Báo cáo khoa học: Characterization of ubiquitin-like polypeptide acceptor protein, a novel pro-apoptotic member of the Bcl2 family pptx

Characterization of ubiquitin-like polypeptide acceptor protein,
a novel pro-apoptotic member of the Bcl2 family
Morihiko Nakamura
1
and Yoshinori Tanigawa
2
1
Cooperative Medical Research Center and
2
Department of Biochemistry, Shimane Medical University, Japan
Monoclonal nonspecific suppressor factor (MNSF) is a
cytokine with antigen nonspecific suppressive activity.
MNSFb (a subunit of MNSF) is a 14.5 kDa fusion protein
consisting of a protein with 36% identity with ubiquitin and
ribosomal protein S30. The ubiquitin-like segment (Ubi-L)
may be cleaved from MNSFb in the cytosol. Recently, we
have observed that Ubi-L covalently binds to intracellular
proteins in mitogen-activated murine T-helper type 2 clone,
D.10 cells. In this study, we purified a 33.5 kDa Ubi-L
adduct from D.10 cell lysates by sequential chromatography
on DEAE, anti-(Ubi-L) Ig–conjugated Sepharose, and
hydroxylapatite. MALDI-TOF-MS fingerprinting revealed
that this Ubi-L adduct consists of an 8.5 kDa Ubi-L and a
Bcl2-like protein, murine orthologue of a previously cloned
human BCL-G gene product with pro-apoptotic function.
Murine Bcl-G mRNA was highly expressed in testis and
significantly in spleen. In addition, the level of Bcl-G mRNA
expression was increased in concanavalin A- and inter-
feron c-activated D.10 cells. The 33.5 kDa Ubi-L adduct
was expressed in spleen but not in testis, even though Bcl-G
protein was highly expressed in this tissue. The antisense

oligonucleotide to Bcl-G significantly decreased the level of
the Ubi-L adduct formation in concanavalin A-activated
D.10 cells and the proliferative response of the D.10 cells.
These results suggest that the post-translational modification
of Bcl-G by Ubi-L might be implicated in T-cell activation.
Keywords: Bcl2; T-cell; ubiquitin-like protein.
The covalent attachment of ubiquitin to proteins and their
subsequent degradation by the 26 S proteasome represents
the most commonly ascribed role for the protein ubiquiti-
nation system. In this respect, ubiquitin conjugation to
target substrates participates in a variety of important
eukaryotic processes, such as DNA repair [1], cell cycle
control [2], ribosome biogenesis [3], and the inflammatory
response [4]. In addition to ubiquitin, it is evident that
several ubiquitin-like proteins have been found to be
covalently or noncovalently attached to target proteins
[5–7].
Monoclonal nonspecific suppressor factor (MNSF), a
lymphokine produced by murine T-cell hybridoma, pos-
sesses pleiotrophic antigen-nonspecific suppressive func-
tions [8]. We have cloned a cDNA encoding a subunit of
MNSF, which was termed MNSFb [9]. MNSFb cDNA
encodes a protein of 133 amino acids (aa) consisting of a
ubiquitin-like protein (36% identity with ubiquitin) fused
to the ribosomal protein S30. The ubiquitin-like moiety
(Ubi-L) of MNSFb shows MNSF-like biologic activity
without cytotoxic action [10]. Interferon c (IFNc)is
involved in the mechanism of action of Ubi-L. We have
demonstrated that Ubi-L specifically binds to cell surface
receptors on mitogen-activated lymphocytes and the T-

helper type 2 clone, the D.10 cells [11].
We have also shown that Ubi-L covalently conjugates to
acceptor proteins and forms Ubi-L adducts including the
33.5 kDa protein in concanavalin A (Con A)- and IFNc-
stimulated D.10 cells [12]. Intracellular function of Ubi-L
remains largely unknown. In this study, we isolated and
characterized the 33.5 kDa Ubi-L adduct in D.10 cells.
Peptide mass fingerprinting using MALDI-TOF MS after
in-gel V8 protease digestion revealed that Ubi-L covalently
binds to a novel protein, a new member of the Bcl2 family,
suggesting that the Ubi-L conjugation might be involved
in the mechanism of survival of T-cells.
Materials and methods
Purification of the 33.5 kDa Ubi-L adduct
D.10 G4.1 cells, a murine T-helper clone type 2, were
cultured in the presence of 3 lgÆmL
)1
Con A (Calbiochem,
La Jolla, CA) as described previously [12,13] to a density of
5 · 10
)6
cellsÆmL
)1
(total volume 5 L). Cells were collected
by centrifugation and solubilized in lysing buffer (0.01
M
sodium phosphate buffer, 1% Triton, 0.5% sodium
deoxycholate, 0.1% SDS, 0.1
M
NaCl, 1 m

M
EGTA,
10 lgÆmL
)1
aprotinin, 10 lgÆmL
)1
leupeptin, 2 m
M
phenyl-
methylsulfonyl fluoride). After sonication, cell debris were
removed by centrifugation at 28 000 g at 4 °C for 60 min.
Supernatants were dialysed against Buffer A (10 m
M
Tris/
HCl, pH 7.2, 1 m
M
phenylmethylsulfonyl fluoride) and
applied to a column of DEAE equilibrated with the
same buffer. The column was washed extensively with
Buffer A containing 50 m
M
NaClandelutedwithBufferA
Correspondence to M. Nakamura, Cooperative Medical Research
Center, Shimane Medical University, 89-1 Enya-cho,
Izumo 693–8501, Japan.
Fax: + 81 853 20 2913, Tel.: + 81 853 20 2916,
E-mail:
Abbreviations: MNSF, monoclonal nonspecific suppressor factor;
Ubi-L, ubiquitin-like moiety of MNSF; Con A, concanavalin A;
IFNc,interferonc; SUMO, small ubiquitin-related modifier.

(Received 5 July 2003, revised 4 August 2003,
accepted 12 August 2003)
Eur. J. Biochem. 270, 4052–4058 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03790.x
containing 75 m
M
NaCl. The eluates were applied to
anti-(Ubi-L) Ig affinity column and Ubi-L adducts were
eluted, as described previously [14]. The eluates containing
anti-(Ubi-L) Ig reactive proteins were dialysed against
10 m
M
sodium phosphate (pH 7.3) and applied to a column
of hydroxylapatite. The column was eluted with 80 m
M
sodium phosphate. To obtain the 33.5 kDa Ubi-L adduct,
each of the fractions were assayed by immunoblotting with
the use of anti-(Ubi-L) Ig as described below.
Antibody preparation
A peptide corresponding to aa 199–208 of Bcl-G underlined
in Fig. 2A was synthesized with the use of the multiple-
antigen peptide system as described previously [15]. After
purification by reverse-phase HPLC, the multiple-antigen
peptide system was used to immunize rabbits. IgG in the
serum was purified by the use of protein A-Sepharose.
Immunoblotting
Cell extracts in SDS sample buffer were subjected to 12.5%
SDS/PAGE, and blotted onto polyvinylidene fluoride
membranes. The membranes were blocked with 5%
ovalbumin in NaCl/P
i

for 1 h and then washed with
NaCl/P
i
containing 1% Triton X-100 (NaCl/P
i
/Triton X).
Subsequently, the membranes were incubated with anti-
(Ubi-L) rabbit IgG (anti-PU1) in the blocking buffer, after
which they were incubated with peroxidase-conjugated
anti-rabbit IgG. Detection was done according to the
enhanced chemiluminescence detection system (Amersham
Biosciences). We have previously demonstrated that
anti-PU1 does not crossreact with ubiquitin [12].
RT-PCR
A mouse multiple tissue cDNA panel used as a template for
semiquantitative RT-PCR to confirm tissue-specific expres-
sion of Bcl-G was obtained from Clontech (Palo Alto, CA).
PCR was performed for 30 cycles according to the
manufacturer’s instructions. The PCR primers used to
detect Bcl-G mRNA shown in Fig. 3 are as follows: sense,
5¢-CCCAAGCTCTCCAGAACAAG-3¢;antisense,5¢-CT
GAGCTCGGATCTCCTTTG-3¢ (213 bp). All short
amplified PCR products were isolated and sequenced to
verify their identity. PCR products were separated by
electrophoresis through 2% agarose gel and stained with
ethidium bromide. In some experiments, signals were
quantitated by densitometry and optical densities for
Bcl-G were normalized to the corresponding values for
glyceraldehyde-3-phosphate dehydrogenase.
In-gel digestion with trypsin and peptide separation

The stained protein band from SDS/PAGE was digested
in-gel, and the peptides were extracted essentially according
to the methods of Rosenfeld et al. [16]. The peptides were
separated by reversed-phase chromatography on C18
column (Nacarai Tesque Inc., Kyoto, Japan) using a linear
gradient of acetonitrile (4–40% in 150 min) in formic
acid. The flow rate was 5 lLÆmin
)1
and detection was at
214 nm. Selected peaks were collected in tubes containing
10 lLÆmin
)1
of 30% acetonitrile, 0.1% formic acid and
subjected to sequence analysis.
In-gel digestion and MALDI-TOF
Silver-stained spots were cut out of the gels for in-gel
digestion and destained with 1 mL 50 m
M
sodium thiosul-
fate, 15 m
M
potassium ferricyanide followed by four washes
in 1 mL H
2
O. The spots were then equilibrated for 20 min
in 500 lLof100m
M
ammonium bicarbonate and then
incubated for 20 min in 500 lL of 50% acetonitrile, 50 m
M

ammonium bicarbonate. The spots were dried, rehydrated
for digestion with 5 lgÆmL
)1
V8 protease (Sigma) in 25 m
M
ammonium bicarbonate, and incubated at 37 °Covernight.
The reaction was stopped by adding 1 lL of 88% formic
acid. The peptides were extracted from the gel matrix by
vortex for 30 min and then concentrated using Zip Tips
(Millipore Corp.). Peptide mass fingerprinting was per-
formed using a PerkinElmer/PerSeptive Biosystems Voy-
ager-DE-RP MALDI-TOF mass spectrometer, operating
in delayed reflector mode at an accelerating voltage of
20 kV. The peptide samples were cocrystallized with matrix
on a gold-coated sample plate using 0.5 lLmatrix
(a-cyano-4-hydroxytranscinnamic acid) and 0.5 lLsample.
Cysteines were treated with iodoacetamide to form carboxy-
amidomethyl cysteine, and methionine was considered to be
oxidized.
Mutagenesis and transfection
Mutant Bcl-G (K110R) was generated by replacing the
codon for lysisne 110 with the codon for arginine by
utilizing QuickChange site-directed mutagenesis (Strata-
gene). cDNA encoding Bcl-G was subcloned in frame into
pQE-31 vector (Qiagen), resulting in the addition of a His
6
tag to the amino terminus. The gene was then subcloned
into the vector pcDNA3.1(+) (Invitrogen Corp.). D.10 cells
were transfected with 5 lg plasmid DNA as described by
Zhang et al.[17].

Oligonucleotide treatments
The oligonucleotides were synthesized as phosphorothioate-
containing two methoxyethyl modifications at positions 1–5
and 15–20. The antisense oligomer was complementary to
nucleotides 318–337 of Bcl-G which encode aa 24–31 of the
protein. Sequences were follows: antisense Bcl-G, 5¢-CG
TAGAAGGCCAGGATTTTG-3¢;senseBcl-G,5¢-CAA
AATCCTGGCCTTCTACG-3¢. The cells were transfected
20 h after Con A-activation using 15 lgÆmL
)1
Lipofectin
(Invitrogen) and 1 l
M
Bcl-G antisense or sense oligomer for
6 h. The cultures were washed with serum-free RPMI 1640
three times to remove Lipofectin. Cells were stimulated with
additional Con A following transfection for 46 h.
Results
Purification of a 33.5 kDa Ubi-L adduct from murine
T-cell line
Previous experiments have shown that Ubi-L conjugates to
acceptor proteins in Con A- and IFNc-stimulated T-cells
Ó FEBS 2003 Ubiquitin-like polypeptide acceptor protein (Eur. J. Biochem. 270) 4053
and T-cell lines. Con A-activated D.10 cells specifically
induces the 33.5 kDa Ubi-L adduct [12]. Thus, we tried to
isolate this adduct to elucidate the function of an unknown
Ubi-L target molecule in D.10 cells. The Ubi-L adduct was
induced in a 1-L shake flask culture for 2 days as described
and the 33.5 kDa Ubi-L adduct was purified to homogen-
eity by a combination of ion exchange chromatography,

anti-(Ubi-L) (anti-PU1) affinity chromatography and hyd-
roxylapatite chromatography (Fig. 1). The 33.5 kDa Ubi-L
adduct present in each fraction was identified by immuno-
blotting using anti-(Ubi-L) Ig. The final preparation gave a
single stained band on SDS/PAGE with mobility corres-
ponding to 33.5 kDa under reducing conditions (Fig. 1,
lane 2). This protein band was the first subjected to
N-terminal sequence analysis after electroblotting. Despite
repeated attempts, ambiguous signals were obtained from
about 100 pmol protein. For internal sequencing the protein
band was digested in-gel with trypsin. Selected peptides were
subjected to sequence analysis with results as shown in
Table 1.
MS of 33.5 kDa Ubi-L adduct
MALDI-TOF-mass fingerprinting was performed by sep-
arating the 33.5 kDa Ubi-L adduct by SDS/PAGE under
reducing conditions. Bands corresponding to the Ubi-L
adduct were excised and subjected to in-gel digestion with
V8 protease as described. Then, the resulting mixtures of
peptides were analysed by MALDI-TOF MS. Table 2
shows the peptide masses of observed by MALDI-TOF
mass fingerprinting of 33.5 kDa Ubi-L adduct purified from
murine D.10 cells. The resulting sets of peptide masses were
then used to search the NCBI database for potential
matches, confirming the Ubi-L adduct as a Ubi-L–Bcl2-like
protein complex. This Bcl2-like protein is a murine ortho-
logue of human Bcl-G, a novel pro-apoptotic member of the
Bcl2 family. Bcl-G possesses the Bcl2 homology domains
(BH2 and BH3) (Fig. 2). Signals were detected at 1241.0
and 1412.1 Da that correspond to aa 38–49 of Ubi-L and

20–32, respectively. Importantly, a pair of signals was
detected at 1556.6 and 2934.7 Da (Table 3). These signals
correspond to digestion fragments in which aa 104–111 of
Bcl-G are covalently linked by an isopeptide bond to aa
67–72 of Ubi-L, and aa 104–124 are linked by an isopeptide
Fig. 1. SDS/PAGE of the 33.5 kDa Ubi-L adduct. Purified fractions of
Ubi-L adduct analysed by SDS/PAGE (12% polyacrylamide gel) and
immunostained for protein. Lanes 1 and 3 contain an aliquot from the
anti-PU1 affinity chromatography purification step; lanes 2 and 4
contain an aliquot from the hydroxylapatite purification step; lanes 1
and 2, silver-stained; lanes 3 and 4, immunostained with anti-(Ubi-L).
Mobilities of the 33.5 kDa Ubi-L adduct and the molecular mass
standards (kDa) are indicated to the right and left, respectively.
Table 1. Internal peptide sequences. The amino acid sequence of Ubi-L
is shown in bold.
Peptide Sequence Residues
1 VACIANR 154–160
2 FEGPCDSK 249–256
3 ALGTWSTDSWTQV 57–69
4 AQELHT 7–12
Table 2. Assignments for peptide fragments from a Staphylococcus V8 protease digest of the 33.5 kDa Ubi-L adduct. The 33.5 kDa Ubi-L adduct was
digested by V8 protease and subjected to MALDI-MS analysis. The data in the second column are the mass values obtained experimentally,
whereas the results in the third column are those calculated from the V8 protease fragmentation of the gene products of Bcl-G and Ubi-L. The
fourth column indicates the number of the first and last amino acid of the identified Bcl-G and Ubi-L peptides, whereas the fifth shows the
corresponding amino acid sequences.
Protein
Mass (MH
+
)
Residues Sequence

Observed Calculated
Bcl-G 897.0 897.1 318–325 KILGISHE
930.1 930.1 208–215 QIISKIVE
1283.9 1283.5 173–184 VIHSQGGSKLKE
1397.0 1396.6 112–124 IRAQGPQGPFPVE
1599.0 1598.7 305–317 YFSPWVQQNGGWE
2065.2 2065.4 288–304 NHPMNRMLGFGTKYLRE
2152.8 2153.3 125–142 RQSGFHNQHWPRSLSSVE
2350.1 2349.8 81–100 KNISLGKKKSSWRTLFRVAE
Ubi-L 1241.0 1241.4 38–49 DQVVLLAGSPLE
1412.1 1411.6 20–32 TVAQIKDHVASLE
4054 M. Nakamura and Y. Tanigawa (Eur. J. Biochem. 270) Ó FEBS 2003
bond to aa 67–72. Collectively, Ubi-L may conjugate to Bcl-
G with a linkage between the C-terminal Gly74 and Lys110.
RT–PCR and immunoblotting experiments
To investigate mRNA levels of Bcl-G in various organs, we
performed PCR on a cDNA reverse transcribed from the
mRNA of different organs. A 213 bp PCR product was
generated by using the primers within the coding sequence.
PCR products were isolated and sequenced to verify their
identity (data not shown). Fig. 3A shows that testis had the
highest expression as described for human Bcl-G [18]. Low
but detectable expression of Bcl-L mRNA was found in
some other tissues including spleen. We also tested Bcl-G
mRNA levels in D.10 cells incubated with or without
Con A and IFNc. As can be seen in Fig. 3B, mRNA level
of Bcl-G in D.10 cells was increased by the treatment with
Con A and IFNc in good agreement with the previous
observations that 33.5 kDa Ubi-L conjugation is increased
in activated T-cells [12]. To confirm that Bcl-G covalently

conjugates to Ubi-L, we performed immunoblotting of the
33.5 kDa Ubi-L adduct using an antibody against synthetic
peptide based on the sequence of Bcl-G. The results of the
Fig. 3. RT-PCR analysis of Bcl-G transcripts in mouse tissues. (A) The
mouse multiple tissue cDNA panel was subjected to PCR using Bcl-G-
specific primers, and the DNA products were analysed by agarose gel
electrophoresis. The expected 213 bp band was prominent in cDNA
from testis. (B) Total RNA was isolated from cultured D.10 cells.
cDNA was synthesized from total RNA and was subjected to PCR as
described in (A). The number under each band is the treated/control
ratiooftheintensityofeachbandnormalizedtothatofglyceralde-
hyde-3-phosphate dehydrogenase measured by densitometry.
Fig. 2. Primary structure of Bcl-G. (A) The predicted amino acid
sequence of murine Bcl-G is presented with the BH2 and BH3 domains
shown in bold and residue numbers indicated. An internal sequence of
10 residues for polyclonal antibody is double underlined. The under-
lined amino acid sequences correspond to peptides whose masses were
detected by MALDI-TOF mass fingerprinting of the extracted in-gel
digest. Lys110 responsible for isopeptide formation is circled.
(B) Amino acid sequence of Ubi-L is presented with the C-terminal
Gly-Gly doublet shown in bold. Internal sequences obtained following
V8 protease digest are underlined.
Table 3. Isopeptide bonds between the C terminal of the glycine residue
of Ubi-L and the lysine of Bcl-G. The 33.5 kDa Ubi-L adduct was
digested by V8 protease and subjected to MALDI-MS analysis. The
data in the first column are the mass values obtained experimentally,
whereas the results in the second column are those calculated from the
V8 protease-fragmented peptide complexes. The third column shows
the corresponding amino acid sequences of Ubi-L and Bcl-G (shown in
bold).

Mass (MH
+
)
Sequence
Observed Calculated
1556.6 1556.9
2934.7 2934.4
Ó FEBS 2003 Ubiquitin-like polypeptide acceptor protein (Eur. J. Biochem. 270) 4055
blotting revealed that this antibody specifically recognized
the authentic 33.5 kDa Ubi-L adduct (Fig. 4A), indicating
that Ubi-L covalently binds to Bcl-G. Thus, the results
of immunoblotting together with the internal peptide
sequences in Table 1 and MALDI-TOF analysis show that
Ubi-L covalently binds to Bcl-G via an isopeptide bond. To
determine how much of endogenous Bcl-G is present as a
Ubi-L adduct in D.10 cells, immunoprecipitation experi-
ments were performed. As can be seen in Fig. 4B the
33.5 kDa Ubi-L was precipitated with anti-(Bcl-G) Ig from
a lysate of Con A-activated D.10 cells, whereas no Ubi-L
adduct was precipitated from unstimulated cells. We next
determined whether transfection of a K110R mutant of a
His-tagged Bcl-G construct would reveal the absence of
Ubi-L modified Bcl-G. As shown in Fig. 4C, Ubi-L linked
Bcl-G could be immunoprecipitated with anti-Bcl-G anti-
body from Con A-activated D.10 cells transfected with
wild-type Bcl-G, but not from cells transfected with the
mutant Bcl-G. These results were consistent with those
of fingerprinting analysis (Table 3). We next carried out
immunoblotting analysis to measure 33.5 kDa Ubi-L and
Bcl-G levels in different organs of mice. As shown in

Fig. 4D the 33.5 kDa Ubi-L adduct formation was repro-
ducibly found in the spleen and thymus. Interestingly, high
level of the Ubi-L adduct was found consistently in the
brain. Unexpectedly, we could not observe Ubi-L adduct in
the testis, even though this tissue expressed higher level of
Bcl-G (Fig. 3A and Fig. 4D).
Antisense oligonucleotide to Bcl-G inhibits the
proliferative response of mitogen-stimulated T-cell line
We next examined the effect of the antisense oligonucleotide
to Bcl-G on T-cell functions. The proliferative response of
Con A-activated D.10 cells was measured 52 h after
addition of the oligonucleotide. Antisense oligonucleotide
to Bcl-G inhibited the response of the mitogen-stimulated
cells by 33 ± 5% when compared with control cells treated
with Lipofectin alone (Fig. 5A). An equal concentration of
the sense oligonucleotide showed no effect. To confirm that
the antisense oligonucleotides were effective in depleting
Bcl-G protein expression, we examined the levels of Bcl-G
protein in unstimulated D.10 cell lysates 42 h after oligo-
nucleotide application. As detected by immunoblotting
analysis (Fig. 5B), the antisense oligonucleotide effectively
decreased the level of Bcl-G when compared with control
cells or to cells treated sense oligonucleotide. In addition, we
tested the levels of 35.5 kDa Ubi-L–Bcl-G complex in Con
A-activated D.10 cell lysates 22 h after oligonucleotide
application. The antisense oligonucleotide to Bcl-G signifi-
cantly decreased the level of the Ubi-L adduct formation.
Thus, it is possible that the post-translational modification
of Bcl-G by Ubi-L might be involved in T-cell activation.
Discussion

We have previously demonstrated that Ubi-L conjugates to
several proteins in Con A- and IFNc-stimulated T-cells [12].
MALDI-TOF mass fingerprinting and immunoblotting by
the use of anti-Bcl-G antibody demonstrate that Ubi-L
covalently binds to Bcl-G via isopeptide bond in activated
T-cells. Thus, Bcl-G might be one of the target molecules of
Fig. 4. Immunoblotting of the 33.5 kDa Ubi-L adduct. (A) Authentic
33.5 kDa Ubi-L adduct was subjected to SDS/PAGE, and blotted
onto nitrocellulose membranes. The membranes were immuno-stained
with antibody to Bcl-G: lane 1, pre-immune (IgG); lane 2, anti-(Bcl-G);
lane 3, anti-(Ubi-L) IgG (positive control). (B) Cell lysate was prepared
from cell cultured with or without Con A for 48 h. An equal amount
of protein (50 lg) from each cell lysate was immunoprecipitated (IP)
with anti-(Bcl-G), and the immunoprecipitant was Western blotted
(WB) for Bcl-G as well as Ubi-L. (C) D.10 cells were transfected with
vectors expressing either wild-type Bcl-G or its mutants (K110R).
Transfected cells were stimulated with Con A for 48 h. Subsequently,
the lysate was immunoprecipitated with anti-(Bcl-G) Ig, and the bound
proteins were analysed by Western blot with the antibodies indicated
on the left of each panel. (D) Tissue distribution of 33.5 kDa Ubi-L–
Bcl-G complex. Tissues homogenate (50 lgofproteineach)obtained
from the indicated organs were subjected to immunoblotting analysis
using anti-(Ubi-L) Ig as well as anti-(Bcl-G) Ig. Molecular mass is
shown in kDa. Anti-actin Ig was used to calibrate the amount of
protein loading and efficient protein transfer.
4056 M. Nakamura and Y. Tanigawa (Eur. J. Biochem. 270) Ó FEBS 2003
Ubi-L. Guo et al. demonstrated that Bcl-G is a novel pro-
apoptotic member of the Bcl2 family [18]. They showed that
Bcl-G induces apoptosis in transfected cells. The present
experiments suggest a new role for Ubi-L as an intracellular

regulator of the proliferation of mitogen-activated T-cell.
Data supporting this conclusion was obtained by antisense
study (Fig. 5). It may be inferred that the formation of
Ubi-L–Bcl-G complex in the early phase might be respon-
sible for T-cell activation. We have previously demonstrated
that the level of the Ubi-L adduct was gradually decreased
during T-cell proliferation [10]. Further studies are required
to more fully define the mechanism of Bcl-G modification
by Ubi-L in T-cell survival.
Bcl2 family proteins are functionally classified into two
groups. Both Bcl2 and Bcl-XL are anti-apoptotic members
of the Bcl2 family protein. In contrast, the other group,
comprising Bax and its related proteins including Bid and
Bad, promotes apoptosis. The activity of Bcl2 family
proteins can be regulated by post-translational modifica-
tions, including proteolysis and phosphorylation. Cleavage
of Bid by caspase 8 results in translocation of the cleaved
Bid to the mitochondria where it induces the release of
cytochrome c [19]. Bad is phosphorylated by the prosur-
vival kinases Akt [20]. Phosphorylation of Bad provides an
important link between extracellular survival factors and the
intrinsic cell death pathway regulated by Bcl2. In this
context, it is interesting that pro-apoptotic Bcl-G can be
modified by Ubi-L protein. We showed that Ubi-L formed
complex with Bcl-G in spleen, thymus and activated T-cells.
Interestingly, the Ubi-L adduct was also observed in brain.
It should be noted that parkin with an N-terminal ubiquitin-
like domain is important for the survival of the neurons that
degenerate in Parkinson’s disease [21,22]. In contrast, we
could not observe the complex in testis, although Bcl-G

protein is expressed in this organ. One might speculate that
enzyme(s) involved in the Ubi-L conjugation may be absent
or noninducible in testis. This phenomenon is being
characterized and will be the topic of another paper.
We could not identify the N-terminal region of Bcl-G
by MALDI-TOF analysis (Table 2) and internal peptide
sequence analysis (Table 1). It is possible that Bcl-G might
be digested by a caspase, because Bcl-G possesses candidate
caspase recognition site at its N-terminal region. Further
support for this hypothesis was obtained using immuno-
blotting. We carried out immunoblotting of the 33.5 kDa
Ubi-L adduct using an antibody against synthetic peptide
based on sequence of N-terminal of Bcl-G. This antibody
recognized Bcl-G but not the 33.5 kDa Ubi-L adduct (data
not shown). Indeed, the migrated position of the Ubi-L
adduct is somewhat smaller than the expected mass
(38 kDa) of Ubi-L–Bcl-G complex.
Ubiquitin-like proteins modify intracellular proteins as
well as ubiquitin. The activities of a number of important
transcription factors, including p53, c-Jun, and androgen
receptor, are regulated by small ubiquitin-like modifier-1
(SUMO-1) modification [23,24]. Thus, one question for
future study is whether Ubi-L also modifies some of these
factors. The nuclear dot protein Sp100 and promyelocytic
leukemia proteins are constituents of nuclear domains,
known as nuclear dots or PML bodies, and are both
covalently modified by SUMO [25]. It is evident that nuclear
dots play a role in autoimmunity [26]. Ubi-L/MNSFb is
also implicated in the mechanism of autoimmune disease.
We showed the presence of Ubi-L in the ascitic fluid of a

patient with systemic lupus erythematosus [27]. Interest-
ingly, the expression of both nuclear dots and Ubi-L are
induced by interferons [12,14,28–30]. Thus, ubiquitin-like
proteins may be involved in the pathogenesis of auto-
immune disease.
References
1. Jentsch, S., McGrath, J.P. & Varshavsky, A. (1987) The yeast
DNA repair gene RAD6 encodes a ubiquitin-conjugating enzyme.
Nature 329, 131–134.
Fig. 5. Effect of Bcl-G antisense oligonucleotide on mitogen-activated
D.10 cells. (A) D.10 cells were activated with Con A and the proli-
ferative response was determined as described [10]. Cells were exposed
to either antisense oligonucleotide or an equal concentration of sense
oligonucleotide: C, Lipofectin only; a, antisense oligonucleotide;
S, sense oligonucleotide. Percent suppression was calculated by com-
parison with the control response. The decrease in the antisense treated
cells was statistically significant compared with control (P <0.05).
Data are expressed as mean ± SD. (B) Immunoblotting of D.10 cells
treated with oligonucleotides as described in (A). Unstimulated D.10
cells were treated with oligonucleotides and blotted with anti-(Bcl-G)
Ig (upper panel); Con A-stimulated D.10 cells were treated with
oligonucleotide and blotted with anti-(Ubi-L) Ig (middle panel). Equal
quantities of protein (15 lg) were loaded on SDS/PAGE gels. The data
represent one of three independent experiments with similar results.
Anti-actin Ig was used to calibrate the amount of protein loading and
efficient protein transfer (lower panel).
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