Effect of Synthetic Sialyl 2→1 Sphingosine and Other Glycosylsphingosines on the Structure and Function of the “Glycosphingolipid Signaling Domain (GSD)” in Mouse Melanoma B16 Cells

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Abstract
Mouse melanoma B16 cells are characterized by a high concentration of GM3 ganglioside, which has been identified as a melanoma-associated antigen and is present as a clustered microdomain organized with major signal transducers, c-Src, small G-protein (Rho A), and focal adhesion kinase (FAK), to form a “glycosphingolipid signaling domain” or “glycosignaling domain” (GSD) separable from cholesterol- and caveolin-enriched microdomain, “caveolae.” Cholesterol-binding reagents, filipin and nystatin, disrupt the structure and function of caveolae, but have no effect on GSD function [Iwabuchi, K., et al. (1998) J. Biol. Chem. 273, 33766−33773]. In this study, we searched for compounds which disrupt the structure and function of GSD in B16 cells. Such compounds should have structural features analogous to those of GM3, destroy or reduce clustering of GM3 in GSD, and inhibit GM3-dependent adhesion and signaling. The simplest compound so far found with these properties is sialyl α2→1 sphingosine (Sph). We describe the synthesis of this compound and its analogues, and their effects on GM3 expression pattern and GSD function, in comparison with effects of lyso-GM3 and other lyso compounds, in B16 cells. Incubation of B16 cells with 0.5−10 μM sialyl α2→1 Sph or 1−5 μM lyso-GM3 reduced GM3 clustering and GM3-dependent adhesion, and inhibited adhesion-dependent cellular FAK activity. The c-Src activation response of GSD isolated from B16 cells was inhibited strongly by sialyl α2→1 Sph. Substitution of the Sph amino group with a chloroacetyl or N,N-dimethyl group strongly reduced the inhibitory effect of sialyl α2→1 Sph on GM3-dependent adhesion, FAK, and c-Src response. Other lyso compounds such as lyso-phosphatidylcholine, galactosyl-Sph (psychosine), and lactosyl-Sph at 0.5−10 μM did not show the same effect as sialyl α2→1 Sph. Thus, adhesion coupled with signal transduction, initiated by clusters of GM3 in GSD, is blocked by sialyl α2→1 Sph or lyso-GM3. Analogues with N-substitution of Sph in sialyl α2→1 Sph, other lyso-phospholipids, and galactosyl- or lactosyl-Sph did not block such adhesion, coupled with activation of c-Src and FAK.