Structure and dynamics in oligomannose-type oligosaccharides

Using a combination of 1H NMR nuclear Overhauser effect measurements, molecular orbital calculations, and molecular dynamics simulations, we have determined the tertiary structure and dynamic properties of the oligomannose oligosaccharide Man alpha 6(Man alpha 3)Man alpha 6(Man alpha 3)Man beta 4GlcNAc beta 4GLcNAc. While the calculated potential surfaces for the majority of the glycosidic linkages could be described by a single deep potential well, similar calculations for the Man alpha 1-6Man alpha and Man alpha 1-6Man beta linkages described a diffuse, shallow well, suggesting that a larger degree of flexibility exists about the latter. These conclusions are supported by the results of molecular dynamics simulations, which suggest that the NMR data should be interpreted in terms of a degree of flexibility about the Man alpha 1-6Man beta and Man alpha 1-6Man alpha linkages. In contrast, a similar series of investigations suggests that the conformation of the Man alpha 1-6Man beta linkage in Man alpha 2Man6(Man alpha 2Man alpha 3)Man alpha 6 (Man alpha 2Man alpha 2Man alpha 3)Man beta 4GlcNAc beta 4GlcNAc is more restricted, resulting in an overall structure that is "restrained".     

Tertiary structure in N-linked oligosaccharides

Distance constraints derived from two-dimensional nuclear Overhauser effect measurements have been used to define the orientation of the Man alpha 1-3Man beta linkage in seven different N-linked oligosaccharides, all containing the common pentasaccharide core Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc. Conformational invariance of the Man alpha 1-3Man beta linkage was found for those structures bearing substitutions on the Man alpha 1-3Man beta antenna. However, the presence of either a GlcNAc residue in the beta 1-4 linkage to Man beta ("bisecting GlcNAc") or a xylose residue in the beta 1-2 linkage to Man beta of the trimannosyl core was found to generate conformational transitions that were similar. These transitions were accompanied by characteristic chemical shift perturbations of proton resonances in the vicinity of the Man alpha 1-3Man beta linkage. Molecular orbital energy calculations suggest that the conformational transition between the unsubstituted and substituted cores arises from energetic constraints in the vicinity of the Man alpha 1-3Man beta linkage, rather than specific long-range interactions. These data taken together with our previous results on the Man alpha 1-6Man beta linkage [Homans, S. W., Dwek R. A., Boyd, J., Mahmoudian, M., Richards, W. G., & Rademacher, T. W. (1986) Biochemistry 25, 6342] allow us to discuss the consequences of the modulation of oligosaccharide solution conformations.     

Solution conformation of asparagine-linked oligosaccharides: alpha(1-2)-, alpha(1-3)-, beta(1-2)-, and beta(1-4)-linked units

The solution conformation is presented for representatives of each of the major classes of asparaginyl oligosaccharides. In this report the conformation of alpha(1-3)-, alpha(1-2)-, beta(1-2)-, and beta(1-4)-linked units is described. The conformational properties of these glycopeptides were determined by high-resolution 1H nuclear magnetic resonance in conjunction with potential energy calculations. The NMR parameters that were used in this analysis were chemical shifts and nuclear Overhauser enhancements. Potential energy calculations were used to evaluate the preferred conformers available for the different linkages in glycopeptides and to draw conclusions about the behavior in solution of these molecules. It was found that the linkage conformation of the Man alpha 1-3 residues was not affected by substitution either at the 2-position by alpha Man or beta GlcNAc or at the 4-position by beta GlcNAc or by the presence of a bisecting GlcNAc on the adjacent beta Man residue.     

A new mannoheptaose containing alpha and beta-(1-->2) linkages isolated from the mannan of Torulaspora delbrueckii: ELISA inhibition studies

Torulaspora delbrueckii starin IFO 0955 was examined with respect to its structural and serological properties of the cell wall mannan (Td-0955-M). Td-0955-M revealed significant reactivities with sera from a commercially available factor serum kit (Candida Check) in ELISA. Td-0955-M was investigated for its chemical structure by acetolysis under conventional and mild conditions. NMR and GC techniques were used as analytical techniques. The mannooligosaccharide fractions eluted from a Bio-Gel P-2 column were found to consist of Man(alpha1-2)Man, M2, Man(alpha1-2)Man(alpha1-2)Man and Man(beta1-2)Man(alpha1-2)Man, M3, Man(alpha1-2)Man(beta1-2)Man(beta1-2)Man(alpha1-2)Man, M5, and a new mannoheptaose, which possesses the structure, Man(alpha1-2)Man(beta1-2)Man(beta1-2)Man(beta1-2)Man(beta1-2)Man(alpha1-2)Man, M7. The results of the inhibition ELISA showed that the M7 oligosaccharide significantly inhibited the reactivities in the Td-0955-M-factor serum systems.     

Substrate specificity of rat liver cytosolic alpha-D-mannosidase. Novel degradative pathway for oligomannosidic type glycans.

The substrate specificity of rat liver cytosolic neutral alpha-D-mannosidase was investigated by in vitro incubation with a crude cytosolic fraction of oligomannosyl oligosaccharides Man9GlcNAc, Man7GlcNAc, Man5GlcNAc I and II isomers and Man4GlcNAc having the following structures: Man9GlcNAc, Man(alpha 1-2)Man(alpha 1-3)[Man(alpha 1-2)Man(alpha 1-6)]Man(alpha 1-6) [Man(alpha 1-2)Man(alpha 1-3)]Man(beta 1-4)GlcNAc; Man5GlcNAc I, Man(alpha 1-3)[Man(alpha 1-6)]-Man(alpha 1-6)Man(alpha 1-3)] Man(beta 1-4)GlcNAc; Man5GlcNAc II, Man(alpha 1-2)Man(alpha 1-2)Man(alpha 1-3) [Man(alpha 1-6)]Man(beta 1-4)GlcNAc; Man4GlcNAc, Man(alpha 1-2)Man(alpha 1-2)Man(alpha 1-3)Man(beta 1-4)GlcNAc. The different oligosaccharide isomers resulting from alpha-D-mannosidase hydrolysis were analyzed by 1H-NMR spectroscopy after HPLC separation. The cytosolic alpha-D-mannosidase activity is able to hydrolyse all types of alpha-mannosidic linkages found in the glycans of the oligomannosidic type, i.e. alpha-1,2, alpha-1,3 and alpha-1,6. Nevertheless the enzyme is highly active on branched Man9GlcNAc or Man5GlcNAc I oligosaccharides and rather inactive towards the linear Man4GlcNAc oligosaccharide. Structural analysis of the reaction products of the soluble alpha-D-mannosidase acting on Man5-GlcNAc I and Man9GlcNAc gives Man3GlcNAc, Man(alpha 1-6)[Man(alpha 1-3)]Man(beta 1-4)GlcNAc, and Man5GlcNAc II oligosaccharides, respectively. This Man5GlcNAc II, Man(alpha 1-2)Man(alpha 1-3)[Man(alpha 1-6)]Man(beta 1-4)GlcNAc, represents the 'construction' Man5 oligosaccharide chain of the dolichol pathway formed in the cytosolic compartment during the biosynthesis of N-glycosylprotein glycans. The cytosolic alpha-D-mannosidase is activated by Co2+, insensitive to 1-deoxymannojirimycin but strongly inhibited by swainsonine in the presence of Co2+ ions. The enzyme shows a highly specific action different from that previously described for the lysosomal alpha-D-mannosidases [Michalski, J.C., Haeuw, J.F., Wieruszeski, J.M., Montreuil, J. and Strecker, G. (1990) Eur. J. Biochem. 189, 369-379]. A possible complementarity between cytosolic and lysosomal alpha-D-mannosidase activities in the catabolism of N-glycosylprotein is proposed.     

Purification and characterization of neutral alpha-mannosidase that is activated by Co2+ from Japanese quail oviduct

An alpha-mannosidase was purified from the magnum section of Japanese quail oviduct by ammonium sulfate precipitation, DEAE-Sephacel chromatography, Sephacryl S-300 chromatography, mannan-Sepharose 4B chromatography, and hydroxyapatite chromatography. The purified alpha-mannosidase (referred to as neutral alpha-mannosidase) showed a single band on polyacrylamide gel with or without sodium dodecyl sulfate. Its molecular weight was found to be 330,000 by gel chromatography. Neutral alpha-mannosidase hydrolyzed p-nitrophenyl alpha-D-mannopyranoside and the pyridylamino derivative of Man alpha 1-6(Man alpha 1-3)Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc (Km value was 3 mM). Mannosyl alpha 1-2 linkages in the pyridylamino derivative of Man alpha 1-2 Man alpha 1-6(Man alpha 1-2Man alpha 1-3)Man alpha 1-6(Man alpha 1-2Man alpha 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc were hardly hydrolyzed. Its optimum pH was found to be 7.0. The activity of the enzyme was activated by CO2+, and was potently inhibited by Cu2+, Hg2+, swainsonine, and 1-deoxymannojirimycin.     

Studies on the substrate specificity of neutral alpha-mannosidase purified from Japanese quail oviduct by using sugar chains from glycoproteins.

The substrate specificity of neutral alpha-mannosidase purified from Japanese quail oviduct [Oku, H., Hase, S., & Ikenaka, T. (1991) J. Biochem. 110, 29-34] was analyzed by using 21 oligomannose-type sugar chains. The enzyme activated with Co2+ hydrolyzed the Man alpha 1-3 and Man alpha 1-6 bonds from the non-reducing termini of Man alpha 1-6(Man alpha 1-3)Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc (M5A), but hardly hydrolyzed the Man alpha 1-2 bonds of Man9GlcNAc2. The hydrolysis rate decreased as the reducing end of substrates became more bulky: the hydrolysis rate for the pyridylamino (PA) derivative of M5A as to that of M5A was 0.8; the values for M5A-Asn and Taka-amylase A having a M5A sugar chain being 0.5 and 0.04, respectively. The end product was Man beta 1-4GlcNAc2. For the substrates with the GlcNAc structure at their reducing ends (Man5GlcNAc, Man6GlcNAc and Man9GlcNAc), the hydrolysis rate was remarkably increased: Man5GlcNAc was hydrolyzed 16 times faster than M5A, and Man2GlcNAc 40 times faster than Man9GlcNAc2. The enzyme did not hydrolyze Man alpha 1-2 residue(s) linked to Man alpha 1-3Man beta 1-4GlcNAc. The end products were as follows: [formula; see text] These results suggest that oligomannose-type sugar chains with the GlcNAc structure at their reducing ends seem to be native substrates for neutral alpha-mannosidase and the enzyme seems to hydrolyze endo-beta-N-acetylgucosaminidase digests of oligomannose-type sugar chains in the cytosol.     

In vitro hydrolysis of oligomannosyl oligosaccharides by the lysosomal alpha-D-mannosidases

In vitro incubation of the oligomannosyl oligosaccharides Man9GlcNAc and Man5GlcNAc with isolated disrupted lysosomes yields different oligosaccharide isomers resulting from mannosidase hydrolysis. These isomers were isolated by HPLC and characterized by 1H-NMR spectroscopy. The first steps of the degradation involve an (alpha 1-2)mannosidase activity and lead to the formation of one Man8GlcNAc, one Man7GlcNAc, two Man6GlcNAc and two Man5GlcNAc isomers. These reactions do not require Zn2+ as activator. On the other hand, the following steps, which lead to the formation of Man3GlcNAc and Man2GlcNAc, are Zn2(+)-dependent. This process is characterized by the preferential action of an (alpha 1-3)mannosidase activity, and the formation of Man(alpha 1-6)Man(alpha 1-6)Man(beta 1-4)GlcNAc and Man(alpha 1-6)Man(beta 1-4)GlcNAc. Therefore, the digestion of Man9GlcNAc inside the lysosome appears to follow a very specific pathway, since only nine intermediate compounds can be identified instead of the 38 possible isomers. Our results are consistent both with the existence of several specific enzymes for alpha 1-2, alpha 1-3 and alpha 1-6 linkages, and with the presence of a unique enzyme whose specificity would be dependent either on Zn2+ or on the spatial conformation of the glycan. Nevertheless, previous work on the structural analysis of oligosaccharides excreted in the urine of patients suffering from mannosidosis, demonstrates the absence of the core alpha 1-6-linked mannosyl residue in the major storage product derived from oligomannosyl oligosaccharides. This observation indicates the presence of a specific (alpha 1-6)mannosidase form, unaffected in mannosidosis.     

Site-specific N-glycosylation of human chorionic gonadotrophin--structural analysis of glycopeptides by one- and two-dimensional 1H NMR spectroscopy.

Glycopeptides representing individual N-glycosylation sites of the heterodimeric glycoprotein hormone human chorionic gonadotrophin (hCG) were obtained from subunits hCG alpha (N-glycosylated at Asn-52 and Asn-78) and hCG beta (N-glycosylated at Asn-13 and Asn-30) by digestion with trypsin and chymotrypsin, respectively. Following purification by reverse-phase HPLC and identification by amino acid sequencing, the glycopeptides were analysed by one- and two-dimensional 1H NMR spectroscopy. The results are summarized as follows: (i) oligosaccharides attached to Asn-52 of hCG alpha comprised monosialylated 'monoantenary' NeuAc alpha 2-3Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3[Man alpha 1-6]Man beta 1-4GlcNAc beta 1-4GlcNAc (N1-4'), disialylated diantennary NeuAc alpha 2-3Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3[NeuAc alpha 2-3-Gal beta 1-4GlcNAc beta 1-2Man alpha 1-6]Man beta 1-4GlcNAc beta 1-4GlcNAc (N2), and the monosialylated hybrid-type structures NeuAc alpha 2-3Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3[Man alpha 1-3Man alpha 1-6]Man beta 1-4GlcNAc beta 1-4GlcNAc (N1-A) and NeuAc alpha 2-3Gal-beta 1-4GlcNAc beta 1-2Man alpha 1-3[Man alpha 1-3(Man alpha 1-6)Man alpha 1-6]Man beta 1-4GlcNAc beta 1-4GlcNAc (N1-AB) in a ratio approaching 5:2:2:1; (ii) Asn-78 of hCG alpha carried N2 and N1-4' almost exclusively (ratio approximately 3:2); (iii) both N-glycosylation sites of hCG beta contained predominantly component N2, partially (approximately 25%) and completely alpha 1-6-fucosylated at the N-acetylglucosamine linked to Asn-13 and Asn-30, respectively. The distinct site-specific distribution of the oligosaccharide structures among individual N-glycosylation sites of hCG appears to reflect primarily the influence of the surrounding protein structure on the substrate accessibility of the Golgi processing enzymes alpha-mannosidase II, GlcNAc transferase II and alpha 1,6-fucosyltransferase.     

Oligosaccharide structure of a functional unit RvH1-b of Rapana venosa hemocyanin using HPLC/electrospray ionization mass spectrometry

In the present study the structures of two glycopeptides (G1 and G1'), isolated from FU RvH(1)-b and two glycopeptides (G2 and G3), isolated from the structural subunit RvH(1) of Rapana venosa hemocyanin, were determined. To structurally characterize the site-specific carbohydrate heterogeneity and binding site of the N-linked glycopeptide(s), a combination of capillary reversed-phase chromatography and ion trap mass spectrometry was used. The amino acid sequences of glycopeptides G1 and G1' determined by Edman degradation and MS/MS sequencing demonstrated that the oligosaccharides are linked to N-glycosylation sites. Two peptides (a glycosylated (G1) and non-glycosylated one) were identified in this fraction and no linkage sites were observed in the latter one. Based on the sequencing of the glycosylated fractions G1, G1', G2 and G3, the carbohydrate structure Man(alpha1-6)Man(alpha1-3)Man(beta1-4)GlcNAc(beta1-4)[Fuc(alpha1-6)]GlcNAc-R could be identified for glycopeptides G1 and G3, and only the typical core structure Man(alpha1-6)Man(alpha1-3)Man(beta1-4)GlcNAc(beta1-4)GlcNAc-R was found for G1' and G2. The Fuc residue found in glycopeptides G1 and G3 is attached to N-acetyl-glucosamine of the carbohydrate core, as often found in other glycoproteins.     

Two positional isomers of sialylheptasaccharides isolated from the urine of a patient with sialidosis

We describe the structures of two positional isomers of sialylheptasaccharide isolated from the urine of a patient with sialidosis with partial deficiency of beta-galactosidase. Based on structural studies including compositional sugar analysis, exoglycosidase digestion, chemical ionization mass spectrometry, proton nuclear magnetic resonance spectrometry, and methylation analysis, their structures were deduced to be as follows: AcNeu alpha 2----6Gal beta 1----4GlcNac beta 1----2Man alpha 1----3(Man alpha 1----6)Man beta 1----4GlcNac; AcNeu alpha 2----6Gal beta 1----4GlcNac beta 1----2Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNac. Sialyloligosaccharide 1 has previously been found in the urine and liver of patients with mucolipidosis I and II and sialidosis, but sialyloligosaccharide 2 has not been found yet in human urine. These two sialyloligosaccharides could not be completely separated by any chromatographic procedures tested. The analytical techniques, including methylation study and NMR spectroscopy, could not clearly detect the differences between them. However, alpha-mannosidase treatment gave important information for the structural analyses of these sialyloligosaccharides.     

Structural study of a cell wall mannan-protein complex of the pathogenic yeast Candida glabrata IFO 0622 strain.

We conducted a structural analysis of the cell wall mannan-protein complex (mannan) isolated from a pathogenic yeast, Candida glabrata IFO 0622 strain. The chemical structure of mannobiose released from this mannan by treatment with 10 mM HCl at 100 degrees C for 1 h was identified as Manp beta 1-2Man. The treatment of this mannan with 100 mM NaOH at 25 degrees C for 18 h gave a mixture of alpha-1,2- and alpha-1,3-linked oligosaccharides, from tetraose to biose, and mannose. The acid- and alkali-stable mannan moiety was subjected to mild acetolysis with a 100:100:1 (v/v) mixture of (CH3CO)2O, CH3COOH, and H2SO4 at 40 degrees C for 36 h. The resultant three novel oligosaccharides, tetraose, hexaose, and heptaose, were identified as Manp beta 1-2Manp alpha 1-2Manp alpha 1-2Man, Manp alpha 1-2Manp alpha 1-2Manp alpha 1-6Manp alpha 1-2Manp alpha 1-2Man, and Manp alpha 1-3Manp alpha 1-2Manp alpha 1-2Manp alpha 1-6Manp alpha 1- 2Manp alpha 1-2Man, respectively, in addition to the three known oligosaccharides, Manp alpha 1-2Man, Manp alpha 1-2Manp alpha 1-2Man, and Manp alpha 1-3Manp alpha 1-2Manp alpha 1-2Man. A sequential analytical procedure involving partial acid hydrolysis with hot 0.3 M H2SO4, methylation, fast atom bombardment mass, and 1H NMR analyses was quite effective in the structural determination of the novel oligosaccharides. The results indicate that this mannan possesses a structure closely resembling that of Saccharomyces cerevisiae X2180-1A wild type strain, with the presence of small amounts of oligomannosyl residue, Manp beta 1-2Manp alpha 1-X, corresponding to one of the epitopes dominating serotype-A specificity of Candida spp., in addition to branches corresponding to hexaose and heptaose each containing one intermediary alpha-1,6 linkage.     

Control of glycoprotein synthesis. UDP-GlcNAc:glycopeptide beta 4-N-acetylglucosaminyltransferase III, an enzyme in hen oviduct which adds GlcNAc in beta 1-4 linkage to the beta-linked mannose of the trimannosyl core of N-glycosyl oligosaccharides

Hen oviduct membranes are shown to catalyze the following enzyme reaction: GlcNAc beta 1-2Man alpha 1-6(GlcNAc beta 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-6)GlcNAc-Asn + UDP-GlcNAc leads to GlcNAc beta 1-2Man alpha 1-6(GlcNAc beta 1-2Man alpha 1-3)GlcNAc beta 1-4)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-6)GlcNAc-Asn + UDP. The enzyme catalyzing this reaction has been named UDP-GlcNAc:glycopeptide beta 4-N-acetylglucosaminyltransferase III (GlcNAc-transferase III) to distinguish it from two other GlcNAc-transferases (I and II) present in hen oviduct and previously described in several mammalian tissues. GlcNAc-transferases I and II, respectively, attach GlcNAc in beta 1-2 linkage to the Man alpha 1-3 and Man alpha 1-6 arms of Asn-linked oligosaccharide cores. A specific assay for GlcNAc-transferase III was devised by using concanavalin A/Sepharose columns to separate the product of transferase III from other interfering radioactive glycopeptides formed in the reaction. The specific activity of GlcNAc-transferase III in hen oviduct membranes is about 5 nmol/mg of protein/h. Substrate specificity studies have shown that GlcNAc-transferase III requires both terminal beta 1-2-linked GlcNAc residues in its substrate for maximal activity. Removal of the GlcNAc residue on the Man alpha 1-6 arm reduces activity by at least 85% and removal of both GlcNAc residues reduces activity by at least 93%. Two large scale preparations of product were subjected to high resolution proton NMR spectroscopy to establish the incorporation by the enzyme of a GlcNAc in beta 1-4 linkage to the beta-linked Man. This GlcNAc residue is called a "bisecting" GlcNAc and appears to play important control functions in the synthesis of complex N-glycosyl oligosaccharides. Several enzymes in the biosynthetic scheme are unable to act on glycopeptide substrates containing a bisecting GlcNAc residue.     

Mutation of Arg(273) to Leu alters the specificity of the yeast N-glycan processing class I alpha1,2-mannosidase

Class I alpha1,2-mannosidases (glycosyl hydrolase family 47) involved in the processing of N-glycans during glycoprotein maturation have different specificities. Enzymes in the endoplasmic reticulum of yeast and mammalian cells remove a single mannose from Man(9)GlcNAc(2) to form Man(8)GlcNAc(2) isomer B (lacking the alpha1, 2-mannose residue of the middle alpha1, 3-arm), whereas other alpha1,2-mannosidases, including Golgi alpha1,2-mannosidases IA and IB, can convert Man(9)GlcNAc(2) to Man(5)GlcNAc(2). In the present work, it is demonstrated that with a single mutation in its catalytic domain (Arg(273) --> Leu) the yeast endoplasmic reticulum alpha1,2-mannosidase acquires the ability to transform Man(9)GlcNAc to Man(5)GlcNAc. High resolution proton nuclear magnetic resonance analysis of the products shows that the order of removal of mannose from Man(9)GlcNAc is different from that of other alpha1, 2-mannosidases that remove four mannose from Man(9)GlcNAc. These results demonstrate that Arg(273) is in part responsible for the specificity of the endoplasmic reticulum alpha1,2-mannosidase and that small differences in non-conserved amino acids interacting with the oligosaccharide substrate in the active site of class I alpha1, 2-mannosidases are responsible for the different specificities of these enzymes.     

Molecular characterization and allergenic activity of Lyc e 2 (beta-fructofuranosidase), a glycosylated allergen of tomato.

Until now, only a small amount of information is available about tomato allergens. In the present study, a glycosylated allergen of tomato (Lycopersicon esculentum), Lyc e 2, was purified from tomato extract by a two-step FPLC method. The cDNA of two different isoforms of the protein, Lyc e 2.01 and Lyc e 2.02, was cloned into the bacterial expression vector pET100D. The recombinant proteins were purified by electroelution and refolded. The IgE reactivity of both the recombinant and the natural proteins was investigated with sera of patients with adverse reactions to tomato. IgE-binding to natural Lyc e 2 was completely inhibited by the pineapple stem bromelain glycopeptide MUXF (Man alpha 1-6(Xyl beta 1-2)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-3)GlcNAc). Accordingly, the nonglycosylated recombinant protein isoforms did not bind IgE of tomato allergic patients. Hence, we concluded that the IgE reactivity of the natural protein mainly depends on the glycan structure. The amino acid sequences of both isoforms of the allergen contain four possible N-glycosylation sites. By application of MALDI-TOF mass spectrometry the predominant glycan structure of the natural allergen was identified as MMXF (Man alpha 1-6(Man alpha 1-3)(Xyl beta 1-2)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-3) GlcNAc). Natural Lyc e 2, but not the recombinant protein was able to trigger histamine release from passively sensitized basophils of patients with IgE to carbohydrate determinants, demonstrating that glycan structures can be important for the biological activity of allergens.     

Primary structure of the glycan chains of normal C 1 esterase inhibitor (C 1-INH) after NMR analysis at 400 MHz

The primary structural analysis of O- and N-linked carbohydrate chains of the C-1-esterase inhibitor purified from normal serum was carried out by 400-MHz 1H-NMR spectroscopy. C-1-esterase inhibitor protein of a molecular weight of 116,000 daltons contains 24 O-glycans: NeuAc (alpha 2-3) Gal (beta 1-3) GalNAc, 4 N-glycans: NeuAc (alpha 2-6) Gal (beta 1-4) (GlcNAc (beta 1-2) Man (alpha 1-3) [NeuAc (alpha 2-6) Gal (beta 1-4) GlcNAc (beta 1-2) Man (alpha 1-6)] Man (beta 1-4) GlcNAc (beta 1-4) GlcNAc and 2 N-glycans: NeuAc (alpha 2-3) Gal (beta 1-4) GlcNAc (beta 1-2) Man (alpha 1-3) [NeuAc (alpha 2-3) Gal (beta 1-4) GlcNAc (beta 1-2) Man (alpha 1-6)] Man (beta 1-4) GlcNAc (beta 1-4) GlcNAc. 30% of the N-glycans are fucosylated.     

Neutral glycosphingolipids of ova of the fresh-water bivalve, Hyriopsis schlegelii

The neutral glycosphingolipids of ova of the fresh-water bivalve, Hyriopsis schlegelii were characterized. The most abundant glycolipid was ceramide monosaccharide, followed by ceramide trisaccharide, ceramide tetrasaccharide, and ceramide disaccharide. More complex neutral glycolipids accounted for almost one-third of the total. The total amount of these glycolipids was 0.59 mg/g of dry weight of the ova preparation, a yield which was one-seventh of that of spermatozoa neutral glycolipids. Structural analyses were performed by enzymatic hydrolysis of the glycolipids with exoglycosidases, permethylation experiments, and also immuno-chemical assays. The proposed structures are as follows: ceramide monosaccharides, Gal-Cer and Glc-Cer; ceramide disacharides, Gal(beta 1-4)Gal-Cer, Gal(beta 1-4)Glc-Cer, and Man(beta 1-4)Glc-Cer; ceramide trisaccharide, Man(alpha 1-3)Man(beta 1-4)Glc-Cer; ceramide tetrasaccharides, Man(alpha 1-3)[Xyl(beta 1-2)]Man(beta 1-4)Glc-Cer, GlcNAc(beta 1-2)Man(alpha 1-3)Man(beta 1-4)Glc-Cer, Man(alpha 1-3)[Gal(beta 1-2)]Man(beta 1-4)Glc-Cer, and Man(alpha 1-2?)Man(alpha 1-3)Man(beta 1-4)Glc-Cer. The latter two ceramide tetrasaccharides were new types of glycosphingolipids. The spectrum of ova glycolipids appeared to be more complicated than that of the spermatozoa glycolipids. The ova glycolipids characterized here, with the exception of ceramide tetrasaccharides, contained considerable amounts of 2-hydroxy fatty acids, which were not observed in the spermatozoa glycolipids. The major sphingosine base was C18-sphingenine in all the ova glycolipids as well as in the spermatozoa glycolipids. However, the content of anteiso type of sphingosine base was 2- to 3-fold higher in the ova than in the spermatozoa.     

Complete structure of the carbohydrate moiety of stem bromelain. An application of the almond glycopeptidase for structural studies of glycopeptides.

Asparagine-linked oligosaccharides of stem bromelain glycopeptides were quantitatively released by digestion with the almond glycopeptidase which cleaves beta-aspartylglycosylamine linkage in glycopeptides with oligopeptide moieties. The primary structures of the two oligosaccharide components, (Man)3(Xyl)1(Fuc)1(GlcNAc)2 and (Man)2-(Xyl)1(Fuc)1(GlcNAc)2 were elucidated as Man alpha 1 leads to 6Man alpha 1 leads to 6[Xyl beta 1 leads to 2]Man beta 1 leads to 4GlcNAc beta 1 leads 4[Fuc alpha 1 leads to 3]GlcNAc and Man alpha 1 leads to 6[Xyl beta 1 leads to 2]Man beta 1 leads to 4 GlcNAc beta 1 leads to 4[Fuc alpha 1 leads to 3] GlcNAc, respectively.     

Biosynthesis of mammary glycoproteins. Partial characterization of the sequence for the assembly of lipid-linked saccharides

Incubation of a membrane preparation from the lactating bovine mammary gland with UDP-[3H]GlcNAc, GDP-[14C]Man, and UDP-[3H]Glc results in the biosynthesis of 15 lipid-linked saccharides that differ from one another by a monosaccharide unit. Pulse and chase kinetics indicate that these glycolipids are related to one another as precursor products for the biosynthesis of asparagine-linked glycoproteins of this tissue. [Man-14C]- and [Man-14C, GlcNAc-3H]saccharides were prepared from corresponding glycolipids by mild acid hydrolysis. Following extensive purification by paper and gel filtration chromatography, structural characterization was conducted on tri-, tetra-, penta-, and undecasaccharides via size determination on calibrated columns of Bio-Gel P-2 and P-4, compositional analysis, exo- and endoglycosidase digestions, methylation, Smith degradation, and acetolysis. These structures were identified as: Man beta 1 leads to 4(3)GlcNAc beta 1 leads to 4(3)Glc-NAc, Man alpha 1 leads to 3Man beta 1 leads to 4(3)GlcNAc beta 1 leads to 4(3)GlcNAc, Man alpha 1 leads to 3(Man alpha 1 leads to 6)Man beta 1 leads to 4(3)Glc NAc beta 1 leads to 4(3)Glc-NAc, and Man alpha 1 leads to 2 Man alpha 1 leads to 2Man alpha 1 leads to 3(Man alpha 1 leads to 2Man alpha 1 leads to 6[Man alpha 1 leads to 2Man alpha 1 leads to 3]Man alpha 1 leads to 6)Man beta 1 leads to 4(3)GlcNAc beta 1 leads to 4(3)GlcNAc.     

Control of glycoprotein synthesis. Detection and characterization of a novel branching enzyme from hen oviduct, UDP-N-acetylglucosamine:GlcNAc beta 1-6 (GlcNAc beta 1-2)Man alpha-R (GlcNAc to Man) beta-4-N-acetylglucosaminyltransferase VI

Hen oviduct membranes were shown to contain high activity of a novel enzyme, UDP-GlcNac:GlcNAc beta 1-6(GlcNAc beta 1-2) Man alpha-R (GlcNAc to Man) beta 4-GlcNAc-transferase VI. The enzyme was shown to transfer GlcNAc in beta 1-4 linkage to the D-mannose residue of GlcNAc beta 1-6 (GlcNAc beta 1-2) Man alpha-R where R is either 1-6Man beta-(CH2)8COOCH3 or methyl. Radioactive enzyme products were purified by several chromatographic steps, including high performance liquid chromatography, and structures were determined by proton nmr, fast atom bombardment-mass spectrometry, and methylation analysis to be GlcNAc beta 1-6 ([14C]GlcNAc beta 1-4) (GlcNAc beta 1-2) Man alpha-R. The enzyme is stimulated by Triton X-100 and has optimum activity at a relatively high MnCl2 concentration of about 100 mM; Co2+, Mg2+, and Ca2+ could partially substitute for Mn2+. A tissue survey demonstrated high GlcNAc-transferase VI activity in hen oviduct and lower activity in chicken liver and colon, duck colon, and turkey intestine. No activity was found in mammalian tissues. Hen oviduct membranes cannot act on GlcNAc beta 1-6Man alpha-R but have a beta 4-GlcNAc-transferase activity that converts GlcNAc beta 1-2Man alpha-R to GlcNAc beta 1-4(GlcNAc beta 1-2) Man alpha-R where R is either 1-6Man beta-(CH2)8COOCH3 or 1-6Man beta methyl. The latter activity is probably due to GlcNAc-transferase IV which preferentially adds GlcNAc in beta 1-4 linkage to the Man alpha 1-3 arm of the GlcNAc beta 1-2Man alpha 1-6(GlcNAc beta 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc-Asn core structure of asparagine-linked glycans. The minimum structural requirement for a substrate of beta 4-GlcNAc-transferase VI is therefore the trisaccharide GlcNAc beta 1-6(GlcNAc beta 1-2) Man alpha-; this trisaccharide is found on the Man alpha 6 arm of many branched complex asparagine-linked oligosaccharides. The data suggest that GlcNAc-transferase VI acts after the synthesis of the GlcNAc beta 1-2Man alpha 1-3-, GlcNAc beta 1-2Man alpha 1-6-, and GlcNAc beta 1-6 Man alpha 1-6-branches by GlcNAc-transferases I, II, and V, respectively, and is responsible for the synthesis of branched oligosaccharides containing the GlcNAc beta 1-6(GlcNAc beta 1-4)(GlcNAc beta 1-2)Man alpha 1-6Man beta moiety.