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Figure 1 Amino acid sequence of XSENP1. (A) Alignment of predicted amino acid sequences of Xenopus SENP1a (XSENP1a), Xenopus
SENP1b ( XSENP1b), human SENP1 ( hSENP1), and rat Axam (rAxam). The expected catalytic residues conserved with these proteases
(H, D, C) are marked with an arrowhead. Amino acids common to all four sequences are boxed in black, and those matching between
XSENP1s ( XSENP1a, XSENP1b) and hSENP1 are boxed with grey. ( B) Estimated evolutionary relationship of XSENP1s with other
SUMO specific proteases. Proteins displaying the greatest sequence similarity are clustered together. (C) Table showing homology
between XSENP1s and hSENP1. The numbers indicate the percentage homology at the amino acid level.
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Figure 2 Temporal and spatial expression of XSENP1s. (A)
Temporal expression analysed by RT-PCR. UF and 4–42 denote
unfertilized egg and stages 4–42, respectively. The amounts of cDNA
were standardized with ornithine decarboxylase (ODC (RT+)).
The experiments without RT were carried out to test whether
genomic DNA was present in the cDNA samples (ODC (RT−)).
(B) Whole-mount in situ hybridization analyses at st.12 (a, b), st.19
(c–f ), st.31 (g, h), and st.41 (i, j). Experiments were done with
XSENP1a anti-sense probe (a, c, e, g, i) or with XSENP1b antisense
probe (b, d, f, h and j). (a, b) animal pole is up (c, d) anterior
is up (e, f ) dorsal is up (g –j ) anterior is left.
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Figure 3 Enzyme activity of XSENP1s
in vitro. (A) Schematic representation of
XSENP1a mutants used in this study.
The SUMO-specific protease homology
(SPH) domain is indicated by the greycoloured
region. (B) GST-fusion-XSENP1
proteins. Purified proteins (0.5 μg of each)
used in the experiments were subjected
to SDS-PAGE followed by Coomassie
Brilliant Blue staining (lanes 1–5). (C)
Hydrolase activity of XSENP1s. After
1.5 μg of GST-SUMO-1-Myc had been
incubated with 0.5 μg of GST-XSENP1a
( lanes 1 and 2), GST-XSENP1b (lanes 3
and 4), GST-N-XSENP1a (lanes 5 and 6),
GST-C-XSENP1a (lanes 7 and 8), or
GST-XSENP1aC602S (lanes 9 and 10) for
0 or 3 h at 30 °C, the mixtures were
subjected to SDS-PAGE and probed
with anti-SUMO-1 (GMP1) antibody.
(D) Cleavage of SUMO-1-Tcf4 by
XSENP1. The lysates of 293 cells
expressing HA-SUMO-1, Flag-PIASy,
and Tcf4 were probed with anti-Tcf3/4
antibody (lane 2). The lysates of COS
cells with the empty vector instead of
HA-SUMO-1 were used as the control
( lane 1). Sumoylated Tcf-4 was incubated
with 0.5 μg of GST ( lane 3), GSTXSENP1a
(lane 4), GST-XSENP1b
(lane 5), GST-N-XSENP1a (lane 6),
GST-C-XSENP1a (lane 7), or GSTXSENP1aC602S
(lane 8) for 3 h at 30 °C.
After incubation, the mixtures were probed
with anti-HA (16B12) antibody. Arrows
indicate the positions of the monomer and
dimer of HA-SUMO-1. Arrowhead
indicates non-sumoylated Tcf4. Ig,
immunoglobulin.
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Figure 4 Injection effects of XSENP1s and XSENP1a mutants. (A) (a–f ) Phenotypes of embryos injected with mRNAs as follows;
EGFP (a), Full-length of XSENP1a ( b), Full-length of XSENP1b (c), C-XSENP1a (d ), N-XSENP1a (e), and XSENP1aC602S (f ). Samples were
injected dorsally (1.0 ng). All embryos shown are at the tadpole stage (st.39–41). (g) Average dorso-anterior index (DAI) of Xenopus embryos
injected with full-length XSENP1s or mutants of XSENP1a mRNA. 2.0 ng of indicated mRNAs were injected dorsally. DAI is a scale of
dorso-anterior development. DAI = 5, normal; DAI = 0, completely ventralized. (B) Suppression of the Wnt target gene expression by XSENP1s
and XSENP1a mutants. Expression of Xnr3 was estimated by RT-PCR. Total RNA was extracted from embryos injected dorsally with
the indicated mRNAs. The amounts of cDNA were standardized with ornithine decarboxylase (ODC (RT+)). The experiments without RT
were carried out to test whether genomic DNA was present in the cDNA samples (ODC (RT−)). ‘FLa’, ‘FLb’, ‘C-1a’, ‘N-1a’, ‘CS-1a’ indicate
full-length XSENP1a, full-length XSENP1b, C-XSENP1a, N-XSENP1b and XSENP1aC602S, respectively. (C) Alteration of β-catenin
stability by XSENP1s injection. myc-β-catenin (1 ng; lane 2), myc-β-catenin and XSENP1a (1 ng each; lane 3), myc-β-catenin and XSENP1b (1 ng
each; lane 4), or myc-β-catenin and GSK-3β (1 ng each; lane 5) were co-injected into the dorsal marginal zone of 4-cell stage embryos.
These embryos were harvested at Stage 9 and the levels of exogenous β-catenin were measured by Western blotting with the anti-myc
antibody (upper column). The same experiment was also performed with anti-α-tubulin antibody (lower column) as a quantity control.
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Figure 5 Effects of XSENP1a on Wnt signalling. (A) Phenotypes
of embryos co-injected with mRNA of XSENP1a and other
canonical Wnt signalling components. All embryos shown are at
tadpole stages (st.39–41). The following mRNAs were injected
ventrally. No mRNA (a); XSENP1a (1.0 ng) ( b); XDvl (500 pg)
(c); XDvl (500 pg) and XSENP1a (1.0 ng) (d); β-catenin (250 pg)
(e); β-catenin (250 pg) and XSENP1a (1.0 ng) (f ); β-cateninSA
(50 pg) (g); β-cateninSA (50 pg) and XSENP1a (1.0 ng) (h); siamois
(25 pg) (i); siamois (25 pg) and XSENP1a (1.0 ng). (B) Frequency
of axis duplication. The results shown in (A) are expressed as the
percentage of ectopic axis duplication. Indicated mRNAs were
injected ventrally at the volume shown in (A). The solid bars show
complete axis duplication, which includes eyes and cement glands.
The open bars indicate incomplete axis duplication characterized
by a distinct branched axis without head structure. (B) Rescue
from head defect of XSENP1a by siamois expression. (a, b)
Phenotypes of embryos injected ventrally with mRNA of
XSENP1a (1.0 ng) (a), or XSENP1a (1.0 ng) and siamois (100 pg)
( b). (c) Average Dorso-anterior index (DAI) of Xenopus embryos
injected with mRNAs indicated in (a) and (b).
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senp1 (SUMO1/sentrin specific peptidase 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 19, anterior view, dorsal up.
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senp1 (SUMO1/sentrin specific peptidase 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.
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