Ganglioside GM2/GM3 complex affixed on silica nanospheres strongly inhibits cell motility through CD82/cMet-mediated pathway

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Table 2. m/z values of precursor and product ions of GM2 (20 nM) plus GM3 (20 nM) with Ca²⁺ (200 nmol/ml) in methanol

*See Fig. 2A

^†See MS/MS spectrum in Fig. 2C.

^‡See MS/MS spectrum in Fig. 2D.

Negative ion spray mass spectrum of GM2 (20 nM) plus GM3 (20 nM) with Ca²⁺ (200 nmol) in methanol (Fig. 2A) showed a ion m/z = 1383 identified as [GM2-H⁺]^- (precursor ion) and a sequence of ions m/z = 1,208, 1,222, 1,236, 1,250, and 1,264 identified as [GM3-H⁺]^- (precursor ions) and representing a mixture of GM3 molecules containing C14, C18, C19, C20, C21, and C22 fatty acids respectively. Such ions (precursor ions) complex with Ca²⁺⁺ originating the Ca²⁺⁺ adducts (product ions) observed on the positive ion spray mass spectrum (Fig. 2B). GM2 complexes with Ca²⁺ generating the ion [GM2-H⁺)^-+Ca⁺⁺]⁺ m/z = 1,423 (Fig. 2Bb). GM3 complex with Ca²⁺ generates the ions [(GM3-H⁺)^-+Ca²⁺]⁺ m/z = 1,248, 1,262, 1,276, 1,290 and 1,304 (Fig. 2Ba). The hetero-dimer of the two gangliosides, [(GM2-H⁺)^-+(GM3-H⁺)^-+2Ca²⁺]⁺⁺ yield the ions m/z = 1,342,1,350, 1,356, 1,364 and 1,372 (Fig. 2Bc).

SI Material and Methods

Antibodies and Reagents. Antibodies used were anti-GM3 DH2 (mouse IgG3) (1), anti-GM2 MK1.8 (mouse IgM) (2), donated by Reiji Kannagi, anti-CD82 (mouse IgG1) (Serotec), HRP-labeled goat anti-mouse IgG (Southern Biotech), and HRP-labeled goat anti-rabbit IgG (Santa Cruz Biotechnology). GSLs GM2, GM3, Gb3, and Gb4 were purchased from Matreya. Normal mouse IgG was from Santa Cruz Biotechnology. GM1 and GD1a were from Calbiochem. Polystyrene beads (1 m m diameter; as pure water suspension) were from Interfacial Dynamics. MicroBCA Protein Assay Reagent Kit and SuperSignal West Pico Chemiluminescent Substrate for detection of HRP were from Pierce. Hepatocyte growth factor was from Calbiochem-Novabiochem. Other reagents: from Sigma, or see legends for figures. GlcCer synthase inhibitor D-threo-1-(3',4'-ethylenedioxy)phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (D-t-EtDO-P4; so-called "P4") was donated by James A. Shayman (University of Michigan).

Cell lines and Cell Culture. Cell line YTS-1, established from invasive human urinary bladder cancer derived from transitional epithelium (3), was provided by Makoto Satoh (Department of Urology, Tohoku University School of Medicine, Sendai, Japan). The HCV29 cell line, established from normal bladder transitional epithelium (4), was obtained from American Type Culture Collection. The YTS1/CD82 was established (5). These cell lines were grown in RPMI MEDIUM 1640 containing 0.4 mM Ca²⁺ and 0.4 mM Mg²⁺, supplemented with 10% FBS (which contains » 4 mM Ca²⁺ and » 2 mM Mg²⁺), 100 Iunits/ml penicillin, and 100 m g/ml streptomycin at 37° C, 5% CO₂.

ldlD cells, a UDP-Glc 4-epimerase deficient mutant of Chinese Hamster Ovary (CHO) cells, were originally donated by Monty Krieger (Massachusetts Institute of Technology, Cambridge, MA). ldlD/pCDNA3 and ldlD/CD82 were established in ref. 6.

Human GM2 synthase (NM_001478) cDNA (a gift from Koichi Furukawa, Nagoya University, Japan) was ligated as 1.7-kilobase NotI fragment into the expression vector pIRESpuro3 (CLONTECH). The plasmid construct was confirmed by DNA sequencing. After linearization with SspI, the plasmid was separately transfected with Fugene 6 (Roche Labs) into ldlD/pCDNA3 and ldlD/CD82 cells according the manufacture's instruction. After culturing in selection media containing puromycin (7.5 m g/ml), the transfectants were screened by cell surface expression of GM2 using anti-GM2 mAb, MK1.8, and cloned by two cycles of colony selection.

ldlD variant cells were maintained in Ham's F-12 medium with 100 units/ml penicillin, 100 m g/ml streptomycin, and 5% (vol/vol) FBS. The glycosylation pattern was altered by changing culture medium from the above to serum-free F-12 medium containing 1% ITS (Collaborative Biomedical Products) on day 0. Gal and GalNAc (0, 20, 100, and 200 m M; see Fig. 5B) were added on day 0. The effect of glycosylation on cellular function was determined on day 3.

SiO₂ Nanosphere Preparation and GSL Coating. (i) A solution containing of gangliosides (25 nmol/ml) with Ca²⁺ (250 nmol/ml) in methanol was dried in a glass tube under a N2 stream. (ii) Nanospheres (50 nm diameter at 16 mg/ml) from Corpuscular were agglutinated by acidification with few drops of HCl (1 N), and centrifuged at 300 rpm, 5 min. The supernatant was discarded and the nanospheres were washed 2 times with 0.5 ml of EtOH:H₂O (90:10 vol/vol). The suspension was sonicated 10 min to resuspend the nanospheres. (iii) 20 m l (0.3 mg containing 18.9 ´ 10¹⁶ nanospheres) of suspension was transferred into the glass tube containing dried gangliosides (25 nmol/0.2 mg of nanospheres) and incubated under agitation at 4° C. The next day the mixture GSL/nanosphere was dried and resuspended in serum-free RPMI to a final volume of 1 ml by sonicating 10 min. The molar ratio of ganglioside/nanosphere binding was determined by using ³H-labeled GM2 by the Rosenthal method (7). The preparations showed concentration-dependent binding. Rosenthal analysis of the data indicates that nanospheres exhibit a maximal binding capacity (Bmax) of 800 nmoles [³H]Gal GM2/mg of nanospheres and a half-maximal binding at 25 nmol. [³H]Gal GM2 was tritiated by a modification of the galactose oxidase-sodium borohydrate method of Novak et al. (8).

Determination of Cell Motility, Its Inhibition by GSL Nanospheres, and Effect of mAb 8E11 That Blocks Motility-Inhibitory Effect of GSL-Nanospheres. Cell motility was determined as the area of phagokinetic tracks on gold sol particle-coated plates, by the method originally described (9) and modified by Todeschini et al. (5). Cells (0.5 ´ 10⁴ per well), in 0.25 ml of RPMI medium containing aqueous GSL solution (25 nmol/ml), or GSL-coated silica nanospheres (prepared as above, 25 nmol/0.2 mg of nanospheres per ml of medium), 5% FBS, were seeded onto gold sol-coated 48-well plates and incubated at 37° C, 5% CO₂. After 18 h cells were observed, and photographed by using a light microscope (Nikon). Motility track area of 15 cells per well were measured by Scion image program and expressed as square pixels.

mAb 8E11 effect on inhibition of cell function induced by ganglioside-coated nanospheres was determined by preincubation of noncoated nanospheres or GM2/GM3-coated silica nanospheres, for 2 h at room temperature, with mAb 8E11 (IgG3) (1 m g/ml). HCV29 cells (0.5 ´ 10⁴ per well), in RPMI medium (0.250 ml) containing 5% of FBS and the antibody/nanospheres suspension, were seeded onto gold sol-coated 48-well plates, incubated and analyzed as above. Normal mouse IgG [1 m g/ml], normal mouse IgM [1 m g/ml], mAb DH2 (IgG3) (1 m g/ml) and mAb MK1.8 (IgM) (1 m g/ml) were used as control for effect of 8E11.

Blocking effect of mAb 8E11 on on p-Src or p-MAPK signaling inhibition by GM2/GM3-coated nanospheres was determined by incubation of HCV29 cells overnight at 37° C, 5% CO₂, with 1 ml of serum-free RPMI medium containing GM2/GM3-coated silica nanospheres preincubated, as above, with mouse IgG [1 m g/ml] or mAb 8E11 (IgG3) (1 m g/ml). Cells were lysated and phosphorylation at position 416 of Src and MAPK of each cell lysate was measured by Western blot analysis as described below.

Determination of GM2/GM3 Heterodimer Effect on LN5-Dependent Cell Adhesion on Met Tyrosine Phosphorylation. LN5-coated plates were prepared as described in ref. 5. Cells were grown on regular culture plates, starved overnight in serum-free RPMI with addition of noncoated nanospheres, GM2-, GM3- or GM2/GM3-coated silica nanospheres (prepared as above). Cells were harvested and (10⁶ cells per ml) were centrifuged, suspended in serum-free medium, tumbled for 1 h at 4° C. Two ml of cell (5 ´ 10⁵ cells) were kept in suspension or added to LN-coated plate, and incubated for 1 h at 37° C. Cells were lysed, 100 m g of lysate was subjected to IP with anti-Met antibody, levels of tyrosine phosphate in IP'd fractions were measured by using anti-phosphotyrosine antibody (Py20), and stripped blots were probed by anti-Met antibody.

Determination of Src and MAPK Tyrosine Phosphorylation. Cells were grown in 6-well culture plates to » 90% confluence, washed, incubated overnight with GSL/nanosphere suspension, washed with PBS containing 1 mM sodium vanadate, and lysed in RIPA buffer (25 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, and 10 mM NaF) containing 75 units aprotinin, 2 mM PMSF, and 5 mM sodium vanadate. Samples with 15 m g of protein, in SDS sample buffer, boiled for 5 min, were subjected to SDS-PAGE with Western blot on PVDF membrane. Levels of P-Src (Tyr-416) or P-p44/42 MAPK (Thr-202/Tyr-204) were measured by using specific antibodies (Cell Signaling). After stripping with Re-Blot Plus solution (Chemicon), membranes were reblotted, respectively, with rabbit anti-c-Src (Santa Cruz) or rabbit anti-p44/42 MAPK (Cell Signaling) antibodies. Intensity of Western blot was determined by densitometry using Scion image program.

Detection of GM2/GM3 Heterodimer by ESI Mass Spectrometry. The existence of GM2/GM3 hetero-dimer was confirmed by mass spectrometry using an ion trap mass spectrometer Esquire LC (Bruker Daltonics) with electrospray ionization source. A solution containing of GM2 (20 nmol/ml) plus GM3 (20 nmol/ml) with Ca²⁺ (200 nmol/ml) in methanol was directly infused into the ion source at a flow rate of 1 m l/min. Spectra were collected in both positive and negative ionization mode.

Production of Monoclonal Antibody 8E11 Directed to GM2/GM3 Heterodimer and Detection of the Heterodimer. A mixture of GM2 (135 m g), GM3 (115 m g), and 200 nmol/ml CaCl₂ was dried under N₂ stream, resuspended in 2 ml of distilled water, and mixed with 2.5 mg of acid-treated Salmonella minnesotae R595 (1:10 ganglioside to bacteria). The suspension was sonicated for 10 min and lyophilized. The material was resuspended in PBS and injected i.v. into BALB/c mice. Each mouse received 5 m g of ganglioside on day 0, 10 m g on day 4, 15 m g on day 7, and 20 m g on day 12. Mice received a booster injection on day 27, and spleens were harvested on day 30. This immunization protocol was similar to that described in ref. 10. Spleen cells were fused with SP2 cells (ATCC) using polyethylene glycol, as described in ref. 11, using Hybridoma Fusion and Cloning Supplement (Roche). Hybridoma secreting Ig were selected by a stronger reaction with GM2/GM3 than with GM2 or GM3 alone, as verified by ELISA. Gangliosides GM2, GM3, and a mixture of GM2 and GM3 in equimolar proportions were dissolved in methanol (1.25 nmol/ml) containing CaCl₂ (200 nmol/ml). A 50-m l aliquot and was added to a 96-well flat-bottom polystyrene plate, air-dried at 37° C, washed in TBS (140 mM NaCl/10 mM Tris× HCl, pH 8.0) containing 2 mM CaCl₂ (TBS+), and binding of mAb 8E11 (1 m g/ml) was determined by ELISA as described in ref. 12. Effect of EDTA on mAb 8E11 binding was assayed by adding 50 mM EDTA to a mixture of GM2-GM3 in methanol solution and using TBS as washing buffer. mAb 8E11 isotype was determined by using IsoStrip mouse mAb Isotyping Kit (Roche) as described by the manufacturer.

mAb 8E11 was used: (i) for detection of GM2/GM3 hetero-dimer at the cell surface by flow cytometry (Figs. 3D, 4A, and 5C and their legends); (ii) in cellular extract by ELISA (Fig. 3A legend); and (iii) for screening of possible Ca²⁺-dependent interaction of various GSLs or gangliosides, using special way of TLC, which was a modification of the method originally used for detection of interaction of GM1 with GD1a (13). Gangliosides, GSLs, and their combinations were placed on upper (*) or lower line (**) of two HPTLC plates, as indicated in Fig. 3C. HPTLC plates were developed with isopropanol, hexane, and 0.2% CaCl₂ in water (75:40:20 vol/vol/vol). One plate was air-dried, immersed in 0.5% poly(isobutyl-metacrylate) in hexane/chloroform (9:1) for 90 sec, dried, blocked with 3% BSA in PBS for 1 h, and reacted with mAb 8E11, DH2, or MK1.8 (for a, b, or c respectively), for 2 h at room temp. After washing, bound antibody was detected by using rabbit anti-mouse IgM + IgG (Pierce, Rockford, IL), followed by detection with ¹²⁵I-Protein A (Perkin-Elmer, Boston, MA). Reactive bands on TLC plates were revealed by autoradiography. Use of isopropanol/hexane/aqueous CaCl₂ system gave much better results than chloroform/methanol/water system, for detection of GSL-to-GSL interaction on HPTLC. Another plate was sprayed with orcinol/H₂SO₄, and heated at 120° C to reveal all GSLs.

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