Nonreductive release of O-linked oligosaccharides from mucin glycoproteins for structure/function assignments as neoglycolipids: application in the detection of novel ligands for E-selectin.

The neoglycolipid technology comprises several microprocedures involving the generation of lipid-linked oligosaccharide probes for carbohydrate recognition studies in conjunction with oligosaccharide sequence determination by mass spectrometry. Although applicable to any desired oligosaccharides, procedures are greatly facilitated if the oligosaccharides are nonreduced, as conjugation is by reductive amination of a reducing end aldehyde to a phosphatidylethanolamine. Using bovine submaxillary mucin as a model for release of O-glycans in the reducing state, and based on yields of neoglycolipids and side-products from "peeling" reactions and degradation, aqueous ethylamine 70% w/v at 22 degrees C for 48 h has been selected in preference to other conditions, triethylamine, sodium hydroxide, and hydrazine. The integrity of the main acidic and neutral oligosaccharides released under these conditions, di- to octasaccharides, was established by analyses of free oligosaccharides by liquid secondary ion mass spectrometry (LSIMS) and of the derived neoglycolipids by TLC-LSIMS; the repertoire compared favorably with that of the oligosaccharide alditols generated by conventional reductive alkaline borohydride treatment. More forcing conditions of ethylamine 70% w/v at 65 degrees C for 6 h were required to release oligosaccharides from porcine gastric mucin; di- to nonasaccharides were obtained of which about one-third had an intact core GalNAc. Relative to yields after reductive alkaline hydrolysis, the overall yields for these two glycoproteins were 20% and 40-50% for acidic and neutral oligosaccharides, respectively. Among O-glycans released from an ovarian cystadenoma glycoprotein using ethylamine, three variants of the sulfated Le(a/x) sequences were identified as ligands for the endothelial adhesion molecule E-selectin, one of which is based on the unusual backbone Gal-3/4GlcNAc-3Gal-3Gal.