MONOSIALOGANGLIOSIDES OF HUMAN MYELOGENOUS LEUKEMIA HL-60 CELLS AND NORMAL HUMAN-LEUKOCYTES .2. CHARACTERIZATION OF E-SELECTIN BINDING FRACTIONS, AND STRUCTURAL REQUIREMENTS FOR PHYSIOLOGICAL BINDING TO E-SELECTIN

E-selectin binding gangliosides were isolated from myelogenous leukemi a HL60 cells, and the E-selectin binding pattern was compared with tha t of human neutrophils as described in the preceding paper in this iss ue, The binding fractions were identified as monosialogangliosides hav ing a series of unbranched polylactosamine cores. Structures of fracti ons 12-3, 13-1, 13-2, and 14, which showed clear binding to E-selectin under the conditions described in the preceding paper, were character ized by functional group analysis by application of monoclonal antibod ies, H-1-NMR, FAB-MS, and electrospray mass spectrometry with collisio n-induced dissociation of permethylated fractions, Fractions 12-3, 13- 1, and 13-2 were characterized by the presence of a major ganglioside with the following structure: NeuAc alpha 2 --> 3Gal beta 1 --> 4GlcNA c beta 1 --> 3Gal beta 1 --> 4(Fuc alpha 1 --> 3)GlcNAc beta 1 --> 3Ga l beta 1 --> 4(Fuc alpha 1 --> 3)GlcNAc beta 1 --> 3Gal beta 1 --> 4Gl cNAc beta 1 --> 3Gal beta 1 --> 4Glc beta Cer. Fractions 12-3 and 13-2 contained, in addition, small quantities (10-15%) of extended SLe(x) with internally fucosylated structures: NeuAc alpha 2 --> 3Gal beta 1 --> 4(Fuc alpha 1 --> 3)GlcNAc beta 1 --> 3Gal beta 1 --> 4(Fuc alpha 1 --> 3)GlcNAc beta 1 --> 3Gal beta 1 --> 4(+/-Fuc alpha 1 --> 3)GlcNA c beta 1 --> 3Gal(b)eta 1 --> 4GlcNAc beta 1 --> 3Gal beta 1 --> 4Glc beta Cer. Fraction 13-1, showing stronger E-selectin binding activity than 12-3 and 13-2, contained only a trace quantity (< 1%) of SLe(x). Fraction 14, which also showed clear binding to E-selectin, was charac terized by the presence of the following structures, in addition to tw o internally monofucosylated structures (XX and XXI, Table 2, text): N euAc alpha 2 --> 3Gal beta 1 --> 4GlcNAc beta 1 --> 3Gal beta 1 --> 4( Fuc alpha 1 --> 3)GlcNAc beta 1 --> 3 Gal beta 1 --> 4(Fuc alpha 1 --> 3)GlcNAc beta 1 --> 3 Gal beta 1 --> 4GlcNAc beta 1 --> 3Gal beta 1 - -> 4GlcNAc beta 1 --> 3Gal beta 1 --> 4Glc beta Cer; and NeuAc alpha 2 --> 3Gal beta 1 --> 4GlcNAc beta 1 --> 3Gal beta 1 --> 4(Fuc alpha 1 --> 3)GlcNAc beta 1 --> 3Gal beta 1 --> 4GlcNAc beta 1 --> 3Gal beta 1 --> 4(Fuc alpha 1 --> 3)-GlcNAc beta 1 --> 3Gal beta 1 --> 4GlcNAc be ta 1 --> 3Gal beta 1 --> 4Glc beta Cer. SLe(x) determinant was complet ely absent. Thus, the E-selectin binding epitope in HL60 cells is carr ied by unbranched terminally alpha 2 --> 3 sialylated polylactosamine having at least 10 monosaccharide units (4 N-acetyllactosamine units) with internal multiple fucosylation at GlcNAc. These structures are he reby collectively called ''myeloglycan''. Monosialogangliosides from n ormal human neutrophils showed an essentially identical pattern of gan gliosides with selectin binding property. Myeloglycan, rather than SLe (x), provides a major physiological epitope in E-selectin-dependent bi nding of leukocytes and HL60 cells.