E-selectin is the inducible adhesion protein on the surface of endothelial cells which has a crucial role in the initial stages of recruitment of leucocytes to sites of inflammation. In addition, it is almost certainly involved in tumor cell adhesion and metastasis. This report is concerned with identification of a new class of oligosaccharide ligand--sulfate-containing--for the human E-selectin molecule from among oligosaccharides on an ovarian cystadenoma glycoprotein. This has been achieved by application of the neoglycolipid technology to oligosaccharides released from the glycoprotein by mild alkaline beta-elimination. Oligosaccharides were conjugated to lipid, resolved by thin-layer chromatography, and tested for binding by Chinese hamster ovary cells which had been transfected to express the full-length E-selectin molecule. Several components with strong E-selectin binding activity were revealed among acidic oligosaccharides. The smallest among these was identified by liquid secondary ion mass spectrometric analysis of the neoglycolipid, in conjunction with methylation analysis of the purified oligosaccharide preparation as an equimolar mixture of the Le(a)- and Le(x)/SSEA-1-type fucotetrasaccharides sulfated at position 3 of outer galactose: [formula: see text] To our knowledge this is the first report of a sulfofucooligosaccharide ligand for E-selectin. The binding activity is substantially greater than those of lipid-linked Le(a) and Le(x)/SSEA-1 sequences and is at least equal to that of the 3'-sialyl-Le(x)/SSEA-1 glycolipid analogue.

Negative ion tandem mass spectrometry (MS/MS) spectra of three isomeric triantennary N-linked glycans provided clear differentiation between the isomers and confirmed the occurrence of an isomer that was substituted with galactose on a bisecting GlcNAc (1 --> 4-substituted on the core mannose) residue recently reported by Takegawa et al. from N-glycans released from human immunoglobulin G (IgG). We extend this analysis of human serum IgG to reveal an analogue of the fucosylated triantennary glycan reported by Takegawa et al. together with a third compound that lacked both the sialic acid and the fucose residues. In addition, we demonstrate the biosynthesis of bisected hybrid-type glycans with the galactose modification, with and without core fucose, on the stem cell marker glycoprotein, 19A, expressed in a partially ricin-resistant human embryonic kidney cell line. It would appear, therefore, that this modification of N-linked glycans containing a galactosylated bisecting GlcNAc residue may be more common than originally thought. Negative ion MS/MS analysis of glycans is likely to prove an invaluable tool in the analysis and monitoring of therapeutic glycoproteins.

The immunoglobulin E-binding protein, epsilon BP (also known as CBP35, Mac-2, L-34, and L-29), is a beta-galactoside-binding protein of approximately 30 kDa and a member of the animal lectin family termed S-type or S-Lac. Multiple biological activities have been attributed to this lectin such as mediation of IgE binding to the surface of Langerhans cells and activation of mast cells through binding to the high affinity IgE receptor. In order to better understand the cell-binding activity and the proposed role for epsilon BP as a biological response modifier, we have studied the specificity of binding of the radioiodinated epsilon BP to a series of lipid-linked, structurally defined oligosaccharide sequences of the lacto/neolacto family. The results show that the minimum lipid-linked oligosaccharides that can support epsilon BP binding are pentasaccharides of the lacto/neolacto series and that the lectin binds more strongly to oligosaccharides of this family that bear the blood group A, B, or B-like determinants than to those bearing blood group H. This preferential binding of epsilon BP is also manifest with whole cells, as erythrocytes of blood groups A and B are more strongly bound by epsilon BP than those of blood group O. Blood group Le(a) and Le(x) sequences are not bound by the lectin.(ABSTRACT TRUNCATED AT 250 WORDS)

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The Lewis X