Monosialogangliosides of Human Myelogenous Leukemia HL60 Cells and Normal Human Leukocytes. 1. Separation of E-Selectin Binding from Nonbinding Gangliosides, and Absence of Sialosyl-Le^x Having Tetraosyl to Octaosyl Core

Abstract

Previous studies suggested that sialosyl-Le(x) (SLex) is a ligand expressed in human neutrophils and myelogenous leukemia HL60 cells which binds to E-selectin and possibly P-selectin. However, clear data on structures of carbohydrate epitopes in these cells were lacking. A systematic study was therefore initiated, employing a large quantity of HL60 cells (> or = 1200 mL packed) and human leukocytes (approximately 100 mL packed). Gangliosides were extracted, followed by extensive fractionation and examination of the E- and P-selectin binding ability of each fraction. The following results were of particular interest: (i) Only monosialogangliosides having a polylactosamine core with > 10 monosaccharide units (or > 4 N-acetyllactosamine units) showed E-selectin binding under static conditions with thin-layer chromatography overlay technique employing 32P-labeled E-selectin-expressing CHO cells. (ii) Sulfate groups were not detectable in the binding fractions, and di- and trisialoganglioside fractions did not show E-selectin binding under these conditions. (iii) None of the fractions showed P-selectin binding under a similar assay system using 32P-labeled P-selectin-expressing CHO cells. (iv) Major gangliosides of HL60 cells were structures I-XI (shown in Table 1 of text), none of which showed E-selectin binding under the above conditions. (v) SLex gangliosides having tetraosyl to octaosyl ceramide core, which are the major gangliosides of epithelial tumors (shown in Table 2), were completely absent from HL60 cells and neutrophils. Isolation and chemical characterization of ganglioside structures I-XI are described in this paper.

Glycosylation-dependent inhibition of cutaneous lymphocyte–associated antigen expression: implications in modulating lymphocyte migration to skin

“…[35][36][37][38] Residual E-selectin ligand activity after bromelain treatment would, therefore, be indicative of activity contributed by a non-PSGL-1 glycolipid component. [40][41][42][43][44] Bromelain-treated cells were then analyzed for both HECA-452 and PSGL-1 expression by flow cytometry. In addition, to further verify the complete disappearance of HECA-452 and PSGL-1 expression from the cell surface after bromelain treatment, membrane proteins were prepared as previously described by our laboratory [35][36][37] and analyzed for HECA-452 antigen and PSGL-1 polypeptide by Western blotting.…”

The α(1,3)-Fucosyltransferase Fuc-TIV, but Not Fuc-TVII, Generates Sialyl Lewis X-like Epitopes Preferentially on Glycolipids

“…Numerous reports have described the binding of E-selectin transfected cells to immobilized sLe xcarrying glycolipids (35,55,56), and it was also shown that E-selectin-transfected cells can roll on immobilized glycolipids under physiologic flow conditions (34). However, the contribution of cell surface-expressed glycolipids in the context of a cell surface glycocalyx to the binding of cells on immobilized Eselectin has not yet been analyzed.…”

Selectin ligands: will the real ones please stand up?

“…It is also noteworthy that E-selectin has been reported to bind with relatively high affinity to certain sialylated polyfucosylated N-linked sugar chains derived from neutrophils, completely independent of any attachment to a polypeptide backbone (52). Likewise, sialylated fucosylated glycolipids from leukocytes can be effective ligands for E-selectin when presented at high density in vitro (53)(54)(55). In all of these instances, definitive proof of in vivo function is currently lacking.…”