Glc Residues Research Articles

Design and Synthesis of Oligosaccharides that Interfere with Glycoprotein Quality‐control systems

Calnexin (CNX) and its soluble homologue calreticulin (CRT) are lectin-like molecular chaperones that help newly synthesized glycoproteins to fold correctly in the rough endoplasmic reticulum (ER). To investigate the mechanism of glycoprotein-quality control, we have synthesized structurally defined high-mannose-type oligosaccharides related to this system. This paper describes the synthesis of the non-natural undecasaccharide 2 and heptasaccharide 16, designed as potential inhibitors of the ER quality-control system. Each possesses the key tetrasaccharide element (Glc1Man3) critical for the CNX/CRT binding, while lacking the pentamannosyl branch required for glucosidase II recognition. These oligosaccharides were evaluated for their ability to bind CRT by isothermal titration calorimetry (ITC). As expected, each of them had a significant affinity towards CRT. In addition, these compounds were shown to be resistant to glucosidase II digestion. Their activities in blocking the chaperone function of CRT were next measured by using malate dehydrogenase (MDH) as a substrate. Their inhibitory effects were shown to correlate well with their CRT-binding affinities, both being critically dependent upon the presence of the terminal glucose (Glc) residue.

Methyl 4-O-β-D-galactopyranosyl α-D-glucopyranoside (methyl α-lactoside)

Methyl alpha-lactoside, C13H24O11, (I), is described by glycosidic torsion angles varphi (O5gal-C1gal-O1gal-C4glc) and psi (C1gal-O1gal-C4glc-C5glc), which have values of -93.52 (13) and -144.83 (11) degrees, respectively, where the ring atom numbering conforms to the convention in which C1 is the anomeric C atom and C6 is the exocyclic hydroxymethyl (-CH2OH) C atom in both residues. The linkage geometry is similar to that observed in methyl beta-lactoside methanol solvate, (II), in which varphi is -88.4 (4) degrees and psi is -161.3 (4) degrees. As in (II), an intermolecular O3glc-H...O5gal hydrogen bond is observed in (I). The hydroxymethyl group conformation in both residues is gauche-trans, with torsion angles omegagal (O5gal-C5gal-C6gal-O6gal) and omega(glc) (O5glc-C5glc-C6glc-O6glc) of 69.15 (13) and 72.55 (14) degrees, respectively. The latter torsion angle differs substantially from that found for (II) [-54.6 (2) degrees; gauche-gauche]. Cocrystallization of methanol, which is hydrogen bonded to O6glc in the crystal structure of (II), presumably affects the hydroxymethyl conformation in the Glc residue in (II).

糖質加水分解酵素ファミリー７４に属するキシログルカン分解酵素の機能と構造

Recently, we isolated and cloned two xyloglucan-specific endoglucanases (XEGs) from Geotrichum sp. M 128 and Gram-positive bacterium sp. KM 21, and a xyloglucan-specific exoglucanase, oligoxyloglucan reducing-end-specific cellobiohydrolase (OXG-RCBH) from Geotrichum sp. M 128, all of which belong to glycoside hydrolase family 74. Geotrichum and KM 21 XEGs have endoglucanase activity toward xyloglucan but not cellulose, while Geotrichum OXG-RCBH is a unique exoglucanase that releases two Glc residue segments from the reducing end of the xyloglucan main chain. Further analysis of the substrate specificities of Geotrichum XEG and OXG-RCBH using various oligosaccharides revealed that they recognize specific xylose branching structures. Although both Geotrichum XEG and OXG-RCBH have at least four subsites (-2 to +2), specific recognition of xylose residues occurs at the +1 and +2 sites in the former and at the -1 site in the latter. Moreover, xylose branching at the reducing end (site +2) eliminates OXG-RCBH activity. This enzymatic activity is very different from those of known glycosidases. Recently, OXG-RCBH was assigned a new EC number (EC 3.2.1.150). To elucidate the molecular mechanism of OXG-RCBH, its three-dimensional structure was analyzed. The X-ray crystal structure of OXG-RCBH revealed a unique feature of this enzyme: OXG-RCBH consists of two similar domains, both of which are folded into seven-bladed β-propeller structures. The cleft between the two domains can accommodate the oligosaccharide substrate and thus constitutes a putative catalytic core. Mutation of either Asp 35 or Asp 465, located in the cleft, to Asn resulted in a protein with no detectable catalytic activity, indicating the critical role of these amino acids in catalysis.

Isolation of Galactoglucomannan from Apple Hemicellulosic Polysaccharides with Binding Capacity to Cellulose

The hemicellulose fraction which is soluble in 24% KOH solution (HC-II) from the cell-walls of apple flesh contained a small amount of galactoglucomannan in addition to a large amount of xyloglucan. A crude galactoglucomannan was isolated from the HC-II fraction with binding capacity to cellulose microfibrils. The crude galactoglucomannan fraction was treated with xyloglucanase to remove concomitant xyloglucan, followed by DEAE-Sephadex A-25 chromatography to give a purified galactoglucomannan fraction. Sugar composition analysis showed that the purified galactoglucomannan fraction consisted of Man, Glc and Gal in the molar ratio of 5.7:3.0:1.1. Methylation analysis suggested that a galactoglucomannan present in apple flesh had β-(1→4)-linked Glc and Man residues, some of which were branched at the O-6 position with Gal residues.

De novo synthesis of bacterial glycogen: Agrobacterium tumefaciens glycogen synthase is involved in glucan initiation and elongation.

Evidence is presented indicating that initiation of glycogen synthesis in Agrobacterium tumefaciens does not require the presence of alpha(1,4)-linked glucans. Crude cell extracts incubated with ADP-glucose (Glc) were able to form alpha(1,4)-linked glucans despite the fact that cells used for extract preparation displayed a genotype that prevented synthesis of Glc-containing sugar nucleotides and thus preformation of alpha(1,4)-linked glucans and that the defined growth medium used contained glycerol as carbon source. A. tumefaciens glycogen synthase (GS) purified to homogeneity from the above-mentioned cells was able to build its own primer by transferring Glc residues from ADP-Glc to an amino acid(s) in the same protein. Primed GS then became the substrate for further GS-catalyzed glucan elongation. It was concluded that, contrary to what happens in mammalian and yeast cells in which two different proteins are required for linear alpha(1,4)-linked glucan formation (glycogenin for initiation and GS for further elongation), in A. tumefaciens and probably in all other bacteria, the same protein is involved in both glycogen initiation and elongation.

Extracellular polysaccharides of Erwinia futululu, a bacterium associated with a fungal canker disease of Eucalyptus spp.

Extracellular polysaccharides (EPSs) produced by an Erwinia spp. associated with a fungal canker disease of Eucalyptus were fractionated into two polysaccharides, one that was identified with that produced by Erwinia stewartii. The other has a similar structure, but with one terminal Glc residue replaced by pyruvic acid to give 4,6- O-[( R)-1-carboxyethylidene)-Gal p. Their structures were determined using a combination of chemical and physical techniques including methylation analysis, periodate oxidation, low-pressure gel filtration and anion-exchange chromatographies, high-pH anion-exchange chromatography, mass spectrometry and 1D and 2D 1H NMR spectroscopy. The new polysaccharides, identified as EPS Futululu FF-1 and FF-2, have the following structures: The molecular weights of the polysaccharides range from 1.3–2.1×10 6 and their hydrodynamic properties are those of polydisperse, polyanionic biopolymers with pseudoplastic, non-thixotropic flow characteristics in aqueous solutions.

SYNTHESIS OF SIALYL-α-(2→3)-NEOLACTOTETRAOSE DERIVATIVES MODIFIED AT C-2 OF THE N-ACETYLGLUCOSAMINE RESIDUE: PROBES FOR INVESTIGATION OF ACCEPTOR SPECIFICITY OF HUMAN α-1,3-FUCOSYLTRANSFERASES, FUC-TVII, AND FUC-TVI 1*

A variety of sialyl-α-(2→3)-neolactotetraose (IV3NeuAcnLcOse4 or IV3NeuGcnLcOse4) derivatives (23, 31–37, 58–60) modified at C-2 of the GlcNAc residue have been synthesized. The phthalimido group at C-2 of GlcNAc in 2-(trimethylsilyl)ethyl (3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-d-glucopyranosyl)-(1→3)-(2,4,6-tri-O-benzyl-β-d-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-d-glucopyranoside (5) was systematically converted to a series of acylamino groups, to give the per-O-benzylated trisaccharide acceptors (6–11). On the other hand, modification of the hydroxyl group at C-2 of the terminal Glc residue in 2-(trimethylsilyl)ethyl (4,6-O-benzylidene-β-d-glucopyranosyl)-(1→3)-(2,4,6-tri-O-benzyl-β-d-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-d-glucopyranoside (42) gave three different kinds of trisaccharide acceptors containing D-glucose (49), N-acetyl-d-mannosamine (50), and D-mannose (51) instead of the GlcNAc residue. Totally ten trisaccharide acceptors (5–11 and 49–51) were each coupled with sialyl-α-(2→3)-galactose donor 12 to afford the corresponding pentasaccharides (14–21 and 52–54) in good yields, respectively, which were then transformed into the target compounds. Acceptor specificity of the synthetic sialyl-α-(2→3)-neolactotetraose probes for the human α-(1→3)-fucosyltransferases, Fuc-TVII and Fuc-TVI, was examined. *Synthetic studies on sialoglycoconjugates, Part 125. For Part 124, see Otsubo, N.; Ishida, H.; Kiso, M. Chemical approach to selectin ligands: total synthesis of O-glycan on GlyCAM-1. Aust. J. Chem.-Int. J. Chem. Sci. 2002, 55, 105–112. †Current address: Basic Research Laboratory, Kanebo Ltd., Kotobuki-cho, Odawara-shi, Kanagawa, Japan.

Structural and serological relatedness of the O-antigens of Proteus penneri 1 and 4 from a novel Proteus serogroup O72.

O-specific polysaccharides (O-antigens) of the lipopolysaccharides (LPS) of Proteus penneri strains 1 and 4 were studied using sugar analysis, (1)H and (13)C NMR spectroscopy, including 2D COSY, H-detected (1)H,(13)C HMQC, and rotating-frame NOE spectroscopy (ROESY). The following structures of the tetrasaccharide (strain 1) and pentasaccharide (strain 4) repeating units of the polysaccharides were established: [reaction: see text]. In the polysaccharide of P. penneri strain 4, glycosylation with the lateral Glc residue (75%) and O-acetylation of the lateral GalNAc residue (55%) are nonstoichiometric. This polysaccharide contains also other, minor O-acetyl groups, whose positions were not determined. The structural similarity of the O-specific polysaccharides was consistent with the close serological relatedness of the LPS, which was demonstrated by immunochemical studies with O-antisera against P. penneri 1 and 4. Based on these data, it was proposed to classify P. penneri strains 1 and 4 into a new Proteus serogroup, O72, as two subgroups, O72a and O72a,b, respectively. Serological cross-reactivity of P. penneri 1 O-antiserum with the LPS of P. penneri 40 and 41 was substantiated by the presence of an epitope(s) on the LPS core region shared by all P. penneri strains studied.

The nematode Caenorhabditis elegans synthesizes unusual O-linked glycans: identification of glucose-substituted mucin-type O-glycans and short chondroitin-like oligosaccharides

The free-living nematode Caenorhabditis elegans is a relevant model for studies on the role of glycoconjugates during development of multicellular organisms. Several genes coding for glycosyltransferases involved in the synthesis of N- and O-linked glycans have already been isolated, but, apart from repetitive dimers of glycosaminoglycans, no detailed structure of either type of component has been published so far. This study aimed to establish the structures of the major O-glycans synthesized by C. elegans to give an insight into the endogenous glycosyltransferase activities expressed in this organism. By the use of NMR and MS, we have resolved the sequence of seven of these components that present very unusual features. Most of them were characterized by the type-1 core substituted on Gal and/or GalNAc by (β1–4)Glc and (β1–6)Glc residues. Another compound exhibited the GalNAc(β1–4)N-acetylglucosaminitol sequence in the terminal position, to which was attached a tetramer of β-Gal substituted by both Fuc and 2-O-methyl-fucose residues. Our experimental procedure led also to the isolation of glycosaminoglycan-like components and oligomannosyl-type N-glycans. In particular, the data confirmed that C. elegans synthesizes the ubiquitous linker sequence GlcA(β1–3)Gal(β1–3)Gal(β1–4)Xyl.

The nematode Caenorhabditis elegans synthesizes unusual O-linked glycans: identification of glucose-substituted mucin-type O-glycans and short chondroitin-like oligosaccharides.

The free-living nematode Caenorhabditis elegans is a relevant model for studies on the role of glycoconjugates during development of multicellular organisms. Several genes coding for glycosyltransferases involved in the synthesis of N- and O-linked glycans have already been isolated, but, apart from repetitive dimers of glycosaminoglycans, no detailed structure of either type of component has been published so far. This study aimed to establish the structures of the major O-glycans synthesized by C. elegans to give an insight into the endogenous glycosyltransferase activities expressed in this organism. By the use of NMR and MS, we have resolved the sequence of seven of these components that present very unusual features. Most of them were characterized by the type-1 core substituted on Gal and/or GalNAc by (beta1-4)Glc and (beta1-6)Glc residues. Another compound exhibited the GalNAc(beta1-4)N-acetylglucosaminitol sequence in the terminal position, to which was attached a tetramer of beta-Gal substituted by both Fuc and 2-O-methyl-fucose residues. Our experimental procedure led also to the isolation of glycosaminoglycan-like components and oligomannosyl-type N-glycans. In particular, the data confirmed that C. elegans synthesizes the ubiquitous linker sequence GlcA(beta1-3)Gal(beta1-3)Gal(beta1-4)Xyl.

Functional characteristics of a protective monoclonal antibody against serotype A and C lipooligosaccharides from Moraxella catarrhalis.

A monoclonal antibody (MAb), designated MAb 8E7 (immunoglobulin G3), specific for Moraxella catarrhalis lipooligosaccharide (LOS) was evaluated for its functional activity in vitro and in a mouse model of colonization. Enzyme-linked immunosorbent assay (ELISA) demonstrated that the MAb 8E7 could be prepared to a high titer against LOS of the homologous strain 035E, and that it had bactericidal activity. MAb 8E7 reacted with M. catarrhalis serotype A and C LOSs but not serotype B LOS, as measured by ELISA and Western blotting. On the basis of published structures of LOSs, this suggests that the epitope recognized by MAb 8E7 is directed to a common sequence of either alpha-GlcNAc-(1-->2)-beta-Glc-(1--> at the branch substituting position 4 of the trisubstituted Glc residue or a terminal tetrasaccharide alpha-Gal-(1-->4)-beta-Gal-(1-->4)-alpha-Glc-(1-->2)-beta-Glc-(1--> at the branch substituting position 6 of the trisubstituted Glc residue. In a whole-cell ELISA, MAb 8E7 reacted with 70% of the 30 wild-type strains and clinical isolates tested. Immuno-electron microscopy demonstrated that MAb 8E7 reacted with a cell surface-exposed epitope of LOS on strain O35E. MAb 8E7 inhibited the adherence of strain O35E to Chang conjunctival epithelial cells by 90%. Passive immunization with MAb 8E7 could significantly enhance the clearance of strain O35E from mouse lungs in an aerosol challenge mouse model. This enhanced bacterial clearance was inhibited when MAb 8E7 was absorbed by M. catarrhalis serotype A LOS, indicating that the M. catarrhalis LOS-directed antibody may play a major role in the enhancement of M. catarrhalis clearance from lungs. These data suggest that MAb 8E7, which recognizes surface-exposed LOS of M. catarrhalis, is a protective antibody against M. catarrhalis.

Characterization of the maltooligosyl trehalose synthase from the thermophilic archaeon Sulfolobus acidocaldarius

We report the molecular characterization and the detailed study of the recombinant maltooligosyl trehalose synthase mechanism from the thermoacidophilic archaeon Sulfolobus acidocaldarius. The mts gene encoding a maltooligosyl trehalose synthase was overexpressed in Escherichia coli using the T7-expression system. The purified recombinant enzyme exhibited optimum activity at 75°C and pH 5 with citrate–phosphate buffer and retained 60% of residual activity after 72 h of incubation at 80°C. The recombinant enzyme was active on maltooligosaccharides such as maltotriose, maltotetraose, maltopentaose and maltoheptaose. Investigation of the enzyme action on maltooligosaccharides has brought much insight into the reaction mechanism. Results obtained from thin-layer chromatography suggested a possible mechanism of action for maltooligosyl trehalose synthase: the enzyme, after converting the α-1,4-glucosidic linkage to an α-1,1-glucosidic linkage at the reducing end of maltooligosaccharide glc( n) is able to release glucose and maltooligosaccharide glc( n−1) residues. And then, the intramolecular transglycosylation and the hydrolytic reaction continue, with the maltooligosaccharide glc( n−1) until the initial maltooligosaccharide is reduced to maltose. An hypothetical mechanism of maltooligosyl trehalose synthase acting on maltooligosaccharide is proposed.

Characterization of the maltooligosyl trehalose synthase from the thermophilic archaeon Sulfolobus acidocaldarius.

We report the molecular characterization and the detailed study of the recombinant maltooligosyl trehalose synthase mechanism from the thermoacidophilic archaeon Sulfolobus acidocaldarius. The mts gene encoding a maltooligosyl trehalose synthase was overexpressed in Escherichia coli using the T7-expression system. The purified recombinant enzyme exhibited optimum activity at 75 degrees C and pH 5 with citrate-phosphate buffer and retained 60% of residual activity after 72 h of incubation at 80 degrees C. The recombinant enzyme was active on maltooligosaccharides such as maltotriose, maltotetraose, maltopentaose and maltoheptaose. Investigation of the enzyme action on maltooligosaccharides has brought much insight into the reaction mechanism. Results obtained from thin-layer chromatography suggested a possible mechanism of action for maltooligosyl trehalose synthase: the enzyme, after converting the alpha-1,4-glucosidic linkage to an alpha-1,1-glucosidic linkage at the reducing end of maltooligosaccharide glc(n) is able to release glucose and maltooligosaccharide glc(n-1) residues. And then, the intramolecular transglycosylation and the hydrolytic reaction continue, with the maltooligosaccharide glc(n-1) until the initial maltooligosaccharide is reduced to maltose. An hypothetical mechanism of maltooligosyl trehalose synthase acting on maltooligosaccharide is proposed.

Thermodynamic Binding Studies of Lectins from the Diocleinae Subtribe to Deoxy Analogs of the Core Trimannoside of Asparagine-linked Oligosaccharides

Lectins from seven different species of the Diocleinae subtribe have been recently isolated and characterized in terms of their carbohydrate binding specificities (Dam, T. K., Cavada, B. S., Grangeiro, T. B., Santos, C. F., de Sousa, F. A. M., Oscarson, S., and Brewer, C. F. (1998) J. Biol. Chem. 273, 12082-12088). The lectins included those from Canavalia brasiliensis, Cratylia floribunda, Dioclea rostrata, Dioclea virgata, Dioclea violacea, and Dioclea guianensis. All of the lectins exhibited specificity for Man and Glc residues, but much higher affinities for the branched chain trimannoside, 3,6-di-O-(alpha-d-mannopyranosyl)-d-mannose, which is found in the core region of all asparagine-linked carbohydrates. In the present study, isothermal titration microcalorimetry is used to determine the binding thermodynamics of the above lectins, including a new lectin from Canavalia grandiflora, to a complete series of monodeoxy analogs of the core trimannoside. From losses in the affinity constants and enthalpies of binding of certain deoxy analogs, assignments are made of the hydroxyl epitopes on the trimannoside that are involved in binding to the lectins. The pattern of binding of the deoxy analogs is similar for all seven lectins, and similar to that of concanavalin A which is also a member of the Diocleinae subtribe. However, differences in the magnitude of the thermodynamic binding parameters of the lectins are observed, even though the lectins possess conserved contact residues in many cases, and highly conserved primary sequences. The results indicate that non-contact residues in the lectins, even those distant from the binding sites, modulate their thermodynamic binding parameters.

The structural heterogeneity of the lipooligosaccharide (LOS) expressed by pathogenic non-typeable Haemophilus influenzae strain NTHi 9274.

Nontypeable Haemophilus influenzae (NTHi) is an important pathogen responsible for otitis media in children and of pneumonitis in adults with depressed resistance. NTHi is acapsular and, therefore, capsular polysaccharide-based vaccines are ineffective for preventing infections by this pathogen. Recently it was found that a detoxified lipooligo-saccharide (LOS) conjugate from NTHi 9274 induced bactericidal antibodies effective against a large number of NTHi isolates, and conferred protection against NTHi otitis media in chinchillas (X.-X.Gu et al., 1996, Infect. Immun.,64, 4047-4053; X. -X.Gu et al., 1997., Infect. Immun.,65, 4488-4493). In this paper we report the chemical character-ization of the LOS from NTHi 9274 LOS. NTHi is capable of expressing a heterogenous population of LOS exhibited by multiple oligosaccharide (OS) epitopes. OSs released from the LOS of NTHi 9274 by mild acid hydrolysis were purified using Bio-Gel P4 gel permeation chromatography. The OSs were characterized by glycosyl composition analysis, glycosyl linkage analysis, nuclear magnetic resonance spectroscopy (NMR), fast atom bombardment mass spectro-metry (FAB-MS), matrix-assisted laser desorption time of flight mass spectro-metry (MALDITOF-MS), and tandem MS/MS. At least 17 different OS molecules were observed. These contained variable glycosyl residues, phosphate (P), and phospho-ethanolamine (PEA) substituents. These molecules contained either three, four, or five hexoses, and all contained four heptosyl residues. The four heptosyl residues consisted of one D,D-Hep and three L,D-Hep. Dephosphorylation of the OSs with aqueous 48% hydrofluoric acid (HF) reduced the number of molecules to about to seven; Hex(1)-(7)Hep(4)Kdo(1). Of these seven, Hex(2)Hep(4)Kdo(1), Hex(3)Hep(4)Kdo(1), and Hex(4)Hep(4)Kdo(1)were the major constituents. Thus, this NTHi LOS preparation is very heterogeneous, and contains structures different from those previously published for Haemophilus influenzae. The tandem MS/MS analysis and glycosyl linkage data suggest that the LOS oligosaccharides have the following structures where Hex is either a Glc or Gal residue.

Analysis of the expression and enzymatic properties of alpha1-->3fucosyltransferase from human lung carcinoma NCI-H69 and PC9 cells.

An analysis of alpha1-->3fucosyltransferase expression and enzyme properties has been conducted in human lung carcinoma NCI-H69 and PC9 cells. The results indicate that multiple forms of alpha1-->3 fucosyltransferase are found in these cells. RT-PCR experiments using total RNA from NCI-H69 and PC9 cells amplified transcripts for three of these enzymes, FucT-IV, -VI, and -VII. Fucose transfer into glycolipid acceptors mediated by truncated chimeric and full length recombinant FucT-IV and -VI enzymes was examined. Both enzymes were found to be type 2 chain specific, but only FucT-VI efficiently transferred fucose to both neutral and sialylated acceptors. A truncated recombinant form of FucT-VI was capable of fucose transfer to the internal Glc residue of a variety of glycolipid acceptors. This property was not observed with the recombinant full length enzyme, suggesting the N-terminal portion of the protein, composed of the intracellular domain, transmembrane domain, and a part of the stem region, is involved in interactions with glycolipid acceptors. Using taurodeoxycholate as the detergent, the distribution of initial fucose transfer into nLc6catalyzed by recombinant full length enzyme indicated 34% of the mono-fucosyl product was fucosylated at the III-GlcNAc and 66% at the V-GlcNAc for FucT-IV, and almost all of the FucT-VI mono-fucosyl product was III-GlcNAc fucosylated. Similar experiments with VI2NeuAcnLc6as the acceptor resulted in predominantly III-GlcNAc monofucosylation, although detectable V-GlcNAc monofucosylation was obtained with FucT-VI. When the cationic detergent G-3634-A was used, substantially greater initial transfer into the V-GlcNAc of both neutral and sialylated acceptors with FucT-VI was observed. Using nonsialylated acceptors, total alpha1-->3 fucosyltransferase activity in NCI-H69 cells was analyzed and found to be diminished 25-30% by exposure to 30 mM NEM, which can be attributed to FucT-VI inactivation. The remaining 70-75% of NEM-resistant activity is attributed to FucT-IV, an NEM-resistant enzyme form capable of fucosylating nonsialylated acceptors. These results suggest that multiple forms of alpha1-->3fucosyltransferase are expressed in NCI-H69 and PC9 cells, which may account for the observed properties of enzyme derived from these cell lines.

Novel Schizosaccharomyces pombe N-linked GalMan9GlcNAc isomers: role of the Golgi GMA12 galactosyltransferase in core glycan galactosylation.

Schizosaccharomyces pombe synthesizes very large N-linked galactomannans, which are elongated from the Man9GlcNAc2 core that remains after the trimming of three Glc residues from the Glc3Man9GlcNAc2 originally transferred from dolichyl pyrophosphate to nascent proteins in the endoplasmic reticulum. Prior to elongation of the galactomannan outer chain, the Man9GlcNAc2 core is modified into a family of Hex10-15GlcNAc2 structures by the addition of both Gal and Man residues (Ziegler et al. (1994) J. Biol. Chem., 269, 12527-12535). To understand the pathway of Man9GlcNAc2 modification, the Hex10GlcNAc-sized pool was isolated by Bio-Gel P-4 gel filtration from the endo H-released N-glycans of S.pombe glycoproteins. This pool yielded four major fractions, a, b, c, and g, on preparative high pH, anion exchange chromatography, that represented 10, 29, 46, and 13% of the total Hex10GlcNAc present, respectively. Structures of the glycan isomers present in each fraction were determined by one- and two-dimensional 1H NMR spectroscopy techniques. Fraction a is principally (approximately 93%) a Man10GlcNAc with a new alpha1,2-linked Man cap on the upper-arm of Man9GlcNAc. Fraction b contained two isomers of GalMan9GlcNAc in which an alpha1,2-linked terminal Gal had been added either to the upper (b1, 30%) or middle-arm (b2, 70%) of Man9GlcNAc. The gma12 - alpha1,2-galactosyltransferase-negative S. pombe strain (Chappell et al. (1994) Mol. Biol. Cell., 5, 519-528) did not make fraction b implying that the gma12p galactosyltransferase is responsible for synthesis of both isomers b1 and b2. Isomer c is Man10GlcNAc in which a new branching alpha1, 6-linked Man had been added to the lower-arm alpha1,3-linked core residue as found earlier in Saccharomyces cerevisiae and Pichia pastoris. Fraction g had less than molar stoichiometry of both Gal and Glc. The major isomer (g1, 85%) is the Man9GlcNAc core with an alpha1,3-linked branching Gal on the penultimate 2-O-substituted Man of the lower arm. This residue is also found on a novel O-linked oligosaccharide recently described in S.pombe; Manalpha1,2(Galalpha1, 3)Manalpha1,2Mannitol (Gemmill and Trimble (1999) Glycobiology, 9, 507-515). The second isomer (g2, 15%) is the partially processed Glc2Man9GlcNAc intermediate. Defining these Hex10GlcNAc structures provides a starting point for understanding the enzymology of N-linked galactomannan core heterogeneity seen on S.pombe glycoproteins.

Histochemical study of skin and gills of Senegal sole, Solea senegalensis larvae and adults.

A battery of horseradish peroxidase-conjugated lectins (Con A, WGA and DBA), as well as conventional histochemical techniques (PAS, saponification, Alcian Blue pH 0.1, 1, 2.5, chlorhydric hydrolisis, neuraminidase, Bromophenol blue, Tioglycollate reduction and Ferric-ferricyanide-FeIII) were used to study the content and distribution of carbohydrates, proteins and glycoconjugate sugar residues on the skin and gills of Senegal sole, Solea senegalensis larvae and adults: During larval development of Solea senegalensis (from hatching until day 45 posthatching), epidermal sacciform, as well as branchial and epidermal chloride cells were unreactive with all cytochemical tests performed in this paper. Mucous or goblet cells of the corporal skin and gills containing strongly sulphated acid glycoproteins were evident on days 15-20 of larval development, as well as in epidermal and branchial mucous cells of adult specimens, which also contained GlcNAc and/or sialic acid. In adult specimen, the proteic content was higher in branchial mucous cells than in epidermal cells. In larvae, variable amounts of glycoproteins containing sialic acid, GlcNAc, GalNAc, Man and/or Glc residues were observed in epithelial cells and/or cuticle. GlcNAc and/or sialic acid sugar residues were only weakly detected in glycoproteins of some epidermal and branchial mucous cells of larvae by day 45, because from hatching until metamorphosis, lectin reactions (WGA, Con A and DBA) were negative in mucous cells.

Identification of a Novel Core Type in SalmonellaLipopolysaccharide: COMPLETE STRUCTURAL ANALYSIS OF THE CORE REGION OF THE LIPOPOLYSACCHARIDE FROM SALMONELLA ENTERICA sv. ARIZONAE O62

For the first time, the complete structure of a lipopolysaccharide (LPS) core region from Salmonella enterica has been identified that is different from the Ra core type generally thought to be present in all Salmonella LPS. The LPSs from two rough mutants and the smooth form of S. enterica sv. Arizonae IIIa O62, which all failed to react with an Ra core type-specific monoclonal antibody and were resistant to phage FO1, were analyzed after chemical modification using monosaccharide analysis, mass spectrometry, and NMR spectroscopy. In the novel core type, the terminal D-GlcNAc residue present in the Ra core type, is replaced by a D-Glc residue. The O-specific polysaccharide is alpha1-->4-linked to the second distal Glc residue of the core. Furthermore, phosphoryl substituents attached to O-4 of L-glycero-D-manno-heptose (Hep) I and II were identified as 2-aminoethyl diphosphate (on Hep I) and phosphate (Hep II). [structure: see text] Abbreviations in Structure I are as follows: Hepp, L-glycero-D-manno-heptopyranose; Kdo, 3-deoxy-D-manno-oct-2-ulopyranosonic acid; PPEA, 2-aminoethyl diphosphate; R, O-specific polysaccharide. The presence of this novel core type in LPS of S. enterica should be taken into account in the development of a general antibody-based diagnostic system for Salmonella.

Developmental and hormonal regulation of protein N-glycosylation in the mammary gland.

Glycosylation represents the most common conjugation of both membrane-bound and secreted proteins of animal cells. Among the different types of glycosylation, the N-linked attachment of sugars to the polypeptide backbone is by far the most abundant modification. The biosynthesis of the precursor carbohydrate unit of these proteins is initiated by a stepwise assembly of Glc3Man9GlcNAc2P-P-Dol in the dolichol cycle, its transfer en bloc to the nascent polypeptide in the rough endoplasmic reticulum (RER), followed by excision of the glucosyl residues by processing-specific enzymes, glucosidase I and II, also resident in the endoplasmic reticulum. Additional posttranslational modifications of the carbohydrate moiety in the RER, Golgi, and trans-Golgi network, differ for individual glycoproteins for the completion of final products as high mannose, complex or hybrid glycoproteins en route to their final destinations in the secretory pathway. The enzyme GlcNAc-1-P transferase (GPT) catalyzes the first and committed step, i.e., the transfer of GlcNAc-1-P from UDP-GlcNAc to Dol-P to form GlcNAc-P-P-Dol, in the assembly of the oligosaccharide precursor. Glucosidase I triggers the maturation phase by clipping the distal alpha 1,2-linked Glc residue on the incipient glycoprotein. The critical juxtaposition of the two enzymes in the multistep pathway makes them excellent candidates for the overall regulation of protein N-glycosylation. The highly elevated needs of glycosylation during lactation demand regulation of glycosylation in the gland over and above the levels in the quiescent, virgin and postlactating, regressed gland.