Báo cáo khoa học: Two short protein domains are responsible for the nuclear localization of the mouse spermine oxidase l isoform pdf

Two short protein domains are responsible for the nuclear localization of the mouse spermine oxidase l isoform Marzia Bianchi 1 , Roberto Amendola 2 , Rodolfo Federico 1 , Fabio Polticelli 1 and Paolo Mariottini 1 1 Dipartimento di Biologia, Universita ` ‘Roma Tre’, Roma, Italy 2 Istituto per la Radioprotezione, ENEA, CR Casaccia, Roma, Italy The polyamines putrescine (Put), spermine (Spm) and spermidine (Spd) are aliphatic amines that are posi- tively charged under physiological conditions and have been shown to be involved in major cellular pro- cesses such as cell growth and proliferation [1,2]. The concerted actions of Spd ⁄ Spm N 1 -acetyl-transferase, vertebrate polyamine oxidase (PAO) (EC 1.5.3.11) and spermine oxidase (SMO) are involved in main- taining polyamine homeostasis in mammalian cells. The cytosolic Spd ⁄ Spm N 1 -acetyl-transferase enzyme is responsible for adding N 1 -acetyl groups to both Spm and Spd [3]. The N 1 -acetylated Spm and Spd are oxidized by the peroxisomal FAD-containing enzyme, PAO, to yield stoichiometric amounts of 3-acetamido- propanal and H 2 O 2 , plus Spd and Put, respectively [4–6]. The last enzyme involved in the mammalian polyamine homeostasis is the flavoprotein SMO, which preferentially oxidizes Spm, producing Spd, 3-aminopropanal and H 2 O 2 [7–9]. Analysis of the expression of the mouse SMO gene (mSMO), encoding at least nine splice variants, as well as biochemical characterization of the canonical alfa isoform (mSMOa), have been reported recently [10,11]. The subcellular localization of the catalytically active isoforms mSMOa and mSMOl has been investigated in the transiently and stably transfected murine neuro- blastoma cell line, N18TG2. Interestingly, mSMOl is present in both nuclear and cytoplasmic compart- ments, while mSMOa is cytosolic. The only structural difference between the two isoforms is the presence of an extra protein domain in mSMO l, encoded by the exon VIa [10]. Comparative analysis of the amino acid sequence of the vertebrate members of the SMO family has revealed a region that is extremely conserved in mam- mals, highly variable and ⁄ or reduced in length in non- mammalian vertebrates, and absent in the aligned PAO sequences. Molecular modeling of mSMO Keywords mouse; nuclear localization; polyamine oxidase; polyamines; spermine oxidase Correspondence P. Mariottini, Dipartimento di Biologia, Universita ` degli Studi ‘Roma Tre’, Viale Guglielmo Marconi 446, 00146 Roma, Italy Fax: +39 06 55176321 Tel: +39 06 55176359 E-mail: mariotpa@bio.uniroma3.it (Received 18 February 2005, revised 7 April 2005, accepted 13 April 2005) doi:10.1111/j.1742-4658.2005.04718.x In mouse, at least two catalytically active splice variants (mSMOa and mSMOl) of the flavin-containing spermine oxidase enzyme are present. We have demonstrated previously that the cytosolic mSMOa is the major iso- form, while the mSMOl enzyme is present in both nuclear and cytoplasmic compartments and has an extra protein domain corresponding to the addi- tional exon VIa. By amino acid sequence comparison and molecular mode- ling of mSMO proteins, we identified a second domain that is necessary for nuclear localization of the mSMOl splice variant. A deletion mutant enzyme of this region was constructed to demonstrate its role in protein nuclear targeting by means of transient expression in the murine neurobla- stoma cell line, N18TG2. Abbreviations MPAO, maize polyamine oxidase; mPAO, mouse polyamine oxidase; hSMO, human spermine oxidase; mSMO, mouse spermine oxidase; NDA, nuclear domain A; NDB, nuclear domain B; PAO, polyamine oxidase; Put, putrescine; SMO, spermine oxidase; Spd, spermidine; Spm, spermine. 3052 FEBS Journal 272 (2005) 3052–3059 ª 2005 FEBS proteins, based on the 3D structure of maize poly- amine oxidase (MPAO), indicated that this region is localized on the tip of the FAD-binding domain, in close spatial proximity to the protein region encoded by exon VIa of the mSMOl isoform. This observation has led us to hypothesize that these two protein domains, named nuclear domain A and nuclear domain B (NDA and NDB, respectively), may have coevolved in mammalian SMOs and that they may cooperate in targeting the mSMOl isoform to the nuc- leus. By means of transient expression of the deletion mutant, mSMOlD, in the murine neuroblastoma cell line, N18TG2, we demonstrated that removal of the NDA amino acid region abolishes proper nuclear targeting of the mSMOl isoform. Results Structural analysis and modeling of vertebrate SMO proteins Comparison of the derived amino acid sequences of vertebrate SMO proteins has revealed that their overall primary structure is well conserved. Taking the sequence of the human SMO (hSMO) as the reference point, the amino acid identity ranges from  99% (chimpanzee) to  67% (pufferfish); as expected, the identity decreases to 40% when compared to the mouse PAO (mPAO) primary sequence (Fig. 1A). The only region that shows a low degree of conserva- tion among SMO proteins, when comparing mammals to other vertebrates, is the central part of the primary sequence, located between positions 277 and 307 in the mSMO sequence (Fig. 1B). This region, of 31 amino acids, has not been shown to contain any residue involved in either the catalytic site or the FAD-binding domain [9,10,12,13]. Interest- ingly, this 31 amino acid region is highly conserved among mammals (human, chimpanzee, dog, cow and rodents), with an identity ranging from 82 to 95%, while there is little, if any, conservation with chicken, frog or fish counterparts. It is interesting to note that the sequence analysis of the mammalian genes encoding SMO (AL121675, human; NW120319, chim- panzee; AF498364, mouse; NW0436471, rat; AAEX01031426, dog; AAFC01101092, cow, partial gene sequence) has revealed the presence of the extra exon VIa [10] (Fig. 1B). By contrast, the same analysis performed on the homolog SMO genes of chicken (M_420872) and pufferfish (http://www.ensembl.org/ Fugu_rubripes/) shows the lack of this extra domain. This observation suggests that the presence of the extra exon VIa is a mammalian feature that is strictly related to the high homology displayed by the 31 amino acid region (residues 277–307; numbering of the human SMO enzyme). The two protein domains may have coevolved, conferring novel properties to mammalian SMOs. Molecular modeling of the 3D structure of the mSMOl isoform was thus carried out in order to test the hypothesis that the 277–307 region and the protein domain encoded by the exon VIa could be spatially contiguous and represent a functional epitope involved in a mammalian-specific function of SMO (e.g. nuclear targeting of the mSMOl isoform). Inspection of the mSMOl modelled structure (Fig. 2) indicates that both regions are located on the tip of the FAD-binding domain, with residues 300–307 located in close spatial proximity to the extra domain of mSMOl . Hence, we postulated that both regions could be involved in the nuclear targeting of the mSMOl enzyme. With this rationale, we made a deletion mutant of the mSMOl isoform, deleting exactly the region 277–307, as described in the Experimental pro- cedures (Fig. 3A). Expression and purification of mSMOlD protein in Escherichia coli cells The recombinant cDNA construct, pmSMOlD-HT, and the controls pmSMOa-HT and pmSMOl-HT, were used to transform E. coli BL21 DE3 cells. After induction and over-expression, the proteins were puri- fied by using a His-Bind chromatography kit (Novagen, Darmstadt, Germany). The SDS ⁄ PAGE electrophoretic analysis performed on purified recombinant mSMO proteins is shown in Fig. 3B. The enzyme activities were measured spectrophotometrically and the catalyti- cally active proteins were expressed at levels ranging from 5 to 15 IUÆL )1 of culture broth. Kinetic properties of the mSMOlD protein The biochemical properties of mSMOa and mSMOl have been reported previously [9,10]. The recombinant mSMOlD isoform also shows catalytic activity. The substrate specificity of mSMOlD for Spm, Spd and N 1 -acetylpolyamines has been investigated under stand- ard conditions at pH 8.5. Purified mSMOlD specifically oxidizes Spm and is not active on Spd, N 1 -acetylSpd or N 1 -acetylSpm. Values of K m , V max and pH optimum were determined by using Spm as the substrate. The purified mSMOlD exhibited biochemical properties very similar to those of mSMOa and mSMOl, in par- ticular a pH optimum of 8.5 in 0.1 m NaP i buffer, a K m value of 220 lm and a k cat value of 1.25 s )1 . M. Bianchi et al. Protein domains involved in mSMOl targeting to the nucleus FEBS Journal 272 (2005) 3052–3059 ª 2005 FEBS 3053 A B Fig. 1. Amino acid sequence comparison of members of the spermine oxidase (SMO) and polyamine oxidase (PAO) family. (A) Amino acid sequence alignment of the SMO and PAO proteins. (B) Alignment of the deduced amino acid sequences corresponding to nuclear domain A and nuclear domain B (exon VIa). Multi-alignment was performed by using the program CLUSTAL W SEQUENCE ALIGNMENT. HsSMO, Homo sap- iens (AAN77119); PtSMO, Pan troglodytes (NW120319); CfSMO, Canis familiaris (AAEX01031426); BtSMO, Bos taurus (AAFC01101092); MmSMO and MmPAO, Mus musculus (AAN32915) and (AAN40705), respectively; RnSMO, Rattus norvegicus (XP_218704); GgSMO, Gallus gallus (XP_420872.1); XlSMO, Xenopus laevis (Q6INQ4); DrSMO, Danio rerio (Q6NYY8); and FrSMO, Fugu rubripes (http://www.ensembl. org/Fugu_rubripes/). Protein domains involved in mSMOl targeting to the nucleus M. Bianchi et al. 3054 FEBS Journal 272 (2005) 3052–3059 ª 2005 FEBS Cell localization of mSMOlD protein in murine neuroblastoma N18TG2 cells The mSMOlD mutant protein was transiently expres- sed in the neuroblastoma cell line, N18TG2, to investi- gate its subcellular localization. Augmented transcript levels for each recombinant protein were detected in transiently transfected neuroblastoma cells, using b- actin as a control housekeeping gene to monitor RNA stability, and amount of processed RNA for each sample (Fig. 3C). To establish where each tagged protein was locali- zed, a confocal microscopy investigation was carried out, using the V5-TAG as epitope to direct primary mAbs. As shown in Fig. 4, in N18TG2 ⁄ pcDNA3 ⁄ mSMOa-V5 and N18TG2 ⁄ pcDNA3 ⁄ mSMOlD-V5 t ra ns- iently transfected cells, we observed a cytoplasmic localization of the tagged recombinant proteins. By contrast, in N18TG2 ⁄ pcDNA3 ⁄ mSMOl-V5 transiently transfected cells, we confirmed a nuclear localization for the mSMOl isoform (Fig. 4). Taken together, these results consistently substanti- ate the hypothesis that these two structural regions are mandatory for the nuclear localization of mSMOl,as the only difference between mSMOl and mSMO lD proteins consists of the lack of the amino acid sequence region 277–307 (Figs 1,2). Discussion In the murine polyamine homeostasis at least two cata- lytically active splice variants of the spermine oxidase enzyme are involved. The cytosolic mSMOa is the major isoform, while the mSMOl enzyme, displaying an extra protein domain corresponding to the addi- tional exon VIa, is localized in both the cytoplasm and the nucleus. The overall primary structure of verteb- rate SMO enzymes is well conserved, with the excep- tion of a region comprising 31 residues (amino acids 277–307). Molecular modeling of the 3D structure of mSMOl indicates that this region (NDA) is localized on the tip of the FAD-binding domain and is located near the protein region encoded by exon VIa (NDB). This amino acid region is highly conserved in mam- mals, while it is highly variable and ⁄ or reduced in length in nonmammalian vertebrates, indicating that a selective evolutionary constrain is operating on it. Interestingly, the presence of exon VIa in vertebrate SMO gene sequences is also a unique mammalian feature. These data suggest that the two domains NDA and NDB, not involved in enzyme activity or FAD bind- ing, could be responsible for the interaction with the nuclear targeting machine. With this hypothesis in mind, we constructed a deletion mutant lacking the amino acid region 277–307, named mSMOlD .We expressed this mutant in E. coli cells and, as expected, the purified recombinant protein showed a catalytic activity comparable to that of the wild-type mSMOl [10]. Notably, by means of transient expression of mSMOlD in the murine neuroblastoma cell line, N18TG2, we demonstrated that deletion of the 277– 307 region abolished nuclear targeting. The presence of the translated region encoded by exon VIa in mSMOl is thus necessary, but not sufficient, for the correct localization of this isoform within the nucleus. In con- clusion, the mSMOl enzyme needs at least two domains to be nuclear localized. Fig. 2. Stereo representation of the mod- elled 3D structure of the mouse spermine oxidase catalytically active splice variant, mSMOl. The molecular surface of the pro- tein is shown in a ‘mesh’ representation. The backbone and the molecular surface of nuclear domains A and B (see the text) are coloured green and blue, respectively. The FAD cofactor is shown as red sticks. The figure was produced by using GRASP [21]. M. Bianchi et al. Protein domains involved in mSMOl targeting to the nucleus FEBS Journal 272 (2005) 3052–3059 ª 2005 FEBS 3055 Experimental procedures Chemicals Spd, Spm, N 1 -acetylspermidine, N 1 -acetylspermine and Put were purchased from Sigma (Milan, Italy). Restriction enzymes and DNA-modifying enzymes were purchased from MBI Fermetas. Taq polymerase and M-MLV reverse transcriptase enzymes were from Promega (Milan, Italy). Other chemicals were from Sigma, Bio-Rad (Milan, Italy) and J. T. Baker (Milan, Italy). DNA methodology and construction of the mSMO expression plasmid DNA manipulation was carried out by using standard techniques [12]. The absence of errors in DNA products generated by the PCR was verified by sequence analysis. The deletion mutant of the mSMOl protein was con- structed by the PCR following the method described by Horton [13] and by using the mSMO l cDNA as a template. The mutagenic primer sequences used are avail- able on request from the first author (M.B.). The intro- duction of the deletion was confirmed by sequence analysis. Amino acid sequence analysis and molecular modeling Overall and local amino acid sequence identity between SMOs and other proteins belonging to the PAO family has been determined from multiple sequence alignments obtained using clustal w [14]. The molecular model of A B C Fig. 3. Amino acid sequence alignment, protein purification and RT-PCR transcript analysis of the mouse spermine oxidase catalytically active splice variants mSMOa, mSMOl and mSMOlD. (A) Amino acid sequence alignment of the region enclosing nuclear domains A and B (exon VIa) of mSMOa, mSMOl, mSMOlD and mouse polyamine oxidase (mPAO) isoforms. Dele- ted residues are marked by dots; the mPAO gap is represented by a dashed region. Amino acid numbering is shown on the right side of the figure. (B) SDS ⁄ PAGE analysis of the recombinant mSMOa, mSMOl and mSMOlD proteins (5–10 lg of the purified enzyme) after staining the gel with Coomas- sie Brilliant Blue. MW, protein molecular mass markers (MBI Fermentas). (C) Total RNA extracted from different homogenates was analyzed by RT-PCR within the linear range. A representative RT-PCR experiment from three independent experiments is shown. M, GeneRuler 1 kb DNA ladder (MBI Fermentas); /, /X174-HaeIII digested DNA marker (MBI Fermentas); NT, untrans- fected cells; P, cells transfected with pcDNA 3 -V5-TAG; Ta, l and lD, cells trans- fected with pcDNA 3 ⁄ mSMOa, l and lD, ⁄ V5-TAG plasmids; C, no-template control. Protein domains involved in mSMOl targeting to the nucleus M. Bianchi et al. 3056 FEBS Journal 272 (2005) 3052–3059 ª 2005 FEBS mSMOl was built by using the crystal structure of MPAO as a template (PDB code: 1B37) [12]. Given the fairly low sequence identity between mSMOl and MPAO (26.5%), a reliable alignment between the two protein sequences was derived from the multiple sequence alignment between mSMOs, MPAO and other PAOs with known amino acid sequence, obtained by using clustal w. In addition, the alignment was manually refined on the basis of mSMOl secondary structure prediction, obtained using the Predict Protein server [17] (available online at http://cubic.bioc. columbia.edu/predictprotein), to avoid the unlikely occur- rence of insertions and deletions within secondary structure elements. Based on this alignment, the 3D structure of mSMOl was then built by using nest, a fast model-build- ing program that applies an ‘artificial evolution’ algorithm to construct a model from a given template and alignment [18]. Expression of mSMOa, mSMOl and mSMOlD isoforms in E. coli cells E. coli BL21 DE3 (Novagen) cells transformed with the pmSMOa and pmSMOl plasmids, as described previously [10], and with the pmSMOlD plasmid, were cultured at 30 °C in Luria–Bertani (LB) medium, containing 50 lgÆmL )1 ampicillin, to an attenuance (D) of 0.6 at 600 nm, and then induced with isopropyl thio-b-d-galactoside (0.4 mm final concentration), followed by further culture for 5 h at 30 °C. The E. coli BL21 DE3 cells were harvested by centrifugation at 4 °C for 10 min at 10 000 g, washed with 0.4 culture vol- umes of 30 mm Tris ⁄ HCl, pH 8.0, containing 20% (w ⁄ v) sucrose and 1 mm EDTA, and then incubated for 5–10 min at room temperature. Each suspension was centrifuged at 10 000 g for 10 min at 4 °C and then the pellets were resus- pended in 0.05 culture volumes of ice-cold 5 mm MgSO 4 , Fig. 4. Subcellular localization of the mouse spermine oxidase catalytically active splice variants mSMOa, mSMOl and mSMOlD in transiently transfected neuroblastoma N18TG2 cells. Transiently transfected cells are indicated on the left side of the figure. Anti-V5 and propidium iodide (PI) dye col- umns indicate the secondary immuno- fluorescence detection and nuclei counterstaining, respectively. Merge column is the result of overlapping images. M. Bianchi et al. Protein domains involved in mSMOl targeting to the nucleus FEBS Journal 272 (2005) 3052–3059 ª 2005 FEBS 3057 with vigorous shaking, for 10 min on ice. The resuspended pellets were then centrifuged at 10 000 g for 10 min at 4 °C. The supernatant, corresponding to the periplasmic fraction, was collected. Rapid affinity purification of mSMOa, mSMOl and mSMOlD isoforms with pET His Tag systems The supernatant from E. coli BL21 DE3 cells transformed with the plasmids pmSMOa-HT, pmSMOl-HT or pmSMOlD-HT was applied to a column (3 mL) with Ni 2+ cations immobilized on the His-Bind resin (Nov- agen), equilibrated with Binding Buffer (5 mm imidazole, 0.5 m NaCl, 20 mm Tris ⁄ HCl pH 7.9). The column was washed with 20 m m Tris ⁄ HCl, pH 7.9, containing 60 mm imidazole and 0.5 m NaCl, and then eluted with 20 mm Tris ⁄ HCl, pH 7.9, containing 750 mm imidazole and 0.5 m NaCl. Determination of the enzyme activity and kinetic constants of recombinant mSMO Enzyme activity was measured by using the spectrophoto- metric assay previously described by Cervelli et al. [10]. The measurements were performed in 0.1 m sodium phos- phate (NaP i ) buffer, pH 8.5, with different substrates at various concentrations. K m and k cat values were deter- mined using Spm as a substrate at concentrations ranging from 50 to 500 lm, at a constant mSMO isoform concen- tration of 2.0 · 10 )8 m. Enzyme activities were expressed in international units (IU: the enzyme concentration that catalyzed the oxidation of 1 lmol of substrateÆmin )1 ) per litre of culture broth. Protein content was estimated by the method of Markwell et al. [19] with BSA as a stand- ard. SDS ⁄ PAGE was performed according to the method of Laemmli [20]. Expression of mSMOa, mSMOl and mSMOlD isoforms in murine neuroblastoma N18TG2 cells All experiments were performed using a pool isolated from three separate transient transfections. mSMOa,-l and -lD cDNA coding sequences were cloned into directional pcDNA 3 -V5-TAG plasmid (Invitrogen, Milan, Italy), accord- ing to the manufacturer’s instructions, to produce recom- binant V5-tagged proteins. Cell culture conditions and transfection procedures of the murine neuroblastoma N18TG2 cell line have been described previously [10]. Aliquots of selected N18TG2 cells were seeded on chamber slides and, 24 h later, fixed with fresh 3.7% (v ⁄ v) parafor- maldehyde in NaCl ⁄ P i (15 min at 4 °C) to evaluate the sub- cellular localization of the various isoforms. Determination of the subcellular localization of mSMOa,-l and -lD⁄V5- tagged proteins was carried out by indirect immunoflures- cence experiments with mouse anti-V5 mAb (Sigma) [1 lgÆ mL )1 ,1%(w⁄ v) BSA in NaCl ⁄ P i ], followed by secon- dary detection using fluorescein isothiocyanate (FITC)-con- jugated goat polyclonal anti-mouse IgG (Sigma) [diluted 1 : 60; 1% (w ⁄ v) BSA in NaCl ⁄ P i ]. Nuclei were counter- stained with propidium iodide and digital images were taken with a LSM510 confocal microscope (Carl Zeiss, Milano, Italy). The transfection efficiency was verified by RT-PCR ana- lysis, utilizing the same experimental conditions as des- cribed previously [10]. The mSMOa-specific primer pairs used were as follows: mSMOa1 forward 5¢-GTACCTGAA GGTGGAGAGC-3¢ and mSMOa2 reverse 5¢-TGCATG GGCGCTGTCTTGG-3¢; mSMOl and mSMOlD specific primer-pairs: mSMOl1 forward 5¢-GATGAGCCGTGG CCTGT-3¢ and mSMOl2 reverse 5¢-CTTTATGGAGCC CCTACTAG-3¢; murine rpS7 control specific primer-pairs: rpS7-forward 5¢-CGAAGTTGGTCGG-3¢ and rpS7-reverse 5¢-GGGAATTCAAAATTAACATCC-3¢; b-actin control specific primer pairs: b-actin-forward 5¢-TGTTACCAACT GGGACGACA-3¢ and b-actin-reverse 5¢-AAGGAAGGC TGGAAAAGAGC-3¢. Three separate experiments were performed from each RNA preparation. Acknowledgements This research was partially supported by the grant PRIN 2003 from ‘Ministero Istruzione, Universita ` e Ricerca’ (MIUR). References 1 Wallace HM, Fraser AV & Hughes A (2003) A perspec- tive of polyamine metabolism. 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