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.
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 alpha2-->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 microM sialyl alpha2-->1 Sph or 1-5 microM 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 alpha2-->1 Sph. Substitution of the Sph amino group with a chloroacetyl or N,N-dimethyl group strongly reduced the inhibitory effect of sialyl alpha2-->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 microM did not show the same effect as sialyl alpha2-->1 Sph. Thus, adhesion coupled with signal transduction, initiated by clusters of GM3 in GSD, is blocked by sialyl alpha2-->1 Sph or lyso-GM3. Analogues with N-substitution of Sph in sialyl alpha2-->1 Sph, other lyso-phospholipids, and galactosyl- or lactosyl-Sph did not block such adhesion, coupled with activation of c-Src and FAK.
Zhang
Y
Y
Division of Biomembrane Research, Pacific Northwest Research Institute, 720 Broadway, Seattle, Washington 98122-4327, USA.
Iwabuchi
K
K
Nunomura
S
S
Hakomori
S
S
D000818:Animals; D000081247:CSK Tyrosine-Protein Kinase; D002240:Carbohydrate Sequence; D002448:Cell Adhesion / Q000187:drug effects; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D005679:G(M3) Ganglioside / Q000502:physiology; D006028:Glycosphingolipids / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D008546:Melanoma, Experimental / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D013110:Sphingosine / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured; D019061:src-Family Kinases
D015815:Cell Adhesion Molecules; D005679:G(M3) Ganglioside; D006028:Glycosphingolipids; C406528:sialyl 2-1 sphingosine; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496051:Ptk2 protein, mouse; D019061:src-Family Kinases; D013110:Sphingosine