Sialosyl-Fucosyl Poly-LacNAc without the Sialosyl-Le^xEpitope as the Physiological Myeloid Cell Ligand in E-Selectin-Dependent Adhesion:  Studies under Static and Dynamic Flow Conditions^,

Abstract

The majority of E- and P-selectin ligands in leukocytes and myelocytic or monocytic leukemia cells are carried by transmembrane glycoproteins having a tandem repeat mucin-like domain through which O-linked carbohydrate ligands are carried. However, determination of structure and adhesive function of carbohydrates in glycoproteins is extremely difficult because of the extensive structural heterogeneity and the scarcity of material for functional analysis. We have overcome this difficulty through use of poly-LacNAc gangliosides isolated from a large quantity of ( approximately 1.2 L packed) HL60 cells [Stroud, M. R., Handa, K., Salyan, M. E. K., Ito, K., Levery, S. B., Hakomori, S., Reinhold, B. B., & Reinhold, V. N. (1996) Biochemistry 35, 758-769, 770-778]. We identified two major types of poly-LacNAc gangliosides without the sialosyl-Lex epitope as being capable of binding to E-selectin: (i) those having a single alpha1-->3 fucosylation at internal GlcNAcs but not at the penultimate GlcNAc and (ii) those having double alpha1-->3 fucosylation at internal GlcNAcs, excluding the penultimate GlcNAc. Gangliosides from group i above did not show any adhesion under static conditions, but showed strong adhesion under dynamic flow conditions. Gangliosides from group ii above showed adhesion under both static and dynamic conditions, as did sialosyl-Lex (SLex)-containing structures in previous studies. However, SLex-containing poly-LacNAc gangliosides are virtually absent or present in only trace quantities in leukocytes and HL60 cells. Poly-LacNAc gangliosides from groups i and ii above, lacking SLex structure, are the major membrane components of leukocytes and HL60 cells. These carbohydrates, bound to lipid or to protein, may therefore be the physiological epitope for E-selectin-dependent binding of these cells, particularly under dynamic flow conditions.

The glycosynapse

“…The ''myeloglycan'' epitope (see Table 1 for structure) found originally in GSLs (35,36) may well be present in O-linked form in PSGL-1 and other mucin-type glycoproteins. Thus, type 2 glycosynapses may play major roles in glycosylationdependent adhesion through selectins and siglecs, and consequent changes in signaling leading to phenotypic changes, e.g., tumor cell invasiveness.…”

ST3Gal-4 is the primary sialyltransferase regulating the synthesis of E-, P-, and L-selectin ligands on human myeloid leukocytes

“…4,7 For example, in the case of GSLs, E-selectin binds gangliosides bearing non-sLe X epitopes. 45 Regardless of the scaffold protein/lipid, our data suggest that a single enzyme ST3Gal-4 may a(2,3)sialylate a majority of these physiologically relevant human E-selectin ligands. Thus, in the knockdown and knockout studies, ST3Gal-4 reduction decreased cell rolling and firm adhesion density by ;90% on stimulated HUVECs under flow.…”

G-CSF Induces Membrane Expression of a Myeloperoxidase Glycovariant that Operates as an E-selectin Ligand on Human Myeloid Cells

“…These findings suggest that G-CSF stimulates de novo expression of HECA-452-reactive moieties predominantly on N-glycans. The residual HECA-452 determinants displayed in the presence of DMJ likely reflect the native contribution(s) of sialofucosylated O-linked glycans found on both glycoproteins and glycolipids (18,19).…”