoccurrence of nonsignificant homologues), resulted in asignificant hit (40% sequence identity on 49 residuesaligned) (Fig. 7A,B), corresponding to a stretch of the ratnucleoporin Nup153, a protein localized on the nucleo-plasmic face of the nuclear pore complex [35].
Discussion
The flavoprotein SMO, which specifically oxidizes Spm, isone of the key regulatory enzymes involved in polyaminehomeostasis in animal cells and has been previouslycharacterized in mouse as a single protein form, named inthis work as mSMOa. We found that the mSMO gene is asingle copy gene coding for at least eight additional splicevariants (namely b, c, d, l, /, e, g and x), and thecorresponding cDNAs from mouse brain were isolated byRT-PCR, cloned and sequenced. Thus, understanding theprecise biochemical and structural properties of thesesplicing variants is essential for a more detailed knowledgeof the expression of this gene. With this in mind, weexpressed, in E. coli cells, the three recombinant isoformsmSMOl, -c and -d. As expected from the primary sequence
comparison between mSMOa and mSMOl proteins, thebiochemical characterization of these proteins revealed thatthe recombinant protein, mSMOl, shows similar propertiesto those previously reported for mSMOa [22–25]. Inparticular, the mSMOl isoform showed very similarsteady-state kinetic parameters, identical substrate specific-ity and a similar pH optimum to mSMOa. The redundancyof these enzymatic activities may be explained relative totheir different subcellular localization (see below). Aspredicted from sequence analysis, the other two recombin-ant proteins (mSMOc and mSMOd) do not display anydetectable SMO activity, probably because they lack someof the protein regions required to bind the FAD prostheticgroup. This is in sharp contrast to the work published byMurray-Stewart et al. [8], who reported that at least threehuman splice variants of the major isoform (PAOh1/SMO)have activity and different substrate specificity. In ouropinion, this is unlikely to occur, mainly because the mostactive variant – PAOh3/SMO, which is the mouse mSMOdcounterpart – is the shortest splice variant and lacks 65%of the coding sequence, including part of the essentialFAD-binding domain.
RT-PCR analysis was carried out to study the relativetranscription levels of the gene splicing variants coding formSMOa, -l, -c and -d in various mature murine organs.Within the linear range of PCR amplification conditions,the isoform transcripts analyzed showed different accumu-lation patterns in various organs; when the experimentswere carried out under saturating conditions, these isoformscan be considered to be expressed in all the organs analyzed.The mSMO activities measured from the same organsamples examined by RT-PCR are in fair agreement withthe transcript level observed for both mSMOa and mSMOlsplice variants. However, the mRNA accumulation levels ofsome organs do not linearly correspond to the enzymaticactivity, the most striking examples being the cerebellum,kidney and lungs. Currently, we cannot assess how themRNA levels relate to the concentration of mSMO proteinsin different organs. Within the linear range of PCRamplification conditions, the mPAO mRNA level observedis in good agreement with that reported by Wu et al. [13].Interestingly, also the mSSAT mRNA level is constant in allthe organs examined.
Fig. 7. Amino acid alignment of exon VIa. (A) Alignment of the
deduced amino acid sequence corresponding to exon VIa of mouse
(mSMO, AF498364), rat (rSMO, NW0436471) and human (PAOh1/
sequence alignment of the region enclosing exon VIa of mSMO and
the homologous region of Nup153. Residues in bold are those con-
served between Nup153 and mSMO. The amino acid sequence of exon
VIa is shaded in gray.
768 M. Cervelli et al. (Eur. J. Biochem. 271) � FEBS 2004
The subcellular localization of mSMOa, -l, -c and -dV5-tagged isoforms was also studied, by microscopy, inthe transiently and stably transfected murine neuro-blastoma cell line, N18TG2. In parallel, the enzymeactivity has been measured on the corresponding sonicat-ed cells. The mSMOa and mSMOl transfected cellsexhibited an enzyme activity of about five times higherthan that shown by pcDNA3, mSMOc and mSMOdtransfected and control cells. The localization experimentsindicate that mSMOa, mSMOc and mSMOd are cyto-plasmic proteins. Very interestingly, the tagged isoformwas present in the nucleus only in the N18TG2/pcDNA3/mSMOl-V5 transfected cells. The translational product,mSMOl, which retains the capability to bind the pros-thetic group, derives from a splicing variant that includesthe extra exon VIa. This alternative exon is present in thehuman PAOh1/SMO gene (accession number AL121675),and utilized to produce the isoform PAOh4/SMO[33], and it is also present in the rat SMO gene (rSMO;accession number NW0436471). Exon VIa corresponds toa 30 amino acid region that, based on the crystal structureof the homologous maize PAO [33] and on the molecularmodel of mSMO [25], appears to be located on a surfaceloop. It is worthy of note that the mSMOl amino acidsequence does not display any known nuclear localizationsequence. Interestingly, a 49 amino acid sequence ofmSMOl, including exon VIa and 19 amino acid residuelong flanking regions, displays 40% sequence identity withthe nucleoporin Nup153, known to be part of thenucleopore complex and to be located on the nucleoplas-mic face of the nuclear pore complex [36]. In conclusion,the availability of the recombinant mSMO enzymespresented here provides an important tool and offerssome unique insights into the regulation and importanceof SMO metabolism.
Acknowledgements
We thank M. T. Carrı and E. Ferraro for their assistance in confocal
microscopy observations. The N18TG2 cell line was a generous gift of
S. Biagioni. This research was supported by grants from Ministero
dell’Universita e della Ricerca Scientifica e Tecnologica (MURST).
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