Abstract
This report describes the preparation of a library of oligosaccharide probes (neoglycolipids) from N-glycosylated proteins, characterization of the probes by liquid secondary ion mass spectrometry, and investigation of their reactions with 125I-labeled bovine serum conglutinin by chromatogram binding assays. The results, together with additional binding studies using neoglycolipids derived from purified complex type bi-, tri-, and tetraantennary oligosaccharides from urine, or their glycosidase-treated products, have shown that the combining specificity of conglutinin includes structures not only on high mannose-type oligosaccharides but also on hybrid- and complex-type chains. With high mannose-type oligosaccharides there is increased reactivity from the Man5 to the Man8 structures, indicating a preference for the terminal Man alpha 1-2 sequence. With complex- and hybrid-type oligosaccharides, the requirements for binding are the presence of nonreducing terminal N-acetylglucosamine or mannose residues, but the presence of a bisecting N-acetylglucosamine residue may inhibit binding. From these results it is deduced that the reactivity of conglutinin with the complement glycopeptide iC3b rather than the intact glycoprotein C3 is due to the oligosaccharide accessibility rendered by proteolysis in the complement cascade.
Highlights
This report describes the preparation of a library of oligosaccharide probes from N-glycosylated proteins, characterization of the probes by liquid secondary ion mass spectrometry, and investigation of their reactions with ‘2SI-labeledbovine serum conglutinin by chromatogram binding assays
In order to study the roles of glycoprotein oligosaccharides as recognition structures, a microtechnique has been under development (Tang et al, 1985; Stoll et al, 1988) which involves the conjugation of oligosaccharides or their alditols to the lipid phosphatidylethanolamine dipalmitoyl
We describe here (a) the generation of the neoglycolipids from oligosaccharides released by hydrazinolysis from several mammalian glycoproteins, or isolated from the urine of patients with GM,gangliosidosis, (6) structural identification of individual neoglycolipid bands by LSIMS’ directly from thin layer silica gel chromatograms, and (c) their reactivities in chromatogram binding assays withlZ5I-labeledconglutinin
Summary
Ferrin were obtained by incubation of the respective asialooligosaccharidemixtureswithjackbean. Areas corresponding t o orcinol-stained bands visualized in parallel lanes were cutand subjected to LSIMS following addition of a matrix mouse IgG, gave (M - H)-ions a t 2340,2502, and 2664 corresponding tothebiantennary agalactosyl,mono-, and digalactosyl, fucosyl oligosaccharides with bisectingN-acetylglucosamine residues (Table I). These areknown to occur in consisting of 3 pl of chloroform/methanol/water,25258 (by volume) human, but notin mouse, IgG. Structures of neoglycolipids identified by LSIMS analysis on silica gel chromatograms
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