Internal N-acetylglucosamine Research Articles

The Structural Difference of Isobaric N-Glycans of Two Microalgae Samples Reveals Taxonomic Distance.

Microalgae of the Chlorella clade are extensively investigated as an environmentally friendly source of renewable biofuels and high-value nutrients. In addition, essentially unprocessed Chlorella serves as wholesome food additive. A recent study on 80 commercial Chlorella preparations revealed an unexpected variety of protein-linked N-glycan patterns with unprecedented structural features, such as the occurrence of arabinose. Two groups of products exhibited a characteristic major N-glycan isobaric to the Man2GlcNAc2XylFuc N-glycan known from pineapple stem bromelain, but tandem mass spectrometry (MS/MS) analysis pointed at two types of N-glycan different from the bromelain structure, as well as from each other. Here we report the exact structures of these two novel N-glycan structures, elucidated by nuclear magnetic resonance spectroscopy and MS/MS, as well as on their phylogenetic context. Despite their humble size, these two N-glycans exhibited a very different design with structural features unrelated to those recently described for other Chlorella-clade strains. The major glycans of this study presented several novel structural features such as substitution by arabinose or xylose of the internal N-acetylglucosamine, as well as methylated sugars. ITS1-5.8S-ITS2 rDNA barcode analyses revealed that the xylose-containing structure derived from a product primarily comprising Scenedesmus species, and the arabinose-containing glycan type related to Chlorella species (SAG211-34 and FACHB-31) and to Auxenochlorella. This is another example where characteristic N-glycan structures distinguish phylogenetically different groups of microalgae.

Regional morphology and mucus composition in the urogenital papilla skin of the blackbelly rosefish Helicolenus dactylopterus (Delaroche, 1809)

Blackbelly rosefish Helicolenus dactylopterus is a zygoparous fish whose males are equipped with the copulating organ named urogenital papilla (UP). This study deals with the morphology and the glycoconjugate pattern of the UP epidermis, which is the male tissue interacting with the female internal body during copulation. The carbohydrate content was studied by means of conventional and lectin histochemistry. The epidermis was shown to be a stratified cuboidal epithelium and to exhibit characteristic intraepithelial pits in the apical zone. The mucous cells are scattered in the epidermis. The epidermal cell layers and their thickness as well as the size of mucous cells varied along the UP. Conventional histochemistry showed that the mucous cells contained i) only neutral glycoproteins in the basal zone; ii) both neutral and acidic non-sulphated glycans as well as only acidic non-sulphated or sulphated glycoconjugates in the intermediate zone; iii) neutral and sulphated glycoconjugates in the apical zone. The mucous cells in the basal region expressed O-linked (mucin type) glycans terminating with αGalNAc, Galβ1,3GalNAc which could be α2,3-linked to sialic acid, and high mannose type N-linked glycans terminating with fucose, lactosamine, and sialic α2,6-linked to galactose/N-acetylgalactosamine; terminal Gal and terminal/internal GlcNAc were also found. The mucous cells in the intermediate zone lacked Galβ1,3GalNAc and showed less terminal α2,3-linked sialic acid, lactosamine, fucose, galactose, and internal N-acetylglucosamine residues. In the apical region, mucous cells only exhibited O-glycans terminating with GalNAc and N-acetylglucosamine. The demonstrated region-specific differences in the UP skin provide new insights into the reproductive biology of fishes with internal fertilization.

Production and Characterization of Transgenic Mice Systemically Expressing Endo- -Galactosidase C

The alphaGal epitope (Galalpha1-3Gal) is a sugar structure expressed on the cell surface of almost all organisms except humans and old-world-monkeys, which express natural anti-alphaGal antibodies. The presence of these antibodies elicits a hyper acute rejection (HAR) upon xenotransplantation of cellular materials, such as from pigs to human beings. Endo-beta-galactosidase C (EndoGalC), an enzyme isolated from Clostridium perfringens, removes the alphaGal epitope by cleaving the Galbeta1-4GlcNAc linkage in the Galalpha1-3Galbeta1-4GlcNAc sequence. To explore the possibility that cells or organs from transgenic pigs systemically expressing EndoGalC might be suitable for xenotransplantation, we first introduced the EndoGalC transgene into the mouse genome via pronuclear injection. The progeny of the resulting transgenics expressed EndoGalC mRNA and protein. Flow cytometry and histochemical analyses revealed a dramatic reduction in the expression of the alphaGal epitope in these mice. They also exhibited abnormal phenotypes, such as occasional death immediately after birth, growth retardation, and transient skin lesions. Interestingly, the phenotypic abnormalities seen in these transgenics were similar to those observed in beta1,4-galactosyltransferase 1 (beta4GalT-1) knockout (KO) mice. Most probably, these phenotypes were caused by exposure of the internal N-acetylglucosamine residue at the end of the sugar chain on the cell surface. The present findings also provide some basis for evaluating possible application of the transgenic approach for xenotranplantation.

SHAP Potentiates the CD44-mediated Leukocyte Adhesion to the Hyaluronan Substratum

CD44-hyaluronan (HA) interaction is involved in diverse physiological and pathological processes. Regulation of interacting avidity is well studied on CD44 but rarely on HA. We discovered a unique covalent modification of HA with a protein, SHAP, that corresponds to the heavy chains of inter-alpha-trypsin inhibitor family molecules circulating in blood. Formation of the SHAP.HA complex is often associated with inflammation, a well known process involving the CD44-HA interaction. We therefore examined the effect of SHAP on the CD44-HA interaction-mediated lymphocyte adhesion. Under both static and flowing conditions, Hut78 cells (CD44-positive) and CD44-transfected Jurkat cells (originally CD44-negative) adhered preferentially to the immobilized SHAP.HA complex than to HA. The enhanced adhesion is exclusively mediated by the CD44-HA interaction, because it was inhibited by HA, but not IalphaI, and was completely abolished by pretreating the cells with anti-CD44 antibodies. SHAP appears to potentiate the interaction by increasing the avidity of HA to CD44 and altering their distribution on cell surfaces. Large amounts of the SHAP.HA complex accumulate in the hyperplastic synovium of rheumatoid arthritis patients. Leukocytes infiltrated to the synovium were strongly positive for HA, SHAP, and CD44 on their surfaces, suggesting a role for the adhesion-enhancing effect of SHAP in pathogenesis.

Pigeon fanciers' lung: identification of disease-associated carbohydrate epitopes on pigeon intestinal mucin.

Pigeon intestinal mucin, a complex high molecular weight glycoprotein, is a key antigen in the development of pigeon fanciers' lung (PFL). We have studied the specificity of antibodies to mucin in patients with PFL and asymptomatic antibody-positive individuals. Extensive papain digestion, which removes the non-glycosylated regions of the mucin leaving the heavily glycosylated 'bottle brush' regions, resulted in a 600-fold decrease in IgG3 antibody titres with little effect on IgG1 and IgG2 titres. This suggests that IgG1 and IgG2 are directed against the region rich in O-linked sugar chains whilst the majority of the IgG3 is directed against epitopes which are proteinase-sensitive. Lectin mapping of the carbohydrates present on pigeon intestinal mucin demonstrated high levels of exposed N-acetyl neuraminic acid, N-acetyl galactosamine and N-acetyl glucosamine, with lower levels of fucose and some galactose. Sera from pigeon fanciers inhibited binding of lectins specific for N-acetyl neuraminic acid, N-acetyl galactosamine, internal N-acetyl glucosamine and fucose. Sera from people with PFL, compared with sera from asymptomatic antibody-positive fanciers, had significantly higher titres of antibody that inhibited binding of four lectins specific for N-acetyl galactosamine and one fucose-specific lectin, suggesting that these sugars may play a dominant role in disease-associated epitopes. The results suggest that different IgG subclasses recognize different epitopes on mucin and that the epitopes recognized by the major subclasses are present on the O-linked oligosaccharides. Further, the carbohydrate-specific anti-mucin antibodies produced by PFL patients may differ in their specificity from those found in asymptomatic individuals.

Evidence for the Covalent Binding of SHAP, Heavy Chains of Inter-α-Trypsin Inhibitor, to Hyaluronan

We previously showed that serum-derived 85-kDa proteins (SHAPs, serum-derived hyaluronan associated proteins) are firmly bound to hyaluronan (HA) synthesized by cultured fibroblasts. SHAPs were then identified to be the heavy chains of inter-alpha-trypsin inhibitor (ITI) (Huang, L., Yoneda, M., and Kimata, K. (1993) J. Biol. Chem. 268, 26725-26730). In this study, the SHAP.HA complex was isolated from pathological synovial fluid from human arthritis patients. The SHAP.HA complex was digested with thermolysin, followed by CsCl gradient centrifugation. The HA-containing fragments thus obtained were further digested with chondroitinase AC II and subjected to TSK gel high performance liquid chromatography (HPLC). Peptide-HA disaccharide-containing fractions (the SHAP.HA binding regions) were further purified by reverse phase HPLC. Major peaks were analyzed by protein sequencing and mass spectrometry (electrospray ionization mass spectrometry and collision induced dissociation-MS/MS). By comparison with the reported C-terminal sequences of the human ITI family, the peptides were found to correspond to tetrapeptides derived from the C termini of heavy chains 1 of and 2 of inter-alpha-trypsin inhibitor (HC1 and HC2), and heavy chain 3 of pre-alpha-trypsin inhibitor (HC3), respectively, and a heptapeptide from HC1. Mass spectrometric analyses suggested that the C-terminal Asp of each heavy chain was esterified to the C6-hydroxyl group of an internal N-acetylglucosamine of HA chain. This report is the first demonstration to give evidence for the covalent binding of proteins to HA.

Spectrum of sialylated and nonsialylated fuco-oligosaccharides bound by the endothelial-leukocyte adhesion molecule E-selectin. Dependence of the carbohydrate binding activity on E-selectin density.

Carbohydrate recognition by the human endothelial-leukocyte adhesion molecule, E-selectin, has been investigated by binding studies using 3H-labeled Chinese hamster ovary cells expressing different levels of the transfected full-length adhesion molecule and a series of structurally defined oligosaccharides linked to the lipid phosphatidylethanolamine dipalmitoate (neoglycolipids) and synthetic glycolipids chromatographed on silica gel plates or immobilized on plastic wells. Evidence is presented for density-dependent binding of the membrane-associated E-selectin not only to 3'-sialyl-lacto-N-fucopentaose II (3'-S-LNFP-II) and 3'-sialyl-lacto-N-fucopentaose III (3'-S-LNFP-III) which express the sialyl Le(a) and sialyl Le(x) antigens, respectively, but also to the nonsialylated analogue LNFP-II; there is a threshold density of E-selectin required for binding to these sialylated sequences, and binding to the nonsialylated analogue is a property only of cells with the highest density of E-selectin expression. The presence of fucose linked to subterminal rather than to an internal N-acetylglucosamine is shown to be a requirement for E-selectin binding, and although the presence of sialic acid 3-linked to the terminal galactose of the LNFP-II or LNFP-III sequences substantially enhances E-selectin binding, the presence of 6-linked sialic acid abolishes binding. E-selectin binding is unaffected in the presence of the blood group H fucose (alpha 1-2 linked to galactose to form the Le(b) antigen). However, the binding is abolished when in addition alpha 1-3-linked N-acetylgalactosamine to the galactose (blood group A antigen) is present. These results indicate that some E-selectin-mediated adhesive events may be influenced by blood group status.

Carbohydrate Composition of β-Core

beta-Core is a major component of the hCG-related molecules found in pregnancy urine. We previously have purified the beta-core molecule and have deduced portions of its carbohydrate structure based on lectin binding data. In the present study we used recently developed technology to determine the carbohydrate composition of beta-core and hCG beta (CR119). For direct compositional analysis, parallel samples were hydrolyzed in trifluoroacetic acid and analyzed for sialic acid and neutral sugars without prior derivatization. Separation of the monosaccharides was achieved by HPLC on a Dionex CarboPac column eluted at high pH, and the resolved monosaccharides were quantified by pulsed amperometric detection. The amounts of sugar that were found relative to peptide indicated the presence of two N-linked oligosaccharides per molecule on both beta-core and hCG beta. hCG beta contained additional sugars consistent with the presence of four O-linked oligosaccharides. Compared to hCG beta, beta-core contained negligible sialic acid, galactose, or N-acetylgalactosamine. The compositional data suggest that beta-core does not contain N-acetylglucosamine at the nonreducing end of the molecule, whereas the trimannosyl-chitobiose core is apparently intact at both glycosylation sites, consistent with the ability of the molecule to bind to Concanavalin-A. Comparable fucose contents and abilities of beta-core and hCG beta to bind to Lens culinaris indicate a similar extent of fucosylation on the internal N-acetylglucosamine in both molecules. We propose that the N-linked oligosaccharides on beta-core closely resemble the underlying N-linked structures of hCG beta with the antennary sialic acid, galactose, and N-acetylglucosamine removed.

The GDP-fucose:N-acetylglucosaminide 3-alpha-L-fucosyltransferases of LEC11 and LEC12 Chinese hamster ovary mutants exhibit novel specificities for glycolipid substrates.

Previous studies have shown that the GDP-fucose:N-acetylglucosaminide 3-alpha-L-fucosyltransferase (alpha (1,3) fucosyltransferase (Fuc-T)) activities expressed by the Chinese hamster ovary cell mutants LEC11 (Fuc-TI) and LEC12 (Fuc-TII) are different enzymes and indicated that Fuc-TI might act on sialylated lactosamine sequences (Campbell, C., and Stanley, P. (1984) J. Biol. Chem. 259, 11208-11214). In this paper we show that CSLEX-1, a monoclonal antibody specific for NeuNac alpha (2,3)Gal beta (1,4)(Fuc alpha (1,3))GlcNAc beta 1 sequences, bound to LEC11 cells but not to LEC12 cells. Direct evidence that Fuc-TI could act on sialylated substrates was sought with a series of glycolipid acceptors. Optimal assay conditions in crude cell extracts were determined with nLc4, a glycolipid which accepted fucose with both Fuc-TI and Fuc-TII to generate the Lex antigenic determinant. The two enzymes differed in their detergent sensitivities, pH optima, Mn2+ requirements, and apparent Km values for nLc4. When sialylated glycolipids were examined as substrates, Fuc-TI added fucose to IV3NeuNAcnLc4 but not to IV6NeuNAcnLc4, whereas Fuc-TII was unable to utilize either glycolipid as a substrate. Further studies showed that Fuc-TI and Fuc-TII possess novel specificities for glycolipids containing two lactosamine sequences as potential fucose acceptors. Fuc-TI exhibited good activities with VI3NeuNAcnLc6 and VI6NeuNAcnLc6 whereas Fuc-TII had very low activity with both substrates. Glycosidase digestions of the labeled products showed that Fuc-TI added fucose primarily to the internal N-acetylglucosamine of both glycolipids. The same preference for the internal N-acetylglucosamine was shown by Fuc-TI when nLc6 was the acceptor. In contrast, Fuc-TII preferred to transfer fucose to the external acceptor site of nLc6, consistent with the low activities of Fuc-TII with sialylated nLc6 derivatives. Thus the two enzymes preferentially add fucose to different N-acetylglucosamines in the same substrate, nLc6. This indicates that the biosynthetic pathway for fucosylation of polylactosamine sequences in glycolipids and glycoproteins will vary depending upon the particular alpha (1,3)fucosyltransferase present.

Structure of a novel sialylated fucosyl lacto-N-norhexaosylceramide isolated from chronic myelogenous leukemia cells.

A novel sialylated fucosyl glycolipid, which is present at an elevated level in chronic myelogenous leukemia cells, was isolated. The structure of this fucoganglioside was elucidated by methylation analysis, fast atom bombardment-mass spectrometry, and enzymatic degradation, followed by reaction with anti-Lex, Gal beta 1----4 (Fuc alpha 1----3) GlcNAc beta 1----, monoclonal antibody. The structure of this ganglioside was found to be: (Formula: see text). This structure is unique in that a fucose is attached to the internal N-acetylglucosamine but not to the subterminal N-acetylglucosamine. Since this glycolipid is apparently absent in normal granulocytes or acute myelogenous leukemia cells, it can be a specific marker for chronic myelogenous leukemia cells. Based on the structures of this fucoganglioside and normal granulocyte glycolipids, a biosynthetic pathway of extension, sialylation, followed by fucosylation is proposed.

Glycoprotein properties of the solubilized atrial muscarinic acetylcholine receptor.

The muscarinic acetylcholine receptor from porcine atria exhibits sialoglycoprotein characteristics based on its sensitivity to neuraminidase digestion and its ability to interact specifically with lectin affinity resins when solubilized with a digitonin/cholate mixed detergent system. Differential lectin binding properties of the neuraminidase-treated and untreated receptor suggest that high-affinity binding to immobilized wheat germ agglutinin is accomplished through the presence of both terminal sialic acid and internal N-acetylglucosamine or its beta(1 leads to 4)-linked oligomers.

Some properties of the lectin from Datura stramonium (thorn-apple) and the nature of its glycoprotein linkages.

The lectin from Datura stramonium (thorn-apple; Solanaceae) has been purified by affinity chromatography and shown to be a glycoprotein containing about 40% (w/w) of carbohydrate. The most abundant amino acids are hydroxyproline, cystine, glycine and serine. Results obtained by gel filtration in 6m-guanidinium chloride on Sepharose 4B suggest that it has a subunit mol.wt. of about 30000 and that it probably associates into dimers. The lectin is inhibited specifically by chitin oligosaccharides and bacterial-cell-wall oligosaccharides, but only weakly by N-acetylglucosamine. Glycopeptides from soya-bean (Glycine max) lectin and fetuin are also strong inhibitors of Datura lectin, indicating that it interacts with internal N-acetylglucosamine residues. Its specificity is similar to, but not identical with, that of potato (Solanum tuberosum) lectin. After prolonged proteolytic digestion of reduced and S-carboxymethylated or S-aminoethylated derivatives of the lectin, glycopeptides of mol.wt. of about 18000 were isolated. The glycopeptides contained all the carbohydrate and hydroxyproline of the original glycoprotein, and lesser amounts of serine, S-carboxymethylcysteine and other amino acids. The arabinose residues of the glycoprotein are present as beta-l-arabinofuranosides linked to the polypeptide chain through the hydroxyproline residues, and can be removed by mild acid treatment; the ratio of arabinose to hydroxyproline is 3.4:1. Some of the serine residues of the polypeptide chain are substituted with one or two alpha-galactopyranoside residues, most of which can be removed by the action of alpha-galactosidase. The galactose residues are more easily removed from the acid-treated glycopeptide (from which arabinose has been removed) than from the complete glycopeptide, indicating a steric hindrance of the galactosidase action by the adjacent chains of arabinosides. There is a slow release of galactose residues by a process of beta-elimination in 0.5m-NaOH (pH13.7) from the complete glycopeptide, and a fairly rapid release of galactose by this process from the acid-treated glycopeptide, which lacks arabinose. This is probably due to the inhibitory effect of the negative charge on the adjacent arabinofuranoside residues. The similarities and differences between the lectins from Datura and potato are discussed, as are their structural resemblance to glycopeptides that have been isolated from plant cell walls.

A lectin from the exudate of the fruit of the vegetable marrow (Cucurbita pepo) that has a specificity for beta-1,4-linked N-acetylglucosamine oligosaccharides.

Lectins are present in the exudate (presumably from the phloem) of the fruits of three species of the Cucurbitaceae, namely vegetable marrow (Cucurbita pepo), melon (Cucumis melo) and cucumber (Cucumis sativus). They are all strongly inhibited in their activities by chitin oligosaccharides, but only weakly by N-acetylglucosamine. Glycopeptides from soya-bean agglutinin and fetuin are also strong inhibitors of Cucurbita pepo lectin, indicating that it interacts with internal N-acetylglucosamine residues. The lectin from Cucurbita pepo fruit was purified by affinity chromatography by using chitin oligosaccharides covalently attached to Sepharose. The lectin is not a glycoprotein, and it consists of a single polypeptide chain of about 20,000 mol.wt. It is a major protein (18% of the total) of the phloem exudate and it is postulated that it may have an anti-parasitic function.

N-Acetylglucosamine-6-sulfate sulfatase from human urine.

N-Acetylglucosamine-6-sulfate sulfatase, which liberates sulfate from the N-acetylglucosamine 6-sulfate residue at the nonreducing terminus of a 3H-labeled trisaccharide prepared from heparan sulfate, was purified 136-fold from human urine. The final N-acetylglucosamine-6-sulfate sulfatase preparation was free of all lysosomal sulfatases known to act on sulfated polysaccharides and gave a single band in polyacrylamide gel electrophoresis. The enzyme appears to be a glycoprotein with a molecular weight of around 97,000 and displays considerable charge heterogeneity. Multiple forms with pI values between 5.4 and 8.3 with a maximum at pH 7.7 were detected. The enzyme acts on the 3H-trisaccharide with a pH optimum at 5.5 and is active towards the sulfated monosaccharides N-acetylglucosamine 6-sulfate and glucose 6-sulfate. Although predominantly in exosulfatase, the enzyme catalyzes hydrolysis of sulfate from internal N-acetylglucosamine 6-sulfate moieties at a low rate. The Km for the 3H-trisaccharide, N-acetylglucosamine 6-sulfate, and glucose 6-sulfate were 0.15, 1.5, and 7.7 mM, respectively. The enzyme is inhibited by albumin, Hg2+, PO43-, SO42-, and CN-. Enzyme activity was highest in kidney and cultured fibroblasts but could be demonstrated in all human tissues tested.

The structure of a glycopeptide purified from porcine thyroblobulin.

1. The structure of a purified glycopeptide isolated from porcine thyroglobulin was studied by sequential hydrolysis with specific glycosidases, by periodate oxidation and by treatment with galactose oxidase. 2. Sequential hydrolysis with several combinations of neuraminidase, alpha-l-fucosidase, beta-d-galactosidase, beta-N-acetyl-d-glucosaminidase and alpha-d-mannosidase presented the evidence for the following structure. 3. The monosaccharide sequence of the peripheral moiety of the heteropolysaccharide chain was sialic acid-->galactose-->N-acetylglucosamine. Some of the galactose residues were non-reducing end-groups with the sequence galactose-->N-acetylglucosamine. 4. After removal of the peripheral moiety composed of sialic acid, fucose, galactose and N-acetylglucosamine, alpha-mannosidase released 1.4mol of mannose/mol of glycopeptide, indicating that two of the three mannose residues were located between peripheral N-acetylglucosamine and internal N-acetylglucosamine or mannose. 5. Periodate oxidation and sodium borohydride reduction confirmed the results obtained by enzymic degradation and gave information concerning the position of substitution. 6. Based on the results obtained by enzymic hydrolysis and periodate oxidation together with the treatment with galactose oxidase, a structure is proposed for the glycopeptide.