evidence for the existence of ganglioside-enriched plasma

Evidence for the existence of ganglioside-enriched plasma membrane domains in human peripheral lymphocytes

Maurizio Sorice, Isabella Parolini,” Tiziana Sansolini, Tina Garofalo, Viicemza Dolo,+ Massimo Sargiacomo,* Tadashi Tai? Cesare Peschle,*.** Maria Rosaria Torrisi, and Antonio Pavan’*+ Dipartimento di Medicina Sperimentale e Patologia, Universih di Roma “La Sapienza”, Rome Italy; Dipartiments di Ematologia-Oncologia e Virologia,* Istituto Superiore di Sanid, Rome, Italy, Dipartimento di Medicina Sperimentale? Universitii di L’Aquila, L’Aquila, Italy; Department of Tumor Immunology,§ Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan; and Thomas Jefferson Cancer Institute,** Thomas Jefferson University, Philadelphia, PA 19107-5541

Abstract In human peripheral blood lymphocytes (PBL) monosialoganglioside GM3 appears to be the major ganglioside on the cell plasma membrane. We have analyzed the expression and distribution pattern of GM3 molecules on the lymphocyte plasma membrane by flow cytometry, immunofluorescence, and immunoelectron microscopy, using an anti- GM3 monoclonal antibody. Both CD4+ and CD8+ T lymphc- cyte subpopulations showed substantial GM3 expression, as determined by thin-layer chromatography and flow cytometric analysis. A clustered distribution of GM3 molecules on the cell surface, revealed by immunofluorescence and immunogold electron microscopy, clearly indicated the presence of GM3 molecule-enriched plasma membrane domains. To bet- ter define these domains, we analyzed the ganglioside and protein composition of buoyant lowdensity Triton-insoluble (LDTI) lymphocyte fractions. The results show that GM3 is enriched -20-fold in LDTI fraction, as compared with total cell lysates. In addition, CD4 and Ick molecules are selectively recovered in the same LDTI fraction isolated from human P B L . I These findings, together with the observation that anti-CD4 co-immunoprecipitated GM3, support the hypothesis of a possible GM3-CD4 interaction and suggest a role for gangliosides as structural components of the membrane multimolecular signaling complex involved in T-cell activation, antigen recognition, and other dynamic lymphocytic plasma membrane functions.-Sorice, M., I. Parolini, T. San- solini, T. Garofalo, V. Dolo, M. Sargiacomo, T. Tai, C. Peschle, M. R Torrisi, and A. Pavan. Evidence for the existence of ganglioside-enriched plasma membrane domains in human peripheral lymphocytes. J. Lipid Res. 1997. 38: 969- 980.

Supplementary key words membrane lipid domain

monosialoganglioside GM3 lymphocyte

Gangliosides are sialic acidcontaining glycosphingolipids located primarily on the outer leaflet of the lipid

bilayer (1). They are composed of hydrophilic carbohy- drate chains linked to ceramide, which are hydrophobic moieties composed of a sphingoid base and a long- chain fatty acid. Gangliosides are ubiquitous constit- uents of cell membranes, where they show cell type- specific and differentiation expression patterns (2, 3) . Functionally, gangliosides can act as specific binding sites for bacterial toxins (4) and viruses (5); they are also involved in membrane receptor modulation (6, 7) and in the control of the cell cycle (8). In T lymphocytes, they appear to be relevant in cell-cell interactions, antigen recognition, cell activation, and signal transduction (9-1 1).

In human peripheral blood lymphocytes (PBL) , monosialoganglioside GM3 represents 72% of the total ganglioside on the cell membranes (12). We recently reported a quantitative and qualitative modification of lymphocyte ganglioside expression in HlV-infected lymphocytes (13, 14). Moreover, exogenous GM3 induced a selective dose-dependent down-modulation and internalization via endocytic pits and vesicles of CD4 molecules on the plasma membrane of human T lymphocytes (15).

Although there is much information available on the chemistry of gangliosides, relatively little is known about their organization and distribution in the cell plasma

Abbreviations: PBL, peripheral blood lymphocytes; LDTI, low density Triton-insoluble; PBS, phosphate-buffered saline; HPTLC, high performance thin-layer chromatography; FITC, fluorescein isothiocyanate; mAb, monoclonal antibody; DIGS, detergent-insoluble glycosphingolipid-enriched complexes.

‘To whom correspondence should be addressed.

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membranes. Lectins (16), cholera toxin (17), anti- glycolipid polyclonal(18,19) and monoclonal antibodies (20) have been used to study the distribution of glycosphingolipids on liposomes and cell surface. In liposomes, monosialoganglioside GM1 is dispersed in phos- pholipid bilayers, whereas asialo GMl has been localized in microdomains (19). The Forssman antigen, a neutral glycosphingolipid, has been observed, by the freeze-etching technique, to be distributed in small clusters and patches on erythrocyte membranes (18). I n MDCK epithelial cells, using pre- and post-embedding immunocytochemical methods, this antigen was sparsely distributed (20, 21). It has recently been reported that in A431 cells, GMl appeared to be nonuni- formly distributed over the plasma membrane, showing a peculiar localization in non-coated invaginations, identified as caveolae by the presence of the VIP-21 protein caveolin (22, 23). Because these membrane struc- tures are enriched in glycosphingolipids (24), they are resistant to solubilization with Triton X-100 (25, 26) and can be easily isolated by a linear sucrose gradient. Although lymphocytes have been shown to lack caveolin, or analogs of caveolin, they contain low density Triton-insoluble (LDTI) microdomains (27, 28) , where src-family nonreceptor protein tyrosine kinases ~ 5 6 " ~ and p53/p56'y", and GPI-anchored proteins are associated (28, 29).

In this report we analyzed the surface expression arid distribution of GM3 on human PBL by flow cytometry, immunofluorescence, and immunoelectron microscopy, using an anti-GM3 (GMR6) monoclonal antibody (30). We also investigated a) the glycosphingolipid composition of LDTI lymphocyte fractions, and 6) the presence of the signaling complex (CD4 and lck proteins) in these microdomains.

MATERIALS AND METHODS

Cells

Human PBL were isolated from fresh heparinized blood by Lymphoprep (Nycomed AS Pharma Diagnos- tic Div., Oslo, Norway) density-gradient centrifugation and washed three times in phosphate-buffered saline (PBS) pH 7.4. The cell preparation was constituted by 75.5 ? 8.1% of CD3+ cells, 53.7 ? 5.5% ofCD4+ cells, 22.3 ? 2.5% of CD8+ cells, 7.5 2 1% ofCD3-/CD56+ (NK) cells, 11 i: 1.5% of CD19+ cells (B lymphocytes), and 5.5 5 0.7% of CD14+ cells (monocytes), as determined by cytofluorimetric analysis, using a FACScan cy- tometer (Becton Dickinson and Co., Mountain View, CA) . All mAbs were purchased from Orthodiagnostic,

Rariton, NJ. CD4+ and CD8+ cells were separated using Dynabeads M-450 coated with affinity-purified sheep anti-mouse IgG covalently bound to the surface (Dynal, Oslo, Norway). CD4+ and CD8+ lymphocytes were up to 80% purified, as determined by cytofluorimetric analysis.

Molt-4 lymphoid cells (31) were cultured in RPMI 1640 medium (Flow Laboratories, Irvine, Scotland) sup- plemented with 10% fetal calf serum (Flow Labora- tories, Irvine, Scotland) and 2% 1.-glutamine.

Anti-GM3 monoclonal antibody

GM3-reactive cells were separated using anti-GM3 1gM mAb GMR6 (30) and Dynabeads M-450 coated with affinity-purified rat anti-mouse IgM covalently bound to the surface (Dynal, Oslo, Norway). The purity of cell preparations was assessed by cytofluorimetric analysis. The epitope recognized by GMR6 is the external disac- charide (Nedca2-3Gal) sequence present in GM3 molecule. It has been reported that GMR6 showed the high- est binding to GMS among the anti-GM3 mAbs generated and characterized (32).

Ganglioside extraction

Gangliosides were extracted from PBL (or fromi the derived LDTI fractions) according to the method of Svennerholm and Fredman ( 3 3 ) , with minor niodifica- tions. Briefly, total cell lysate or LDTI fraction (100 pg of proteins) was extracted twice in chloroform-methanol-water 4: 8: 3 ( V / V / V ) and subjected to Folch parti- tion 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 (34). The eluted glycosphingolipids were dried down and separated by high-performance thin- layer chromatography (HPTLC), using silica gel 60 HPTLC plates (Merck, Darmstadt, Germany), preacti- vated by heating to 100°C for 30 min. Chromatography was performed in chloroform-methanol-0.25% aque- ous KCl 5:4: 1 (v/v/v) . GM3 (Fidia Research Lab., A h aiio Terme, Padua, Italy); GM1, GDla, GDlb, GTlb, (Sigma Chemical Co., St. Louis, MO) were used as stan- dards; plates were then air-dried and gangliosides were visualized with resorcinol (3.5). Quantitative analysis of GM3-scraped band WAS performed by measuring thc lipid-bound sialic acid, using the periodate-resoi-cinol method (36). Absorbance was measured at 630 nrn with a Perkin-Elmer Lambda 4B spectrophotometer.

TLC immunostairiing was performed on aluminium- backed silica gel plates (Merck, Darmstadt, Germany),

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as previously described (37), using the anti-GM3 mAb GMRG (30).

Immunofluorescence and flow cytometric analysis

Cells (1 X lo6 in 1 ml of PBS) were fixed in 4% formaldehyde in PBS for 2 h at 4°C. After washing three times in PBS, cells were incubated with anti-GM3 mAb GMRG for 1 h at 4"C, followed by the addition of fluorescein isothiocyanate (F1TC)-conjugated goat anti- mouse IgM (p-chain specific, Sigma) for 30 min at 4°C. In parallel experiments, double immunofluorescence on PBL was performed with anti-GM3 mAb followed by FITC-conjugated goat anti-mouse IgM as above and then with phycoerythrinconjugated DAKO T3 (anti- CD3), T4 (anti-CD4), or T8 (anti-CD8) (Dakopatts, Glostrup, Denmark). After washing with PBS at 4"C, fluorescence intensity was analyzed by a FACScan cyto- meter (Becton Dickinson and Co., Mountain View, CA). Cells were gated on the bases of forward angle light scatter and 90" light scatter parameters. Five thousand cells were counted for each histogram.

Formaldehyde-fixed lymphocytes, labeled with anti- GM3 mAb and FITGconjugated goat anti-mouse IgM as above, were also analyzed by immunofluorescence microscopy.

Immunoelectron microscopy

Lymphocytes and Molt-4 cells, fixed in formaldehyde (4% in PBS for 2 h at 4°C) were incubated with anti- GM3 mAb for 1 h at 4°C. After incubation with rabbit anti-mouse IgM (Sigma) ( 1 : 10 in PBS, for 1 h at 4"C), cells were fixed with glutaraldehyde (1 % in PBS for 1 h at 4"C), extensively washed, and then labeled with colloidal gold (18 nm, prepared by the citrate method) conjugated with protein A (Pharmacia Fine Chemicals, Uppsala, Sweden) (38) for 3 h at 4°C. Separately, in parallel experiments, cells were fixed with glutaraldehyde (1 % in PBS for 1 h at 4°C) immediately after the incubation with anti-GM3 mAb and before the secondary anti-mouse IgM (39). Control experiments were performed omitting the mAb from the immunolabeling procedure. All samples were post-fixed in osmium te- troxide 1% in Verona1 acetate buffer, pH 7.4, for 2 h at 4"C, stained with uranyl acetate (5 mg/ml), dehy- drated in acetone, and embedded in Epon 812.

Isolation of low-density Triton-insoluble complexes (LDTI)

LDTI complexes were isolated as previously described (40). Briefly, 5 X 10' PBL were washed and re- suspended in 2 ml MESbuffered saline (MBS: 25 mM MES, pH 6.5,0.15 M NaCl) containing 1% Triton X-100 and 1 mM phenylmethylsulfonyl fluoride and homoge- nized with 10 strokes of a Dounce homogenizer, ad-

justed to 40% sucrose and placed at the bottom of an ultracentrifuge tube. A 5-30% linear sucrose gradient was then placed above the lysate, and centrifuged at 39,000 rpm for 16 h at 4°C in a SW41 rotor (Beckman Instruments, Palo Alto, CA) . The visible band migrating at 20% sucrose was harvested and washed twice with MBS at 14,000 rpm for 30 min at 4°C. Five X 10' cells (5-7 mg total protein), yielded 15-17 pg of LDTI complexes, i.e., 0.26% of the initial homogenate.

For negative staining in immunoelectron microscopy, fresh suspension of LDTI in PBS was applied to collo- dion-coated grids and fixed with 2% formaldehyde in PBS for 15 min at room temperature. The LDTI preparation was immunolabeled with anti-GM3 mAb in PBS containing 1% BSA for 1 h in a humidified chamber. After washing, the sample was incubated with gold- conjugated anti-mouse IgM, 10 nm (Sigma) for 1 h. The sample was then negatively stained with 1% phospho- tungstic acid, brought to pH 7.0 with NaOH, and exam- ined by transmission electron microscopy (CMlO Philips).

Quantitative analysis of cholesterol

The amount of cholesterol in microsamples of LDTI fractions was evaluated as previously described (41). Cholesterol was quantitated from TLC plates by densitometric scanning and comparison with standard. The density of the bands used to quantitate cholesterol concentration fell within the linear range of compound concentration versus absorbance.

Immunoblotting

LDTI fractions and total cell lysate were normalized for protein content (Bradford Assay), then analyzed for CD4 and lck expression by SDSPAGE under reducing conditions, followed by transfer to 0.22 p nitrocellulose paper (Nitrobind, MSI). Blots were blocked with 10 mM Tris, pH 8.0, 0.15 M NaC1, 0.05% Tween-20, 5% non-fat dry milk (Carnation), for 45 min at 25°C. After repeated washes, the filter was incubated for 45 min at 25°C with 1 pg/ml monoclonal anti-CD4 (kindly provided by Dr. Malavasi) , 1 pg/ ml polyclonal anti-lck (Transduction Laboratories, KY), or 1 pg/ml monoclonal antLCD3 (Ancell Corp., Bayport, MN), under the same conditions. Bound antibodies were then visualized with a monoclonal or a polyclonal HRP-conjugated secondary antibody diluted 1 :5000, followed by incubation with the enhanced chemiluminescence (ECL) Western blotting detection reagent (Amersham Corp.) according to the manufacturer's instructions. The fold-enrichment of the proteins was determined by densitometric quanti- tation (Ultroscan XL-Pharmacia L). The density of the bands scanned to quantitate lck, CD4, and CD3 expres-

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sion fell within the linear range of compounds concentration to density.

GM3 + Immunoprecipitation Human PBL were lysed in lysis buffer (20 mM HEPES,

pH 7.2/ 1 % Nonidet P40/ 10%) glycerol/50 mM NaF/ 1 mM phenylmethylsulfonyl fluoride (PMSF)/ 1 mM Na3V0,/10 pg of leupeptin per ml). Cell free lysates were normalized for proteins, immunoprecipitated with anti-CD4 mAb, electrophoretically transferred to nitrocellulose membrane (Bio-Rad, Hercules, CA) after SDS PAGE with 10% polyacylamide gels, and then probed with the anti-GM3 mAb. Bound antibodies were then visualized with anti-mouse IgM HRF'conjugated secondary antibody diluted 1 :5000, followed by incubation with the enhanced chemiluminescence Western blotting detection reagent (Amersham Corp.) . Manufac- turer-specified protocols were used to strip anti-GM3 antibody from the blots (ECL Manual, Amersham) and to reprobe the membranes with antibody anti-CD4.

A B For the analysis of the gangliosides present in the Fig. 1. HPTLC analysis of ganglioside pattern in CD4+ and CD8+

an ti-CD4 immunoprecipitated, we performed extrac- cells. Gangliosides were extracted in chloroform-methanol-water from 2 X 10: cells, separated by Dpaheads M 4 O coated with affinity-

tion in chloroform-methanol-water followed by purified sheep anti-mouse I@; conalently tmund to the surface: lane HPTLC as reported above. A, gangliosides obtained from CD4+ cells: lane R. gangliosides o h

tainetl from CD8+ cells. The plate w a q stained with resorcinol (gangli- osideapecific stain). G M 3 double band is due t o the heterogeneity of fatty acid composition. The GMS expression was virtually the same in the T cell suhpopulation.

RESULTS

Ganglioside pattern of PBL HPTLC analysis of acidic glycosphingolipids, ex-

tracted from human PBL and visualized by resorcinol, showed a GM3 comipt ing double band (-70% of total ganglioside content) in both CD4+ and CD8+ cells, separated using Dynabeads M-450 coated with covalent surface bound affinity-purified sheep anti-mouse IgG (Fig. 1). In both cell types, the GM3 double band is duc to the heterogeneity of fatty acid composition, as described (42). The identity of the GM3 comipt ing band was verified by gas chromatographic analysis, as previously reported ( 13). Both lymphocytic and standard GM3 showed the same retention time peaks (a and p anomers of methylketosides of sialic acid) as those of standard N-acetylneuraminic acid (13). Speci- ficity of the anti-GM3 mAb was assessed by immunostaining of the GM3 band with the GMR6 mAb (not shown).

The GM3 content, determined as lipid-bound sialic acid, was 0.864 5 0.1 pg/mg of protein. Two other main bands, migrating between GMl and GDla (0.168 5 0.02 pg/mg) and between GM3 and GMl (0.084 5 0.01 pg/mg) were also detected. N o significant difference was found between CD4+ and CD8+ cells (Fig. 1).

Flow cytometric and immunofluorescence analysis Cytofluorimetric analysis, performed with anti-GM3

mAb (GMR6) on human PBL, showed the majority of GM3 expression on the cell plasma membrane

To analyze whether the cell plasma membrane reactivity w a s related to a T-lymphocyte subpopulation, lymphocytes were immunostained with anti-GM3 mAb followed by FITCkonjugated goat anti-mouse IgM and then with phycoerythrinconjugated anti-CD3, anti- CD4, or anti-CD8 mAbs, respectively. The difference in reactivity among these lymphocyte subpopulations was not statistically significant, revealing that GM3 expression was virtually the same in T cell subpopulations (not shown).

The distribution pattern of GM3 on the cell surface was analyzed by immunofluorescence microscopy. In lymphocytes immiinostained with anti-GM3 mAb, the anti-GM3 signal appeared uneven and punctate over the plasma membrane (Fig. 3 A-D). As a control, parallel experiments were performed on M o l t 4 cells, a lymphoblastoid T cell line that is known to express a high amount of GM3 (43). Results showed a higher fluorescence intensity in M o l t 4 cells as compared with PBL, but with the same uneven signal distribution (Fig. 3 E-F).

(Fig. 2).



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A P Ganglioside content dependent immunoreactivity w

Fig. 2. Cytofluorimetric analysis of antiCMJ reactivity on PRL. Reac- tivity of the antiCMJ monoclonal antibody with PRL, followed by incubation with FITCdon.jngated goat anti-mouse IgM. A aspecific binding of only secondaT antibody; B reactivity of the antiCM3 antibody. Histograms represent log fluorescence versus cell number, gated on lymphocyte population ora side scatter/forward scatter (SS/ FS) histogram. Cell number is indicated on the y-axisand fluorescence intensity is shown in three logarithmic unit? at the x-axis. The figure reveals that GMJ expression was at the plasma membrane level.

To evaluate whether different anti-GM3 reactivity was related to ganglioside content, GM3 content was analyzed in anti-GM3 reactive and unreactive Molt4 cells, separated by Dynabeads M-450 and coated with rat anti- mouse IgM covalently bound to the surface. This analysis showed that anti-GMS reactive cells had a GM3 content significantly higher as compared with unreactive cells: 280 pmol/g (5.86 pg/108 cells) compared to 115 pmol/g (2.42 pg/lO* cells) of packed cells (not shown).

Immunoelectron microscopic analysis To investigate the distribution of GM3, formalde-

hyde-fixed PBL were immunolabeled with anti-GM3 mAb followed by anti-IgM antibodies and by protein A-colloidal gold. Immunoelectron microscopic observations showed an uneven distribution of GM3 on the lymphocyte plasma membrane (Fig. 4A); the gold immunolabeling appeared in small clusters and localized either over the microvilli (Fig. 4A, arrows) or over the nonvillous portion of the membrane (Fig. 4A, arrowheads). Gold clusters were also observed inside both uncoated and coated pits (not shown). To rule out the possibility that this distribution pattern was an artefact due to anti-

Fig. 3. Immunofluorescence analysis of CMJ distribution on cell plasma membranes. A-D) In human PBL anti-GMJ immunolabeling appears uneven and punctate over the cell surface. E,F) Anti-GMJ immunostained Molt4 lymphoid cells show an intense and uneven distribution over the cell plasma membranes. A-D: X1.500: E,F X1200: bar: 10 pm.

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6.' Fig. 4. A) Imnitinolahrling of' GMJ on the lymphocyte plasma membranes. The sitfiace distrihiition of the immimogold particles is not uniform. Small clusten are Incalixd over the microvilli (arrow) and over the nonvillous portion (arrowhcads) of the plwma membranes. R) Immunolahrling of CDJ antigc-ns on the lymphocyte plasma membranes perlormetl tinder the same experimental conditions used for GM.8 iiiiiiiunolabeling. <mid particles are uniformly distrihutrtl over the lymphocyte plasma memhmnes. A X2700; R: X27000; Imr: I pni.

body-crosslinking or aggregated gold particles, CD3 antigens were immunolabeled under the same experimental conditions. Figure 4B shows that CD3 immunolabeling was uniformly distributed over the cell surface.

In parallel experiments on Molt4 cells, the density of GM3 immunolabeling was more intense than that o h

served on lymphocytes. In this case the presence of large GM3 clusters and a very low number of isolated gold particles were observed on the cell surface (Fig. 5A). To exclude the possibility that the clustered distribution might result from cross-linking by secondary antibody of only partially immobilized ganglioside mole-



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Fig. 5. Immiinolaheling of GM3 on the pla..ma membranes of M o l t 4 lymphoid cells. A) lmmiinogold lahcling of GM.7 on formaldehyde prc- fixed M o l t 4 cells, pnst-fixed with gltitaraltlehytle immediately after the inciihation with antiCM3 mAh and hrfnre treatment with the second antihody: gold panicles are non-uniformly tli5trihuted and reveal the presence of large GM3 clusters over the crll suf i~cr . 13) <zllswere incnhiitrtl with anti4X.7 mAh. followed hy anti-mousc IgM and then fixed with glutaraldeliyde hefore protein A colloirlal gold incuhation: the GM.7 tlistrihution appean in clusters similar t o thosc. clescrihcd in (A). A: X2O(H)O: R: X'LOoOO; har: I pm.

cules, a parallel control experiment was performed. Thus, formaldehyde-fixed M o l t 4 cells were incubated with anti-GM3 mAb and then fixed with glutaraldehyde before the addition of the second antibody (38). Cells were then incubated with anti-IgM antibodies, followed by protein A-colloidal gold as above. Our observations confirmed that the immunogold clustering represents the native distribution of GMS molecules over the cell plasma membrane (Fig. 5B).

Ganglioside pattern of LDTI plasma membrane domains

We investigated the ganglioside composition of LDTI fractions of lymphocyte plasma membrane, according to the method previously described (25, 40). which is based on resistance to solubilization by Triton X-100 at

4OC and buoyancy at a specific density in a linear sucrose gradient. 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, / I ) between GM3 and GMl, and c) behveen GM1 and GDla (Fig. 6). The GMS content, determined as lipid-bound sialic acid, was 17.5 2 1.4 pg/mg of protein in LDTI fraction, as compared with 0.864 t 0.1 pg/mg of protein in total lymphocytes. These results confirm the hypothesis of an enrichment of gangliosides on LDTI fractions.

Cholesterol content, quantified by densitometric scanning, was increaqed more than 20-fold in the same fraction as compared with total lymphocytes (7.75 t 2.40 pg/mg of protein in LDTI fraction, 0.35 t 0.17 pg/ mg of protein in total lymphocytes).

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CDS content was similar in both total cell lysate and LDTI complexes (not shown).

GM3 detection in CD4 immunoprecipitates To determine the possible CD4/GM3 interaction,

cell-free lysates from human PBL were immunoprecipitated with anti-CD4 mAb. Acidic glycosphingolipids were then extracted from these immunoprecipitates and HPTLC analysis showed only GMS; no other resorcinol-positive bands were detected (Fig. 9A).

In a parallel experiment we performed Western blot analysis of the anti-CD4 immunoprecipitates with anti- GMS mAb. Our results revealed the presence of a 5 8 kDa band (Fig. 9B). To confirm the positive band as CD4, the anti-GMS antibody was stripped from the poly- vinylidene difluoride membrane, and the membrane was then reprobed with an anti-CD4 antibody. The results showed the positive band as CD4 (not shown).

GM3 +

GM1 + G D l a -b

G D l b

G T l b -b

1.. . *

A B Fig. 6. HPTLC analysis of the ganglioside pattern of Triton-insolu- hlc plasma memhrane domains. Human PRI. ohtained from healthy donors were lysed and subjected t o sucrose densitv p d i e n t for LDTI fraction isolation. Gangliosides were then extrnctetl in chloroform- methanol-water from LDTI fraction, obtained from lymphocytes: lane A, standard gangliosides CM.7. GMI. GDla, GDlh. GTlh: lane B, gangliosides obtained from lymphocyte LDTI fraction, 100 pg. The plate \vas smined with resorcinol (ganglioside-pecific stain). The figure shows the presence of three resorcitiol-positive.hands comipt ing with GM.7. hetween CM.7 and C M I and hetireen CMI and GDla, rc- spectirely.

Negatively stained fresh preparations of LDTI complexes were analyzed by electron microscopic analysis. Negative staining is an efficient method for assessing the quality of fractions during isolation of particular cell components or isolated organelles. Briefly, the applica- tion of heaw metal salt solution dries down support film, resulting in an electron-translucent membrane domain on a dark surrounding background. At low magnification, the presence of many dispersed membrane fragments were observed that frequently appeared as curved membrane (Fig. 7A). The LDTI fraction was then labeled with anti-GMS mAb: gold particles indicated the existence of GM.knriched domains on these membrane fragments (Fig. 7B.C).

CD4 and Ick enrichment in LDTI plasma membrane domains

We evaluated the enrichment of CD4 and Ick in PBL and in LDTI complexes. The amount of protein in each fraction was first quantified, by Bradford aqsay, and then serially diluted to determinate the relative enrichment of CD4 and Ick (Fig. 8). The enrichment was then quantified by densitometric analysis (not shown). CD4 and Ick enrichment in LDTI complexes was approximately 35fold and 50-fold, respectively. On the other hand.

DISCUSSION

We analyzed GM3 expression and distribution on lymphocyte plasma membrane by HPTLC, cytofluorimetric, and immunoelectron microscopic analyses. In agreement with previous reports (1 2,43), HPTLC analysis of the PBL ganglioside pattern showed that GMS is the major ganglioside constituent, although not corre- lated with a particular lymphocyte population, i.e.. both CD4+ and CD8+ cells express a similar amount of GMS. This finding is consistent with our previous data on GM3 expression in T cells ( 13).

Immunofluorescence microscopy revealed an uneven and punctate anti-GM3 immunostaining on the cell plasma membrane of both human PBL and Molt- 4 lymphoblastoid T cell line. Immunolabeling density varied from cell to cell, reflecting heterogeneous ganglioside molecule expression. It has been reported that the reactivity of gangliosides with specific mAbs is strictly dependent on their concentration at the cell surface (44, 45). This discrepancy may be explained by a threshold value for immunological staining of cellular glycosphingolipids and the antibody reactivity may be controlled by an “all or none” phenomenon. Particu- larly, it has been reported that cells containing GMS at a concentration greater than 200 pmol/g of packed cells were positive by immunofluorescence analysis (45). Our findings are in agreement with these data, showing that anti-GM3 reactive Molt4 lymphoid cells had a GMS content of 280 pmol/g, which was significantly higher than anti-GMS unreactive cells (1 15 pmol/g). Immuno- logical staining may also depend on the degree of expo- sure at the cell surface, on the possibility that ganglia-



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t i

b

Fig. 7. Electron microscopy negative staining of LDTl complexes. A) Low magnification shows mainly dispersed membrane portions in LDTI preparation. R,C) Immunogold laheling of GMS on LDTI complexes. The presence of gold particles on the memhnne fractions identifies domains enriched in GMS. A X30.000: bar: 0.5 pm; B X 1 10.00(1; bar: 1 pm: C: X 144.000: bar: 1 pm.

side molecules are cryptic (44) and the total ganglioside composition on the cell plasma membrane (32). In addition, modulation of ganglioside surface expression has been reported to be strictly related to the cell cycle (46) and cell density (47) in adherent cells.

Immunoelectron microscopic observations showed uneven GMS distribution on the cell surface of PBL and Mol t4 cells and indicated the presence of GM3 clusters. As very few isolated gold particles were present on the cell membrane, our results strongly suggest the existence of ganglioside domains with GM3 molecule concentration. These observations are consistent with previously reported thermodynamic results (48), showing that gangliosides form clusters when their concentration is higher than a critical value. In these clusters, par- ticularly when their formation is supported by proteins or ions, gangliosides are packed in a more effective way, due to a less active ganglioside-ganglioside repulsive interaction, as confirmed by their decreased mobility

(49). The clustered distribution of GM3 molecules on the lymphocyte plasma membrane is in agreement with previous immunofluorescence data (47), showing uneven distribution of GM3 on the surface of human skin fibroblasts. The existence of gangliosidecontaining domains suggests a possible functional association of glycosphingolipids with membrane-bound proteins. In fact, GMS has been observed to decrease the EGF- induced growth of KB and A431 cells (7). the FGF- induced proliferation of BHK fibroblasts (50). and the PDGF-induced growth of STS cells (6,51), by inhibiting autophosphorylation of the corresponding receptors. We recently reported that exogenous GMS selectively induces redistribution, clustering, and internalization via endocytic pits and vesicles of CD4 molecules on the lymphocyte plasma membrane (15). Therefore, GM3 molecule clusters, on both the cell surface and inside endocytic pits observed here, suggest a possible GMS CD4 interaction in ganglioside-enriched domains. This

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CD4 A

58 kDa - - 0

A B C GM3 *

I c k

-- 56 kDa e

A B C Fig. 8. CD4 and Ick enrichment in lymphocyte LDTI frxtion. Hit- miin peripheral 1y”hocytes obtained from healthy donors were lysed and subjected to sucrose density gradient for LDTl fraction isolation. I’roteins from 1,DTI fraction and total cell homogenates were quantified, serially diluted, separated on 10% SDSPAGE. and transferred to filter. I s k and CD4 expression were detectetl hv blotting with 1 pg/ ml anti-lck or antiCD4. Enrichment wr\s quantified by densitometric analysis. CD4: lane A LDTI complexes, 2 pg; lane R: LDTI complexes, 20 pg; lane (2 total lyate, 20 pg. Ick lane A LDTl complexes, I pg; lane I): LDTl complexes, 10 pg; lane (2 total Ivsate. 1 0 pg. CD4 and Ick enrichment in LDTl complrxes is -3.5foId and -50-folt1, respec- tiucly, as compared with total cell lysate.

hypothesis is supported by our Western blot and TLC analyses showing that anti-CD4 Abs selectively co- immunoprecipitated GMS, although we cannot exclude the possibility that GM3 could co-immunoprecipitate with other protein(s).

A strong indication in favor of the possible GM3CD4 interaction also derives from the results of LDTI membrane fraction analysis. The observation of a real enrichment of gangliosides in these fractions supports the view of a ganglioside-protein(s) association in the same membrane domains. In particular, the demonstration that CD4 and Ick are selectively recovered in LDTI complexes isolated from human PBL, together with the quantitative data of 20-fold enriched GMS, suggests an involvement of this ganglioside in signal transduction mechanisms of human PBL. In human leukemic cell lines, several Src-family nonreceptor protein tyrosine kinases are associated with cell surface receptors in large detergent-resistant complexes (28). In particular, molecules participating in T-cell receptor activation, e.g., Ick and lyn, are highly enriched in the LDTI fraction, as compared with the total cell lysate in T-lymphoid and promyelocytic cell lines. On the other hand. the associa-

58 kDa-

Fig. 9. Human PRL were lysed in lysis huffer. Cell-free lysate were normalized for proteins and immunoprecipitated with anti-CD4 mAb. A) HPTIG analysis of CD4 immunoprecipitite. C;angliosides were extracted from the immunoprecipitates and the plate w i s stained with resorcinol. The figure shows that the immunoprecipitates contained selectively GMS. N o other resorcinol-positi\’e bands were detected. R) U’estcrn hlot analysis of CD4 immrtnoprecipitate. I’roteins from the immunoprecipitate were separated on 10%) SDSPACE and probed with the anti-GMS mAb. The figure show the presence of a 58 kDa band. corresponding to CD4.

tion of glycosphingolipids with GPI-anchored proteins and proteintyrosine kinases suggests a role for gangliosides as components of a cell membrane multimolecular signaling complex (29, 52, 53). Our results further support the view that lymphocyte surface glycosphingolipids are present in relatively detergent-resistant areas of the membrane (microdomains) associated with some intracellular proteins, including tyrosine kinases. We also show that cholesterol is highly enriched in these lymphocyte membrane fractions, confirming that interaction between glycolipids and cholesterol is critical for maintaining integrity of these membrane domains, as observed in other cell types (54,55). Finally, our results support the existence of detergent-insoluble complexes in cells that, by both morphological criteria and a b sence of VIP2lcaveolin protein or caveolin analogs, do not show caveolae on the cell plasma membrane (27); only transfection of VIP-21 caveolin induces new caveo-



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lae membrane structural formation (56). Microdo- mains of distinct proteins (src family and CD4) and lipids reported here seem to correspond to the rafts of detergent-insoluble glycosphingolipid-enriched complexes (DIGs) that have been recently reported to exist on caveolin/ caveolae independent cell plasma membrane microdomains (57). The clustered surface distribution of GMS molecules, observed in our immunogold experiments, is in accordance with the existence of DIGs in lymphocyte plasma membrane. Thus, the presence of glycosphingolipids (GMS) , src-family proteins, and CD4 proteins indicates that DIGs in lymphocytes may be involved in, or contribute to, lymphocytic functions such as signal trasduction, cell activation, and/ or endocytic process of specific membrane antigens. Fur- ther studies are required to verify the direct lipid- protein interactions and clarify their role in cell membrane dynamic events.U

We thank Dr. Angelo Del Nero for excellent graphic and pho- tographic work, and Mr. Giuseppe Lucania and Ms. Lucia Cu- tini for skillful technical assistance. This work was partially supported by grants from MURST, Consiglio Nazionale delle Ricerche PF ACRO and Associazione Italiana Ricerca sul Can- cro (AIRC), Italy. Manuscript receiwd 7 October 1996 and in revised form 30 , Januq 1997.

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evidence for the existence of ganglioside-enriched plasma

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