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:
(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 ( />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 (embl.
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 c.
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. Biochem J 376, 1–14.
2 Seiler N (2003) Thirty years of polyamine-related
approaches to cancer therapy. Retrospect and prospect.
Part 2. Structural analogues and derivatives. Curr Drug
Targets 4, 565–585.
3 Casero RA Jr & Pegg AE (1993) Spermidine ⁄ spermine
N
1
-acetyltransferase – the turning point in polyamine
metabolism. FASEB J 7, 653–661.
4 McIntire WS & Hartman C (1993) Copper containing

amine oxidases. In Principle and Application of Quino-
proteins (Davison, VL, ed.), pp. 97–171. Marcel Dekker
Inc., New York.
5 Seiler N (1995) Polyamine oxidase, properties and func-
tions. Prog Brain Res 106, 333–344.
6 Van den Munckhof RJ, Denyn M, Tigchelaar-Gutter W,
Schipper RG, Verhofstad AA, Van Noorden CJ & Fre-
deriks WM (1995) In situ substrate specificity and ultra-
structural localization of polyamine oxidase activity in
unfixed rat tissues. J Histochem Cytochem 43, 1155–1162.
7 Wang Y, Devereux W, Woster PM, Stewart TM,
Hacker A & Casero RA Jr (2001) Cloning and charac-
Protein domains involved in mSMOl targeting to the nucleus M. Bianchi et al.
3058 FEBS Journal 272 (2005) 3052–3059 ª 2005 FEBS
terization of a human polyamine oxidase that is induci-
ble by polyamine analogue exposure. Cancer Res 61,
5370–5373.
8 Vujcic S, Diegelman P, Bacchi CJ, Kramer DL & Porter
CW (2002) Identification and characterization of a novel
flavin-containing spermine oxidase of mammalian cell
origin. Biochem J 367 , 665–675.
9 Cervelli M, Polticelli F, Federico R & Mariottini P
(2003) Heterologous expression and characterization of
mouse spermine oxidase. J Biol Chem 278 , 5271–5276.
10 Cervelli M, Bellini A, Bianchi M, Marcocci L, Nocera
S, Polticelli F, Federico R, Amendola R & Mariottini P
(2004) Mouse spermine oxidase gene splice variants:
nuclear sub-cellular localization of a novel active iso-
form. Eur J Biochem 271, 760–770.
11 Bellelli A, Stefano Cavallo S, Nicolini L, Cervelli M,

Bianchi M, Mariottini P, Zelli M & Federico R (2004)
A model of the catalytic cycle and its inhibition by
N,N
1
-bis (2,3-butadienyl)-1,4-butanediamine. Biochem
Biophys Res Commun 322, 1–8.
12 Binda C, Coda A, Angelini R, Federico R, Ascenzi P &
Mattevi A (1999) A 30 A
˚
long U-shaped catalytic tunnel
in the crystal structure of polymine oxidase. Structure 7,
265–276.
13 Binda C, Newton-Vinson P, Hubalek F, Edmondson
DE & Mattevi A (2002) Structure of human monoamine
oxidase B, a drug target for the treatment of neurologi-
cal disorders. Nat Struct Biol 9, 22–26.
14 Sambrook J, Fritsch EF & Maniatis T (1989) Molecular
Cloning: a Laboratory Manual, 2nd edn. Cold Spring
Harbor Laboratory, Cold Spring Harbor, NY.
15 Horton RM (1993) In vitro recombination and mutagen-
esis of DNA. In Methods in Molecular Biology, Vol. 15:
PCR Protocols: Current Method and Applications
(White BA, ed.), pp. 251–261. Humana Press Inc.,
Totowa, N J.
16 Thompson JD, Higgins DG & Gibson TJ (1994) CLUS-
TAL W: improving the sensitivity of progressive multi-
ple sequence alignment through sequence weighting,
position-specific gap penalties and weight matrix choice.
Nucleic Acids Res 22, 4673–4680.
17 Rost B (1996) PHD: predicting one-dimensional protein

structure by profile-based neural networks. Methods
Enzymol 266, 525–539.
18 Petrey D, Xiang Z, Tang CL, Xie L, Gimpelev M, Mitros
T, Soto CS, Goldsmith-Fischman S, Kernytsky A, Schles-
singer A et al. (2003) Using multiple structure alignments,
fast model building, and energetic analysis in fold recog-
nition and homology modeling. Proteins 53, 430–435.
19 Markwell MA, Haas SM, Bieber LL & Tolbert NE
(1978) A modification of the Lowry procedure to
simplify protein determination in membrane and
lipoprotein samples. Anal Biochem 87, 206–210.
20 Laemmli UK (1970) Cleavage of structural proteins
during assembly of the head of bacteriophage T4.
Nature 277, 680–685.
21 Nicholls A, Sharp K & Honig B (1991) Protein folding
and association: insights from the interfacial and ther-
modynamic properties of hydrocarbons. Proteins 11,
281–296.
M. Bianchi et al. Protein domains involved in mSMOl targeting to the nucleus
FEBS Journal 272 (2005) 3052–3059 ª 2005 FEBS 3059