Johanna H. G. M. Mutsaers

A 500-MHz 1H-NMR study on the N-linked carbohydrate chain of bromelain. 1H-NMR Structural-reporter-groups of fucose α(1-3)-linked to asparagine-bound N-acetylglucosamine

The 500-MHz1H-NMR characteristics of theN-linked carbohydrate chain Manα1-6[Xylβ1-2]Manβ1-4GlcNAcβ1-4[Fucα1-3]GlcNAcβ1-NAsn of the proteolytic enzyme bromelain (EC 3.4.22.4) from pineapple stem were determined for the oligosaccharide-alditol and the glycopeptide, obtained by hydrazinolysis and Pronase digestion, respectively. The1H-NMR structural-reporter-groups of the α(1–3)-linked fucose residue form unique sets of data for the alditol as well as for the glycopeptide.The 500-MHz1H-NMR characteristics of theN-linked carbohydrate chain Manα1-6[Xylβ1-2]Manβ1-4GlcNAcβ1-4[Fucα1-3]GlcNAcβ1-NAsn of the proteolytic enzyme bromelain (EC 3.4.22.4) from pineapple stem were determined for the oligosaccharide-alditol and the glycopeptide, obtained by hydrazinolysis and Pronase digestion, respectively. The1H-NMR structural-reporter-groups of the α(1–3)-linked fucose residue form unique sets of data for the alditol as well as for the glycopeptide.

Determination of the structure of the carbohydrate chains of acid α-glucosidase from human placenta

Acid alpha-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20) from human placenta (70 and 76 kDa) was found to contain 4 N-glycosidic carbohydrate chains per molecule. Sugar analysis of purified enzyme revealed the presence of mannose, N-acetylglucosamine and fucose at a molar ratio of 5.0:2.0:0.6. In addition, trace amounts of galactose and N-acetylneuraminic acid were detected. The sugar chains were liberated from the polypeptides by the hydrazinolysis procedure and subsequently fractionated by gel filtration and HPLC. Purified compounds were investigated by 500-MHz 1H-NMR spectroscopy. Oligomannoside-type chains of intermediate size, e.g., Man5GlcNAcGlcNAc-ol and Man7GlcNAcGlcNAc-ol, and N-type chains of smaller size e.g., Man2-3GlcNAc[Fuc]0-1GlcNAc-ol, were demonstrated to be present at a ratio of 2:3. In addition, a small amount of sialylated N-acetyllactosamine-type chains has been found. The possible biosynthetic route of the fucose-containing small-size chains is discussed.

Primary structure of O- and N-glycosylic carbohydrate chains derived from murine submandibular mucin (MSM)

The carbohydrate moiety of mouse submandibular mucin (MSM) contains mainly D-mannose and 2-acetamido-2-deoxy-D-glucose together with sialic acid, D-galactose, and 2-acetamido-2-deoxy-D-galactose. O-Glycosylically bound saccharides, obtained by treatment of MSM with alkaline borohydride, were shown by methylation analysis to have the structure: alpha-NeuAc-(2----3)-beta-Gal-(1----3)-GalNAc-ol. N-Glycosylically bound saccharides obtained from MSM by hydrazinolysis, and analysed by 500-MHz 1H-n.m.r. spectroscopy, were shown to have the following comprehensive structures. (Formula: see text).

Structural analysis of dansyl glyco-asparagines from quail ovalbumin

Abstract The carbohydrate moiety of quail ovalbumin was isolated in the form of dansyl glyco-asparagines. The various components were separated by high-performance liquid chromatography, yielding two major fractions (90% of the total carbohydrate material). By 500-MHz 1 H-NMR spectroscopy they were found to be of the oligomannoside type, containing eight and seven d -mannose residues. The largest one possesses the following structure: while the other one is its analogue missing mannose D 1 . Dansyl glyco-asparagines turned out to be suitable derivatives for 1 H-NMR spectroscopic analysis; in combination with HPLC, heterogeneity of sugar chains on glycoproteins can be elegantly characterized.

The structures of N- and O-glycosidic carbohydrate chains of a chondroitin sulfate proteoglycan isolated from the media of the human aorta

A large Mr chondroitin sulfate proteoglycan was extracted from the media of human aorta under dissociative conditions and purified by density-gradient centrifugation, ion-exchange chromatography, and gel filtration chromatography. Removal of a contaminating dermatan sulfate proteoglycan was accomplished by reduction, alkylation and rechromatography on the gel filtration column. After chondroitinase ABC treatment, the proteoglycan core was separated from a residual heparan sulfate proteoglycan by a third gel filtration chromatography step. As assessed by radioimmunoassay, the isolated proteoglycan core was free of link protein, but possessed epitopes that were recognized by antisera against the hyaluronic acid binding region of bovine cartilage proteoglycan as well as those that were weakly recognized by anti-keratan sulfate antisera. Following beta-elimination of the protein core, the liberated low Mr oligosaccharides were partially resolved by Sephadex G-50 chromatography, and their primary structure was determined by 500-MHz1H NMR spectroscopy in combination with compositional sugar analysis. The N-glycosidic carbohydrate chains, which were obtained as glycopeptides, were all biantennary glycans containing NeuAc and Fuc; microheterogeneity in the NeuAc----Gal linkage was detected in one of the branches. The N-glycosidic glycans have the following overall structure: (Formula: see text). The majority of the O-glycosidic carbohydrate chains bound to the protein core were found to be of the mucin type. They were obtained as glycopeptides and oligosaccharide alditols, and possessed the following structures: NeuAc alpha(2----3)Gal beta(1----3)GalNAc-ol, [NeuAc alpha(2----3)Gal beta(1----3)[NeuAc alpha(2----6)]GalNAc-ol, and NeuAc alpha-(2----3) Gal beta(1----3)[NeuAc alpha(2----3)Gal beta(1----4)GlcNAc beta(1----6)] GalNAc-ol. The remainder of the O-glycosidic carbohydrate chains bound to the isolated proteoglycan were the hexasaccharide link regions of the chondroitin sulfate chains that remained after chondroitinase ABC treatment of the native molecule. These latter glycans, which were obtained as oligosaccharide alditols, had the following structure (with GalNAc free of sulfate or containing sulfate bound at either C-4 or C-6): delta 4,5GlcUA beta(1----3)GalNAc beta(1----4)GlcUA beta(1----3)Gal beta(1----3)Gal beta(1----4)Xyl-ol.