Characterization of a novel type of chain‐terminator Galβ1–6Galβ1–4)GlcNAc in an oligosaccharide related to N‐glycosylated protein glycans isolated from GM₁ the urine of patients with gangliosidosis

Abstract

Two new oligosaccharides were isolated from the urine of a patient with G M gangliosidosis. Final purification of the oligosaccharides was accomplished by capillary supercritical fluid chromatography. Structural analysis was by chemical analysis, chemical-ionization mass spectrometry and 400-MHz H-NMR spectroscopy, leading to two primary structures. The first is derived from a classical triantennary N-acetyllactosamine-type glycan :Gal/31-4GlcNAc~1-4(Gal~1-4GlcNAc~1-2)Mancrl-3Man~1-4GlcNAc.The second is unusual with a terminal disaccharide GalPl-6Ga1, which had not yet been described for glycans of the N-acetyllactosamine type:Gal~~1-6Gal~1-4GlcNAc~1-2Man~l-6Man~1-4GlcNAc.Urine of patients suffering from glycoproteinosis contains a large amount of oligosaccharides or glycopeptides which originate from incomplete lysosomal catabolism of glycoprotein glycans [I -31. Isolation and structural analysis of compounds accumulated in this kind of diseases are of interest and importance since they provide valuable information on the broad spectrum of glycan structures which normally exist in human cellular glycoproteins [4,5]. In this context, studying minor oligosaccharides present in glycoproteinosis urine may provide structural information on some unusual carbohydrate determinants naturally occurring in glycoconjugate glycans. As a result of the P-galactosidase deficiency occuring in GM1 gangliosidosis [6], a number of different galactosyl oligosaccharides are excreted in the urine of patients [7-91. We report here detailed structural analysis of two minor urinary oligosaccharides, one of which presents a new type of galactosyl linkage never described before for the N-acetyllactosamine-type glycans. MATERIALS AND METHODS UrineUrine (8 1) was collected from a patient with G M I gangliosidosis type 1. The urine was chilled at -20 'T immediately after collection and was stored without preservatives. Ahhreviutions. SFC-CI, supercritical-fluid-chromatography chemical ionization; FAB-MS, fast-atom-bombardment mass spectrometry; Hex, hexose; HexNAc, 2-deoxy-N-acetylamino-~-hexose cosy; correlated spectroscopy. Isolation of neutral oligosaccharidesNeutral oligosaccharides were obtained as previously described [lo] and directly applied to an activated charcoalCelite (Sigma) column (10 cm x 100 cm). The column was washed with distilled water and adsorbed material stepwise eluted with 10 1 ethanol of increased concentration (1 .5 -50%).Further separation and purification of the oligosaccharides were carried out by descending paper chromatography (Whatman no 3 paper) using a solvent system of ethyl acetate/pyridine/acetic acid/water (5 : 5 : 1 : 3, by vol.), performed over 4 -12 days. Analytical proceduresThe molar ratios of hexoses and N-acetylhexosamines were determined by GLC analysis of the trifluoroacetyl derivatives obtained after methanolysis of oligosaccharides (methanol/ 0.5 M HC1; 24 h, 80°C). Methylation analysis was performed according to [Ill. Supercritical-fluid-chromatography mass spectrometryThe oligosaccharides w...

A mass spectrometric strategy for profiling glycoproteinoses, Pompe disease, and sialic acid storage diseases

“…As shown in Fig. 3C Hex-NAc 2 ) has a monosaccharidic composition consistent with a monoantennary structure and may also correspond to the atypical Gal-(b1,6)-terminated N-glycan described by Michalski et al [41]. N-glycans with tri-(m/z 2071, HexNAc 4 Hex 5 ; m/z 2275, HexNAc 4 Hex 6 ) and tetraantennary structures (2521, HexNAc 5 Hex 6 ; 2725, HexNAc 5 Hex 7 ) were also observed.…”

Green Solvents in Carbohydrate Chemistry: From Raw Materials to Fine Chemicals

“…IL-based, surfactant-improved, dispersive liquid–liquid microextraction and derivatization have also been reported on aminoglycosides in milk samples . Supercritical fluid chromatography can also be used in glycan purification . Two new oligosaccharides isolated from the urine of a patient with GM gangliosidosis were purified by capillary supercritical fluid chromatography using a Brownlee syringe pump equipped with a 60-nL Valco injection valve.…”

“…329 Supercritical fluid chromatography can also be used in glycan purification. 330 Two new oligosaccharides isolated from the urine of a patient with GM gangliosidosis were purified by capillary supercritical fluid chromatography using a Brownlee syringe pump equipped with a 60-nL Valco injection valve.…”