Acquisition of P-selectin binding activity by en bloc transfer of sulfo Le(x) trisaccharide to the cell surface: comparison to a sialyl Le(x) tetrasaccharide transferred on the cell surface.
Sialyl Le(x), NeuNAcalpha2 --> 3Galbeta1 --> 4(Fucalpha1 --> 3)GlcNAcbeta --> R, is known to be a ligand for E-selectin in various assays. The sulfated counterpart of sialyl Le(x), sulfo Le(x), (Sulfo --> 3) Galbeta1 --> 4 (Fucalpha1 --> 3) GlcNAcbeta --> R, was also shown to be a ligand for E-selectin in solid-phase assays employing immobilized oligosaccharides. In order to determine whether sulfo Le(x) structure on the cell surface also works as E-selectin or P-selectin ligand, a novel approach for in vitro transfer of oligosaccharides (S. Tsuboi, Y. Isogai, N. Hada, J. K. King, O. Hindsgaul, and M. Fukuda (1996) J. Biol. Chem. 271, 27213-27216) was utilized. A synthetic GDP-fucose harboring sialyl Le(x) or sulfo Le(x) oligosaccharide was enzymatically transferred to Chinese hamster ovary (CHO) cells with a milk fucosyltransferase. The resultant cells, CHO-sialyl Le(x) and CHO-sulfo Le(x) were tested for adhesion to E-selectin. IgG or P-selectin. IgG chimeric protein coated on plates. The results indicate that CHO-sialyl Le(x) adhered efficiently to E-selectin, while adhesion of CHO-sulfo Le(x) was very poor despite the fact that near equal number of the ligands had been attached to the cell surface. In contrast, CHO-sulfo Le(x) adhered efficiently to P-selectin, while CHO-sialyl Le(x) adhered modestly to P-selectin. These results demonstrate that sialyl Le(x) and sulfo Le(x) structures on the cell surface differ substantially in their ability to adhere to E- and P-selectin.
Tsuboi
S
S
Glycobiology Program, Cancer Research Center, La Jolla, California, 92037, USA.
Srivastava
O P
OP
Palcic
M M
MM
Hindsgaul
O
O
Fukuda
M
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D019556:COS Cells; D002240:Carbohydrate Sequence; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D005647:Fucosyltransferases / Q000378:metabolism; D007700:Kinetics; D016256:Lewis X Antigen / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism; D019007:P-Selectin / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D013431:Sulfates / Q000378:metabolism; D014312:Trisaccharides / Q000737:chemistry / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D016256:Lewis X Antigen; D008562:Membrane Glycoproteins; D009844:Oligosaccharides; D019007:P-Selectin; D013431:Sulfates; D014312:Trisaccharides; D005647:Fucosyltransferases