Functional role of glycosphingolipids and gangliosides in control of cell adhesion, motility, and growth, through glycosynaptic microdomains

Abstract

At cell surface microdomains, glycosyl epitopes, carried either by glycosphingolipids, N- or O-linked oligosaccharides, are recognized by carbohydrate-binding proteins or complementary carbohydrates. In both cases, the carbohydrate epitopes may be clustered with specific signal transducers, tetraspanins, adhesion receptors or growth factor receptors. Through this framework, carbohydrates can mediate cell signaling leading to changes in cellular phenotype. Microdomains involved in carbohydrate-dependent cell adhesion inducing cell activation, motility, and growth are termed "glycosynapse". In this review a historical synopsis of glycosphingolipids-enriched microdomains study leading to the concept of glycosynapse is presented. Examples of glycosynapse as signaling unit controlling the tumor cell phenotype are discussed in three contexts: (i) Cell-to-cell adhesion mediated by glycosphingolipids-to-glycosphingolipids interaction between interfacing glycosynaptic domains, through head-to-head (trans) carbohydrate-to-carbohydrate interaction. (ii) Functional role of GM3 complexed with tetraspanin CD9, and interaction of such complex with integrins, or with fibroblast growth factor receptor, to control tumor cell phenotype and its reversion to normal cell phenotype. (iii) Inhibition of integrin-dependent Met kinase activity by GM2/tetraspanin CD82 complex in glycosynaptic microdomain. Data present here suggest that the organizational status of glycosynapse strongly affects cellular phenotype influencing tumor cell malignancy.

Fig. 1

Overview of key steps during the development of glycosynapse concept. 1. First observations of polarized sorting of GSL [–14] and glycoproteins [15, 16] in epithelial cells. 2. Observation of GSL organization associated to biological function in cell surface membranes [17]. 3. Demonstration of GSL clustering in cell surface membranes by freeze-fracture [18], and at the surface of GSL-phosphatidylcholine liposome [19, 20]. 4. Concomitantly, it was found that clustered GSL were insoluble in 1 % Triton X-100 due to their association with cytoskeleton components and extra cellular matrix [5, 97]. 5. Demonstration that tyrosine phosphorylation of growth factor receptor is affected by neighboring GSLs in microdomains [28, 29]. 6. Stefanova and coworkers [30] found that proteins anchored to the membrane by glycophosphatidylinositol (GPI) anchor associate with protein tyrosine kinase related to Src family. 7. Presentation of rafts as new structure of cell membranes [23, 27]. 8. Current work.

Fig. 2

Illustration of three types of glycosynapses proposed by Hakomori [32]. A. Glycosyl epitopes involved in cell-to-cell adhesion are found in glycosynapse 1 (B) and 2 (C), while carbohydrates implicated in cell-to-ECM adhesion compose glycosynapse 3 (D). B. Glycosynapse 1 is based on GSL-to-GLS interaction (Bi) or GLS-to-binding protein interaction (Bii), in which GSL are clustered with signal transducers (TD). C. Glycosynapse 2 is based on interaction between O-linked oligosaccharides of mucin like glycoproteins and carbohydrate binding proteins associated with TD. D. Glycosynapse 3 is established by carbohydrate dependent association of N-glycosylated adhesion receptor (e.g. integrin receptor), TSP, GLS and TD mediating cell adhesion to ECM. Cell adhesion and motility are greatly inhibited when glycosynapse 3 complex is stabilized by glycosylation of integrin receptor or TSP.

Fig. 3

A. Solution conformation of bis-Le^x calcium complex. GlcNAc (a, black), Fuc (b, blue) Gal (c, red) and the methyllene spacer, linking the GlcNAc, is shown in green. The same conformational minimum is shown from two different points of view. Observe that rings a and a’ are coplanar (left), and ring b is located approximately 90° from ring b’ assuming a cross like orientation which is more visible from a different direction (right). B. Proposed Le^x-Le^x interaction between glycolipids anchored to the same microdomain (cis-CCI) or anchored to adjacent microdomains (trans-CCI). Two Le^x pentasacharides, a Le^x trisaccharide (filled pyranose rings) plus a lactose moiety (open pyranose rings), were sketched with the relative orientation of the Le^x epitopes as demonstrated in A, above, were the GlcNac (a and a) rings are coplanar and the Fuc ring (b) is located approximately 90° from Fuc ring (b). The orientation proposed in A allows for cis- and trans-CCI between Le^x glycoconjugates. Data from Geyer et al. [40].

Fig. 4

Le^x mediates homotypic adhesion of embryonal cells independently from E-cad. Adhesion of: (1) F9 Ecad (−/−), (2) D3M Ecad (−/−) and control (3) PYS-2 cells to plates coated with Le^x GSL (Le^x GSL (Galβ4(Fucα3)GlcNacβ3Galβ4GlcβCer). Both cells F9 Ecad (−/−), (2) D3M Ecad, in the absence of Ecad expression, adhered in a dose-dependent manner to plates coated with 0.25, 0.5, 1.0, or 2.0 µg/ well of Le^x GSL. PYS-2 cells, a cell line derived from mouse 129 embryonal carcinoma OTT6050 (from which F9 cells were derived) but do not express Le^x glycan, did not show adhesion to Le^x GSL-coated plates. Data from Handa et al. [43].

Fig. 5

Illustration of cis-CCI controlling glycosylation-dependent cell motility and/or growth. Integrin subunits α and β, TSP and GRF interaction mediated by N-linked glycans and surrounding GSL. GSL associate with Scr family kinases and other signal transducers. a. GFR and its interaction with surrounding gangliosides. b. TSP interaction with gangliosides.

Fig. 6

Interaction of EGRF with GM3 but not GM1 or Gb4 and inhibition of EGRF/GM3 interaction by Fr. B but not by cellobiose. EGRF, from A431 cell lysate, shows strong binding to GM3 coated polystyrene beads (lane d), weak or no binding to beads coated with GM1 (lane b), Gb4 (lane c) or control beads. Binding of GM3 to EGRF was abrogated by co-incubation with a N-linked glycan with five to six GlcNAc termini, Fr.B (lane f) but was not inhibited by the Glc disaccharide, cellobiose (lane e) [61].

Fig. 7

Effects of GM3 levels, in bladder cancer cells, glycosynapse. A. Exogenous addition of GM3 (50 µM) resulted in significant inhibition of phagokinetic cell motility in YTS1 cells as compared wit control and GM1 (50 µM) treated cells. B. GM3 mediates interaction of α3 integrin with CD9. Exogenous addition of GM3 to YTS1 cells causes enhanced co-immunoprecipitation of α3 integrin and CD9. No effect was observed when GM1 was added to the cells. Upper panel: Cell lysates were immunoprecipitated with anti-α3 or anti-CD9, as control, and analyzed by Western blot with anti-CD9. Bottom panel: Densitometry of α3 band co-immunoprecipitated with CD9 in control and GM3- or GM1-trated cells. C. Increased GM3 levels on c-Src phosphorylation and csk localization in fractions obtained by sucrose density centrifugation. Enhanced GM3 levels did not cause significant change in Src phosphorylation or csk level in the total extract (PNF). However, an increased phosphorylation at Tyr-527, with clear translocation of csk to GEM fractions 4–6 (maximum at 3 h) associated with a simultaneous decrease in phosphorylation of Tyr-416 was observed [79].

Fig. 8

GM2 complexes with CD82 and promotes CD82 interaction with Met A. CD82, specifically interacts with GM2-coated polystyrene beads (d). No interaction was observed when YTS1/CD82⁺ cell lysate was incubated with noncoated (a) Gb4-coated (b) or GM3-coated (c) beads. (e) protein load (30 µg). B. Effect of GM2 on CD82 and Met interaction on CD82 expressing cells HCV29 and YTS1/CD82 cells. Exogenous addition of GM2 to HCV29 and YTS1/CD82 cells enhances interaction between CD82 and Met (b and e) while ganglioside depletion by P4 significantly decreases this interaction (c and f) when compared with medium treated cells (a and d). Data from Todeschini et al. [86].

Fig. 9

Hypothetical associations among components of glycosynapse from bladder epithelial cells. Bladder epithelial cells express two major receptors as follows: (i) HGF receptor Met and its kinase (shown at left), which is inhibited by GM2-CD82 complex ("a"); (ii) integrin receptor α3β1, which binds to extra cellular matrix component LN5/10–11 upon cell adhesion (shown at right). α3β1 activation is blocked by GM3-CD9 complex in bladder epithelial cells ("b") [79]. The functional interaction between systems i and ii is blocked by GM2-CD82 complex ("c"). Signaling shown for both systems is arbitrary, based on a few previous reviews or studies by others and by our group [79, 85]. Grb2 and Gab1 are initial signaling molecules that may lead to activation of extra cellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK), phosphatidylinositol 3-kinase (PI3K), or focal adhesion kinase (FAK) [85], controlling cell growth and motility. α3β1 may act through Src family kinases (which are inhibitable by Csk) [78, 79], and lead to Rak/phosphatidylinositol 3-kinase/Akt signaling [99], controlling cell adhesion and motility. From Todeschini et al. [86].

Fig. 10

Effect of integrin mediated adhesion of HCV29, YTSI, or YTS1/CD82 cells to Ln5 on Met kinase activation. The degree of Met activation (rate of Met phosphorylation relative to total Met level) of HCV29 (A), YTS1 (B), and YTS1/CD82 cells (C) was determined for cells in suspension (lanes 1 and 2) versus adhesion to LN5-coated plates (lanes 3 and 4). Met activation was also determined after GLS depletion by P4 (black versus gray columns). First row at bottom: intensity of Met tyrosine phosphorylation probed by Py20 in Western blot. Second row: Met level by Western blot. Third row: suspension without (−) or with (+) LN5 adhesion. Fourth row: pre-incubation without (−) or with (+) P4. Data were expressed as mean ± SD. Significance of differences indicated by bracket: *** p< 0.001. Data from Todeschini et al. [86]. Observe that adhesion to LN5 of normal HCV29 cells did not affect Met tyrosine kinase phosphorylation, apparently due to ganglioside inhibition since, ganglioside depletion increases Met phosphorylation upon adhesion to LN5 (lane 4 versus 1–3). Adhesion of YTS1 and YTS1/ CD82 cells to LN5-coated plate greatly increased Met tyrosine phosphorylation, as compared to suspension culture (lane 3 vs. 1 B and C). The degree of enhancement of Met kinase activity by LN5-dependent adhesion was increased when gangliosides were depleted by P4 treatment (lane 4 vs. 3 in B and C).