GlcNAc Beta Research Articles

A serum-sensitive, sialyltransferase-deficient mutant of Neisseria gonorrhoeae defective in conversion to serum resistance by CMP-NANA or blood cell extracts

A stable, sialyltransferase-deficient mutant of Neisseria gonorrhoeae strain F62 totally defective in CMP-NANA-dependent lipopolysaccharide (LPS) sialylation was isolated by insertion mutagenesis with transposon Tn 1545-delta 3 and screened for unlabelled colonies following incubation with CMP-14C-NANA. In contrast to the parental strain which became serum resistant on incubation with CMP-NANA or blood cell extracts, the mutant, JB1, remained serum sensitive. French press extracts of strain F62 catalysed LPS sialylation, but corresponding extracts of the mutant were inactive. Five LPS components were detected by SDS-PAGE in the parental strain. Five components of the same Mr were also found in the mutant. Three identical components were detected by Western blotting using MAb 3F11, which recognizes the Gal beta 1-4GlcNAc groups in the conserved LPS components of F62 which can be sialylated. The mutant, JB1, is therefore deficient in the sialyltransferase that is essential for both LPS sialylation and conversion of serum-sensitive gonococci to serum resistance by either CMP-NANA or blood cell extracts. No evidence was obtained for an LPS sialylation pathway by blood cell extracts that is independent of CMP-NANA.

Carbohydrate Structure Analysis of Batroxobin, a Thrombin-Like Serine Protease from Bothrops moojeni Venom

The carbohydrate side chains of batroxobin were liberated from tryptic glycopeptides by treatment with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F, pyridylaminated and separated by two-dimensional HPLC. Neutral oligosaccharide derivatives obtained after desialylation were characterized by methylation analysis, liquid secondary-ion mass spectrometry, digestion with exoglycosidases and endoglycosidases and, in part, by acetolysis, whereas sialic acid constituents were identified by reverse-phase HPLC after conjugation with 1,2-diamino-4,5-methylene-dioxybenzene. The overall glycosylation status of the protein was studied by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The results revealed that batroxobin is heterogeneously glycosylated carrying predominantly diantennary, partially incomplete complex-type glycans in addition to hybrid-type species. Most glycans were core-fucosylated at C6 of the innermost GlcNAc. As a characteristic feature, galactose was completely replaced by GalNAc beta 4-substituents in complex-type antennae, the GlcNAc-residues of which were, in part, fucosylated at C3. Furthermore, evidence was obtained that suggested the presence of a novel type of glycoprotein-N-glycan comprising two GalNAc beta 4GlcNAc beta 4GlcNAc beta 2Man-antennae. Sialic acid residues represented a mixture of N-acetylneuraminic acid (Neu5Ac) and N-acetyl-4-O-acetylneuraminic acid (Neu4,5Ac2), which were exclusively linked to C3 of subterminal GalNAc. A precise assignment of these sialic acid derivatives to distinct oligosaccharide structures or antennae, however, was not carried out. Finally, MALDI-TOF-MS demonstrated that both potential N-glycosylation sites of batroxobin are substituted by carbohydrate chains. In conclusion, our studies revealed that this snake venom glycoprotein is characterized by a unique oligosaccharide pattern partly comprising novel structural elements.

Relationship between colonial morphology and adherence of Streptococcus pneumoniae.

Phase variants in colonial opacity of pneumococci differ in the ability to colonize the nasopharynx of infant rats. To explain this observation at a cellular level, we compared the ability of opacity variants to adhere to buccal epithelial cells, type II pneumocytes, or vascular endothelial cells and to the glycoconjugates that represent the cognate receptors at each of these sites. The transparent phenotype was associated with enhanced adherence to buccal cells (approximately 100%) and their receptor relative to that of the opaque variants. Only modest differences in adherence (< 45%) were demonstrated to resting lung and vascular cells. In contrast, adherence of transparent variants increased by 90% to lung cells stimulated with interleukin-1 and by 130% to endothelial cells stimulated with tumor necrosis factor. In contrast, cytokine stimulation did not influence the adherence of opaque pneumococci. This difference correlated with the unique ability of transparent variants to adhere to immobilized GlcNAc and to cells bearing transfected platelet-activating factor receptors. These results suggest that the mechanism of enhanced colonization of the nasopharynx in vivo by transparent as compared with opaque phase variants involves a greater ability to adhere to both GlcNAc beta 1-3Gal on buccal epithelial cells and GlcNAc and PAF receptors on cytokine-activated, as opposed to resting, lung and endovascular cells.

?1,3-Fucosylation of branched blood group I-type oligo-(N-acetyllactosamino)glycans by human milk transferases is restricted to distalN-acetyllactosamine units: The resulting isomers are separated by WGA-agarose chromatography

A partially purified preparation of alpha 1,3-fucosyltransferase(s) from human milk was used to [14C]fucosylate oligosaccharides containing Gal beta 1-4GlcNAc units. Substitution of N-acetyllactosamine at position 3' with a beta-linked N-acetyl-glucosamine enhanced the reactivity of the acceptor, whereas similar substitution at position 6' was inhibitory. Thus, the trisaccharide GlcNAc beta 1-6Gal beta 1-4GlcNAc (5), the branched tetrasaccharide GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc (11) and the triply branched decasaccharide GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-3[GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-6]Gal beta 1-4GlcNAc (26) gave remarkably poor yields of alpha 1,3-fucosylated products in comparison to GlcNAc beta 1-3Gal beta 1-4GlcNAc (3). beta 1,4-Galactosyl derivatives of 5 and 11, however, gave good yields of alpha 1,3-fucosylated products, but the fucosylation was restricted to the distal N-acetyllactosamine units of Gal beta 1-4GlcNAc beta 1-6Gal beta 1-4GlcNAc (16), Gal beta 1-4GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc (18) and also in Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3(Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc (22). Immobilized wheat germ agglutinin (WGA), possessing high affinity for 16 [1], revealed no affinity for the fucosylated derivative Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-6Gal beta 1-4GlcNAc (17). The isomeric heptasaccharides Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc (19) and Gal beta 1-4GlcNAc beta 1-3[Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-6]Gal beta 1-4GlcNAc (20) were readily separated from each other on WGA agarose, and so were the isomeric nonasaccharides Gal alpha 1-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3(Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc (23) and Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3[Gal alpha 1-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-6]Gal beta 1-4GlcNAc (24).

Enzymic remodelling of the N- and O-linked carbohydrate chains of human chorionic gonadotropin. Effects on biological activity and receptor binding.

The effects of altered terminal sequences in human chorionic gonadotropin (hCG) N- and O-linked glycans on receptor binding and signal transduction were analyzed using forms of hCG with remodelled carbohydrate chains. hCG derivatives were obtained by enzymic removal of the alpha 3-linked sialic acid residues followed by alpha 6-sialylation, alpha 3-galactosylation or alpha 3-fucosylation of uncovered Gal beta 1-->4GlcNAc (LacNAc) termini, or alpha 3-sialylation of Gal beta 1-->3GalNAc sequences. Also a form that carried GalNAc beta 1-->4-GlcNAc units, which are typical for pituitary hormone oligosaccharides, was derived by enzymic desialylation and degalactosylation followed by beta 4-N-acetylgalactosaminylation. The potency to stimulate testosterone production and the binding to the lutotropin/choriogonadotropin receptor of the preparations were compared with those of native and desialylated hCG (as-hCG). The decrease in bioactivity caused by desialylation of hCG was only restored upon alpha 6-sialylation of the Gal beta 1-->4GlcNAc beta 1-->-2Man alpha 1-->3Man branch of the N-linked glycans. This was without a major effect on receptor binding. Further alpha 6-sialylation, occurring at the Gal beta 1-->4GlcNAc beta 1-->2Man alpha 1-->6Man branch, resulted in a bioactivity below a level found with as-hCG, concomitant with a decreased receptor binding affinity. Similarly alpha 3-galactosylation of the Gal beta 1-->4GlcNAc beta 1-->2-Man alpha 1-->6Man branch yielded a hCG derivative that showed decreased bioactivity and receptor binding. alpha 3-Fucosylation of native as well as as-hCG also led to a decreased activity. Re-alpha 3-sialylation of the O-linked chains on as-hCG had little effect on the bioactivity and receptor binding. Hormone preparations with GalNAc beta 1-->4GlcNAc termini showed lower bioactivity and receptor affinity than as-hCG. It is concluded that the Gal beta 1-->4GlcNAc beta 1-->2Man alpha 1-->3Man- rather than the Gal beta 1-->4GlcNAc beta 1-->2-Man alpha 1-->6Man branch of the N-linked glycans on hCG plays an essential role in signal transduction, whereas the latter branch can potentially interfere with receptor binding. Furthermore attachment of sialic acid, but not of other sugars, to the first branch fulfils the requirement for the full expression of bioactivity, while sialylation of the O-linked chains is of minor importance.

The asparagine-linked carbohydrate of honeybee venom hyaluronidase

Hyaluronidase from the venom of the honeybee (Apis mellifera) has been purified by gelpermeation and cation exchange chromatography. Its asparagine-linked carbohydrate chains were released from tryptic glycopeptides with N-glycosidase A and reductively aminated with 2-aminopyridine. Separation of the fluorescent derivatives by size-fractionation and reversed-phase HPLC afforded eighteen fractions which were analysed by two-dimensional HPLC mapping combined with exoglycosidase digestions. The bulk of the N-linked glycans of hyaluronidase consisted of small oligosaccharides (Man1-3GlcNAc2), most of which were either alpha 1,3-monofucosylated or alpha 1,3-(alpha 1,6-)difucosylated at the innermost GlcNAc residue. High-mannose type structures constituted the minor fractions, together making up about 5% of the oligosaccharide pool from hyaluronidase. Four fractions, making up 8% of the N-linked glycans, contained the terminal trisaccharide GalNAc beta 1-4[Fuc alpha 1-3]GlcNAc beta 1- in beta 1,2-linkage to the core alpha 1,3-mannosyl residue. No evidence for the presence of O-glycans or sialic acids could be found.

Characterization of neutral glycosphingolipids in rat lens

Neutral glycosphingolipids were purified from non-cataractous lenses of Sprague-Dawley rats by a combination of solvent extraction, Folch's partition, and column chromatography using DEAE-Sephadex and Iatrobeads. Six major GSLs from monohexosylceramide to pentahexosylceramide were identified by sugar composition analysis, methylation analysis and glycosidase digestion. Structural relationships among the six neutral glycosphingolipids revealed metabolic pathways leading to the synthesis of Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1 ceramide (IV3Gal alpha nLc4), instead of a Lewis(x) glycolipid (Gal beta 1- 4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1 ceramide, III3FucnLc4), from neolactotetraosylceramide (nLc4), together with isoglobotriaosylceramide (iGb3). The alpha-galactosyl epitope, Gal alpha 1-3Gal-R, is evolutionarily conserved in many types of cells of non-primate mammals, prosimians and New World monkeys, but not in those of Old World monkeys or humans. This evolution-related difference in carbohydrate epitopes suggests different cell-to-cell attachments, which may be mediated through cell surface glycosphingolipids, between rat and human lenses.

Conformational analysis of the xylose-containing N-glycan of pineapple stem bromelain as part of the intact glycoprotein.

The conformational behavior of the N-glycan Man alpha 1-6(Xyl beta 1-2)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-3)GlcNAc beta of stem bromelain as part of the intact glycoprotein was investigated and compared with that of the same N-glycan as part of a bromelain-derived glycopeptide. Proton chemical shifts of the glycoprotein N-glycan were determined by 2D HOHAHA and 2D NOESY measurements, making use of the glycopeptide 1H NMR data. During each 2D NMR experiment about 4% of the glycoprotein denatured. Experimental data concerning interproton distances of the intact glycoprotein N-glycan were obtained by NOESY 1H NMR spectroscopy. Several theoretical models for the N-glycan, obtained by molecular dynamics simulations of the glycopeptide, were investigated. Comparison of experimental and theoretical NOESY cross peak intensities was performed with the program CROSREL. In comparison with the glycopeptide, the distribution of populations between two main conformations of the Fuc alpha 1-3GlcNAc linkage was altered. In addition, the omega = 60 degrees (gt) rotamer of the Man alpha 1-6Man linkage seems to be present for a significant period of time, whereas in the glycopeptide the omega = -60 degrees (gg) conformation exists exclusively. Except for the Xyl beta 1-2Man linkage, the mobilities around the glycosidic linkages in the glycoprotein were reduced compared with those in the glycopeptide, especially concerning the Fuc alpha 1-3GlcNAc and Man alpha 1-6Man linkages. These findings might be the result of an interaction of the polypeptide chain with the Fuc alpha/Man alpha side of the N-glycan. A qualitative analysis of the NMR spectra showed a larger degree of mobility in the denatured glycoprotein N-glycan than in the intact glycoprotein.

Identification of a novel UDP-GalNAc:GlcNAc beta-R beta 1-4 N-acetylgalactosaminyltransferase from the albumen gland and connective tissue of the snail Lymnaea stagnalis.

Both the albumen gland, one of the female accessory sex glands, and connective tissue of the freshwater snail Lymnaea stagnalis contain N-acetylgalactosaminyltransferase activity, capable of transferring GalNAc from UDP-GalNAc in beta 1-4 linkage to the terminal GlcNAc residue of GlcNAc beta-R. The albumin gland enzyme was partially purified by affinity chromatography on UDP-hexanolamine-Sepharose 4B. Using GlcNAc beta 1-2Man alpha 1-6(GlcNAc beta 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc or GlcNAc beta 1-OMe as substrates, the enzyme showed an absolute requirement for Mn2+ with an optimum concentration of 12.5-50 mM. The optimal pH was approximately pH 7.0. The enzyme activity was independent of the Triton X-100 concentration in the range 0.25-2.5%, and no activation effect was found. The more labile connective tissue microsomal enzyme, subjected to the same optimization procedure, gave comparable results. Both enzyme activities have similar substrate specificities towards GlcNAc or GlcNAc beta 1-OMe, and towards oligosaccharides or glycopeptides with a non-reducing terminal beta-GlcNAc unit, but cannot act on GlcNAc alpha 1-OMe. Saccharides with non-reducing terminal Gal or GalNAc residues, and free GalNAc, Gal or Glc residues are not acceptors. Product analysis was carried out for albumen gland N-acetylgalactosaminyltransferase and four acceptors having GlcNAc beta 1-R as the terminal non-reducing unit, and for connective tissue N-acetylgalactosaminyltransferase with GlcNAc beta 1-OMe as acceptor. In all instances, products with GalNAc beta 1-4-linked to GlcNAc were obtained, showing that the connective tissue and the albumen gland activities are probably from one enzyme. This enzyme activity can be identified as UDP-GalNAc:GlcNAc beta-R beta 1-4 N-acetylgalactosaminyltransferase, and is probably involved in the biosynthesis of N,N'-diacetyllactosediamine-containing glycoproteins, like hemocyanin, in the snail L. stagnalis.

Monoclonal antibody LU-BCRU-G7 against a breast tumour-associated glycoprotein recognizes the disaccharide Gal beta 1-3GlcNAc.

The monoclonal antibody LU-BCRU-G7, that was generated by in vitro immunization, shows clinical value as a prognostic marker in early stage breast carcinoma. It has now been characterized with regard to its binding epitope. Using a recently described method based on the construction of N-substituted polyacrylamide (PAA) derivatives of carbohydrates (pseudopolysaccharides), the structure of the epitope for the monoclonal antibody LU-BCRU-G7 has been determined. Competitive binding assays and inhibitory enzyme-linked immunosorbent assays (ELISAs) using these pseudopolysaccharides have shown the LU-BCRU-G7 epitope to be a disaccharide Gal beta 1-3GlcNAc (Lec; where Gal is D-galactose, Glc is D-glucose and GlcNAc is N-acetyl-D-glucosamine). Both galactose and N-acetyl glucosamine moieties are essential for binding. Substitution on C-2 or C-3 of the terminal galactose abolished binding, as did galactose-alpha terminated oligosaccharides. The galactose moiety alone, as expressed by the Gal beta-PAA conjugate, appeared to be a more important feature of the epitope than the GlcNAc-PAA conjugate, which failed to bind or inhibit the LU-BCRU-G7 antibody. In the N-acetyl glucosamine moiety, binding was decreased but not eliminated by fucose substitution, as in Lea, or change in configuration of C-4, as in Gal beta 1-3GlcNAc. Omission of the NAc group resulted in complete loss of activity. The tetrasaccharide lacto-N-tetraose, although containing the terminal Lec disaccharide, does not react with the antibody, suggesting conformational interference of the binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

The biosynthesis of Lewis X in Helicobacter pylori.

The biosynthesis of the Lewis X determinant (Gal beta 1-4 [Fuc alpha 1-3]GlcNAc beta-) in three strains of Helicobacter pylori has been investigated. Strains UA 861, UA 802 and UA 1182 contain alpha 1,3 fucosyltransferase and beta 1,4 galactosyltransferase activities that synthesize the Lewis X structure by the transfer of monosaccharides from GDP-fucose and UDP-galactose donors, respectively. The enzyme reaction products that formed were characterized by capillary zone electrophoresis and by 1H-NMR spectroscopy. The biosynthetic pathway is therefore identical to that found in humans. In the three strains, the fucosyltransferase and galactosyltransferase activities differed in various cellular fractions. Km values for their donor and acceptor substrates also differed.

Characterization of a novel mucin sulphotransferase activity synthesizing sulphated O-glycan core 1,3-sulphate-Gal beta 1-3GalNAc alpha-R.

A novel sulphotransferase (sulpho-T) activity from rat colonic mucosa was characterized using O-glycan core 1 substrate, Gal beta 1-3GalNAc alpha-benzyl. Derivatives of Gal beta 1-3GalNAc- were used to demonstrate that the 3- and 4-hydroxyl of Gal and the 2-acetamido group of the GalNAc residue of Gal beta 1-3GalNAc alpha-benzyl substrates were important for activity. Sulphated product using Gal beta 1-3GalNAc alpha-benzyl as substrate was analysed by ion spray mass spectrometry, methylation analysis, high-pH anion-exchange chromatography and beta-galactosidase digestion. The results suggested that sulphate was added to the 3-position of the Gal residue. The synthesis of core 2 from core 1 by UDP-GlcNAc: Gal beta 1-3GalNAc beta 6-GlcNAc-transferase was inhibited by sulphation of the Gal residue, indicating that GlcNAc beta 1-6 branching has to precede sulphation in the O-glycan core 1 processing pathway. These data demonstrate several novel pathways in the synthesis of sulphated mucin-type oligosaccharides.

Characterization of the epitope recognized by a mAb that reacts differentially with murine suppressor T cells.

Although reliable antibodies are available that distinguish human suppressor T (Ts) cells from CTL and other T cells, few are available for murine Ts cells. We have developed a mAb (984D4.6.5) that, in the presence of complement, depletes alloantigen-specific Ts cells but not CTL. This antibody recognizes activated Ts cells but not their precursors. In these studies, flow cytometric analysis demonstrates that 984D4.6.5 reacts with several Ts cell hybridomas, cloned Ts cell lines and WEHI-3 (a myelomonocytic tumor cell line). Reactivity was not detected with BW5147, Th cell hybridomas, cloned Th cells, CTL lines and hybridomas, B cell lines, thymocytes, splenocytes, bone marrow cells nor a variety of tumor cells. Among 984D4.6.5 positive lines, expression is heterogeneous and the number of cells expressing high levels of the epitope is increased when the hybridomas are maintained at a relatively high cell density. Neuriminidase and pronase deplete the epitope recognized by mAb 984D4.6.5. Protein synthesis and glycosylation inhibitors also reduce expression of this epitope. These observations suggest that the epitope recognized by 984D4.6.5 is a carbohydrate linked to a polypeptide. This antibody was tested by ELISA for binding to a large panel of carbohydrates and glycolipids coupled to BSA. The only one that bound 984D4.6.5 was LS tetrasaccharide c (NeuNAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc), an O-linked carbohydrate. Comparative analysis shows that both the sequence and the linkage of these sugars are essential to the reactivity with the 984D4.6.5 antibody. This epitope is expressed by a glycoprotein of approximately 200 kDa, as shown by Western blots. The identity of this glycoprotein remains to be determined, but indirect evidence suggests that it is not CD45.

Carbohydrate binding specificity of the B-cell maturation mitogen from Artocarpus integrifolia seeds.

Artocarpin, a mannose-specific lectin, is a homotetrameric protein (M(r) 65,000) devoid of covalently attached carbohydrates and consists of four isolectins with pI in the range 5-6.5. Investigations of its carbohydrate binding specificity reveal that among monosaccharides, mannose is preferred over glucose. Among mannooligosaccharides, mannotriose (Man alpha 1-3[Man alpha 1-6]Man) and mannopentaose are the strongest ligands followed by Man alpha 1-3Man. Extension of these ligands by GlcNAc at the reducing ends of mannooligosaccharides tested remarkably improves their inhibitory potencies, while substitution of both the alpha 1-3 and alpha 1-6 mannosyl residues of mannotriose and the core pentasaccharide of N-linked glycans (Man alpha 1-3[Man alpha 1-6]Man beta 1-4GlcNAc beta 1-4GlcNAc) by GlcNAc or N-acetyllactosamine in beta 1-2 linkage diminishes their inhibitory potencies. Sialylated oligosaccharides are non-inhibitory. Moreover, the substitution of either alpha 1-3 or alpha 1-6 linked mannosyl residues of M5Gn or both by mannose in alpha 1-2 linkage leads to a considerable reduction of their inhibitory power. Addition of a xylose residue in beta 1-2 linkage to the core pentasaccharide improves the inhibitory activity. Considering the fact that artocarpin has the strongest affinity for the xylose containing hepasaccharide from horseradish peroxidase, which differs significantly from all the mannose/glucose-specific lectins, it should prove a useful tool for the isolation and characterization of glycoproteins displaying such structure.

Genetic locus for the biosynthesis of the variable portion of Neisseria gonorrhoeae lipooligosaccharide.

A locus involved in the biosynthesis of gonococcal lipooligosaccharide (LOS) has been cloned from gonococcal strain F62. The locus contains five open reading frames. The first and second reading frames are homologous, but not identical, to the fourth and fifth reading frames, respectively. Interposed is an additional reading frame which has distant homology to the Escherichia coli rfaI and rfaI genes, both glucosyl transferases involved in lipopolysaccharide core biosynthesis. The second and fifth reading frames show strong homology to the lex-1 or lic2A gene of Haemophilus influenzae, but do not contain the CAAT repeats found in this gene. Deletions of each of these five genes, of combinations of genes, and of the entire locus were constructed and introduced into parental gonococcal strain F62 by transformation. The LOS phenotypes were then analyzed by SDS-PAGE and reactivity with monoclonal antibodies. Analysis of the gonococcal mutants indicates that four of these genes are the glycosyl transferases that add GalNAc beta 1-->3Gal beta 1-->4GlcNAc beta 1-->3 Gal beta 1--4 to the substrate Glc beta 1-->4Hep--R of the inner core region. The gene with homology to E. coli rfaI/rfaI is involved with the addition of the alpha-linked galactose residue in the biosynthesis of the alternative LOS structure Gal alpha 1-->4Gal beta 1-->4Glc beta 1-->4Hep-->R. Since these genes encode LOS glycosyl transferases they have been named lgtA, lgtB, lgtC, lgtD, and lgtE. The DNA sequence analysis revealed that lgtA, lgtC, and lgtD contained poly-G tracts, which, in strain F62 were, respectively, 17, 10, and 11 bp. Thus, three of the LOS biosynthetic enzymes are potentially susceptible to premature termination by reading frame changes. It is likely that these structural features are responsible for the high-frequency genetic variation of gonococcal LOS.

Lectin-histochemical analysis of glycans in ovine and bovine near-term placental binucleate cells.

Chorionic binucleate cells (BNC) occur in several ruminants including cow, deer, goat and sheep. They migrate through the chorionic tight junction to fuse with uterine epithelial cells and discharge their granules into maternal connective tissue. We have compared the BNC of near-term, resin-embedded, ovine and bovine placentae using 15 biotinylated lectins and an avidinperoxidase revealing system. There was pronounced conservation of saccharides between the two species. Several sub-types of N-glycan were present, with highly branched structures being abundant, as shown by Galanthus nivalis, Pisum sativum and Phaseolus vulgaris (leuko) agglutinins. Among the non-reducing terminal saccharides conserved were GalNAc alpha 1,3(Fuc alpha 1,2)-Gal beta 1,4GlcNAc beta 1-, GalNAc alpha 1,6Gal beta 1-, Gal beta 1-, Gal beta 1,4GlcNAc- and Gal beta 1,3GalNAc alpha 1- shown by Dolichos biflorus, Wisteria floribunda, Erythrina cristagalli, and Maclura pomifera agglutinins, respectively. Arachis hypogaea and Glycine max agglutinins tended to bind to bovine BNC at different stages of maturity, while fucosyl residues detectable by Tetragonolobus purpureus and Ulex europaeus-1 agglutinins were not observed in either species. The only major difference related to sialyl residues, with alpha 2,3-linked sialic acid being present in bovine (Maackia amurensis, Limax flavus) and alpha 2,6 sialic acid being present in ovine (Sambucus nigra agglutinin) cells. This conservation of glycan may be related to glycosylation of peptide hormones in the granules, and may thus be important in the targeting of these hormones to their receptors.

Demonstration of a Novel UDPGalNAc:GlcNAc ß1-4 N-Acetylgalactosaminyltransferase in Extracts of Schistosoma mansoni

It was recently demonstrated that complex-type N-linked oligosaccharides in glycoproteins synthesized by Schistosoma mansoni contain the unusual terminal sequence GalNAc beta 1-4GlcNAc beta 1-2Man alpha 1-2R. This structure suggests that the parasite might contain a novel glycosyltransferase that can add GalNAc to terminal GlcNAc residues in N-linked oligosaccharides. This report describes an assay for this enzyme, designated UDPGalNAc:GlcNAc beta 1-4 N-acetylgalactosaminyltransferase (beta 1-4GalNAcT), and demonstrates the presence of the enzyme activity in schistosome extracts. As an acceptor for the beta 1-4GalNAcT, we prepared from human fibrinogen a truncated biantennary glycopeptide that contained terminal GlcNAc residues. When this acceptor was incubated with schistosome extracts in the presence of UDP-[3H]GalNAc, Mn2+, and detergent, [3H]GalNAc was transferred to the glycopeptide acceptor. Approximately 75% of the radioactivity in the product isolated by lectin affinity chromatography was recovered as [3H]GalNAc following hydrolysis; likewise, a majority of the isolated product was bound by immobilized Wisteria floribunda agglutinin, a lectin that binds to schistosome-derived oligosaccharides containing terminal beta 1-4-linked GalNAc residues. The activity of the beta 1-4GalNAcT in schistosome extracts was dependent on time, protein, and UDPGalNAc.

Characterization of the major sulfated protein of mouse pancreatic acinar cells: A high molecular weight peripheral membrane glycoprotein of zymogen granules

The major sulfated protein of the mouse pancreatic acinar cell, gp300, has been identified and characterized with monoclonal and polyclonal antibodies. gp300 is a glycoprotein of M(r) = 300,000 which contains approximately 40% of metabolically incorporated [35S]sulfate in the acinar cell. Sulfate on gp300 is resistant to hot 1N HCl, but sensitive to alkaline hydrolysis, demonstrating that the sulfate is carbohydrate-linked rather than tyrosine-linked. gp300 metabolically labeled with [3H]glucosamine and [35S]sulfate was chemically and enzymatically treated followed by Bio-Gel P-10 gel filtration. Both labels were resistant to treatments which degrade glycosaminoglycans. Treatment of dual-labeled gp300 with PNGase F to cleave N-linked oligosaccharides released approximately 17% of [3H] and little [35S]. Mild alkaline borohydride treatment after removal of N-linked sugar released the remainder of both labels, indicating the presence of sulfated O-linked oligosaccharides. Biosynthesis studies and PNGase F digestion indicate that the core protein is approximately 210 kDa, with apparent contributions of approximately 35 kDa N-linked sugar, and approximately 55 kDa O-linked sugar. Lectin blotting and glycosidase digestion demonstrated the presence of Gal beta(1-3)GalNAc and sialic acid alpha(2-3)Gal in O-linked oligosaccharide, and Gal beta(1-4)GlcNAc in N-linked oligosaccharide. Immunolocalization and subcellular fractionation showed that gp300 is a peripheral membrane protein localized to the lumenal face of the zymogen granule membrane. gp300 was not secreted in response to hormone stimulation of acini, so it is not a secretory product. Immunoblot analysis showed that gp300 is present in other gastrointestinal tissues and parotid glands. Localization of this nonsecreted sulfated glycoprotein to exocrine secretory granule membranes suggests that gp300 may have a role in granule biogenesis.

The recognition of three different epitopes for the H-type 2 human blood group determinant by lectins of Ulex europaeus, Galactia tenuiflora and Psophocarpus tetragonolobus (winged bean).

The chemical mapping of the regions of H-type 2 human blood group-related trisaccharide (Fuc alpha (1-2)Gal beta (1-4)GlcNAc beta Me) that are recognized by three different lectins, the so-called epitopes, are reviewed together with an account of how and why oligosaccharides form specific complexes with proteins as presently viewed in this laboratory. The occasion is used to report the synthesis of the various mono-O-methyl derivatives of the above trisaccharide that were used in these investigations. Also, Fuc alpha (1-2)Gal beta (1-4)Xyl beta Me was synthesized in order to examine whether or not the hydroxymethyl group of the GlcNAc residue participates in the binding reaction.

The Immunologically Reactive O‐Linked Polysaccharide Chains Derived from Circulating Cathodic Antigen Isolated from the Human Blood Fluke Schistosoma Mansoni have Lewis x as Repeating Unit

The gut-associated excretory antigen circulating cathodic antigen (CCA) was isolated by immunoaffinity chromatography from adult Schistosoma mansoni worms, which were collected from infected golden hamsters. This antigen is probably involved in protection of the schistosome gut and is increasingly used in highly sensitive and specific immunodiagnostic assays. Amino acid analysis before and after alkaline borohydride treatment of CCA and monosaccharide analysis indicated that CCA is O-glycosylated mostly via GalNAc-Thr. After reductive alkaline treatment, the O-linked carbohydrate chains were fractionated by gel-permeation chromatography, followed by normal-phase HPLC on LiChrosorb-NH2. Carbohydrate-positive fractions were investigated by one-dimensional and two-dimensional 1H-NMR spectroscopy, fast atom bombardment mass spectrometry and collision-induced-dissociation tandem mass spectrometry. The analyses showed that the low-molecular-mass O-linked oligosaccharide alditols (the minor fraction) consist of disaccharides to hexasaccharides having the Gal beta (1-3)GalNAc-OL core in common. The major carbohydrate fraction comprises a population of polysaccharides, containing Lewis x repeating units (-3)Gal beta (1-4)[Fuc alpha (1-3)]GlcNAc beta (1-). CCA-specific monoclonal antibodies and IgM antibodies in patient sera recognized the fucosylated O-linked carbohydrate antigenic structures. Since CCA evokes a strong IgM antibody response and carbohydrate structures containing repeating Lewis x units are found on circulating neutrophils, it is proposed that the antigenic poly-Lewis x polysaccharide of CCA is involved in the induction of auto-immunity against granulocytes, resulting in the mild to moderate neutropenia observed during schistosome infection.