From the Department of Biochemistry, Shimane Medical University, Izumo 693, Japan
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ABSTRACT |
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Monoclonal nonspecific suppressor factor (MNSF), a
lymphokine produced by murine T cell hybridoma, possesses pleiotrophic antigen-nonspecific suppressive functions. A cDNA clone encoding MNSF- Ubiquitin, a highly conserved 76-amino acid protein present in all
eukaryotic cells, is involved in the degradation of short lived or
structurally abnormal proteins. The process is accomplished through a
unique posttranslational modification in which the carboxyl Gly-Gly
terminus of ubiquitin is ligated covalently to lysine residues in
acceptor proteins. Ubiquitin-dependent proteolysis is
conducted via a multienzyme, ATP-dependent degradative
pathway (1). Other cellular processes in which the ubiquitin system is
also involved include antigen processing (2), ribosome biogenesis (3),
cell cycle progression (4), and regulation of the transcriptional nuclear factor- The monoclonal nonspecific suppressor factor (MNSF) is a product of a
concanavalin A (ConA)-activated murine hybridoma that inhibits the
generation of lipopolysaccharide (LPS)-induced immunoglobulin-secreting cells, proliferation of mitogen-activated T and B cells, and
interleukin (IL)-4 secretion by bone marrow-derived mast cells (10,
11). We have cloned a cDNA encoding a subunit of MNSF (12). The
subunit, termed MNSF- Most recently, we have demonstrated that Ubi-L covalently conjugates to
intracellular acceptor proteins in vitro (15) and in
vivo (16). MNSF- Several other ubiquitin-like proteins have been isolated and
characterized. Sentrin (also called SUMO-1), for instance,
preferentially modifies nuclear proteins (18). Like ubiquitin, the
ubiquitin cross-reactive protein (UCRP) conjugates to a number of
intracellular proteins (19). Interestingly, UCRP and Ubi-L are
subjected to induction by interferon (IFN) (11, 20). Furthermore, they show type specificity for IFN and have immunoregulatory properties (14,
21), although they have opposite functions (22). Accordingly, ubiquitin-like proteins may be involved in many biological reactions such as immune responses.
We have presented evidence that mitogen-activated T and B cells, and
murine lymphoid cell lines, may have an MNSF receptor (23). Although
further biochemical and functional analysis of this receptor protein
had been prevented because of the lack of a recombinant ligand, Ubi-L
enabled us to isolate and characterize the receptor for MNSF. In the
present study, we describe how Ubi-L specifically binds to cell surface
receptors on mitogen-activated lymphocytes and the T helper type 2 clone, the D.10 cell. Studies were also performed to characterize the
biochemical nature of Ubi-L receptor protein.
Materials--
Ubiquitin and rabbit anti-ubiquitin antibody (Ab)
were obtained from Sigma (St. Louis, MO). Mouse recombinant IFN- Tumor Cell Lines--
D.10 (type 2 helper T cell clone), BW5147
(T lymphoma), EL4 (T lymphoma), MOPC-31C (plasmacytoma), NFS-5C-1
(pre-B lymphoma), L929 (fibroblast), and B16 (melanoma) were maintained
in our laboratory. D.10 cells were maintained by biweekly stimulation
with 100 µg/ml conalbumin in the presence of 0.5 unit/ml recombinant
human IL-2. D.10 cells were used 10-12 days after stimulation with antigen.
Preparation of T Cells and B Cells--
T cells were enriched
from splenocytes by Lymphoprep centrifugation, passage over nylon wool,
and treatment with a mixture of monoclonal Abs against B220 and MAC-1,
and complement. All Ab depletions resulted in >99% of T cells as
assayed by flow cytometry. For B cell preparation, splenocytes were
incubated with a mixture of monoclonal Abs to Thy-1.2, L3T4, Lyt-2,
MAC-1, and a granulocyte-specific antigen, and complement. Live cells
were separated from dead cells and erythrocytes on Histopaque. With
this procedure, the T-depleted population was consistently more than
95% B cells (B220+).
MNSF- Biotinylation of Ubi-L--
500 µg of Ubi-L was mixed with a
20-fold molar excess of
sulfo-N-hydroxysulfosuccinimide-biotin in 1 ml of 0.01 M sodium phosphate, 0.15 M NaCl, and 0.1 mM phenylmethylsulfonyl fluoride, pH 7.2. Incorporation of
biotin into Ubi-L was determined by the
2-(4'-hydroxyazobenzene)benzilic acid assay (24), performed according
to the manufacturer's instructions (Pierce, Rockford, IL). In some
experiments, MNSF- Binding Experiments of Biotinylated Ubi-L to Target
Cells--
Binding experiments were performed at 24 °C.
Biotinylated Ubi-L (bio-Ubi-L; 20 nM) and the cells (2 × 106) to be tested were incubated in 200 µl of the
binding medium (RPMI 1640 containing 1% bovine serum albumin and 20 mM Hepes, pH 7.4) for 2 h. Nonspecific binding, unless
stated otherwise, was determined by measuring binding in the presence
of 100-fold molar excess unlabeled Ubi-L. Specific binding was
determined by subtracting nonspecific binding from total binding. The
cells were washed with binding medium five times and incubated with 200 µl of streptavidin-horseradish peroxidase conjugate in the same
medium for an additional hour. The cells were then washed five times
with 0.01 M sodium phosphate, 0.15 M NaCl, pH,
7.4; the substrate, turbo-3,3',5,5'-tetramethylbenzidine (100 µl;
Pierce), was added for 5 min. The color reaction was halted by the
addition of 1 M phosphoric acid (100 µl), and bound
biotinylated proteins were quantified by measuring the absorbance of
the reaction mixture at A450 nm using a
spectrometer. Bio-Ubi-L to cells was qualified by converting absorbance
units into mol of bio-Ubi-L using a standard curve made relating the
absorbances of known amounts of bio-Ubi-L.
Labeling of Cell Surface Receptors by
Biotinylation--
Unstimulated and ConA-stimulated D.10 cells (5 × 107) were washed three times with PBS and incubated in 2 ml of PBS containing 1 mg/ml
sulfo-N-hydroxysulfosuccinimide-biotin at 4 °C for 1 h. Thereafter, cells were washed three times with cold PBS containing 15 mM glycine to quench the reaction. The packed cells were
suspended in homogenization buffer containing 10 mM
Tris-HCl, pH 7.5, 1% Nonidet P-40, 0.1% SDS, 150 mM NaCl,
10 µg/ml aprotinin, 5 µg/ml leupeptin, and 1 mM
phenylmethylsulfonyl fluoride. The cells were homogenized in a glass
homogenizer. Detergent-insoluble materials was removed by
centrifugation at 12, 000 × g for 30 min at 4 °C. The supernatant fraction was either subjected immediately to affinity chromatography or stored at Preparation of Cell Membranes--
All procedures were performed
at 4 °C. D.10 cells (2 × 108 cells) were washed
with Hanks' balanced salt solution and suspended in a hypotonic buffer
(10 mM Tris-HCl, pH 7.5, 1 mM
MgCl2, 1 mM CaCl2, 1 mM
phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin). The suspension was
mixed vigorously on a Vortex mixer and allowed to stand for 5 min
before being spun at three different stages: 700 × g
for 5 min, 3,500 × g for 10 min, and 40,000 × g for 1 h. The 40,000 × g precipitate
was collected. Membranes were suspended in a solubilization buffer (1%
Triton X-100, 10 mM 4-Hepes, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, pH
7.5) and left at 4 °C for 1 h with occasional shaking. The mixture was then spun (10,000 × g, 15 min, 4 °C),
and the supernatant was collected and spun in an ultracentrifuge
(100,000 × g, 60 min, 4 °C). The supernatant was
subjected to ligand blot assay as described below.
Affinity Column Chromatography--
Ubi-L was coupled to
CNBr-activated Sepharose 4B (Amersham Pharmacia Biotech) according to a
procedure provided by the manufacturer. Detergent extract of membrane
fraction (13 mg of protein) was applied to the Ubi-L column and
incubated overnight under gentle agitation at 4 °C. The beads were
then packed into a column and washed with sodium phosphate buffer, pH
7.5, containing 100 mM NaCl and 0.1% Triton X-100, and
then the receptor was eluted with 1 M NaCl. The elutes were
concentrated in Centricon-10 (Amicon, Beverly, MA) and subjected to
reverse phase HPLC (Cosmosil, Nacalai tesq, Kyoto, Japan).
To obtain small peptides suitable for amino acid sequence analysis,
purified Ubi-L receptor was digested with trypsin at an enzyme:substrate molar ratio of 1:100 at 37 °C for 4 h. Tryptic peptides were separated by reverse phase HPLC using a C18 column equilibrated with 5% acetonitrile in 0.1% trifluoroacetic acid. The
column was developed with a 5-60% linear gradient of acetonitrile for
120 min at 25 °C with a flow rate of 1.0 ml/min. Peptides were
sequenced directly from polyvinylidene difluoride membranes using an
ABI477 protein sequencer (Applied Biosystems Inc.).
Immunoblotting--
The biotinylated cell surface proteins (250 µg of protein) were applied to the Ubi-L column and incubated as
described above. The receptor was eluted with a 2 × SDS sample
buffer, subjected to 10% SDS-PAGE, and blotted onto nitrocellulose
membranes. The membranes were blocked with 5% bovine serum albumin in
PBS for 1 h and then washed three times with PBS containing 0.5%
Tween 20 (PBS/Tween 20). Subsequently, the membranes were incubated with streptavidin conjugated to horseradish peroxidase (1:1,000) in
PBS/Tween 20 for 45 min. Membranes were washed five times in PBS/Tween
20 and developed using the ECL reagents (Amersham Pharmacia Biotech).
In some experiments, MNSF- Ligand Blot Assay--
Detergent-extracted membrane proteins
were prepared from antigen-stimulated D.10 cells that were not labeled
with biotin. The proteins (730 µg) were blotted onto nitrocellulose
sheets as described above. The nitrocellulose sheets were incubated in buffer containing 50 mM Tris-HCl, pH 7.5, 0.1% Tween 20, and 50 µg/ml biotin-labeled Ubi-L or 200 µg/ml biotin-labeled
ubiquitin for 16 h at 4 °C. The sheets were washed twice with
binding buffer without the Ubi-L for 5 min and then incubated with
streptavidin conjugated to horseradish peroxidase in PBS/Tween 20 for
30 min and subsequently washed three times for 5 min with the same
buffer. Detection of labeled proteins was performed with ECL reagents.
Immunization of Mice--
A protein band (82 kDa) was excised
from polyacrylamide slab gels, minced, and injected subcutaneously
together with complete Freund's adjuvant. Additional immunizations
were given 3 and 4 weeks later.
Immunoprecipitation of a Cross-linked 125I-Ubi-L
Receptor Complex with Anti-receptor Serum--
Ubi-L was labeled with
125I as described previously for GST-Ubi-L (15). Aliquots
of the purified receptor (500 ng) were mixed with
125I-Ubi-L and left for 1 h at 24 °C.
Disuccinimidyl suberate was added to a final concentration of 0.3 mM. The cross-linking was stopped after 30 min at 4 °C
by the addition of Tris-HCl buffer, pH 7.5, to a final concentration of
20 mM. Mouse anti-receptor serum or control serum was added
and incubated for 2 h at 24 °C. The antigen-antibody complex
was adsorbed on protein A-Sepharose and analyzed by SDS-PAGE followed
by autoradiography. In some experiments, ConA-activated D.10 cells were
biotinylated and solubilized as described under "Labeling of Cell
Surface Receptors by Biotinylation." To determine whether proteins
other than the 82-kDa polypeptide are associated with Ubi-L receptor,
lysates were immunoprecipitated with anti-Ubi-L receptor Ab.
Affinity Chromatography on a Wheat Germ Agglutinin (WGA)
Column--
Ubi-L receptor purified by a Ubi-L column was incubated at
4 °C for 6 h with 5 ml of WGA-Sepharose equilibrated in 50 mM Tris-HCl, pH 7.4, 140 mM NaCl, 10%
glycerol, and 0.1% Triton X-100. After incubation, the mixture was
transferred to a column, the beads allowed to settle, and the column
was washed with 50 ml of 0.5 M KCl, 50 mM
Tris-HCl, pH 7.4, 140 mM NaCl, and 0.1% Triton X-100. The
absorbed Ubi-L receptor was then eluted with 10 ml of 0.5 M
N-acetyl-D-glucosamine, 50 mM
Tris-HCl, pH 7.4, 0.5 M NaCl, 10% glycerol, and 0.1%
Triton X-100.
Deglycosylation of Ubi-L Receptor Proteins--
The membrane
fraction of ConA-activated D.10 cells was acidified by the addition of
sodium citrate buffer, pH 6.2, to a final concentration of 50 mM. Neuraminidase (0.7 unit) was added, and the mixture was
incubated at 37 °C for 4 h. SDS sample buffer was added to
deglycosylated samples and boiled for 5 min. SDS-PAGE and
immunostaining with anti-Ubi-L receptor Ab were carried out as
described above.
Neutralizing Tests--
Determination of IgE production by
LPS-stimulated B cells was done as described previously (14). Briefly,
the purified B cells (5 × 105/ml) were cultured with
20 µg/ml LPS. Ubi-L, recombinant IFN- Binding Experiments of Recombinant Ubi-L to Target
Cells--
Previous experiments have shown that Ubi-L acts on murine
helper T cell clone, D.10 cells (25). To investigate whether or not
Ubi-L would bind specifically to D.10 cells, a binding assay was
performed. Purified recombinant Ubi-L enabled acquisition of bio-Ubi-L
with a high specific activity comparable to that of unlabeled Ubi-L. As
shown in Fig. 1, bio-Ubi-L bound to
ConA-activated (48 h), but not to unstimulated D.10 cells. Bio-Ubi-L
bound to the activated D.10 cells most rapidly at 24 °C (data not
shown). We also tested the possibility that 14.5-kDa MNSF- Ubi-L Receptor Expression Is Limited to Lymphoid Cells--
We next investigated the cellular distribution of the Ubi-L
receptor. Binding experiments were carried out using various cells of
murine and human origin. Among a series of mouse cell lines tested, EL4
and MOPC-31C cells apparently carried the Ubi-L receptors (Table
I). Of note, both cell lines are
sensitive to Ubi-L in terms of inhibition of proliferation (13). D.10
cells were stimulated with antigen (conalbumin) and ConA, as described
previously (25). Ubi-L bound to both stimulated D.10 cells. Together,
the expression of Ubi-L receptor should be limited to lymphoid cells.
In contrast, Ubi-L did not bind to any human cell lines such as Jurkat,
K562, MOLT-3, Namalwa, HL60, U937, Detroit 562 (data not shown),
suggestive of the species specificity of Ubi-L action.
Specificity of Ubi-L Binding to Its Receptor--
To clarify the
specificity of the Ubi-L binding, competitive assay experiments were
performed by adding to the experimental system the nonlabeled Ubi-L,
MNSF- Affinity Chromatography and Immunoblotting--
We next
investigated biochemical nature of the receptor protein for Ubi-L.
Cross-linking experiments with the use of recombinant Ubi-L were
insufficient for identification of Ubi-L receptor protein(s). We
observed heterogeneous bands on SDS-PAGE (data not shown), which seemed
to be self-aggregation of Ubi-L probably because of its strong
hydrophobicity. We also employed 125I-GST-Ubi-L, which is a
stable fusion protein. It should be noted that the activity of
GST-Ubi-L is lower than that of
Ubi-L.2 A trace amount of protein
(approximately 120 kDa) was reproducibly cross-linked by
125I-GST-Ubi-L (data not shown). Therefore, we decided to
use affinity chromatography on an immobilized Ubi-L column as the main
step of Ubi-L receptor purification. D.10 cells were stimulated with ConA, biotinylated, and lysed. Biotinylated membrane proteins (250 µg/0.1 ml) were incubated with Ubi-L-Sepharose, and bound proteins
were eluted as described under "Experimental Procedures." The
eluates were subjected to SDS-PAGE, blotted onto nitrocellulose membranes, and visualized by ECL system. As depicted in Fig.
4A, only a single band of 82 kDa
under nonreducing conditions was observed (lane 2). The
migrated position of this band was unchanged under reducing conditions
(lane 3). These results are consistent with the
cross-linking experiments with the use of 125I-GST-Ubi-L
(34 kDa) in terms of the molecular mass. On the contrary, no band was
recovered from the biotinylated membrane proteins from unstimulated
D.10 cells (lane 1), in good accordance with the results of
binding assay (Fig. 1) and the previous observations that
antigen-activated, but not unstimulated, D.10 cells are sensitive to
Ubi-L (25). Additionally, we made MNSF-
We next performed immunoblotting analysis by the anti-receptor Ab.
Detergent extract of ConA-activated D.10 cells was resolved by SDS-PAGE
and subjected to electrophoretic transfer and immunochemical staining
for Ubi-L receptor. As shown in Fig.
5A, a major protein band at 82 kDa
and a minor band at 65 kDa under nonreducing conditions were detected
by the anti-receptor Ab (lane 2). This finding indicates sequence homology between 82-kDa and 65-kDa proteins. We speculate that
a protease, for instance, endoproteinase, has cleaved up to 17 kDa from
the intact 82-kDa receptor, which results in the generation of receptor
fragment that has lost the ability to bind Ubi-L but not the capacity
to interact with the anti-receptor Ab. The migrated positions of these
bands were unchanged under reducing conditions (lane 3). The
same results were obtained from antigen (conalbumin)-stimulated D.10
cells (not shown). In contrast, the Ubi-L receptor was not seen in
detergent extract of unstimulated D.10 cells (lane 4). We
next performed deglycosylation analysis of Ubi-L receptor proteins
because the 82-kDa protein may be glycosylated (Fig. 4C).
The membrane fraction of ConA-activated D.10 cells was treated with
neuraminidase and subjected to SDS-PAGE and immunoblotting as described
above. Deglycosylation converted 82-kDa and 65-kDa molecules to 74-kDa
and 60-kDa molecules, respectively (Fig. 5B). The mobility
on a gel was changed further by additional treatment with
N-glycanase, albeit a product was very faint (data not
shown). These findings indicate that Ubi-L receptor protein may be a
glycoprotein.
Immunoprecipitation of Ubi-L Receptor Complex--
We next
determined whether antiserum prepared against the 82-kDa protein band
would immunoprecipitate the cross-linked complex of
125I-Ubi-L (8 kDa) and its receptor. A band of 90 kDa was
obtained (Fig. 6A), although
several bands including 98-, 106-, 114-kDa were also observed
presumably because of self-aggregation of 125I-Ubi-L.
Control serum did not immunoprecipitate any complexes. To investigate
whether proteins other than the 82-kDa polypeptide are associated with
Ubi-L receptor complex, extracts of biotinylated D.10 cells were
immunoprecipitated with anti-Ubi-L receptor Ab. As can be seen in Fig.
6B, the 105-kDa protein was coimmunoprecipitated by the
anti-receptor (82-kDa polypeptide) Ab. We speculate that this 105-kDa
protein may be involved in the signal transduction of Ubi-L, although
it lacks an ability to bind Ubi-L itself.
Neutralization of Ubi-L Activity by Anti-Ubi-L Receptor Ab--
To
demonstrate that the 82-kDa protein is involved in Ubi-L-mediated
signal transduction, a neutralizing test was carried out by the use of
specific Ab (IgG) to this protein. As shown in Table
II, this Ab could neutralize the Ubi-L
activity, confirming that the 82-kDa protein is bioactive Ubi-L
receptor. Interestingly, this Ab abolished the synergism between Ubi-L
and IFN- Determination of the Amino Acid Sequence of Ubi-L Receptor
Protein--
We next attempted to analyze the amino acid sequence of
the 82-kDa Ubi-L receptor protein (Table
III). The NH2-terminal amino acid sequence of the receptor protein could not be determined probably
because of acetylation. Thus this protein was digested with trypsin,
separated by reverse phase HPLC, and tryptic peptides were sequenced.
As shown below, four of the five peptide sequences derived from Ubi-L
receptor are in alignment with a related sequence found in the open
reading frame predicted by the DNA sequence of the cDNA
encoding mouse IL-11 receptor (28). The similarity of Ubi-L receptor
peptide sequences to sequences in mouse IL-11 receptor as well as the
similarity in size of the two proteins suggests that the Ubi-L receptor
might be an another closely related protein. It should be noted that
tryptic peptide 2 contains a WSXWS motif commonly seen in a
cytokine receptor family (29).
Several lines of evidence have emerged from our studies indicating
that a ubiquitin-like polypeptide specifically binds to target cells
such as D.10 cells. We employed D.10 cells throughout the experiments
because the response to Ubi-L had been well characterized in our
previous study (25). The binding of Ubi-L to its receptor might be
specific for the following reasons. (i) Ubi-L bound to antigen-stimulated but not to unstimulated D.10 cells. (ii) The binding
of bio-Ubi-L was inhibited by the addition of unlabeled Ubi-L but not
by irrelevant proteins. (iii) The binding showed species specificity.
(iv) The molecular mass of affinity-purified Ubi-L receptor was the
same as that of the protein obtained from ligand blot assay. (v) The
antigenicity of these proteins was much the same. Finally (vi) Ubi-L
activity was neutralized by anti-Ubi-L receptor Ab. On SDS-PAGE under
nonreducing conditions, the apparent molecular weight of the Ubi-L
receptor was 82,000. This apparent size was unchanged by the disulfide
reducing agent dithiothreitol, indicating a lack of disulfide bonds to
other proteins. This 82-kDa protein may be associated with the 105-kDa protein lacking an ability to bind Ubi-L (Fig. 6B).
Characterization of this 105-kDa protein is under way to clarify its
function. Scatchard analysis of Ubi-L receptor was unsuccessful because of an aggregative property of recombinant Ubi-L. Of note, Ubi-L has a
secondary structure containing the tandem largely hydrophobic structural units. This likely accounts for our consistently poor yield
of Ubi-L after cleavage from the fusion partner GST. It is difficult to
handle a small amount of Ubi-L even in the presence of a carrier
protein such as bovine serum albumin. In contrast, ubiquitin has a
stable secondary structure containing only a low percentage of
It has been claimed that suppressor factors consist of effector and
accessory molecules (31). Like antigen-specific suppressors, hybridoma-derived (native) 70-kDa MNSF consists of 8-kDa Ubi-L (MNSF- It is evident that several cell surface receptor proteins are
ubiquitinated. For instance, platelet-derived growth factor receptor is
a ubiquitin acceptor (8, 9). The intracellular domain of this receptor
is ubiquitinated, which leads to intracellular signaling. Similarly,
cytosolic regions of the TNF- Ubiquitin is not the only molecular tag for protein modification.
Sentrin (18) and UCRP (19) have been shown to be conjugated to other
proteins in a process analogous to ubiquitination. The COOH termini of
UCRP and sentrin are processed efficiently, which allows for subsequent
protein conjugation. Similarly, Ubi-L is cleaved from a fusion partner,
ribosomal protein S30, in cytosol. Like UCRP, Ubi-L conjugates to
intracellular proteins in vitro (15) and in vivo
(16). Ubi-L conjugates to intracellular acceptor proteins including
MNSF- A number of biotinylated cytokines have been employed for receptor
analysis instead of iodination (35-37). Bio-Ubi-L shows biological
activity similar to that of unlabeled
Ubi-L.3 Nevertheless,
cross-linking experiments with the use of biotinylated or iodinated
Ubi-L were unsuccessful because of the nature of self-aggregation, as
described above. Therefore, affinity chromatography on an immobilized
Ubi-L column was used for the identification of Ubi-L receptor protein.
This Ubi-L column allowed us to purify the Ubi-L receptor and prepare
antiserum against it. We are attempting to investigate whether or not
soluble Ubi-L receptor may affect the immune responses in
vitro and in vivo and isolate a cDNA encoding this
receptor protein. Recombinant receptor may pave the way for further
clarification of the mechanism of action of ubiquitin-like polypeptide(s).
Peptide sequence analysis suggests that this 82-kDa protein should be a
member of a cytokine receptor family. Ubi-L receptor peptide sequences
are similar to those in IL-11 receptor (Table III), although the mode
of action of Ubi-L is different from that of IL-11. Most recently we
observed that Ubi-L receptor ligation rapidly induces protein tyrosine
phosphorylation of distinct proteins in D.10 cells.2
We have reported previously that IFN-, an isoform of the MNSF, has been isolated and characterized. MNSF-
cDNA encodes a fusion protein consisting of a
ubiquitin-like segment (Ubi-L) and ribosomal protein S30. Ubi-L appears
to be cleaved from the ribosomal protein and released extracellularly in association with T cell receptor-like polypeptide. In the current study we have characterized the biochemical nature of the Ubi-L receptor on D.10 G4.1, a murine T helper clone type 2. Biotinylated Ubi-L bound preferentially to concanavalin A-stimulated but not to
unstimulated D.10 cells. Detergent-extracted membrane proteins were
applied to an immobilized Ubi-L column. SDS-polyacrylamide gel
electrophoresis of eluted fraction revealed a band of
Mr = 82,000. Biotinylated Ubi-L specifically
recognized this band, confirming that the 82-kDa protein is the Ubi-L
receptor. A complex of Mr = 90,000 was
visualized by immunoprecipitation of 125I-Ubi-L
cross-linked to the purified receptor followed by SDS-polyacrylamide gel electrophoresis and autoradiography. In addition, a 105-kDa protein
was coimmunoprecipitated by anti-Ubi-L receptor (82-kDa polypeptide)
antibody, indicative of the association of this protein with the Ubi-L
receptor complex. Amino acid sequence analysis of the 82-kDa
polypeptide revealed that the Ubi-L receptor may be a member of a
cytokine receptor family.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B (5). In addition, several signal transducing receptors, in particular the
-subunit of the
TCR1-CD3 complex (6), the high
affinity IgE receptor (7), and platelet-derived growth factor receptor
(8, 9), are ubiquitinated receptors.
, encodes a protein of 133 amino acids
consisting of a ubiquitin-like protein (36% identity with ubiquitin)
fused to the ribosomal protein S30. We have reported evidence showing that the ubiquitin-like segment of MNSF-
(Ubi-L) is responsible for
its activity (13). Ubi-L inhibits IgE and IgG1 production by
LPS-activated B cells and division in various tumor cell lines of
murine origin (14).
, a subunit of MNSF, was identified as an acceptor protein for Ubi-L. It is probable that Ubi-L might be released
in a posttranslationally modified form (i.e. conjugation of
Ubi-L to MNSF-
) because it lacks a signal peptide (12). Partially
purified isopeptidase dissociates Ubi-L from MNSF-
, suggesting that
the COOH-terminal Gly-Gly doublet of Ubi-L may covalently ligate to the
lysine residue in MNSF-
(17).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(1 × 107 units/mg) was purchased from Genzyme
(Cambridge, MA). Mouse IFN-
(1 × 107 units/ml) was
obtained from SeroTech (Tokyo, Japan). Specific Ab against synthetic
peptides corresponding to the ubiquitin-like region (PU1) was raised in
rabbits as described previously (12). Peroxidase-conjugated goat
anti-mouse IgG Ab was from Capel (Durham, NC).
and Ubi-L--
Recombinant MNSF-
and Ubi-L were
obtained as described previously (12). Briefly, either MNSF-
or
Ubi-L was expressed as a fusion protein with glutathione
S-transferase (GST) using the pGEX-2T vector (Amersham
Pharmacia Biotech). Ubiquitin-like segment was cleaved from the fusion
partner by thrombin and purified by using anti-PU1 Ab coupled to
Sepharose 4B (Amersham Pharmacia Biotech).
was also biotinylated as described for Ubi-L.
Each molecule of Ubi-L was labeled with three molecules of biotin,
albeit self-aggregation occurred during the procedure.
80 °C.
or ubiquitin was coupled to
CNBr-activated Sepharose 4B and used for affinity chromatography as
described above.
, and anti-Ubi-L receptor
serum (IgG) were added at the initiation of the cultures. Supernatants
were harvested 7 days after initiation of the cultures, and IgE
production was detected by enzyme-linked immunosorbent assay.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(Ubi-L-ribosomal protein S30), which shows Ubi-L-like activity (26),
might bind to the D.10 cells. It should be noted that the COOH-terminal
Gly-Gly doublet of Ubi-L is followed by ribosomal protein S30. Despite of the blocked COOH terminus, biotinylated MNSF-
bound to
ConA-activated D.10 cells (Fig. 1), as did Ubi-L. The pattern of
MNSF-
binding was much the same. In contrast, ribosomal protein S30
did not bind to the cells (data not shown). These findings indicate
that Ubi-L may not bind to its receptors via the COOH-terminal glycyl doublet responsible for ubiquitination process. Because Ubi-L shows a
36% homology with ubiquitin (12), we tested the possibility of the
binding of ubiquitin to D.10 cells. Fig. 1 shows that 10 mM
biotinylated ubiquitin, 500-fold the amount of Ubi-L used in the
binding experiments, bound slightly to antigen-stimulated D.10 cells
probably because of the homology. We also examined whether Ubi-L would
recognize mitogen-activated lymphocytes. T cells and B cells were
separated from splenocytes as described under "Experimental
Procedures." As shown in Fig. 2, exposure of the T cells to 3 µg/ml ConA for 2 days led to maximal binding of
bio-Ubi-L on the cell surface. Bio-Ubi-L bound to 20 µg/ml LPS-activated B cells almost in the same manner as ConA-activated T
cells. In contrast, neither unstimulated (day 0) T cells nor B cells
showed any significant bio-Ubi-L binding. These results are consistent
with those of binding experiments of native MNSF (23).
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Fig. 1.
Binding of bio-Ubi-L to D.10 cells. The
specific binding of 20 nM bio-Ubi-L ( ,
), 20 nM MNSF-
(
],
), and 100 nM ubiquitin
(
) at 24 °C to D.10 cells (2 × 106) was
determined.
,
, unstimulated D.10 cells;
,
,
,
ConA-stimulated (48 h). Each biotinylated polypeptide was added in the
absence or presence of a 100-fold molar excess of unlabeled ligand.
Specific binding was presented in the figure. The data shown are the
means ± S.D. of three independent experiments.
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Fig. 2.
Binding of bio-Ubi-L to B cells and T
cells. The specific binding of bio-Ubi-L (20 nM) at
24 °C to 3 µg/ml ConA-stimulated (48 h) T cells (5 × 106) ( ) and 20 µg/ml LPS-stimulated B cells (5 × 106) (
) was determined. Specific binding is presented in
the figure as described in the legend to Fig. 1. The data shown are the
means ± S.D. of three independent experiments.
Cellular distribution of murine Ubi-L receptor
, ubiquitin, and other suppressive cytokines such as IFN-
and IL-10. As can be seen in Fig. 3, Ubi-L and MNSF-
exclusively inhibited the binding of bio-Ubi-L to
ConA-activated D.10 cells. However, the irrelevant ligands (IFN-
and
IL-10) did not show any competition, indicative of the specificity for Ubi-L binding. Ubiquitin showed a slight but significant inhibition of
the Ubi-L binding in agreement with previous finding that ubiquitin inhibits Ubi-L-induced suppression (13).
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Fig. 3.
Specificity of bio-Ubi-L binding to
ConA-activated D.10 cells. D.10 cells (2 × 106
cells) were incubated with 20 nM bio-Ubi-L in the presence
of unlabeled Ubi-L ( ), MNSF-
(
), ubiquitin (
), IFN-
(
), and IL-10 (
). The binding of bio-Ubi-L to D.10 cells was
determined as described under "Experimental Procedures." The data
shown are the means ± S.D. of the three independent
experiments.
and ubiquitin affinity columns to isolate Ubi-L receptor protein(s). The same band of 82 kDa
was obtained from MNSF-
column, whereas no significant amount of
protein band could be recovered from the ubiquitin column (data not
shown). To confirm that the 82-kDa protein is a Ubi-L receptor, a
ligand blot assay was carried out. Detergent-extracted membrane
proteins were prepared from ConA-stimulated D.10 cells and blotted onto
nitrocellulose membranes. Bio-Ubi-L specifically recognized the 82-kDa
band (Fig. 4B, lane 1), suggesting that it should
be a ligand for this receptor protein. In contrast, Ubi-L did not bind
to any membrane proteins from unstimulated D.10 cells (Fig.
4B, lane 3). Interestingly, ubiquitin slightly bound to the Ubi-L receptor (Fig. 4B, lane 2). We
also examined whether the 82-kDa Ubi-L receptor binds to lectins. Ubi-L
receptor purified by a Ubi-L column was incubated with WGA-Sepharose.
After 6 h, the absorbed Ubi-L receptor was eluted with
N-acetyl-D-glucosamine. The eluate was
electrophoresed and silver stained. As can be seen in Fig.
4C, the 82-kDa Ubi-L receptor was recovered in the eluate fraction, suggesting that Ubi-L receptor may bind to WGA.
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Fig. 4.
Biochemical analysis of Ubi-L receptor by
SDS-PAGE. Panel A, SDS-PAGE of the purified Ubi-L
receptor. D.10 cells (5 × 107 cells) were stimulated
with ConA for 48 h, biotinylated, and lysed as described under
"Experimental Procedures." Biotinylated membrane proteins were
applied to a Ubi-L-Sepharose column, and bound proteins were eluted.
The eluates were subjected to 10% SDS-PAGE, blotted onto
nitrocellulose membrane, and visualized by the ECL system. Lane
1, unstimulated D.10 cells; lanes 2 and 3,
ConA-stimulated (48 h); lanes 1 and 2, eluate
from the Ubi-L Sepharose column under nonreducing conditions;
lane 3, same as lane 2 but under reducing
conditions. The positions of molecular mass markers (kDa) are shown on
the left. Panel B, ligand blot assay for Ubi-L
receptor protein. Detergent-extracted membrane proteins were prepared
from D.10 cells as described under "Experimental Procedures." The
proteins were blotted onto nitrocellulose sheets and incubated with
bio-Ubi-L (lanes 1 and 3) or biotinylated
ubiquitin (lane 2). Lanes 1 and 2,
extract from ConA-stimulated D.10 cells; lane 3,
unstimulated cells. The positions of molecular mass markers (kDa) are
shown on the left. Panel C, affinity
chromatography on WGA. Ubi-L receptor purified by a Ubi-L Sepharose
column was applied to a WGA column. Fractions from WGA were
electrophoresed on 10% gel and silver stained. Lane 1,
effluent; lane 2, eluate.
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Fig. 5.
Immunoblotting analysis by anti-receptor
Ab. Panel A, determination of anti-82-kDa protein
Ab-reactive proteins. Detergent extract of the membrane fraction of
ConA-activated (48 h) D.10 cells was subjected to SDS-PAGE.
Immunoblotting was performed by using peroxidase-conjugated goat
anti-mouse IgG as a second Ab. Detection of biotin-labeled proteins was
carried out with the ECL system. Lanes 1-3, ConA-stimulated
cells; lane 4, unstimulated; lane 1, control Ab;
lanes 2-4, anti-receptor Ab. Panel B,
deglycosylation of Ubi-L receptor proteins. The membrane fraction of
ConA-activated D.10 cells was treated with neuraminidase as described
under "Experimental Procedures." SDS-PAGE and immunostaining were
performed as described above. Lane 1, untreated; lane
2, treated with neuraminidase.
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Fig. 6.
Immunoprecipitation by specific Ab to Ubi-L
receptor protein. Panel A, immunoprecipitation of
125I-Ubi-L receptor complex by anti-82-kDa protein Ab.
125I-Ubi-L cross-linked to the purified receptor was
immunoprecipitated with Ab (IgG) and analyzed by SDS-PAGE followed by
autoradiography. Lane 1, IgG to the 82-kDa band; lane
2, IgG to complete Freund's adjuvant. The positions of molecular
mass markers (kDa) are shown on the left. Panel
B, immunoprecipitation of biotinylated D.10 cells by anti-82-kDa
protein Ab. Extracts of biotinylated D.10 cells were immunoprecipitated
by the Ab and analyzed by SDS-PAGE followed by the ECL system.
Lane 1, unstimulated D.10 cells; lanes 2 and
3, ConA-stimulated (48 h); lanes 1 and
2, anti-82-kDa protein IgG; lane 3, control
IgG.
. This finding supports the notion that IFN-
might
enhance the expression of Ubi-L receptor protein on the target cells
(27).
Abrogation of Ubi-L activity by specific antibody to its receptor
Alignment between amino acid sequence derived from sequencing Ubi-L
receptor (R) and the corresponding sequences from the open reading
frame of cloned murine IL-11 receptor
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix or
-sheet (30).
) and 62-kDa MNSF-
, a polypeptide serologically related to
TCR-
chain (17). We speculate that the Ubi-L may possibly be a
candidate for an effector molecule. Support for this theory is the fact
that the Ubi-L itself bound directly to the target cells. On the other
hand, MNSF-
is necessary for the extracellular release and stability
of Ubi-L. Whether MNSF-
might contribute to the Ubi-L binding to its
target cells remains to be determined. To date, isolation of cDNA
encoding MNSF-
has been unsuccessful because of a transient and
faint expression of the mRNA.
receptor (32),
-subunit of the
TCR-CD3 complex (6), and high affinity IgE receptor (7) are
ubiquitinated. Interestingly, Ab specific for ubiquitin detected
antigenic determinants on the cells expressing lymphocyte horming
receptor, indicating that ubiquitin covalently binds to the
extracellular domain of this receptor (33, 34). Although Ubi-L also
recognizes cell surface receptor proteins, it seems unlikely that it
binds covalently to the receptors in a manner similar to ubiquitination
because MNSF-
, which lacks the free COOH-terminal glycyl doublet
responsible for isopeptide bond formation, bound to target cells (Fig.
1).
. The Ubi-L conjugation is similar but not identical to the
ubiquitination process because acceptor proteins for Ubi-L are
different from those for ubiquitin (15). The Ubi-L conjugation is
thought to occur via isopeptide bond formation because isopeptidase
prepared from murine livers dissociates MNSF-
from Ubi-L (17). In
this context, we speculate that the intracellular and extracellular
mode of actions of Ubi-L may differ.
is involved in the mode of
action of Ubi-L (14). However, neither IFN-
nor IL-10, an
immunosuppressive cytokine, shared the Ubi-L receptor (Fig. 3).
Interestingly, ubiquitin slightly but significantly bound to the
receptor probably because of its homology with Ubi-L. Indeed, we have
observed recently that high dose ubiquitin (100 µM)
showed a Ubi-L-like activity in vitro and that low dose (10 nM) ubiquitin, which is insufficient for the suppression of
the antibody response, could inhibit the Ubi-L-suppressive activity
(13). In the current study, we showed evidence that the inhibition is
caused, at least in part, by a competitive action at receptor level.
Most recently, we observed that Ubi-L exerts its suppressor activity
in vivo (38). High dose administration of ubiquitin (5 µg/body) showed a mimetic activity. Pancré et al. (39)
mentioned that ubiquitin shows an inhibitory effect on IgE-induced
platelet cytotoxicity. Additionally, ubiquitin is thought to be
involved in the mechanism of hypersensitivity to Hymenoptera venom and
aspirin-sensitive asthma (40). Taken together, ubiquitin-like
protein(s) and ubiquitin may play an important role in keeping the
immune system in balance.
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FOOTNOTES |
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* This work was supported in part by Grant-in-aid 08670367 (to M. N.) for Scientific Research from the Ministry of Education, Science, and Culture, Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biochemistry,
Shimane Medical University, 89-1 Enya-cho, Izumo 693, Japan. Tel.:
81-853-23-2111; Fax: 81-853-22-4963; E-mail:
nkmr0515{at}shimane-med.ac.jp.
2 M. Nakamura and Y. Tanigawa, unpublished data.
3 M. Nakamura, T. Tsunematsu, and Y. Tanigawa, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: TCR, T cell receptor; MNSF, monoclonal nonspecific suppressor factor; ConA, concanavalin A; LPS, lipopolysaccharide; IL, interleukin; Ubi-L, ubiquitin-like segment of MNSF; UCRP, ubiquitin cross-reactive protein; IFN, interferon; Ab, antibody; GST, glutathione S-transferase; bio-Ubi-L, biotinylated Ubi-L; PBS, phosphate-buffered saline; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; ECL, enhanced chemiluminescence; WGA, wheat germ agglutinin.
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REFERENCES |
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