Ganglioside GM2/GM3 complex affixed on silicananospheres strongly inhibits cell motilitythrough CD82/cMet-mediated pathwayAdriane Regina Todeschini*, Jose Nilson Dos Santos*, Kazuko Handa, and Sen-itiroh Hakomori†

Division of Biomembrane Research, Pacific Northwest Research Institute, and Departments of Pathobiology and Microbiology,University of Washington, Seattle, WA 98195

Contributed by Sen-itiroh Hakomori, October 9, 2007 (sent for review September 13, 2007)

Ganglioside GM2 complexed with tetraspanin CD82 in glycosyn-aptic microdomain of HCV29 and other epithelial cells inhibitshepatocyte growth factor-induced cMet tyrosine kinase. In addi-tion, adhesion of HCV29 cells to extracellular matrix proteins alsoactivates cMet kinase through ‘‘cross-talk ’’ of integrins with cMet,leading to inhibition of cell motility and growth. Present studiesindicate that cell motility and growth are greatly influenced byexpression of GM2, GM3, or GM2/GM3 complexes, which affectcMet kinase activity of various types of cells, based on the follow-ing series of observations: (i) Cells expressing CD82, cultured withGM2 and GM3 cocoated on silica nanospheres, displayed strongerand more consistent motility inhibition than those cultured withGM2 or GM3 alone or with other glycosphingolipids. (ii) GM2-GM3,in the presence of Ca2� form a heterodimer, as evidenced byelectrospray ionization (ESI) mass spectrometry and by specificreactivity with mAb 8E11, directed to GM2/GM3 dimer structure.(iii) Cells expressing cMet and CD82 were characterized by en-hanced motility associated with HGF-induced cMet activation. BothcMet and motility were strongly inhibited by culturing cells withGM2/GM3 dimer coated on nanospheres. (iv) Adhesion of HCV29 orYTS-1/CD82 cells to laminin-5-coated plate activated cMet kinase inthe absence of HGF, whereas GM2/GM3 dimer inhibited adhesion-induced cMet kinase activity and inhibited cell motility. (v) Inhib-ited cell motility as in i, iii, and iv was restored to normal level byaddition of mAb 8E11, which blocks interaction of GM2/GM3 dimerwith CD82. Signaling through Src and MAP kinases is activated orinhibited in close association with cMet kinase, in response toGM2/GM3 dimer interaction with CD82. Thus, a previously unchar-acterized GM2/GM3 heterodimer complexed with CD82 inhibitscell motility through CD82-cMet or integrin-cMet pathway.

glycosphingolipid � growth factor receptor � ldlD cells � tyrosine kinase

G lycosphingolipids (GSLs), including gangliosides, interactwith specific membrane proteins, such as growth factor

receptors, integrins, tetraspanins (TSPs), and nonreceptor cy-toplasmic kinases (e.g., Src family kinases and small G proteins),to form glycosynaptic microdomains controlling GSL-dependentor -modulated cell adhesion, growth, and motility (for review,see refs. 1–3).

Our previous studies indicate the following: (i) Gangliosidethe GM3/TSP CD9 complex interacts with integrin �3�1 or �5�1and inhibits motility of CD9-expressing tumor cells (4, 5). (ii)The GM3/CD9/CD81 complex inhibits tyrosine kinase associ-ated with fibroblast growth factor receptor (FGFR) (6) andblocks functional interaction of integrins with FGFR (7). (iii)Enhancement of GM3 or CD9 level in bladder cancer cell lineYTS-1 causes reversion to normal phenotype, whereby Srckinase activity is strongly inhibited (8).

In contrast to these previous studies, focused on GM3-CD9interaction that inhibits tumor cell motility, our recent studies (9)addressed the functional role of the GM2/CD82 complex thatinhibits (i) hepatocyte growth factor (HGF)-induced cMet ty-rosine kinase and (ii) laminin-5-mediated activation of �3 and its

cross-talk with cMet tyrosine kinase, leading to inhibition of cellmotility and growth.

In the present study, the motility-inhibitory effect of GM2 inCD82-expressing cells was greatly enhanced when nanomolar-level GM3 was added. The inhibitory effect was stronger andmore consistent when nanospheres cocoated with GM2 andGM3 were added in the presence of Ca2�. No such inhibitoryeffect was observed for other GSLs or their combinations. Aquestion then arose regarding the possibility that a GM2/GM3heterodimer distinct from GM2 or GM3 alone is formed and thatsuch heterodimer interacts with CD82. Here, we report variousexperiments designed to test this possibility. A similar possibilityof ganglioside heterodimer was described based on specificity ofanti-ganglioside antibodies present in sera of patients withGuillain–Barre Syndrome (10) (see Discussion).

ResultsSpecific Inhibitory Effect of Gangliosides GM2 and GM3 on Motilityand Signaling of Normal vs. Cancer Bladder Cells. Based on theincidental observation that GM3 addition enhanced the inhib-itory effect of GM2 on HGF-induced motility of HCV29 cells,expressing cMet and CD82, we observed the following:

The motility inhibitory effect of GM2 on HCV29 cell motilitywas enhanced when GM3 was added (Fig. 1Aa Left), and thiseffect was more clearly observed when GM2 and GM3 wereadded together, cocoated on silica nanospheres. No such effectwas observed for other GSL combinations (Fig. 1 Ab Left).Motility of highly malignant YTS-1 cells, lacking CD82, wasindependent of HGF (9) and not affected by soluble GSLs or byGSLs coated on nanospheres (Fig. 1 A a and b Right).

The above results suggest that CD82 (expressed in HCV29, butnot in YTS-1) interacts preferentially with the GM2-GM3mixture over GM2 or GM3 alone. This possibility was tested bybinding of CD82 to polystyrene beads coated with GSLs, usingthe procedure described in ref. 11. CD82 bound more stronglyto beads coated with GM2-GM3 mixture than to beads coatedwith GM2 or GM3 alone or GM2-LacCer mixture (Fig. 1B).

We showed previously (9) that the GM2/CD82 complexinhibits cMet tyrosine phosphorylation induced by HGF, andcurrent results suggest that inhibitory effect of the GM2/GM3/CD82 complex on cMet kinase is enhanced in CD82-expressingcells. GM2- or GM3-coated nanospheres caused minor inhibi-tion (Fig. 1C, lanes 2 and 3), whereas GM2/GM3 cocoated

Author contributions: A.R.T., K.H., and S.-i.H. designed research; A.R.T., J.N.D.S., and K.H.performed research; A.R.T. analyzed data; and A.R.T. and S.-i.H. wrote the paper.

The authors declare no conflict of interest.

*On leave from: Instituto de Biofısica Carlos Chagas Filho, Universidade Federal de Rio deJaneiro, 21941-590 Rio de Janeiro, Brazil.

†To whom correspondence should be addressed. E-mail: hakomori@u.washington.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0709619104/DC1.

© 2008 by The National Academy of Sciences of the USA

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nanospheres strongly inhibited (lane 4) cMet kinase inducedby HGF (50 ng/ml), compared with noncoated nanospheres(lane 1).

Adhesion of YTS-1/CD82 cells to LN5-coated plate in theabsence of exogenous HGF greatly enhanced cMet tyrosinephosphorylation (Fig. 1D Right, lane 1), compared with the samecells in suspension culture without adhesion (Left). LN5-inducedactivation of cMet tyrosine phosphorylation was reduced greatlyby pretreatment of cells with GM2 and GM3 together (Right,lane 4) and, to a lesser degree, with GM2 or GM3 alone (lanes2 and 3). LN5-induced activation of cMet tyrosine phosphory-lation may be based on functional interaction (‘‘cross-talk’’)between integrin �3 and cMet.

Noncoated nanospheres (lane 1), nanospheres coated withGM1 (lane 2), GM2 (lane 3), GM3 alone (lane 4), or theGM1-GM2 mixture (lane 5) did not change tyrosine phosphor-ylation at Src416. In contrast, GM2-GM3 cocoated on nano-spheres (lane 6) strongly inhibited Src tyrosine phosphorylation

(Fig. 1E) and MAPK phosphorylation at Thr-202 and Tyr-204(Fig. 1F).

Presence of GM2/GM3 Heterodimer in the Presence of Ca2�, Indicatedby Positive Ion Spray Mass Spectrometry (MS). Physical associationof GM2 and GM3 (20 nmol/ml, 1:1) was studied by electrosprayionization (ESI)-MS in the presence of Ca2� (200 nmol/ml) inmethanol (12). The negative ion spectrum of the mixture pre-sented the ion expected for GM2, m/z � 1,383,corresponding to[GM2-H�]� (Fig. 2 A–b) plus GM3, m/z � 1,208, 1,222, 1,236,1,250, and 1,264, which correspond to [GM3-H�]� arising fromGM3 molecules containing C14, C18, C19, C20, C21, and C22fatty acids, respectively (Fig. 2 Aa).

The positive ion spectrum of GM2 and GM3 in presence ofCa2� (Fig. 2B) showed a high abundant ion at m/z � 1,423 ion(Fig. 2 Bb), suggesting that GM2 complexes with Ca2� gener-ating the adduct [(GM2-H�)� � Ca2�]�.

Ions at m/z � 1,248, 1,262, 1,276, 1,290, and 1,304 from the

Fig. 1. Inhibitory effect of GSLs on cell motility and inhibition of cMet kinase pathway, and interaction of GSLs with CD82. (A) Comparative effect of solubleGSL (a) vs. GSL-coated nanospheres (b) on haptotactic motility. (a) Effect of RPMI medium (column 1) or medium containing 25 nmol/ml of GM2 (column 2), GM3(column 3), or GM2/GM3 (column 4) on haptotactic motility of HCV29 vs. YTS-1 cells. (b) Effect of noncoated (column 1), GM2-coated (column 2), GM3-coated(column 3), GM2/GM3-cocoated (column 4), Gb3-coated (column 5), Gb3/GM2-cocoated (column 6), LacCer-coated (column 7), or LacCer/GM2-cocoated (column8) silica nanospheres on cell motility. The type of cell is indicated at the top. Cells (0.5 � 104 per well), in medium with addition of aqueous GSL solution orGSL-coated silica nanospheres, containing 5% FBS and 50 ng/ml HGF, were seed onto gold sol-coated 48-well plates and haptotactic motility was assessed as inMaterials and Methods. For methods for preparation of SiO2 nanospheres and GSL coating, see Materials and Methods. (B) Interaction of GSLs with CD82, probedby binding of CD82 on polystyrene beads alone (lane a), GM2-coated (lane b), GM2-GM3-cocoated (lane c), GM3-coated (lane d), LacCer-coated (lane e), orLacCer-GM2-cocoated (lane f) polystyrene beads. Beads (6.85 � 107, 1-�m diameter) prepared as described in ref. 9 were incubated with 100 �g of YTS-1/CD82cell lysate overnight at 4°C, washed three times with TBS [140 mM NaCl and 10 mM Tris�HCl (pH 8.0)] (�), resuspended in SDS/PAGE sample buffer, and analyzedby Western blotting with anti-CD82 Ab. (C) Effect of GSL-coated nanospheres on HGF-induced cMet phosphorylation of HCV29 and YTS-1/CD82 cells. Cells wereincubated overnight with 1 ml of serum-free RPMI medium 1640, containing 18.9 � 1016 nanospheres (50-nm diameter) coated with ganglioside GM2 (column2), GM3 (column 3), GM2/GM3 (column 4) or uncoated nanospheres (column 1), with final concentration of 25 nmol/ml. Cells were then treated with 50 ng/mlHGF for 10 min, washed, and lysed, and 200 �g of protein was immunoprecipitated with anti-Met antibody as described in ref. 9. Levels of tyrosine phosphatein immunoprecipitated fractions were measured by using anti-phosphotyrosine antibody (Py20) (upper row), and stripped blots were probed by anti-Metantibody (lower row). Ratios of phospho-Met/Met are shown. (D) The difference in ratio of cMet tyrosine phosphorylation relative to total cMet level ofYTS-1/CD82 cells pretreated with GM2 (lane 2), GM3 (lane 3), GM2/GM3 (lane 4), or no GSL (lane 1) was compared for cells in suspension (Left) vs. cells adheredto LN5-coated plate (Right). (E) Effect of GSL-coated nanospheres on Src phosphorylation of HCV29 cells. Cells were incubated overnight with 1 ml of serum-freeRPMI medium 1640 containing 18.9 � 1016 nanospheres (50-nm diameter) noncoated (lane 1); coated with ganglioside GM1 (lane 2), GM2 (lane 3), GM3 alone(lane 4), or GM1-GM2 (lane 5); or cocoated with GM2-GM3 (lane 6) at final concentrations of 25 nmol/ml. Cells were washed and lysed. Cell lysate containing15 �g of protein was Western blotted by using anti-P-Src (Tyr-416), and stripped blots were probed by anti-c-Src antibody. Intensity of Western blot wasdetermined by densitometry, using Scion image program. Ratio of P-Src (top row) relative to total c-Src level (lower row) is shown in each column. (F) Ratio ofphospho-p44/42 MAPK (top row) relative to p44/42 MAPK (lower row), analyzed as above. Data in A, and C–F are presented as means � SD. Significance ofdifference versus control cells: **, P � 0.01; *, P � 0.001. The results in B –D are representative of three experiments.

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positive mode ESI mass (Fig. 2 Ba) arise from the [(GM3-H�)�

� Ca2�]� adducts. However, in addition to the peaks arisingfrom GM2 and GM3 Ca2� adducts, the positive mode spectrumpresented intense ions corresponding to heterodimers of the twogangliosides, [(GM2-H�)� � (GM3-H�)� � 2Ca2�]2�, at m/z �1,342 ,1,350, 1,366, 1,364, and 1,372 (Fig. 2 Bc). Supportinginformation (SI) Table 2 summarizes the precursor ions, theproduct ions generated by complexing with Ca2� and productions arising from the heterodimer [(GM2-H�)� � (GM3-H�)�

� 2Ca2�]2�.Formation of such a complex was supported by collision-

induced decomposition of the heterodimer by selecting the ionsat m/z � 1,350 (Fig. 2C). Analysis of the positive ion spectrumconfirms that the noncovalent dissociation of the complex resultsin formation of both GM2 and GM3, generating the ions m/z �1,423 and 1,275 respectively. Decomposition of the ion m/z �1,364 (Fig. 2D) generates ions m/z � 1,423 and 1,303, corre-sponding to GM2 and GM3, respectively.

Specific Reactivity of mAb 8E11 Determined with Gangliosides andwith HCV29 Cells Under Various Conditions. The mAb 8E11 wasestablished after immunization of BALB/c mice with dried residueof GM2-GM3 solution with 200 nmol of CaCl2 per milliliter ofmethanol, which is assumed to contain the heterodimer adsorbedon acid-treated Salmonella minnesotae. For the method of immu-nization and cloning of hybridoma, see Materials and Methods. 8E11was highly reactive with evaporated residue of methanol solution asabove but was not reactive with the heterodimer residue treatedwith 50 mM EDTA, nor with GM2 or GM3 alone (Fig. 3A). 8E11reacted strongly with GM2-GM3 mixture, but did not react withother GSLs or their mixtures as shown in Fig. 3B (abscissa). OnHigh-performance (HP) TLC developed with isopropanol, hexane,

and 0.2% CaCl2 in water (75:40:20 vol/vol/vol), 8E11 reactedspecifically with GM2/GM3 heterodimer but not with GM2 orGM3 alone or with GM2 or the GM3 mixture with GD1a, GM1,or Gb3 (Fig. 3Ca Lower). On TLC immunostaining under the samecondition as above, GM2/GM3 heterodimer also bound to anti-GM2 mAb MK1.8 (Fig. 3Cc Lower), and very weakly to anti-GM3mAb DH2 (Fig. 3Cb Lower).

To confirm the specificity and reactivity of mAb 8E11 to GSLsexpressed at the HCV29 cell surface, endogenous GSLs weredepleted by treatment of cells with GlcCer synthase inhibitor P4(13), followed by addition of GM2, GM3, or the GM2-GM3mixture. Then, cells were immunostained with 8E11 or anti-GM3 DH2. Only P4-treated, GM2/GM3-incubated cells reactedstrongly with 8E11 in flow cytometry. Control IgG3-, GM2-, andGM3-incubated cells showed no binding to 8E11 (Fig. 3D Left).In contrast, DH2 showed highest reactivity with P4-treated,GM3-incubated cells (Fig. 3D Right).

GM2/GM3 Heterodimer Expressed in HCV29 and YTS-1 and Effect ofmAb 8E11 to Restore Haptotactic Cell Motility and Signaling. HCV29and YTS-1 cells express equal level of high-fluorescence ’’peak2,’’ in addition to low-fluorescence ‘‘peak 1’’ (Fig. 4 Ab). Peak 2,defined by mAb 8E11, must correspond to GM2/GM3 het-erodimer, because reactivity decreased greatly when cells weretreated with 50 mM EDTA (Fig. 4 Ac).

Motility of HCV29 cells, inhibited by incubation with GM2/GM3 heterodimer coated on nanospheres (see Fig. 1 Aand B),was restored to control level (i.e., that of cells incubated withnoncoated nanospheres) by coincubation with mAb 8E11 but notwith normal mouse IgG3, IgM, or anti-GM2 mAb MK1.8 (Fig.4B). Anti-GM3 DH2 only weakly inhibited the motility.

The inhibitory effect of GM2/GM3 heterodimer on P416 Src

Fig. 2. GM2/GM3 heterodimer indicated by mass spectrometric pattern. (A) Negative ion spray mass spectrum of GM2 (20 nM) plus GM3 (20 nM) with Ca2�

(200 nmol) in methanol. Ion m/z � 1,383 (region b) corresponds to [GM2-H�]�. Ions m/z � 1,208, 1,222, 1,236, 1,250, and 1,264 (region a) correspond to [GM3-H�]�

and represent a mixture of GM3 molecules containing C14, C18, C19, C20, C21, and C22 fatty acids, respectively. (B) (Region b) Positive ion spray mass spectrumof GM2 (20 nmol) plus GM3 (20 nmol) with Ca2� (200 nmol) in methanol. GM2 complexes with Ca2� generating the ion [(GM2-H�)� � Ca2�]� m/z � 1,423. (Regiona) The GM3 complex with Ca2� generates the ions [(GM3-H�)� � Ca2�]� m/z � 1,248, 1,262, 1,276, 1,290, and 1,304. Region c, which is not observed in the negativespectrum in A, corresponds to the heterodimer GM2/GM3 ions [(GM2-H�)� � (GM3-H�(1237))� � 2Ca2�]2� m/z � 1,342, 1,350, 1,356, 1,364, and 1,372.Declustering potential was 80 V. (C) MS/MS spectrum of the GM2/GM3 complex [(GM2-H�)� � (GM3-H�(1237))� � 2Ca2�]2� m/z � 1,350. (D) MS/MS spectrumof the GM2/GM3 complex [(GM2-H�)� � (GM3-H�(1237))� � 2Ca2�]2� m/z � 1,364.

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(Fig. 4C) and MAPK Thr-202 and Tyr-204 phosphorylation (Fig.4D) in HCV29 cells was abolished by preincubation of theheterodimer coated on nanospheres with 8E11, i.e., the inhibitedlevel was returned to the control level (Fig. 4 C and D, column4 vs. column 1).

Endogenous GM2/GM3 Heterodimer Expression and Its Effect onMotility and Signaling in ldlD Cells. ldlD, a mutant cell line defectivein UDP-Glc 4-epimerase (14), is incapable or capable of syn-thesizing GM3 or extended N-linked glycan when cultured in theabsence or presence of Gal in serum-free insulin/transferring/selenium (ITS) medium. We further established ldlD cell vari-ants by transfection of CD82 gene or GM2 synthase gene (seeMaterials and Methods). When ldlD/GM2syn/pCDNA3 and ldlD/GM2syn/CD82 cells were cultured in ITS with Gal and GalNAcaddition (conditions 1–5 in Table 1; see Materials and Methodsand Fig. 5 legend), optimal GM2 and GM3 synthesis occurredunder condition 4 (Fig. 5B, patterns 4 and 5). Using ldlD/GM2syn/CD82, which is capable of synthesizing both GM2 andCD82, and ldlD/GM2syn/pCDNA3, which is capable of synthe-sizing GM2 but not CD82, we compared (i) expression levels ofGM2/GM3 heterodimer, defined by mAb 8E11, and (ii) hapto-tactic motility of these two mutant cell lines under five growthconditions as in Table 1. Cell surface reactivity with mAb 8E11increased significantly for both these mutant cell lines undercondition 4, in which both GM2 and GM3 synthesis occurred,and, therefore, the heterodimer became highly reactive with8E11 regardless of CD82 expression (Fig. 5B).

Haptotactic motility was inhibited in ldlD/GM2syn/CD82(Fig. 5C Left) but not in ldlD/GM2syn/pCDNA3 under condi-

tions 3, 4, and 5 (Fig. 5C Right), in which GM2 and GM3 synthesisoccurred, and, therefore, the GM2/GM3 heterodimer complexwith CD82 was formed. Motility inhibition was not observed forldlD/GM2syn cells without CD82.

DiscussionSpecific carbohydrates interact with the same or different car-bohydrate, as initially observed for Lex-to-Lex interaction me-diating self-adhesion of mouse embryonic stem cells (15) and foracidic oligosaccharides having GlcNAc�4Fuc� core in proteo-glycans mediating self-adhesion of sponge cells (16). A numberof subsequent studies indicate that processes of carbohydrate–carbohydrate interaction (CCI) mediating cell adhesion alwaysrequire �1 mM level of bivalent cation (Ca2� or Mg2�) (forreview, see refs. 17 and 18).

Results of the present study clearly indicate three points: (i)only the GM2/GM3/CD82 complex (but not CD82, GM2, orGM3 alone) efficiently inhibits cell motility through blocking ofHGF-induced cMet activation in HCV29 and YTS-1/CD82 cells;(ii) adhesion of YTS-1/CD82 cells to LN5-coated plate activatescMet independently from HGF, based on integrin �3 interactionwith cMet (‘‘cross-talk’’), which is preferentially blocked by theGM2/GM3 complex with CD82; and (iii) motility of ldlD cellsthat express CD82 but not cMet was still inhibited when endog-enous GM2 and GM3 were coexpressed. Finding iii suggests thatthe presence of the GM2/GM3/CD82 complex blocks integrin-dependent signaling.

In the motility-inhibitory process described above, the GM2/GM3 complex was more effective and consistent when coated onnanospheres than when present in solution. The effect was

Fig. 3. GM2/GM3 heterodimer defined by mAb 8E11. (A) Specificity of mAb 8E11 and DH2, determined by ELISA. Various concentrations of gangliosides GM2(rectangles), GM3 (triangles), and GM2/GM3 (circles) in methanol plus 2 mM CaCl2 were dried into 96-well flat-bottom polystyrene plates at 37°C and washedin TBS containing 2 mM CaCl2 (TBS�). Binding of mAb 8E11 (1 �g/ml) was determined by ELISA as described in ref. 35. Effect of EDTA on binding of 8E11 toGM2/GM3 plus 2 mM CaCl2 was determined by addition of 50 mM EDTA to a mixture of GM2/GM3 in methanol solution and washing in TBS. 8E11 reactivity isshown by the diamonds. (B) Single GSL (1.25 nmol) and GSL mixtures with GM2 or GM3 (0.65 nmol of each) in methanol plus 2 mM CaCl2 were tested for reactivitywith mAb 8E11, as described for determination of IgG3 Ab reactivity (35). Data are presented as mean � SD. Significance of difference versus GM2/GM3-treatedwells: *, P � 0.001. (C) High-performance TLC (HPTLC) patterns of various gangliosides and their combinations. (Upper) Immunostained with mAb 8E11 (a), DH2(b), or MK1.8 (c), as described in SI Materials and Methods. (Lower) Detected by 0.2% orcinol in 10% H2SO4. (D) Reactivity of GM2/GM3 or GM3 expressed onHCV29 cells was analyzed by flow cytometry, using mAb 8E11 or DH2. Cells were pretreated with P4 for 72 h (1 �M) to deplete endogenous GSLs, washed, andincubated overnight with serum-free medium alone (a) or with 25 �M GM2 (b), 25 �M GM3 (c), or GM2/GM3 (12.5 �M each) (d). Cells were released in 0.01%trypsin and 0.1 mM EDTA; incubated with 1 �g/ml normal mouse IgG (a), 1 �g/ml mAb 8E11 (b–d Left), or 1 �g/ml DH2 mAb (b–d Right); and labeled with AlexaFluor 488-labeled anti-mouse IgG. The experiments shown were performed multiple times with comparable results.

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reversed, i.e., motility was restored to control level, when cellswere pretreated with mAb 8E11, which binds specifically to theGM2/GM3 complex. The GM2/GM3 complex was identified asa heterodimer resulting from Ca2�-dependent CCI, based onelectrospray ionization mass spectrometry. The same methodwas used successfully to elucidate homotypic Lex-to-Lex (19) and

heterotypic sulfatide-to-galactosylceramide interaction (12),which both mediate specific cell adhesion. The molecular modelof Lex-to-Lex compared with Lex-to-lactosyl interaction, medi-ated by Ca2�, was studied by 1H-NMR (20).

Nanotechnology has progressed greatly and has been appliedwith various target molecules (e.g., refs. 21 and 22). GSLs andgangliosides have not been used so far. The use of silicananospheres could increase stability of the GM2/GM3 het-erodimer, resulting in efficient interaction of the heterodimerwith target CD82. Extension of this study with other TSPs, incombination with other GSLs, will be useful in suppression ofcancer metastasis, diabetes, and other diseases in which GSLsplay a major role.

Ganglioside heterodimer was also found associated withGuillain–Barre Syndrome. The process is caused by paralysis ofperipheral motor nerves by anti-ganglioside antibodies directedto lipopolysaccharide of Campylobacter jejuni. The major anti-bodies were identified as directed to GM1 (23). Antibodiescausing more serious motor nerve paralysis were found to bedirected to heterodimer structures, such as GD1a/GM1, GD1b/GT1b, and GM1/GT1b, rather than single ganglioside structures(10, 24).

Thus, various cell adhesion processes mediated by GSLs and TSPcause extensive phenotypic changes, including reversion from ma-lignant to normal cell phenotype (8, 9, 25). In analogy, Bissell andcolleagues (26, 27) showed that functional antibodies directed to �1integrin of breast cancer cells caused reversion, in vitro and in vivo,to normal cellular organization, indicating that malignancy mayarise from disorganization of glycosynaptic microdomain ratherthan from changes in gene structure and expression.

Materials and MethodsCell Lines and Cell Culture. Cell line YTS-1 (28) was provided by M. Satoh(Department of Urology, Tohoku University School of Medicine, Sendai, Japan).

Fig. 4. Reactivity of mAb 8E11 and its reversing effect on motility inhibitionand signaling by GM2/GM3 heterodimer. (A) Fluorescence-activated flowcytometric pattern of HCV29 and YTS-1 cells. (a) IgG control. (b) Cells with 8E11by the method as in Fig. 3D. (c) Cells released by 50 mM EDTA. Peak 1, negativecells. Peak 2, 8E11 reactive cells. Note that the peak positive for mAb 8E11(peak 2) is absent in EDTA-treated cells, suggesting that this peak is endoge-nous GM2/GM3 heterodimer. (B) Antibody effect on inhibition of haptotacticmotility of HCV29 cells induced by GM2/GM3-coated silica nanospheres (opencolumns), compared with noncoated nanospheres (filled column). GM2/GM3-coated nanospheres were incubated for 2 h with medium, mouse IgG (1�g/ml), normal mouse IgM (1 �g/ml), mAb 8E11 (IgG3) (1 �g/ml), mAb DH2(IgG3) (1 �g/ml) or mAb MK1.8 (IgM) (1 �g/ml). HCV29 cells (0.5 � 104 per well),treated as above, were suspended in 0.250 ml of RPMI medium 1640 with 5%FBS, containing noncoated nanospheres or nanospheres/antibody suspension,seeded onto gold sol-coated 48-well plates and incubated, and haptotacticmotility was analyzed as in Materials and Methods. (C) Reversing effect ofmAb 8E11 on inhibition of p-Src phosphorylation of HCV29 cells induced by 2 hof incubation with GM2/GM3-coated nanospheres in medium containing noIgG (column 2), mouse IgG (1 �g/ml) (column 3), or mAb 8E11 (IgG3) (1 �g/ml)(column 4). Cells were incubated overnight at 37°C in 5% CO2 and lysed, andphosphorylation at position 416 of Src of each cell lysate was measured byWestern blot analysis as described in Materials and Methods and comparedwith lysate from cells incubated with noncoated nanospheres (column 1).Note that only 8E11 restored Src phosphorylation induced by GM2/GM3. (D)Reversing effect of mAb 8E11 on MAPK phosphorylation inhibition by GM2/GM3-coated nanospheres. Cells were treated as in C. Data are expressed asmean � SD. and analyzed by one-way ANOVA (Dunnett test). Note that only8E11 restored MAPK phosphorylation induced by GM2/GM3. Significance ofdifference versus GM2/GM3-coated nanospheres-treated cells: **, *, P � 0.01;

*, P � 0.001.

Table 1. Different combinations and concentrations of galactose(Gal) and N-acetylgalactosamine (GalNAc) in five differentconditions of ldlD cell growth in ITS medium

Condition Gal, �M GalNAc, �M

1 0 02 20 03 20 204 20 1005 20 200

Fig. 5. Ganglioside expression patterns of ldlD cells expressing GM2 synthasegrown under five different conditions, with or without expression of CD82. (A)Ganglioside expression in ldlD cells grown under five different conditions asin Table 1 determined by HPTLC/immunostaining with anti-GM3 mAb DH2 (a)or anti-GM2 MK1.8 (b). (B) Flow cytometric patterns with mAb 8E11. Shownare differences in ldlD/GM2syn cell variants with vs. without CD82 grownunder five different conditions (as in Table 1). Procedures are as described inMaterials and Methods and Fig. 4A legend. (D) Haptotactic motility of ldlD/GM2syn cell variants with vs. without CD82, grown under five differentconditions (as in Table 1). Cells were cultured as above, and motility wasdetermined as in Materials and Methods. Data were expressed as mean � SD.Significance of differences: *, P � 0.001.

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The HCV29 cell line (29), was obtained from American Type Culture Collection.The YTS-1/CD82 was established in ref. 9. These cell lines were grown in RPMImedium 1640, containing 0.4 mM Ca2� and 0.4 mM Mg2�, supplemented with10% FBS (FBS) (which contains �4 mM Ca2� and �2 mM Mg2�), 100 internationalunits/ml penicillin, and 100 �g/ml streptomycin at 37°C, 5% CO2.

ldlD cells and their variants (see below) were maintained in Ham’s F-12 me-dium with 100 units/ml penicillin, 100 �g/ml streptomycin, and 5% (vol/vol) FBS.The glycosylation pattern was altered by changing culture medium from theabove to serum-free F-12 medium containing 1% ITS (Collaborative BiomedicalProducts) on day 0. Gal and GalNAc (0, 20, 100, and 200 �M; see Fig. 5B) wereadded on day 0. The effect of glycosylation on cellular function was determinedon day 3.

ldlD Cells and Establishment of Their Variants. ldlD cells, a UDP-Glc 4-epimerasedeficient mutant of Chinese hamster ovary (CHO) cells (14), were originallydonated by M. Krieger (Massachusetts Institute of Technology, Cambridge, MA).The CD82 transfectant, ldlD/CD82, and its vector control, ldlD/pCDNA3, wereestablished in ref. 30. A new ldlD cell variant, a transfectant of human GM2synthase gene, was prepared as follows. Human GM2 synthase (NM�001478)cDNA (31) (a gift from K. Furukawa, Nagoya University, Japan) was ligated as a1.7-kilobase Not1 fragment into the expression vector pIRESpuro3 (Clontech).The plasmid construct was confirmed by DNA sequencing. After linearizationwith SspI, the plasmid was separately transfected with Fugene 6 (Roche) intoldlD/pCDNA3 and ldlD/CD82 cells according the manufacture’s instruction. Afterculturing in selection media containing puromycin (7.5 �g/ml), the transfectantswere screened by cell surface expression of GM2, using anti-GM2 mAb, MK1.8,and cloned by two cycles of colony selection.

SiO2 Nanosphere Preparation and GSL Coating. A solution containing ganglio-sides (25 nmol/ml) with Ca2� (250 nmol/ml) in methanol was dried in a glass tubeunderaN2stream.Nanospheres (50-nmdiameterat16mg/ml) fromCorpuscular,Inc. were agglutinated by acidification with few drops of HCl (1 N) and centri-fuged at 300 rpm (21 � g) for 5 min. The supernatant was discarded and thenanospheres were washed two times with 0.5 ml of EtOH:H2O (90:10 vol/vol). Thesuspension was sonicated 10 min to resuspend the nanospheres. Twenty micro-liters (0.3 mg containing 18.9 � 1016 nanospheres) of suspension was transferredinto the glass tube containing dried gangliosides (25 nmol/0.2 mg nanospheres)and incubated under agitation at 4°C. The next day, the mixture GSL/nanospherewas dried and resuspended in serum-free RPMI medium 1640 to a final volume of1 ml by sonicating 10 min. The molar ratio of ganglioside/nanosphere bindingwas determined by using 3H-labeled GM2 by the Rosenthal method (32). Thepreparations showed concentration-dependent binding. Rosenthal analysis ofthedata indicates thatnanospheresexhibitamaximalbindingcapacity (Bmax)of800 nmoles [3H]Gal GM2 per milligram of nanospheres and a half-maximalbinding at 25 nmol. [3H]Gal GM2 was tritiated by a modification of the galactoseoxidase-sodium borohydrate method of Novak et al. (33).

Determination of Cell Motility, Its Inhibition by GSL-Nanospheres, and the Effectof mAb 8E11 That Blocks Motility-Inhibitory Effect of GSL-Nanospheres. Cellmotility was determined as the area of phagokinetic tracks on gold sol

particle-coated plates, as described in ref. 9, except that cells (0.5 � 104 perwell) in 0.25 ml of RPMI medium 1640 containing aqueous GSL solution (25nmol/ml) or GSL-coated silica nanospheres (prepared as above; 25 nmol per 0.2milligrams of nanospheres per milliliter of medium) and 5% FBS were seededonto gold sol-coated 48-well plates.

Determination of GM2/GM3 Heterodimer Effect on LN5-Dependent Cell Adhe-sion on cMet Tyrosine Phosphorylation. Effect of LN5 cell adhesion on cMettyrosine phosphorylation was determined as described in ref. 9. Cells were grownon regular culture plates, starved overnight in serum-free RPMI with addition ofnoncoated-, GM2-, GM3-, or GM2/GM3-coated silica nanospheres (prepared asabove). Cells were harvested, and cMet kinase activation of cells kept in suspen-sion or added to LN5-coated plates was analyzed as described in ref. 9.

Determination of Src and MAPK Tyrosine Phosphorylation. Cells were grown insix-well culture plates to �90% confluence, washed, incubated overnight withGSL/nanosphere suspension, washed with PBS containing 1 mM sodium vana-date, and lysed, and levels of P-Src (Tyr-416) or P-p44/42 MAPK (Thr-202/Tyr-204)were measured as described in ref. 8. Blocking effect of mAb 8E11 on p-Src orp-MAPK signaling inhibition by GM2/GM3-coated nanospheres was determinedby incubation of HCV29 cells overnight at 37°C with 5% CO2 and 1 ml ofserum-free RPMI medium 1640, containing GM2/GM3-coated silica nanospherespreincubated, as above, with mouse IgG (1 �g/ml) or mAb 8E11 (IgG3) (1 �g/ml).

Detection of GM2/GM3 Heterodimer by ESI Mass Spectrometry. The existence ofGM2/GM3 heterodimer was confirmed by mass spectrometry, using an iontrap mass spectrometer Esquire LC (Bruker Daltonics) with electrospray ion-ization source. A solution containing GM2 (20 nmol/ml) plus GM3 (20 nmol/ml)with Ca2� (200 nmol/ml) in methanol was directly infused into the ion sourceat a flow rate of 1 �l/min. Spectra were collected in both positive- andnegative-ionization modes.

Production of Monoclonal Antibody 8E11 Directed to GM2/GM3 Heterodimer andDetection of the Heterodimer. A mixture of GM2 (135 �g), GM3 (115 �g), and200 nmol/ml CaCl2 was dried under N2 stream, resuspended in 2 ml of distilledwater, and mixed with 2.5 mg of acid-treated S. minnesotae R595 (1:10ganglioside to bacteria). The suspension was sonicated for 10 min and lyoph-ilized. The material was resuspended in PBS and injected i.v. into BALB/c mice.The immunization protocol was similar to that described in ref. 34.

ACKNOWLEDGMENTS. We thank Reiji Kannagi (Program of ExperimentalPathology, Aichi, Cancer Center, Nagoya, Japan) for donation of anti-GM2antibody MK1.8, James A. Shayman (Department of Internal Medicine, Uni-versity of Michigan, Ann Arbor, MI) for donation of P4, Daniel Gillespie fortechnical assistance with plasmid construct and DNA sequence in study of ldlDtransfectants, and Steve Anderson for help with preparation of the manu-script and figures. This work was supported by National Institutes of HealthGrants GM070593 and CA080054 (to S.H.) and a Conselho Nacional de Des-envolvimento Cientıfico e Tecnologico (Brazil) Postdoctoral Fellowship (toA.R.T.).

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