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Association of GM3 with Zap-70 Induced by T Cell Activation in Plasma Membrane Microdomains

2002; Elsevier BV; Volume: 277; Issue: 13 Linguagem: Inglês

10.1074/jbc.m109601200

ISSN

1083-351X

Autores

Tina Garofalo, Luisa Lenti, Agostina Longo, Roberta Misasi, Vincenzo Mattei, G. M. Pontieri, Maurizio Sorice, Antonio Pavan,

Tópico(s)

Cell Adhesion Molecules Research

Resumo

Recent evidence demonstrated that T cell activation leads to the redistribution of membrane and intracellular kinase-rich raft microdomains at the site of TCR engagement. In this investigation we demonstrated by high performance thin layer chromatography, gas chromatographic, and mass spectrometric analyses that GM3 is the main ganglioside constituent of these microdomains in human lymphocytes. Then we analyzed GM3 distribution and its interaction with the phosphorylation protein Zap-70. Human T lymphocytes were stimulated with anti-CD3 and anti-CD28. Immunofluorescence microscopy analysis revealed a clustered GM3 distribution over the cell surface and an intracellular localization resembling specific cytoplasmic compartment(s). Scanning confocal microscopy showed that T cell activation induced a significant association between GM3 and Zap-70, as revealed by nearly complete colocalization areas; very few colocalization areas were detected in unstimulated cells. Coimmunoprecipitation experiments revealed that GM3 was immunoprecipitated by anti-Zap-70 only after co-stimulation through CD3 and CD28 as detected by both thin layer chromatography and immunoblotting. Therefore, T cell activation does not promote a redistribution of glycosphingolipid-enriched microdomains but induces Zap-70 translocation in selective membrane domains in which Zap-70 may interact with GM3. These findings suggest that GM3 is a component of a multimolecular signaling complex involved in T cell activation. Recent evidence demonstrated that T cell activation leads to the redistribution of membrane and intracellular kinase-rich raft microdomains at the site of TCR engagement. In this investigation we demonstrated by high performance thin layer chromatography, gas chromatographic, and mass spectrometric analyses that GM3 is the main ganglioside constituent of these microdomains in human lymphocytes. Then we analyzed GM3 distribution and its interaction with the phosphorylation protein Zap-70. Human T lymphocytes were stimulated with anti-CD3 and anti-CD28. Immunofluorescence microscopy analysis revealed a clustered GM3 distribution over the cell surface and an intracellular localization resembling specific cytoplasmic compartment(s). Scanning confocal microscopy showed that T cell activation induced a significant association between GM3 and Zap-70, as revealed by nearly complete colocalization areas; very few colocalization areas were detected in unstimulated cells. Coimmunoprecipitation experiments revealed that GM3 was immunoprecipitated by anti-Zap-70 only after co-stimulation through CD3 and CD28 as detected by both thin layer chromatography and immunoblotting. Therefore, T cell activation does not promote a redistribution of glycosphingolipid-enriched microdomains but induces Zap-70 translocation in selective membrane domains in which Zap-70 may interact with GM3. These findings suggest that GM3 is a component of a multimolecular signaling complex involved in T cell activation. Gangliosides are synthesized by virtually all the cells of peripheral blood (1.Kiguchi K. Henning-Chubb B.C. Huberman E. J. Biochem. 1990; 107: 8-14Crossref PubMed Scopus (89) Google Scholar). However, patterns of ganglioside cell expression depend on the species, cell type, and age of the individual. In human peripheral blood lymphocytes (PBL) 1The abbreviations used are: PBLperipheral blood lymphocyte(s)GEMglycosphingolipid-enriched microdomainsGSLglycosphingolipidTCRT cell receptorHPTLChigh-performance thin layer chromatographyMSmass spectrometryFITCfluorescein isothiocyanatemAbmonoclonal antibodyGLCgas liquid chromatographyPBSphosphate-buffered salineHPLChigh pressure liquid chromatography 1The abbreviations used are: PBLperipheral blood lymphocyte(s)GEMglycosphingolipid-enriched microdomainsGSLglycosphingolipidTCRT cell receptorHPTLChigh-performance thin layer chromatographyMSmass spectrometryFITCfluorescein isothiocyanatemAbmonoclonal antibodyGLCgas liquid chromatographyPBSphosphate-buffered salineHPLChigh pressure liquid chromatography monosialoganglioside GM3 represents the main ganglioside constituent of cell plasma membrane (72% of total ganglioside content) (1.Kiguchi K. Henning-Chubb B.C. Huberman E. J. Biochem. 1990; 107: 8-14Crossref PubMed Scopus (89) Google Scholar), although not correlated with a particular lymphocyte subpopulation, i.e. both CD4+ and CD8+ cells express a similar amount of GM3 (2.Misasi R. Sorice M. Griggi T. d'Agostino F. Garofalo T. Masala C. Pontieri G.M. Lenti L. Clin. Immunol. Immunopathol. 1993; 67: 216-223Crossref PubMed Scopus (24) Google Scholar). Minor ganglioside constituents of human PBL include sialosyl paragloboside (about 14%) and sialosyl lactohexaosyl ceramide (about 7%) (1.Kiguchi K. Henning-Chubb B.C. Huberman E. J. Biochem. 1990; 107: 8-14Crossref PubMed Scopus (89) Google Scholar). In addition, disialoganglioside GD3 is also a minor component on a small subset of human peripheral blood T cells (3.Merrit W.D. Taylor B.J. Der-Minassian V. Reaman G.H. Cell. Immunol. 1996; 173: 131-148Crossref PubMed Scopus (19) Google Scholar).Previous immunofluorescence and immunogold electron microscopic studies revealed a clustered distribution of GM3 molecules on the cell surface of human PBL, clearly indicating the presence of glycosphingolipid (GM3)-enriched microdomains (GEM) (4.Sorice M. Parolini I. Sansolini T. Garofalo T. Dolo V. Sargiacomo M. Tai T. Peschle C. Torrisi M.R. Pavan A. J. Lipid Res. 1997; 38: 969-980Abstract Full Text PDF PubMed Google Scholar, 5.Parolini I. Topa S. Sorice M. Pace A. Ceddia P. Montesoro E. Pavan A. Lisanti M.P. Peschle C. Lisanti M. J. Biol. Chem. 1999; 274: 14176-14187Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). They are involved in modulating signal transduction by GSL-GSL interaction, binding with specific antibodies, or assembly with signal transducer molecules (6.Hakomori S. Handa K. Iwabuchi K. Yamamura S. Prinetti A. Glycobiology. 1998; 8: xi-xixCrossref PubMed Scopus (186) Google Scholar). The variety of proteins detected in these domains isolated from different cell types is extremely wide (7.Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8019) Google Scholar). The presence of tyrosine kinase receptors, mono (Ras, Rap)- and heterotrimeric G proteins, Src-like tyrosine kinases (LCK, LYN, FYN), protein kinase C isozymes, and glycosylphosphatidylinositol-anchored proteins (8.Parolini I. Sargiacomo M. Lisanti M.P. Peschle C. Blood. 1996; 87: 3783-3794Crossref PubMed Google Scholar,9.Horejsi V. Drbal K. Cebecauer M. Cerny J. Brdicka T. Angelisova P. Stockinger H. Immunol. Today. 1999; 20: 356-361Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar) allows these portions of the plasma membrane to be considered as “glycosignaling domains” (6.Hakomori S. Handa K. Iwabuchi K. Yamamura S. Prinetti A. Glycobiology. 1998; 8: xi-xixCrossref PubMed Scopus (186) Google Scholar). We previously demonstrated that, in lymphocytes, not only GM3 and cholesterol but also CD4 and p56lck are selectively recovered in GEM (4.Sorice M. Parolini I. Sansolini T. Garofalo T. Dolo V. Sargiacomo M. Tai T. Peschle C. Torrisi M.R. Pavan A. J. Lipid Res. 1997; 38: 969-980Abstract Full Text PDF PubMed Google Scholar). The CD4-p56lck complex represents one of the most important receptor systems in the T cell function, and CD4 is considered the TCR co-receptor in thymic selection, T cell activation, and cellular response (10.Blue M.L. Hafter D.A. Craig K.A. Levine H. Schlossman S.F. J. Immunol. 1987; 139: 3949-3954PubMed Google Scholar, 11.Gay D. Buus S. Pasternak J. Kappler J. Marrak P. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5629-5634Crossref PubMed Scopus (52) Google Scholar). Most of the CD4 functions are due to CD4 interaction with p56lck (12.Veillette A. Bookman N.A. Horak E.N. Samelson L.E. Bolen J.B. Nature. 1989; 338: 257-259Crossref PubMed Scopus (527) Google Scholar). Interestingly, in human T lymphocytes exogenous GM3 induces CD4 phosphorylation (13.Garofalo T. Sorice M. Misasi R. Cinque B. Giammatteo M. Pontieri G.M. Cifone M.G. Pavan A. J. Biol. Chem. 1998; 273: 35153-35160Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), dissociation from p56lck, and internalization via endocytic pits and vesicles (14.Sorice M. Pavan A. Misasi R. Sansolini T. Garofalo T. Lenti L. Pontieri G.M. Frati L. Torrisi M.R. Scand. J. Immunol. 1995; 41: 148-156Crossref PubMed Scopus (28) Google Scholar).The role of GEM in lymphocyte activation became obvious after the demonstration that T cell activation by co-stimulation through CD28 led to the redistribution and clustering of membrane and intracellular kinase-rich microdomains at the site of TCR engagements (15.Viola A. Schroeder S. Sakakibara Y. Lanzavecchia A. Science. 1999; 283: 680-682Crossref PubMed Scopus (840) Google Scholar). The hypothesis for the role of GEM in initiating TCR signaling has gained further support from the identification in them of two other crucial molecules, phosphatidylinositol 4,5-biphosphate (16.Pike L.J. Casey L. J. Biol. Chem. 1996; 271: 26453-26456Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar) (a substrate of phospholipase Cγ) and LAT (17.Zhang W. Trible R.P. Samelson L.E. Immunity. 1998; 9: 239-246Abstract Full Text Full Text PDF PubMed Scopus (749) Google Scholar, 18.Leyton L. Quest A.F.G. Bron C. Mol. Immunol. 1999; 36: 755-768Crossref PubMed Scopus (30) Google Scholar) (one of the earliest and major tyrosine-phosphorylated proteins found following TCR triggering). LAT phosphorylated following Zap-70 activation. Zap-70, a Syk family kinase, is activated via both a self- and a Lck-dependent phosphorylation mechanism (19.Quian D. Weiss A. Curr. Opin. Cell Biol. 1997; 9: 205-212Crossref PubMed Scopus (286) Google Scholar) and translocate from cytoplasmic compartment to the cell surface. It phosphorylates substrates, which in turn leads to the subsequent docking and activation of other Src homology 2 (SH2)-containing molecules involved in the amplification and diversification of TCR-initiated signaling. Zap-70 becomes GEM-associated (20.Montixi C. Langlet C. Bernard A.M. Thimonier J. Dubois C. Wurbel M.A. Chauvin J.P. Pierres M. He H.T. EMBO J. 1998; 17: 5334-5348Crossref PubMed Scopus (558) Google Scholar, 21.Janes P.W. Ley S.C. Magee A.I. J. Cell Biol. 1999; 147: 447-461Crossref PubMed Scopus (694) Google Scholar) and binds phospholipase Cγ, which, after phosphorylation, cleaves phosphatidylinositol 4,5-biphosphate.Most of the studies aimed to the study of GEM during T cell activation were performed using cholera toxin as a marker of these microdomains (15.Viola A. Schroeder S. Sakakibara Y. Lanzavecchia A. Science. 1999; 283: 680-682Crossref PubMed Scopus (840) Google Scholar, 20.Montixi C. Langlet C. Bernard A.M. Thimonier J. Dubois C. Wurbel M.A. Chauvin J.P. Pierres M. He H.T. EMBO J. 1998; 17: 5334-5348Crossref PubMed Scopus (558) Google Scholar, 21.Janes P.W. Ley S.C. Magee A.I. J. Cell Biol. 1999; 147: 447-461Crossref PubMed Scopus (694) Google Scholar, 22.Harder T. Simons K. Eur. J. Immunol. 1999; 29: 556-562Crossref PubMed Scopus (305) Google Scholar, 23.Yashiro-Ohtani Y Zhou XY. Toyo-oka K. Tai X.G. Park C.S. Hamaoka T. Abe R. Miyake K. Fujiwara H. J. Immunol. 2000; 164: 1251-1259Crossref PubMed Scopus (139) Google Scholar, 24.Ebert P.J.R. Baker J.F. Punt J.A. J. Immunol. 2000; 165: 5435-5442Crossref PubMed Scopus (52) Google Scholar, 25.Khoshnan A. Bae D. Tindell C.A. Nel A.E. J. Immunol. 2000; 165: 6933-6940Crossref PubMed Scopus (123) Google Scholar, 26.Saint-Ruf C. Panigada M. Azogui O. Debey P. von Boehmer H. Grassi F. Nature. 2000; 406: 524-527Crossref PubMed Scopus (137) Google Scholar, 27.Tuosto L. Parolini I. Schroeder S. Sargiacomo M. Lanzavecchia A. Viola A. Eur. J. Immunol. 2001; 31: 345-349Crossref PubMed Scopus (135) Google Scholar, 28.Millan J. Qaidi M. Alonso M. Eur. J. Immunol. 2001; 31: 467-473Crossref PubMed Scopus (33) Google Scholar), taking advantage of its capability to bind monosialoganglioside GM1. However, this molecule has been shown to be virtually undetectable (1.Kiguchi K. Henning-Chubb B.C. Huberman E. J. Biochem. 1990; 107: 8-14Crossref PubMed Scopus (89) Google Scholar) or present at very low levels (2 × 10−18 g/cell) in resting peripheral blood T lymphocytes (27.Tuosto L. Parolini I. Schroeder S. Sargiacomo M. Lanzavecchia A. Viola A. Eur. J. Immunol. 2001; 31: 345-349Crossref PubMed Scopus (135) Google Scholar). Thus, in this investigation we preliminarily analyzed the ganglioside pattern and composition of GEM and then focused on the distribution of the main ganglioside constituent of these domains and its interaction with the phosphorylation protein Zap-70 after T cell activation by CD3 and CD28 engagements.MATERIALS AND METHODSCellsHuman PBL were isolated from fresh heparinized blood by Lymphoprep (Nycomed AS Pharma Diagnostic Div., Oslo, Norway) density gradient centrifugation and washed three times in phosphate-buffered saline (PBS), pH 7.4. Human T lymphocytes were stimulated with anti-CD3 (10 μg/ml, Ortho-Clinical Diagnostics, Raritan, NJ) and anti-CD28 (10 μg/ml, PharMingen, La Jolla, CA) antibodies for 1 h at 37 °C.Isolation and Analysis of Glycosphingolipid-enriched Microdomain FractionGEM fraction from human PBL were isolated as described previously (29.Rodgers W. Rose J.K. J. Cell Biol. 1996; 135: 1515-1523Crossref PubMed Scopus (285) Google Scholar). Briefly, 2 × 108 cells were suspended in 1 ml of lysis buffer containing 1% Triton X-100, 10 mm Tris-HCl (pH 7.5), 150 mm NaCl, 5 mm EDTA, 1 mm NaVO4, and 75 units of aprotinin and allowed to stand for 20 min. The cell suspension was mechanically disrupted by Dounce homogenization (10 strokes). The lysate was centrifuged for 5 min at 1300 × g to remove nuclei and large cellular debris. The supernatant fraction (postnuclear fraction) was subjected to sucrose density gradient centrifugation, i.e. the fraction was mixed with an equal volume of 85% sucrose (w/v) in lysis buffer (10 mm Tris-HCl, pH 7.5, 150 mm NaCl, 5 mm EDTA). The resulting diluent was placed at the bottom of a linear sucrose gradient (5–30%) in the same buffer and centrifuged at 200,000 × g for 16–18 h at 4 °C in a SW41 rotor (Beckman Instruments, Palo Alto, CA). After centrifugation, the gradient was fractionated, and 11 fractions were collected starting from the top of the tube. All steps were done at 0–4 °C. The amount of protein in each fraction was first quantified by Bio-Rad protein assay (Bio-Rad). The amount of cholesterol was evaluated as described previously (30.Huber L.A. Xu Q. Jurgens G. Bock G. Buhler E. Gey K.F. Schonitzer D. Trall K.N. Wick G. Eur. J. Immunol. 1991; 21: 2761-2765Crossref PubMed Scopus (89) Google Scholar). Free cholesterol was quantitated from TLC plates by densitometric scanning and comparison with the standard. The density of the bands used to quantitate cholesterol concentration fell within the linear range of compound concentration versus absorbance.Finally, the GEM fraction was subjected to ganglioside extraction according to the method of Svennerholm and Fredman (31.Svennerholm L. Fredman P. Biochim. Biophys. Acta. 1980; 617: 97-109Crossref PubMed Scopus (637) Google Scholar) with minor modifications. Briefly, the GEM fraction was extracted twice in chloroform:methanol:water (4:8:3, v:v:v) and subjected to Folch partition by the addition of water resulting in a final chloroform:methanol:water ratio of 1:2:1.4. The upper phase, containing polar glycosphingolipids, was purified of salts and low molecular weight contaminants using Supelclean LC-18 columns, 3 ml (Supelco Inc., Bellefonte, PA) according to the method of Williams and McCluer (32.Williams M.A. McCluer R.H. J. Neurochem. 1980; 35: 266-269Crossref PubMed Scopus (405) Google Scholar). The eluted glycosphingolipids were dried and separated by high performance thin-layer chromatography (HPTLC) using silica gel 60 HPTLC plates (Merck). Chromatography was performed in chloroform:methanol:0.25% aqueous KCl (5:4:1, v:v:v). Plates were then air-dried, and gangliosides were visualized with resorcinol (33.Svennerholm L. Biochim. Biophys. Acta. 1957; 24: 604-611Crossref PubMed Scopus (2156) Google Scholar).Isolation and Gas Chromatographic Analysis of GM3 from GEMGM3 was isolated by HPTLC from the GEM fraction ganglioside extract. After exposure to iodide vapors, the band comigrating with standard GM3 was scraped, eluted from silica with chloroform:methanol (2:1, v:v), and dried under nitrogen.The identification of carbohydrates was carried out by methanolysis with 3 n methanolic HCl as reported (34.Esselman W.J. Laine R.A. Sweeley C.C. Methods Enzymol. 1972; 28: 149-163Google Scholar). Trimethylsilylmethylglycosides were injected into a SPB-5 fused silica capillary column programmed at 5 °C/min from 150 to 210 °C, maintained isothermally for 5 min, and programmed at 5 °C/min from 210 to 280 °C. As standards, p-nitrophenyl β-d-galactopyranoside and p-nitrophenyl β-d-glucopyranoside (Sigma) were used.The identification of sialic acid was performed as described (35.Schauer R. Methods Enzymol. 1978; 50: 83-84Google Scholar) after methanolysis in 0.05 n methanolic-HCl. The thoroughly dried products were converted to trimethylsilyl ester derivatives with 50 μl of trimethylsilylimidazole (Supelchem) at 70 °C for 20 min. Aliquots of the reaction mixture were injected into a SPB-5 fused silica capillary column programmed at 3 °C/min from 220 to 280 °C. As standard, 2-O-(p-nitrophenyl)-α-d-N-acetylneuraminic acid (Sigma) was used.The types and the percentages of fatty acids were determined as methyl esters after vigorous methanolysis with 0.5 n anhydrous HCl at 80 °C for 18 h. After cooling, the solution was extracted four times with hexane. The hexane phases containing the fatty acids methyl esters were collected, dried, and injected (0.5 μl in CH2Cl2) into an SPB-2380 fused silica capillary column programmed at 5 °C/min from 140 to 170 °C.Dry ganglioside was hydrolyzed using 5 n aqueous HCl:methanol:1:4, as described (36.Barenholz Y. Gatt S. Methods Enzymol. 1975; 35: 529-530Crossref PubMed Scopus (4) Google Scholar). The sphingosine bases were derivatized and analyzed as trimethylsilyl esters under the same conditions reported for the identification of carbohydrates.Purification and Mass Spectrometric Analysis of GM3 from GEMGM3 was isolated from the GEM fraction ganglioside extract by HPLC on amino silica columns, as already reported (37.Previti M Dotta F. Di Pontieri G.M. Mario U. Lenti L. J. Chromatogr.aphy. 1992; 605: 221-225Crossref Scopus (9) Google Scholar). The use of an on-line variable wavelength diode array detector allows the identification and quantitative determination of GM3 on the basis of its UV spectrum and accurately acquired analytical data. The GM3 peak was collected and analyzed by mass spectrometry (MS) using an Applied Biosystems Sciex API III tandem mass spectrometer (Applera Italia, Monza, Italy), equipped with articulated ion spray source. Mass calibration and resolution were checked with a polypropylene glycol solution. The MS experiments were run with a resolution better than 0.8 atomic mass unit; for the MS/MSMS runs, resolution for both quadrupoles was set at 1 atomic mass unit. All of the instrumental operating parameters were standard except the orifice voltage, which was operated at 50 V. MS/MSMS gas collision fragmentation was run at a collision energy of 50 eV with argon at a thickness of 280 × 1012 molecules/cm2. Sample was dissolved in an aqueous solution of 50% ethanol containing 0.1% formic acid and 2 mm ammonium acetate. The introduction was operated by a Harvard infusion pump at a flow rate of 2 μl/min. MS and MSMS spectra were collected at a rate of 10 ms/atomic mass unit with a step size of 0.1 atomic mass unit for MS and 1 atomic mass unit for MSMS experiments. Acquired data were processed by MacSpec software; the Hypermass option was used to handle data concerning multiply charged ions.TLC Immunostaining AnalysisImmunostaining was performed using as antigen about 2 μg of the ganglioside extract from either untreated or anti-CD3 and anti-CD28-treated lymphocytes. Gangliosides were separated by TLC using HPTLC aluminum-backed silica gel 60 plates (Merck). Plates were soaked in a 0.2% solution of polyisobutylmethacrylate in hexane for 90 s, air-dried, and incubated in the blocking solution (3% bovine serum albumin in 20 mm Tris, 0.5 m NaCl, pH 7.5) for 1 h at room temperature. The blocking solution was removed and replaced by washing buffer (TBS). The plates were then incubated for 1 h at room temperature with anti-GD3 mAb (R24, Matreya Inc., Pleasant Gap, PA) in 1% bovine serum albumin-TBS. The antibody was removed, and plates were washed three times for 10 min with TBS. Horseradish peroxidase-conjugated sheep anti-mouse IgG (Sigma) in 1% bovine serum albumin-TBS was added and incubated for 1 h at room temperature. The color reaction was obtained by adding 200 mg of sodium nitroprusside (Sigma) and 80 mg of o-dianisidine (Sigma) dissolved in 100 ml of H20 containing 35 μl of 30% H2O2. The reaction was stopped by washing in distilled water. As a control for nonspecific reactivity, parallel blots were performed as described above using an anti-mouse IgG3 (Sigma).Immunofluorescence AnalysisHuman PBL were incubated with GMR6 anti-GM3 mAb (38.Kotani M. Ozawa H. Kawashima I. Ando S. Tai T. Biochim. Biophys. Acta. 1992; 1117: 97-103Crossref PubMed Scopus (143) Google Scholar) (a gift from Dr. T. Tai, Tokyo Metropolitan Institute of Medical Science, Tokyo) for 1 h at 4 °C followed by three washes in PBS and the addition (30 min at 4 °C) of fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgM (Sigma). Cells were then fixed in 4% formaldehyde in PBS and analyzed by immunofluorescence microscopy. In parallel experiments, cells were permeabilized with 0.5% Triton X-100 in PBS for 5 min at room temperature before incubation with anti-GM3 mAb.Analysis of GM3-Zap-70 Colocalization on the Cell Surface by Scanning Confocal MicroscopyHuman PBL were fixed with 4% formaldehyde in PBS for 30 min at 4 °C and labeled with anti-Zap-70 mAb (Upstate Biotechnology, Lake Placid, NY) for 1 h at 4 °C followed by the addition (30 min at 4 °C) of Texas red-conjugated anti-mouse IgG (Calbiochem, La Jolla, CA). After three washes in PBS, cells were incubated with GMR6 anti-GM3 mAb (38.Kotani M. Ozawa H. Kawashima I. Ando S. Tai T. Biochim. Biophys. Acta. 1992; 1117: 97-103Crossref PubMed Scopus (143) Google Scholar) for 1 h at 4 °C followed by three washes in PBS and the addition (30 min at 4 °C) of FITC-conjugated goat anti-mouse IgM (Sigma). In parallel experiments, cells were stained with anti-GM3 mAb before fixing the cells. Cells were finally washed three times in PBS and resuspended in glycerol/Tris-HCl, pH 9.2. The images were acquired through a confocal laser scanning microscope (Sarastro 2000, Molecular Dynamics) equipped with a Nikon Optiphot microscope and an argon ion laser. Simultaneously, the green (FITC) and the red (Texas red, which reduces greatly overlapping) fluorophores were excited at 488 and 518 nm. Acquisition of single FITC-stained samples in dual fluorescence scanning configuration did not show overlapping. Images were collected at 512 × 512 pixels.Co-immunoprecipitation of GM3 and Zap-70Co-immunoprecipitation of GM3 and Zap-70 was performed according to Iwabuchi et al. (39.Iwabuchi K. Handa K. Hakomori S.I. Methods Enzymol. 2000; 312: 448-494Google Scholar). Briefly, human PBL were lysed in lysis buffer (20 mm HEPES, pH 7.2, 1% Nonidet P-40, 10% glycerol, 50 mm NaF, 1 mmphenylmethylsulfonyl fluoride, 10 μg of leupeptin/ml). Cell-free lysates (containing 20–25 μg of protein) were mixed with protein G-acrylic beads and stirred by a rotary shaker for 2 h at 4 °C to preclear nonspecific binding. After centrifugation (500 ×g for 1 min), the supernatant was immunoprecipitated with anti-Zap-70 mAb (Upstate Biotechnology) plus protein G-acrylic beads. A mouse IgG isotypic control (Sigma) was employed.The immunoprecipitates were split into two aliquots. A portion was then subjected to ganglioside extraction as reported above. Another portion of the immunoprecipitates was electrophoretically transferred to a nitrocellulose membrane (Bio-Rad) after 10% SDS-PAGE and then probed with polyclonal anti-Zap-70 (Santa Cruz Biotechnology, Santa Cruz, CA) or, alternatively, with GMR6 anti-GM3 mAb. The bound antibodies were then visualized with peroxidase-conjugated anti-rabbit IgG or anti-mouse IgM (Sigma), respectively. Immunoreactivity was assessed by chemiluminescence reaction using the ECL Western blocking detection system (Amersham Biosciences).RESULTSGanglioside Pattern and Composition of GEM in Human PBLWe investigated the ganglioside composition of GEM fraction of lymphocyte plasma membrane. Gangliosides were extracted in chloroform:methanol:water and separated in HPTLC as reported above. Three main resorcinol-positive bands were shown: (a) comigrating with GM3, (b) between GM3 and GM1, and (c) between GM1 and GD1a. The GM3 content, determined as lipid-bound sialic acid, was 17.5 ± 1.4 μg/mg of protein in the GEM fraction as compared with 0.864 ± 0.1 μg/mg of protein in total lymphocytes (Table I). The two other main bands, migrating between GM1 and GD1a (4.375 ± 0.3versus 0.168 ± 0.02 μg/mg of protein) and between GM3 and GM1 (1.2 ± 0.1 versus 0.084 ± 0.01 μg/mg of protein) also revealed a significant enrichment in the GEM fraction as compared with the total cell lysate.Table IGanglioside composition of GEMGlycosphingolipidsGEMTotal lymphocytesμg/mg of proteinμg/mg of proteinGM317.5 ± 1.40.864 ± 0.1Band x4.375 ± 0.30.168 ± 0.02Band y1.2 ± 0.10.084 ± 0.01Cholesterol/total cell phospholipid ratio7.75 ± 2.400.35 ± 0.17Quantitative analysis of ganglioside scraped bands was performed by measuring the lipid-bound sialic acid. Cholesterol was quantified according to the method described by Huber et al. (30.Huber L.A. Xu Q. Jurgens G. Bock G. Buhler E. Gey K.F. Schonitzer D. Trall K.N. Wick G. Eur. J. Immunol. 1991; 21: 2761-2765Crossref PubMed Scopus (89) Google Scholar). Band x, migrating between GM1 and GD1a, was immunostained by anti-GD3 mAb. It does not exclude the presence of sialosyl paragloboside, as described (1.Kiguchi K. Henning-Chubb B.C. Huberman E. J. Biochem. 1990; 107: 8-14Crossref PubMed Scopus (89) Google Scholar, 40.Sekine M. Ariga T. Miyatake T. Kuroda Y. Suzuki A. Yamakawa T. J. Biochem. 1984; 95: 155-160Crossref PubMed Scopus (20) Google Scholar). Band y, migrating between GM3 and GM1, was characterized as sialosyl lactohexaosyl ceramide, as reported (1.Kiguchi K. Henning-Chubb B.C. Huberman E. J. Biochem. 1990; 107: 8-14Crossref PubMed Scopus (89) Google Scholar, 40.Sekine M. Ariga T. Miyatake T. Kuroda Y. Suzuki A. Yamakawa T. J. Biochem. 1984; 95: 155-160Crossref PubMed Scopus (20) Google Scholar). Open table in a new tab The identity of the GM3 comigrating band was verified by gas liquid chromatographic (GLC) analysis as reported previously (2.Misasi R. Sorice M. Griggi T. d'Agostino F. Garofalo T. Masala C. Pontieri G.M. Lenti L. Clin. Immunol. Immunopathol. 1993; 67: 216-223Crossref PubMed Scopus (24) Google Scholar). In the hydrophilic head galactose, glucose (Fig. 1a), and sialic acid in a molar ratio of 1:1:1 were found. Lymphocytic GM3 showed the same retention time peaks (α and β anomers of methylketosides of sialic acid) as those of standard N-acetylneuraminic acid (Fig. 1b); in the hydrophobic part, the main fatty acids were C16:0, C18:0, C18:1, and C15:0 (Fig. 1c). Long-chain bases were analyzed by GLC as O-trimethysilyl derivatives. GM3 exclusively gave n-C18-sphingenine, which accounted for more than 95% of the long-chain bases.The MS analysis of GM3 from GEM fraction ganglioside extract confirmed the identity of the molecule (Fig. 2a). The linkage of sialic acid and sugars was analyzed both by GM3 fragmentation after mild alkaline hydrolysis at pH 7 (Fig. 2b) and by GM3 fragmentation under acidic conditions (Fig. 2c).Figure 2MS analysis of GM3 from lymphocytic GEM fraction ganglioside extract. a, MS spectrum of GM3 from HPLC containing phospholipids contaminants. b, MSMS spectrum of GM3 fragmented after mild alkaline hydrolysis at pH 7 (M+Na)+ 1204. c, MSMS spectrum of GM3 fragmented under acidic conditions (M+H)+ 1182. The experimental conditions are described in the text.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Expression and Distribution of GM3 after T Cell Activation by Co-stimulation through CD28We investigated the ganglioside composition of human PBL in the absence and presence of anti-CD3 and CD28 stimulation. Gangliosides were extracted in chloroform:methanol:water and separated in HPTLC as reported above. Again, three main resorcinol-positive bands were shown: (a) comigrating with GM3, (b) between GM3 and GM1, and (c) between GM1 and GD1a (Fig. 3a). In both cell types, the GM3 double band is due to the heterogeneity of fatty acid composition as described (40.Sekine M. Ariga T. Miyatake T. Kuroda Y. Suzuki A. Yamakawa T. J. Biochem. 1984; 95: 155-160Crossref PubMed Scopus (20) Google Scholar). No significant differences in the amount of single gangliosides were observed (Fig. 3a).Figure 3a, HPTLC analysis of the ganglioside pattern of anti-CD3- and CD28-treated and untreated human PBL. Gangliosides were extracted in chloroform/methanol/water. The plate was stained with resorcinol (ganglioside-specific stain). Lane A, standard gangliosides GM3, GM1, GD1a, GD1b, GT1b; lane B, ganglioside

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