Abstract
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.
Highlights
From The Hiomembrane Institute, Seattle, Washington 98119 and the Department of Pathobiology, University of Washington, Seattle, Washington 9819.5
GM3-expressing cellosn Gg3, LacCer, and Gb4-coated Le” interaction [11].Subsequent systematic studies [12] resolid phaseis based oninteraction of GM3 with Gg, and, vealed that interaction ofGM:3- and Gg3-expressing cells was to a lesser extent, with LacCer and Gbs, as demon- based on interactionof these component GSLs
Strated by: ( i )the interactionof the GM3 liposome with In this paperw, e extend these studies to interactiofnGM:i, solid phase coated with Gg, LacCer, and Gbr, respec- with other GSLs and to cell spreading and motility induced tively; ( i i )the abolitionof cell adhesion on each GSL- by GSL-GSL interaction
Summary
Preparation of Liposomes and Glycolipid-coated Solid Phase-Liadherent cells were harvested and counted ina scintillation counter. Glycolipid- washed withPBS;photographs of adherent cells (five fields/well, coated solid phase was incubatedwith 1% BSAfor 1 h a t room magnification X 200) were taken; and spread cells as well as total temperature andwashed twice with T B S or PBS before use. LysLys conjugate (10,000 cpm/100 nmol) was dissolved in the same removed, and both chambers were washed with PBS. The column free RPMI 1640 medium (500 pl) was added to the lower chamber, was eluted with 7.5 ml each of T B S (Ca'+, Mg"), T B S containing 10 and 100 p1 of cell suspension (5 X 105/ml) in serum-free RPMI 1640.
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