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• W1965171248 abstract "Monoclonal nonspecific suppressor factor (MNSF), a lymphokine produced by murine T cell hybridoma, possesses pleiotrophic antigen-nonspecific suppressive functions. A cDNA clone encoding MNSF-β, an isoform of the MNSF, has been isolated and characterized. MNSF-β 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 ofM r = 82,000. Biotinylated Ubi-L specifically recognized this band, confirming that the 82-kDa protein is the Ubi-L receptor. A complex of M r = 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. Monoclonal nonspecific suppressor factor (MNSF), a lymphokine produced by murine T cell hybridoma, possesses pleiotrophic antigen-nonspecific suppressive functions. A cDNA clone encoding MNSF-β, an isoform of the MNSF, has been isolated and characterized. MNSF-β 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 ofM r = 82,000. Biotinylated Ubi-L specifically recognized this band, confirming that the 82-kDa protein is the Ubi-L receptor. A complex of M r = 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. 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 (1Jentsch S. Seufert W. Hauser H.-P. Biochim. Biophys. Acta. 1991; 1089: 127-139Crossref PubMed Scopus (122) Google Scholar). Other cellular processes in which the ubiquitin system is also involved include antigen processing (2Hochstrasser M. Cell. 1996; 84: 813-815Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar), ribosome biogenesis (3Finley D. Bartel B. Varshavsky A. Nature. 1989; 338: 394-401Crossref PubMed Scopus (550) Google Scholar), cell cycle progression (4Glotzer M. Murray A.W. Kirschner M.W. Nature. 1991; 349: 132-138Crossref PubMed Scopus (1881) Google Scholar), and regulation of the transcriptional nuclear factor-κB (5Palombella V.J. Rando O.J. Goldberg A.L. Maniatis T. Cell. 1994; 78: 773-785Abstract Full Text PDF PubMed Scopus (1908) Google Scholar). In addition, several signal transducing receptors, in particular the ζ-subunit of the TCR 1The 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, glutathioneS-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. 1The 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, glutathioneS-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.-CD3 complex (6Cenciarelli C. Hou D. Hsu K.-C. Rellahan B.L. Wiest D.L. Smith H.T. Fried V.A. Weissman A.M. Science. 1992; 257: 795-797Crossref PubMed Scopus (183) Google Scholar), the high affinity IgE receptor (7Paolini R. Kinet J.-P. EMBO J. 1993; 12: 779-786Crossref PubMed Scopus (108) Google Scholar), and platelet-derived growth factor receptor (8Yarden Y. Escobedo J.A. Kuang W.-J. Yang-Feng T.L. Daniel T.O. Tremble P.M. Chen E.Y. Ando M.E. Harkins R.N. Francke U. Fried V.A. Ullrich A. Williams L.T. Nature. 1986; 323: 226-232Crossref PubMed Scopus (763) Google Scholar, 9Mori S. Heldin C.-H. Claesson-Welsh L. J. Biol. Chem. 1992; 267: 6429-6434Abstract Full Text PDF PubMed Google Scholar), are ubiquitinated receptors. 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 (10Nakamura M. Ogawa H. Tsunematsu T. J. Immunol. 1986; 136: 2904-2909PubMed Google Scholar,11Nakamura M. Ogawa H. Tsunematsu T. J. Immunol. 1987; 138: 1799-1803PubMed Google Scholar). We have cloned a cDNA encoding a subunit of MNSF (12Nakamura M. Xavier R.M. Tsunematsu T. Tanigawa Y. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3463-3467Crossref PubMed Scopus (57) Google Scholar). The subunit, termed MNSF-β, 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-β (Ubi-L) is responsible for its activity (13Nakamura M. Xavier R.M. Tanigawa Y. J. Immunol. 1996; 156: 532-538PubMed Google Scholar). Ubi-L inhibits IgE and IgG1 production by LPS-activated B cells and division in various tumor cell lines of murine origin (14Nakamura M. Nagata T. Xavier R.M. Tanigawa Y. Int. Immunol. 1996; 8: 1659-1665Crossref PubMed Scopus (11) Google Scholar). Most recently, we have demonstrated that Ubi-L covalently conjugates to intracellular acceptor proteins in vitro (15Nagata T. Nakamura M. Kawauchi H. Tanigawa Y. Biochim. Biophys. Acta. 1998; 1401: 319-328Crossref PubMed Scopus (7) Google Scholar) and in vivo (16Nakamura M. Tanigawa Y. Biochem. J. 1998; 330: 683-688Crossref PubMed Scopus (14) Google Scholar). MNSF-α, 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-α) because it lacks a signal peptide (12Nakamura M. Xavier R.M. Tsunematsu T. Tanigawa Y. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3463-3467Crossref PubMed Scopus (57) Google Scholar). Partially purified isopeptidase dissociates Ubi-L from MNSF-α, suggesting that the COOH-terminal Gly-Gly doublet of Ubi-L may covalently ligate to the lysine residue in MNSF-α (17Nakamura M. Tsunematsu T. Tanigawa Y. Immunology. 1998; 94: 142-148Crossref PubMed Scopus (5) Google Scholar). Several other ubiquitin-like proteins have been isolated and characterized. Sentrin (also called SUMO-1), for instance, preferentially modifies nuclear proteins (18Kamitani T. Nguyen H.P. Yeh E.T.H. J. Biol. Chem. 1997; 272: 14001-14004Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Like ubiquitin, the ubiquitin cross-reactive protein (UCRP) conjugates to a number of intracellular proteins (19Haas A.L. Ahrens P. Bright P.M. Ankel H. J. Biol. Chem. 1987; 262: 11315-11323Abstract Full Text PDF PubMed Google Scholar). Interestingly, UCRP and Ubi-L are subjected to induction by interferon (IFN) (11Nakamura M. Ogawa H. Tsunematsu T. J. Immunol. 1987; 138: 1799-1803PubMed Google Scholar, 20Blomstrom D.C. Fahey D. Kutny R. Korant B.D. Knight Jr., E. J. Biol. Chem. 1986; 261: 8811-8816Abstract Full Text PDF PubMed Google Scholar). Furthermore, they show type specificity for IFN and have immunoregulatory properties (14Nakamura M. Nagata T. Xavier R.M. Tanigawa Y. Int. Immunol. 1996; 8: 1659-1665Crossref PubMed Scopus (11) Google Scholar,21Knight Jr., E. Cordova B. J. Immunol. 1991; 146: 2280-2284PubMed Google Scholar), although they have opposite functions (22D'Cunha J. Knight Jr., E. Haas A.L. Truitt R.L. Borden E.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 211-215Crossref PubMed Scopus (283) Google Scholar). 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 (23Nakamura M. Ogawa H. Tsunematsu T. Cell. Immunol. 1990; 130: 281-290Crossref PubMed Scopus (12) Google Scholar). 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. Ubiquitin and rabbit anti-ubiquitin antibody (Ab) were obtained from Sigma (St. Louis, MO). Mouse recombinant IFN-γ (1 × 107 units/mg) was purchased from Genzyme (Cambridge, MA). Mouse IFN-α (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 (12Nakamura M. Xavier R.M. Tsunematsu T. Tanigawa Y. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3463-3467Crossref PubMed Scopus (57) Google Scholar). Peroxidase-conjugated goat anti-mouse IgG Ab was from Capel (Durham, NC). 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. 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+). Recombinant MNSF-β and Ubi-L were obtained as described previously (12Nakamura M. Xavier R.M. Tsunematsu T. Tanigawa Y. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3463-3467Crossref PubMed Scopus (57) Google Scholar). Briefly, either MNSF-β or Ubi-L was expressed as a fusion protein with glutathioneS-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). 500 μg of Ubi-L was mixed with a 20-fold molar excess of sulfo-N-hydroxysulfosuccinimide-biotin in 1 ml of 0.01m 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 (24Green N.M. Biochem. J. 1965; 94: 23-24Crossref PubMed Google Scholar), performed according to the manufacturer's instructions (Pierce, Rockford, IL). In some experiments, MNSF-β 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. 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 A 450 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. 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 mmTris-HCl, pH 7.5, 1% Nonidet P-40, 0.1% SDS, 150 mm NaCl, 10 μg/ml aprotinin, 5 μg/ml leupeptin, and 1 mmphenylmethylsulfonyl 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 −80 °C. 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 mmMgCl2, 1 mm CaCl2, 1 mmphenylmethylsulfonyl 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 × gfor 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. 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.). 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-β or ubiquitin was coupled to CNBr-activated Sepharose 4B and used for affinity chromatography as described above. 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. 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. Ubi-L was labeled with125I as described previously for GST-Ubi-L (15Nagata T. Nakamura M. Kawauchi H. Tanigawa Y. Biochim. Biophys. Acta. 1998; 1401: 319-328Crossref PubMed Scopus (7) Google Scholar). Aliquots of the purified receptor (500 ng) were mixed with125I-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. 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 mmTris-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 mmTris-HCl, pH 7.4, 0.5 m NaCl, 10% glycerol, and 0.1% Triton X-100. 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. Determination of IgE production by LPS-stimulated B cells was done as described previously (14Nakamura M. Nagata T. Xavier R.M. Tanigawa Y. Int. Immunol. 1996; 8: 1659-1665Crossref PubMed Scopus (11) Google Scholar). Briefly, the purified B cells (5 × 105/ml) were cultured with 20 μg/ml LPS. Ubi-L, recombinant IFN-γ, 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. Previous experiments have shown that Ubi-L acts on murine helper T cell clone, D.10 cells (25Nakamura M. Xavier R.M. Tanigawa Y. Eur. J. Immunol. 1995; 25: 2417-2419Crossref PubMed Scopus (16) Google Scholar). 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-β (Ubi-L-ribosomal protein S30), which shows Ubi-L-like activity (26Suzuki K. Nakamura M. Dekio S. Nariai Y. Tanigawa Y. Immunobiology. 1996; 195: 187-198Crossref PubMed Scopus (10) Google Scholar), 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-β bound to ConA-activated D.10 cells (Fig. 1), as did Ubi-L. The pattern of MNSF-β 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 (12Nakamura M. Xavier R.M. Tsunematsu T. Tanigawa Y. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3463-3467Crossref PubMed Scopus (57) Google Scholar), we tested the possibility of the binding of ubiquitin to D.10 cells. Fig. 1 shows that 10 mmbiotinylated 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 (23Nakamura M. Ogawa H. Tsunematsu T. Cell. Immunol. 1990; 130: 281-290Crossref PubMed Scopus (12) Google Scholar).Figure 2Binding 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.View Large Image Figure ViewerDownload (PPT) 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 (TableI). Of note, both cell lines are sensitive to Ubi-L in terms of inhibition of proliferation (13Nakamura M. Xavier R.M. Tanigawa Y. J. Immunol. 1996; 156: 532-538PubMed Google Scholar). D.10 cells were stimulated with antigen (conalbumin) and ConA, as described previously (25Nakamura M. Xavier R.M. Tanigawa Y. Eur. J. Immunol. 1995; 25: 2417-2419Crossref PubMed Scopus (16) Google Scholar). 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.Table ICellular distribution of murine Ubi-L receptorDesignationCharacteristicsUbi-L boundaSpecifically bound Ubi-L was determined as described in the legend to Fig. 1.pmol/10E6 cellsEL4T lymphoma0.6 ± 0.3MOPC-31CPlasmacytoma0.5 ± 0.1D.10Type 2 helper T cells cloneUnstimulated—bLess than 0.05 pmol/10E6 cells.AntigencD.10 cells were stimulated with conalbumin (100 μg/ml) for 48 h.0.7 ± 0.2ConAdD.10 cells were costimulated with 2 μg/ml ConA and 0.5 unit/ml IL-1β for 48 h.1.1 ± 0.1BW5147T lymphoma—NFS-5C-1Pre-B lymphoma—L929Fibroblast—B16Melanoma—a Specifically bound Ubi-L was determined as described in the legend to Fig. 1.b Less than 0.05 pmol/10E6 cells.c D.10 cells were stimulated with conalbumin (100 μg/ml) for 48 h.d D.10 cells were costimulated with 2 μg/ml ConA and 0.5 unit/ml IL-1β for 48 h. Open table in a new tab 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-β, ubiquitin, and other suppressive cytokines such as IFN-γ and IL-10. As can be seen in Fig. 3, Ubi-L and MNSF-β exclusively inhibited the binding of bio-Ubi-L to ConA-activated D.10 cells. However, the irrelevant ligands (IFN-γ 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 (13Nakamura M. Xavier R.M. Tanigawa Y. J. Immunol. 1996; 156: 532-538PubMed Google Scholar). 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. 2M. Nakamura and Y. Tanigawa, unpublished data. A trace amount of protein (approximately 120 kDa) was reproducibly cross-linked by125I-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.4 A, 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 (25Nakamura M. Xavier R.M. Tanigawa Y. Eur. J. Immunol. 1995; 25: 2417-2419Crossref PubMed Scopus (16) Google Scholar). Additionally, we made MNSF-β and ubiquitin affinity columns to isolate Ubi-L receptor protein(s). The same band of 82 kDa was obtained from MNSF-β 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. 4 B, 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" @default.
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