GM3 ganglioside inhibits CD9-facilitated haptotactic cell motility: coexpression of GM3 and CD9 is essential in the downregulation of tumor cell motility and malignancy

A cooperative inhibitory effect of GM3, together with CD9, on haptotactic cell motility was demonstrated by a few lines of study as described below. (i) Haptotactic motility of colorectal carcinoma cell lines SW480, SW620, and HRT18, which express CD9 at a high level, is inhibited by exogenous GM3, but not by GM1. (ii) Motility of gastric cancer cell line MKN74, which expresses CD9 at a low level, was not affected by exogenous GM3. Its motility became susceptible to and inhibited by exogenous GM3, but not GM1, when the CD9 level of MKN74 cells was converted to a high level by transfection with CD9 cDNA. Findings i and ii suggest that haptotactic tumor cell motility is cooperatively inhibited by coexpression of CD9 and GM3. (iii) This possibility was further demonstrated using cell line ldlD 14, and its derivative expressing CD9 through transfection of its gene (termed ldlD/CD9). Both of these cell lines are defective in UDP-Gal 4-epimerase and cannot synthesize GM3 unless cultured in the presence of galactose (Gal(+)), whereas GM3 synthesis does not occur when cells are cultured in the absence of Gal (Gal(-)). Haptotactic motility of parental ldlD cells is low, and shows no difference in the presence and absence of Gal. In contrast, the motility of ldlD/CD9 cells is very high in Gal(-) whereby endogenous GM3 synthesis does not occur, and is very reduced in Gal(+) whereby endogenous GM3 synthesis occurs. (iv) Photoactivatable (3)H-labeled GM3 added to HRT18 cells, followed by UV irradiation, causes cross-linking of GM3 to CD9, as evidenced by (3)H labeling of CD9, which is immunoprecipitated with anti-CD9 antibody. These findings suggest that CD9 is a target molecule interacting with GM3, and that CD9 and GM3 cooperatively downregulate tumor cell motility.     

Regulation of Epidermal Growth Factor Receptor Through Interaction of Ganglioside GM3 with GlcNAc of N-Linked Glycan of the Receptor: Demonstration in ldlD Cells

We investigated interaction of GM3 with N-acetylglucosamine (GlcNAc) termini of N-linked glycans of epidermal growth factor receptor (EGFR), as the underlying mechanism for inhibitory effect of GM3 on EGFR activation, using ldlD cells transfected with EGFR gene. These cells, defective in UDP-Gal/UDP-GalNAc 4-epimerase, are incapable of synthesizing galactose (Gal)-containing glycans, unless Gal is provided in culture (+Gal). Key observations: (1) Expression of GlcNAc termini was high in -Gal cells, and strongly reduced in +Gal cells. (2) Comparative study of inhibitory effect of exogenously-added GM3 on EGFR activation in +Gal versus -Gal cells indicated that higher level of GlcNAc termini on EGFR is correlated with greater inhibitory effect of GM3. (3) GM3-, but not GM1-, coated beads bound to EGFR in lysate of -Gal cells, which have highly exposed GlcNAc termini. Such binding was inhibited in the presence of EDTA, similarly to other carbohydrate-carbohydrate interactions.     

Glycosylation effect on membrane domain (GEM) involved in cell adhesion and motility: a preliminary note on functional alpha3, alpha5-CD82 glycosylation complex in ldlD 14 cells

Laminin (LN)- or fibronectin (FN)-dependent adhesion in Krieger's ldlD 14 (D14) cells is enhanced significantly in the presence vs absence, of galactose (Gal), whereas LN- or FN-induced haptotactic cell motility is barely affected unless cells express CD82 by its gene transfection (cells termed D14/CD82). The effect of CD82 on LN- or FN-induced motility is based on its ability to associate with alpha3 or alpha5 integrin to form a complex associated with a low-density lipid membrane domain (termed GEM or GSD). Complex formation is greatly affected by N-glycosylation of both integrin and CD82, as well as by concurrent GM3 ganglioside synthesis. The effect of glycosylation on alpha5-CD82 complex was also studied in D14 cells expressing mutant CD82, defective in all three N-glycosylation sites. LN-induced motility was greatly inhibited, whereas FN-induced motility was enhanced, with complete N-glycosylation in D14/CD82 cells in Gal-added medium, whereby alpha5-CD82 complex formation did not occur or occurred at a minimal level. Both LN- and FN-induced motility were inhibited when N-glycosylation was impaired, or N-glycosylation of CD82 was deleted, whereby alpha5-CD82 complex formation occurred strongly. Thus, glycosylation profoundly affects interaction of integrin with CD82, leading to significant inhibition or promotion of cell motility.     

Glycosylation affects translocation of integrin, Src, and caveolin into or out of GEM

Endogenous GM3 synthesis and full N-glycosylation in membrane receptors occurred in "4-epimerase-less" ldlD (Krieger's CHO mutant) cells cultured in Gal-containing medium, whereby components of detergent-insoluble, low-density, buoyant membrane fraction, termed "glycolipid-enriched microdomain (GEM)," varied significantly by translocation into or out of GEM. Integrins alpha3 and alpha5 were translocated into GEM in the presence of 0.5 or 0.25% Triton X-100, particularly in the absence of Gal, whereby integrins are underglycosylated and GlcCer is the major glycolipid component in GEM. Src family kinase was translocated into and enriched in GEM fractions when prepared in 0.5 or 0.25% Triton X-100 from cells grown in Gal-containing medium, whereby GM3 synthesis is induced. In contrast, caveolin is highly enriched in GEM when GM3 synthesis does not occur, and is translocated into high-density membrane fraction when GM3 synthesis occurs. The results suggest that levels of key molecules controlling cell adhesion and signaling are defined by translocation into or out of GEM, which depends on glycosylation state.     

A specific microdomain ("glycosynapse 3") controls phenotypic conversion and reversion of bladder cancer cells through GM3-mediated interaction of alpha3beta1 integrin with CD9

Cell motility is highly dependent on the organization and function of microdomains composed of integrin, proteolipid/tetraspanin CD9, and ganglioside (Ono, M., Handa, K., Sonnino, S., Withers, D. A., Nagai, H., and Hakomori, S. (2001) Biochemistry 40, 6414-6421; Kawakami, Y., Kawakami, K., Steelant, W. F. A., Ono, M., Baek, R. C., Handa, K., Withers, D. A., and Hakomori, S. (2002) J. Biol. Chem. 277, 34349-34358), later termed "glycosynapse 3" (Hakomori, S., and Handa, K. (2002) FEBS Lett. 531, 88-92, 2002). Human bladder cancer cell lines KK47 (noninvasive and nonmetastatic) and YTS1 (highly invasive and metastatic), both derived from transitional bladder epithelia, are very similar in terms of integrin composition and levels of tetraspanin CD9. Tetraspanin CD82 is absent in both. The major difference is in the level of ganglioside GM3, which is several times higher in KK47 than in YTS1. We now report that the GM3 level reflects glycosynapse function as follows: (i) a stronger interaction of integrin alpha3 with CD9 in KK47 than in YTS1; (ii) conversion of benign, low motility KK47 to invasive, high motility cells by depletion of GM3 by P4 (D-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol) treatment or by knockdown of CD9 by the RNA interference method; (iii) reversion of high motility YTS1 to low motility phenotype like that of KK47 by exogenous GM3 addition, whereby the alpha3-to-CD9 interaction was enhanced; (iv) low GM3 level activated c-Src in YTS1 or in P4-treated KK47, and high GM3 level by exogenous addition caused Csk translocation into glycosynapse, with subsequent inhibition of c-Src activation; (v) inhibition of c-Src by "PP2" in YTS1 greatly reduced cell motility. Thus, GM3 in glycosynapse 3 plays a dual role in defining glycosynapse 3 function. One is by modulating the interaction of alpha3 with CD9; the other is by activating or inhibiting the c-Src activity, possibly through Csk translocation. High GM3 level decreases tumor cell motility/invasiveness, whereas low GM3 level enhances tumor cell motility/invasiveness. Oncogenic transformation and its reversion can be explained through the difference in glycosynapse organization.     

Effect of ganglioside and tetraspanins in microdomains on interaction of integrins with fibroblast growth factor receptor

The functional interaction ("cross-talk") of integrins with growth factor receptors has become increasingly clear as a basic mechanism in cell biology, defining cell growth, adhesion, and motility. However, no studies have addressed the microdomains in which such interaction takes place nor the effect of gangliosides and tetraspanins (TSPs) on such interaction. Growth of human embryonal WI38 fibroblasts is highly dependent on fibroblast growth factor (FGF) and its receptor (FGFR), stably associated with ganglioside GM3 and TSPs CD9 and CD81 in the ganglioside-enriched microdomain. Adhesion and motility of these cells are mediated by laminin-5 ((LN5) and fibronectin (FN) through alpha3beta1 and alpha5beta1 integrin receptors, respectively. When WI38 cells or its transformant VA13 cells were adhered to LN5 or FN, alpha3beta1 or alpha5beta1 were stimulated, giving rise to signaling to activate FGFR through tyrosine phosphorylation and inducing cell proliferation under serum-free conditions without FGF addition. Types and intensity of signaling during the time course differed significantly depending on the type of integrin stimulated (alpha3beta1 versus alpha5beta1), and on cell type (WI38 versus VA13). Such effect of cross-talk between integrins and FGFR was influenced strongly by the change of GM3 and TSPs. (i) GM3 depletion by P4 caused enhanced tyrosine phosphorylation of FGFR and Akt followed by MAPK activation, without significant change of ceramide level. GM3 depletion also caused enhanced co-immunoprecipitation of FGFR with alpha3/alpha5/beta1 and of these integrins with CD9/CD81. (ii) LN5- or FN-dependent proliferation of both WI38 and VA13 was strongly enhanced by GM3 depletion and by CD9/CD81 knockdown by siRNA. Thus, integrin-FGFR cross-talk is strongly influenced by GM3 and/or TSPs within the ganglioside-enriched microdomain.     

A critical role for tetraspanin CD151 in alpha3beta1 and alpha6beta4 integrin-dependent tumor cell functions on laminin-5

The basement membrane protein laminin-5 supports tumor cell adhesion and motility and is implicated at multiple steps of the metastatic cascade. Tetraspanin CD151 engages in lateral, cell surface complexes with both of the major laminin-5 receptors, integrins alpha3beta1 and alpha6beta4. To determine the role of CD151 in tumor cell responses to laminin-5, we used retroviral RNA interference to efficiently silence CD151 expression in epidermal carcinoma cells. Near total loss of CD151 had no effect on steady state cell surface expression of alpha3beta1, alpha6beta4, or other integrins with which CD151 associates. However, CD151-silenced carcinoma cells displayed markedly impaired motility on laminin-5, accompanied by unusually persistent lateral and trailing edge adhesive contacts. CD151 silencing disrupted alpha3beta1 integrin association with tetraspanin-enriched microdomains, reduced the bulk detergent extractability of alpha3beta1, and impaired alpha3beta1 internalization in cells migrating on laminin-5. Both alpha3beta1- and alpha6beta4-dependent cell adhesion to laminin-5 were also impaired in CD151-silenced cells. Reexpressing CD151 in CD151-silenced cells reversed the adhesion and motility defects. Finally, loss of CD151 also impaired migration but not adhesion on substrates other than laminin-5. These data show that CD151 plays a critical role in tumor cell responses to laminin-5 and reveal promotion of integrin recycling as a novel potential mechanism whereby CD151 regulates tumor cell migration.     

Glycosynaptic microdomains controlling tumor cell phenotype through alteration of cell growth, adhesion, and motility

Glycosphingolipids (GSLs) GM3 (NeuAcα3Galβ4Glcβ1Cer) and GM2 (GalNAcβ4[NeuAcα3]Galβ4Glcβ1Cer) inhibit (i) cell growth through inhibition of tyrosine kinase associated with growth factor receptor (GFR), (ii) cell adhesion/motility through inhibition of integrin-dependent signaling via Src kinases, or (iii) both cell growth and motility by blocking “cross-talk” between integrins and GFRs. These inhibitory effects are enhanced when GM3 or GM2 are in complex with specific tetraspanins (TSPs) (CD9, CD81, CD82). Processes (i)-(iii) occur through specific organization of GSLs with key molecules (TSPs, caveolins, GFRs, integrins) in the glycosynaptic microdomain. Some of these processes are shared with epithelial-mesenchymal transition induced by TGFβ or under hypoxia, particularly that associated with cancer progression.     

N-Acetylneuraminyllactosylceramide, GM3-NeuAc, a new influenza A virus receptor which mediates the adsorption-fusion process of viral infection. Binding specificity of influenza virus A/Aichi/2/68 (H3N2) to membrane-associated GM3 with different molecular species of sialic acid

Agglutinates of native chicken erythrocytes caused by influenza virus A/Aichi/2/68 (H3N2) at 4 degrees C were potently fused and lysed at low pH (optimum pH 5.3) at 37 degrees C. Exogenous gangliosides GM3 (Sia alpha 2-3Gal beta 1-4Glc beta 1-ceramide) and GM2 (GalNAc beta 1-4(Sia alpha 2-3)-Gal beta 1-4Glc beta 1-ceramide) were integrated into the membranes of chicken asialoerythrocytes within 5-min incubation at 37 degrees C. We found that the incorporation of ganglioside GM3 containing N-acetylneuraminic acid into asialoerythrocytes restored the biological responsiveness to the virus as established by agglutination at 4 degrees C and fusion and hemolysis at 37 degrees C at pH 5.3. Biological responsiveness of GM3-NeuAc-erythrocytes to the virus was considerably higher than that of GM3-NeuGc-erythrocytes under the same experimental conditions. Treatment of the GM3-NeuAc-erythrocytes with neuraminidase again resulted in the complete abolishment of the response to the virus. Erythrocytes containing GM2-NeuAc showed no detectable biological responses toward the virus. The above results indicate that the hemagglutinin of influenza virus A/Aichi/2/68 (H3N2) recognizes the sialyloligosaccharide chain of ganglioside GM3 as its receptor which mediates the adsorption and fusion process on the virus entry into the host cells and has more preferential specificity for binding to N-acetylneuraminic acid-containing GM3 than that to N-glycolyl type in the target cell membranes.     

EWI-2 regulates alpha3beta1 integrin-dependent cell functions on laminin-5

EWI-2, a cell surface immunoglobulin SF protein of unknown function, associates with tetraspanins CD9 and CD81 with high stoichiometry. Overexpression of EWI-2 in A431 epidermoid carcinoma cells did not alter cell adhesion or spreading on laminin-5, and had no effect on reaggregation of cells plated on collagen I (alpha2beta1 integrin ligand). However, on laminin-5 (alpha3beta1 integrin ligand), A431 cell reaggregation and motility functions were markedly impaired. Immunodepletion and reexpression experiments revealed that tetraspanins CD9 and CD81 physically link EWI-2 to alpha3beta1 integrin, but not to other integrins. CD81 also controlled EWI-2 maturation and cell surface localization. EWI-2 overexpression not only suppressed cell migration, but also redirected CD81 to cell filopodia and enhanced alpha3beta1-CD81 complex formation. In contrast, an EWI-2 chimeric mutant failed to suppress cell migration, redirect CD81 to filopodia, or enhance alpha3beta1-CD81 complex formation. These results show how laterally associated EWI-2 might regulate alpha3beta1 function in disease and development, and demonstrate how tetraspanin proteins can assemble multiple nontetraspanin proteins into functional complexes.     

Cell growth regulation through GM3-enriched microdomain (glycosynapse) in human lung embryonal fibroblast WI38 and its oncogenic transformant VA13

Cell growth control mechanisms were studied based on organization of components in glycosphingolipid-enriched microdomain (GEM) in WI38 cells versus their oncogenic transformant VA13 cells. Levels of fibroblast growth factor receptor (FGFR) and cSrc were 4 times and 2-3 times higher, respectively, in VA13 than in WI38 GEM, whereas the level of tetraspanin CD9/CD81 was 3-5 times higher in WI38 than in VA13 GEM. Csk, the physiological inhibitor of cSrc, was present in WI38 but not in VA13 GEM. Functional association of GEM components in control of cell growth in WI38 is indicated by several lines of evidence. (i) Confluent, growth-inhibited WI38 showed a lower degree of FGF-induced MAPK activation than actively growing cells in sparse culture. (ii) The level of inactive cSrc (with Tyr-527 phosphate) was higher in confluent cells than in actively growing cells. Both processes i and ii were inhibited by GM3 since they were enhanced by GM3 depletion with d-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (P4). (iii) The high level of inactive cSrc associated with growth-inhibited cells was caused by coexisting Csk in WI38 GEM. (iv) Interaction of GM3 with FGFR was demonstrated by binding of GM3 to FGFR in the GEM fraction, as probed with GM3-coated beads, and by confocal microscopy. In contrast to WI38, both cSrc and MAPK in VA13 were strongly activated regardless of FGF stimulation or GM3 depletion by P4. Continuous, constitutive activation of both cSrc and MAPK was due to (i) a much higher level of cSrc and FGFR in VA13 than in WI38 GEM, (ii) their close association/interaction in VA13 GEM as indicated by clear coimmunoprecipitation between cSrc and FGFR, and (iii) the absence of Csk in VA13 GEM, making GEM incapable of inhibiting cSrc activation.     

Characterization of the ligand-binding specificities of integrin alpha3beta1 and alpha6beta1 using a panel of purified laminin isoforms containing distinct alpha chains

Integrins alpha3beta1 and alpha6beta1 are two major laminin receptors expressed on the surface of mammalian cells. Interactions of cells with laminins through these integrins play important roles in cell adhesion, differentiation, motility, and matrix assembly. To determine the binding specificity and affinity of these integrins toward various types of laminins at the level of direct protein-protein interactions, we purified integrins alpha3beta1 and alpha6beta1 from human placenta, and examined their binding to a panel of laminin isoforms, each containing distinct alpha chains (i.e., laminin-1, laminin-2/4, laminin-5, laminin-8, and laminin-10/11). Integrin alpha3beta1 showed clear specificity for laminin-5 and laminin-10/11, with no significant binding to laminin-1, laminin-2/4, and laminin-8. In contrast, integrin alpha6beta1 showed a broad spectrum of specificity, with apparent binding affinity in the following order: laminin-10/11 > laminin-5 > laminin-1 > laminin-2/4 congruent with laminin-8. Integrin titration assays demonstrated that laminin-10/11 was the most preferred ligand among the five distinct laminin isoforms for both alpha3beta1 and alpha6beta1 integrins. Given that laminin-10/11 is the major basement membrane component of many adult tissues, the interaction of laminin-10/11 with these integrins should play a central role in the adhesive interactions of epithelial cells with underlying basement membranes.     

Cell adhesion, spreading, and motility of GM3-expressing cells based on glycolipid-glycolipid interaction

Cell lines expressing varying levels of ganglioside GM3 at the cell surface show different degrees of adhesion and spreading on solid phase coated with such glycosphingolipids (GSLs) as Gg3 (GalNAc beta 1----4Gal beta 1----4Glc beta 1----1Cer), LacCer (Gal beta 1----4Glc beta 1----1Cer), or Gb4 (GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1Cer) (where Cer is ceramide), which may have structures complementary to GM3, but not on solid phase coated with various other GSLs. The degree of cell adhesion and spreading on Gg3 was correlated with the degree of cell-surface GM3 expression, as defined by reactivity with anti-GM3 monoclonal antibody (mAb) DH2. Only cells with high GM3 expression adhered on solid phase coated with LacCer or Gb4. Adhesion of GM3-expressing cells on Gg3-, LacCer-, and Gb4-coated solid phase is based on interaction of GM3 with Gg3 and, to a lesser extent, with LacCer and Gb4, as demonstrated by: (i) the interaction of the GM3 liposome with solid phase coated with Gg3, LacCer, and Gb4, respectively; (ii) the abolition of cell adhesion on each GSL-coated solid phase by treatment of cells with mAb DH2 or sialidase; and (iii) the inhibition of cell adhesion by treatment of GSL-coated solid phase with mAb specific to each GSL. Sialosyllactosyl-lysyllysine conjugate was bound to Gg3 adsorbed on a C18 silica gel column in the presence of bivalent cation, suggesting that the carbohydrate moiety of GM3 is involved in GM3-Gg3 interaction. Not only the adhesion and spreading of GM3-expressing cells, but also their cell motility was greatly enhanced on Gg3-coated solid phase, as determined by Transwell assay and phagokinetic track assay on a gold sol-coated surface. Spreading and motility of GM3-expressing cells on Gg3-coated solid phase were both inhibited by treatment of cells with mAb DH2 or sialidase. These results provide evidence that not only cell adhesion, but also spreading and motility in these cell lines are controlled by complementary GSL-GSL interaction.     

Identification of erythrocyte Gal alpha 1-3Gal glycosphingolipids with a mouse monoclonal antibody, Gal-13

The Gal alpha 1-3Gal structural determinant has been found to have a unique distribution in mammals. Although this determinant is abundantly expressed by erythrocytes and nucleated cells of many mammals, it has not been detected in human cells. However, our previous studies (Galili, U., Rachmilewitz, E. A., Peleg, A., and Flechner, I. (1984) J. Exp. Med. 160, 1519-1531; Galili, U., Clark, M. R., and Shohet, S. B. (1986) J. Clin. Invest. 77, 27-33) have suggested that this epitope is present in small amounts and may be involved in immune-mediated destruction of senescent human erythrocytes. To have a means for exploring this possibility and for studying the species and tissue distribution of this epitope we have raised a monoclonal antibody (Gal-13) which specifically binds to glycoconjugates with a nonreducing terminal Gal alpha 1-3Gal disaccharide. Mice were immunized with rabbit erythrocytes, which express an abundance of glycoconjugates with Gal alpha 1-3Gal epitopes. Clones were screened with a solid-phase binding assay (enzyme-linked immunosorbent assay) for antibodies which bound to ceramide pentahexoside (Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3-Gal beta Gal beta 1-4Glc1-1Cer) but not to ceramide trihexoside (Gal alpha 1-4Gal beta 1-4Glc1-1Cer). Gal-13 bound to a number of neutral glycosphingolipids from rabbit and bovine erythrocytes. These glycosphingolipids have previously been shown to be a family of linear and branched polylactosamine structures, which have non-reducing terminal Gal alpha 1-3Gal epitopes. The antibody did not bind to the human blood group B glycolipid, Gal alpha 1-3(Fuc alpha 1-2)Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc1-1Cer, and, therefore, branching at the penultimate galactose blocks Gal-13 binding. However, after removal of the fucose from the B antigen Gal-13 recognized the resulting derivative. Other Gal alpha 1-3Gal glycosphingolipids with an isogloboside or globoside core structure were not recognized by Gal-13 suggesting that the antibody binds to Gal alpha 1-3Gal carried by a lactosamine core structure. Gal-13 has been used to demonstrate that the Gal alpha 1-3Gal ceramide pentahexoside has been evolutionarily conserved in red cells of animals up to the stage of New World monkeys but is not found in Old World monkey red cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Colon cancer cells adhesion and spreading on autocrine laminin-10 is mediated by multiple integrin receptors and modulated by EGF receptor stimulation

Epidermal growth factor (EGF) receptor ligands such as EGF and transforming growth factor-alpha (TGF-alpha) play an important role in controlling the proliferation, survival, morphology, and motility of colonic epithelial cells. There is also increasing evidence that growth factors and extracellular matrix (ECM) proteins cooperate to regulate these cellular processes. We have reported previously that autocrine TGF-alpha and an unidentified ECM protein in the serum-free conditioned medium of the human colon carcinoma cell line LIM1215 synergize to induce spreading of these cells in low-density cultures. We have now purified the ECM protein secreted by LIM1215 cells and show that it synergizes with EGF to induce spreading of LIM1215 cells and other human cell lines from the colon and other tissues. The purified ECM migrated as a single protein band with an apparent molecular mass of approximately 800 kDa on SDS-PAGE under nonreducing conditions and, under reducing conditions, as three protein bands of approximately 360, 210, and 200 kDa. Immunoblotting experiments and mass spectrometry analysis of tryptic digests on the purified protein identified the 360-, 210-, and 200-kDa protein bands as laminin alpha5, beta1, and gamma1 chains, respectively, indicating that LIM1215 cells secrete laminin-10 (alpha5 beta1 gamma1). In serum-free medium, LIM1215 cells adhere to laminin-10 primarily via alpha2 beta1 and alpha3 beta1 integrin receptors. EGF-induced spreading of LIM1215 cells on laminin-10 is partially inhibited by pretreatment of the cells with blocking antibodies directed against integrin alpha3 or beta1 but not alpha2, alpha6, or beta4 subunits. Spreading is almost completely inhibited by blocking alpha3 + alpha2, alpha3 + alpha6, or beta1 + beta4 integrin chains and results in cell death. Increased spreading in the presence of EGF correlates with up-regulation of alpha6 beta4 integrins in these cells after exposure to EGF. These results indicate that colon cancer cells attach and spread on laminin-10 via multiple integrin receptors and suggest a critical role for alpha3 beta1 integrins in the spreading response. Together, our results support the concept that the adhesive properties of colon cancer cells are modulated by autocrine production of TGF-alpha and laminin-10 and autocrine induction of appropriate integrins.     

Laminin alpha3 LG4 module induces keratinocyte migration: involvement of matrix metalloproteinase-9

Laminin alpha3 chain, a functionally key subunit of laminin-5, contains a large globular module (G module) which consists of a tandem repeat of five homologous LG modules (LG1-5). We previously demonstrated that the LG4 module of laminin alpha3 chain (alpha3 LG4) induces a matrix metalloproteinase-1 (MMP-1) expression through the interaction with syndecans leading to MAPK activation/IL-1beta expression signaling loop (Utani et al., J. Biol. Chem. 278, 34483-34490, 2003). Here, we show that a recombinant alpha3 LG4 and synthetic peptides containing syndecan binding motif induced a cell motility and a MMP-9 expression in ketarinocytes. The synthetic peptide (A3G756)-induced cell migration and MMP-9 upregulation were inhibited by each application of a heparin and an IL-1 receptor antagonist (IL-1RA), suggesting the involvement of syndecans and IL-1beta autocrine. Furthermore, the A3G756-induced cell motility was inhibited by an MMP-9 inhibitor and a neutralizing antibody of MMP-9, indicating induced cell motility was dependent on an MMP-9 activity. Taken these together, laminin-5 alpha3 LG4 module may play an important role in re-epithelialization at tissue remodeling.     

Tetraspanin CD151 regulates glycosylation of (alpha)3(beta)1 integrin

The tetraspanin CD151 forms a stoichiometric complex with integrin alpha3beta1 and regulates its endocytosis. We observed that down-regulation of CD151 in various epithelial cell lines changed glycosylation of alpha3beta1. In contrast, glycosylation of other transmembrane proteins, including those associated with CD151 (e.g. alpha6beta1, CD82, CD63, and emmprin/CD147) was not affected. The detailed analysis has shown that depletion of CD151 resulted in the reduction of Fucalpha1-2Gal and bisecting GlcNAc-beta(1-->4) linkage on N-glycans of the alpha3 integrin subunit. The modulatory activity of CD151 toward alpha3beta1 was specific, because stable knockdown of three other tetraspanins (i.e. CD9, CD63, and CD81) did not affect glycosylation of the integrin. Analysis of alpha3 glycosylation in CD151-depleted breast cancer cells with reconstituted expression of various CD151 mutants has shown that a direct contact with integrin is required but not sufficient for the modulatory activity of the tetraspanin toward alpha3beta1. We also found that glycosylation of CD151 is also critical; Asn(159) --> Gln mutation in the large extracellular loop did not affect interactions of CD151 with other tetraspanins or alpha3beta1 but negated its modulatory function. Changes in the glycosylation pattern of alpha3beta1 observed in CD151-depleted cells correlated with a dramatic decrease in cell migration toward laminin-332. Migration toward fibronectin or static adhesion of cells to extracellular matrix ligands was not affected. Importantly, reconstituted expression of the wild-type CD151 but not glycosylation-deficient mutant restored the migratory potential of the cells. These results demonstrate that CD151 plays an important role in post-translation modification of alpha3beta1 integrin and strongly suggest that changes in integrin glycosylation are critical for the promigratory activity of this tetraspanin.     

Structure of novel glyceroglycolipids in Adzuki bean (Vigna angularis) seeds

In addition to monoglycosyl diacylglycerol, three glyceroglycolipids with two to four hexose units were isolated from Adzuki bean seeds. Their structures were determined using enzymatic hydrolysis, permethylation analysis, nuclear magnetic resonance spectroscopy, and reversed-phase high pressure liquid chromatography, as folows: Gal(beta 1----3')- for the monoglycosyl diacyglycerol, Gal(alpha 1----6)-Gal(beta 1----3')- and Gal(beta 1----6)-Gal(beta1----3')- for the diglycosyl diacylglycerol, [Gal(beta 1----6]2-Gal(beta 1----3')- and Gal(beta 1----6)-Gal(alpha 1----6)-Gal(beta 1----3')- for the triglycosyl diacylglycerol, and [Gal(beta 1----6)]3-Gal(beta 1----3')- and [Gal(beta 1----6)]2-Gal(alpha 1----6)-Gal(beta 1----3')- for the tetraglycosyl diacylglycerol. Except for Gal(alpha 1----6)-Gal(beta 1----3')-diacylglycerol, all of the glycolipids with oligosaccharide moieties had novel glycosidic linkage patterns differing from those in the well-known glyceroglycolipids in nature. The predominant diacylglycerol species component was characterized as dilinolenin. However, the molecular species composition of the diacylglycerol moieties of the two diglycosyl isomers with different glycosidic linkage patterns were found to be qualitatively the same but different quantitatively, as follows: in the well-known isomer Gal(alpha 1----6)-Gal(beta 1----3')-, 39% dilinolenin, 15% palmitoyl linolenin, and 14% linoleoyl linolenin; and in the novel isomer Gal(beta 1----6)-Gal(beta 1----3')-, 35% dilinolenin, 24% dilinolein, and 21% linoleoyl linolenin.     

The c-series gangliosides GT3, GT2 and GP1c are formed in rat liver Golgi by the same set of glycosyltransferases that catalyse the biosynthesis of asialo-, a- and b-series gangliosides.

Biosynthesis of the c-series gangliosides GT3, GT2 and GP1c was studied in Golgi derived from rat liver. Competition experiments show that the synthesis of ganglioside GT2 (GalNAc beta 1----4-(NeuAc alpha 2----8NeuAc alpha 2----8NeuAc alpha 2----3)Gal- beta 1----4Glc beta 1----1Cer) from GT3 (NeuAc alpha 2----8NeuAc alpha 2----8-NeuAc alpha 2----3Gal beta 1----4Glc beta 1----1Cer) seems to be catalysed by the same N-acetylgalactosaminyl-transferase (GalNAc-T), which converts GM3 (NeuAc alpha 2----3Gal beta 1----4Glc beta 1----1Cer) to GM2 (GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----4Glc beta 1----1Cer). Similar competition experiments suggest moreover that the sialytransferase V (SAT V), which catalyses the synthesis of GT1a (NeuAc alpha 2----8NeuAc alpha 2----3Gal beta 1----3GalNAc beta 1----4- (NeuAc alpha 2----3)-Gal beta 1----4Glc beta 1----1Cer) from GD1a (NeuAc alpha-2----3Gal beta 1----3GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----4Glc beta 1----1-Cer) appears to be identical to the enzyme that catalyses the synthesis of GP1c (NeuAc alpha 2----8NeuAc alpha 2----3Gal beta 1----3-GalNAc beta 1----4(NeuAc alpha 2----8-NeuAc alpha 2----8NeuAc alpha 2----3)Gal beta-1----4Glc beta 1----4Glc beta 1----1Cer) from GQ1c (NeuAc alpha 2----3Gal beta 1----3Gal-NAc beta 1----4 (NeuAc alpha 2----8NeuAc alpha 2----8NeuAc alpha 2----3)Gal beta 1----4-Glc beta 1----1Cer).(ABSTRACT TRUNCATED AT 250 WORDS)     

Ganglioside GM2-tetraspanin CD82 complex inhibits met and its cross-talk with integrins, providing a basis for control of cell motility through glycosynapse

Glycosphingolipids (GSLs) at the cell surface membrane are associated or complexed with signal transducers (Src family kinases and small G-proteins), tetraspanins, growth factor receptors, and integrins. Such organizational framework, defining GSL-modulated or -dependent cell adhesion, motility, and growth, is termed "glycosynapse" (Hakomori, S., and Handa, K. (2002) FEBS Lett. 531, 88-92; Hakomori, S. (2004) Ann. Braz. Acad. Sci. 76, 553-572). We describe here the functional organization of the glycosynaptic microdomain, and the mechanisms for control of cell motility and invasiveness, in normal bladder epithelial HCV29 cells versus highly invasive bladder cancer YTS1 cells, both derived from transitional epithelia. (i) Ganglioside GM2, but not GM3 or globoside, interacted specifically with tetraspanin CD82, and such a complex inhibited hepatocyte growth factor (HGF)-induced activation of Met tyrosine kinase in a dose-dependent manner. (ii) Depletion of GM2 in HCV29 cells by treatment with D-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (P4), or reduction of CD82 expression by RNA interference, significantly enhanced HGF-induced Met tyrosine kinase and cell motility. (iii) In contrast, YTS1 cells, lacking CD82, displayed HGF-independent activation of Met tyrosine kinase and high cell motility. Transfection of the CD82 gene to YTS1 inhibited HGF dose-dependent Met tyrosine kinase activity and cell motility, due to formation of the GM2-CD82 complex. (iv) Adhesion of YTS1 or YTS1/CD82 cells to laminin-5-coated plates, as compared with noncoated plates, strongly enhanced Met activation, and the degree of activation was further increased in association with GSL depletion by P4. Laminin-5-dependent Met activation was minimal in HCV29 cells. These findings indicate that GSL, particularly GM2, forms a complex with CD82, and that such complex interacts with Met and thereby inhibits HGF-induced Met tyrosine kinase activity, as well as integrin to Met cross-talk.