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

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-

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

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

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

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

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-

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].

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

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