Sugar Chains Of Glycoproteins Research Articles

SiaNAl can be efficiently incorporated in glycoproteins of human mesenchymal stromal cells by metabolic glycoengineering.

Metabolic glycoengineering involves the stimulation of cells with functionalized monosaccharides. Glucosamine, galactosamine, and mannosamine derivatives are commercially available, but their application may lead to undirected (i.e., chemical) incorporation into proteins. However, sialic acids are attached to the ends of complex sugar chains of glycoproteins, which might be beneficial for cell surface modification via click chemistry. Thus, we studied the incorporation of chemically synthesized unnatural alkyne modified sialic acid (SiaNAl) into glycoproteins of human telomerase-immortalized mesenchymal stromal cells (hMSC-TERT) and we show that SiaNAl can be efficiently incorporated in glycoproteins involved in signal transduction and cell junction.

糖タンパク質糖鎖のはたらき : 新しい生物情報の担い手( 話題を探る)

糖タンパク質糖鎖のはたらき : 新しい生物情報の担い手( 話題を探る)

The Metabolic Pathways for the Sugar Chains of Glycoproteins in Bifidobacteria

The Metabolic Pathways for the Sugar Chains of Glycoproteins in Bifidobacteria

Introduction of H-antigens into oligosaccharides and sugar chains of glycoproteins using highly efficient 1,2-α-l-fucosynthase.

Fucα1-2 Gal linkages, or H-antigens, constitute histo-blood group antigens and are involved in various physiological processes. In addition, recent studies have shown that the H-antigen-containing glycans play an important role, not only in establishing harmonious relationship between gut microbes and the host, but also in preventing gut dysbiosis-related diseases. Therefore, development of an efficient method for introducing Fuc residue at Gal residue at the nonreducing end of glycans via α-(1→2) linkage is desired for research as well as medicinal purposes. In this study, we succeeded in derivatizing inverting 1,2-α-l-fucosidase (AfcA) into a highly efficient 1,2-α-l-fucosynthase. The synthase specifically synthesized H type 1-, type 2-, type 3- and type 4-chain-containing oligosaccharides with yields of 57-75% based on acceptor depletion. The synthase was also able to specifically introduce Fuc residues into Lewis a/x antigens to produce Lewis b/y antigens, with yields of 43% and 62%, respectively. In addition, the enzyme efficiently introduced H-antigens into sugar chains of porcine gastric mucins, as revealed by lectin blotting and mass spectroscopy analysis of the sugars. Detailed acceptor specificity analysis using various monosaccharides and oligosaccharides unraveled unique substrate recognition feature of this synthase at the subsite (+1), which can be explained by our previous X-ray crystallographic study of AfcA. These results show that the synthase developed in this study could serve as an alternative to other H-antigen synthesis methods involving α-1,2-fucosyltransferases and retaining α-fucosidase.

Review: Prize-awarded article] The Study of the Glycoside Hydrolases Acting on Sugar Chains of Glycoproteins

Review: Prize-awarded article] The Study of the Glycoside Hydrolases Acting on Sugar Chains of Glycoproteins

Mammalian ER mannosidase I resides in quality control vesicles, where it encounters its glycoprotein substrates.

Endoplasmic reticulum α1,2 mannosidase I (ERManI), a central component of ER quality control and ER-associated degradation (ERAD), acts as a timer enzyme, modifying N-linked sugar chains of glycoproteins with time. This process halts glycoprotein folding attempts when necessary and targets terminally misfolded glycoproteins to ERAD. Despite the importance of ERManI in maintenance of glycoprotein quality control, fundamental questions regarding this enzyme remain controversial. One such question is the subcellular localization of ERManI, which has been suggested to localize to the ER membrane, the ER-derived quality control compartment (ERQC), and, surprisingly, recently to the Golgi apparatus. To try to clarify this controversy, we applied a series of approaches that indicate that ERManI is located, at the steady state, in quality control vesicles (QCVs) to which ERAD substrates are transported and in which they interact with the enzyme. Both endogenous and exogenously expressed ERManI migrate at an ER-like density on iodixanol gradients, suggesting that the QCVs are derived from the ER. The QCVs are highly mobile, displaying dynamics that are dependent on microtubules and COP-II but not on COP-I vesicle machinery. Under ER stress conditions, the QCVs converge in a juxtanuclear region, at the ERQC, as previously reported. Our results also suggest that ERManI is turned over by an active autophagic process. Of importance, we found that membrane disturbance, as is common in immunofluorescence methods, leads to an artificial appearance of ERManI in a Golgi pattern.

Glycoproteins isolated from scallop shells inhibit differentiation of 3T3-L1 preadipocyte cells

We previously demonstrated that the organic components isolated from scallop shells (scallop shell extract) inhibit the differentiation of 3T3-L1 preadipocyte cells. In this study, we show that a pronase-treated scallop shell extract inhibited differentiation, but degradation of sugar chains in the scallop shell extract with trifluoromethane sulfonic acid resulted in the loss of the differentiation-inhibiting activity, suggesting that the sugar chain modifications were responsible for the inhibitory activity. To identify the bioactive substance in the scallop shell extract, we isolated glycoproteins from the scallop shell extract via affinity chromatography using concanavalin A (Con A), wheat germ agglutinin, lens culinaris agglutinin (LCA), and ricinus communis agglutinin. LCA and Con A binding fractions inhibited the differentiation of 3T3-L1 preadipocyte cells. In addition, a glycoprotein with a molecular weight of 16 kDa that was purified from the LCA binding fraction reduced lipid accumulation in 3T3-L1 cells during differentiation. The glycoprotein inhibited differentiation-associated mitotic clonal expansion and suppressed the expression of an adipocyte-specific protein, fatty acid binding protein. These results suggest that the sugar chains of glycoproteins in the scallop shell extract inhibit the differentiation of 3T3-L1 preadipocyte cells.

The Lewis X-related α1,3-Fucosyltransferase, Fut10, Is Required for the Maintenance of Stem Cell Populations

Lewis X (Le(X), Galβ1-4(Fucα1-3)GlcNAc) is a carbohydrate epitope that is present at the nonreducing terminus of sugar chains of glycoproteins and glycolipids, and is abundantly expressed in several stem cell populations. Le(X) antigen can be used in conjunction with fluorescence-activated cell sorting to isolate neurosphere-forming neural stem cells (NSCs) from embryonic mouse brains. However, its function in the maintenance and differentiation of stem cells remains largely unknown. In this study, we examined mice deficient for fucosyltransferase 9 (Fut9), which is thought to synthesize most, if not all, of the Le(X) moieties in the brain. We found that the number of NSCs was increased in the brain of Fut9(-/-) embryos, suggesting that Fut9-synthesized Le(X) is dispensable for the maintenance of NSCs. Another α1,3-fucosyltransferase gene, fucosyltransferase 10 (Fut10), is expressed in the ventricular zone of the embryonic brain. Overexpression of Fut10 enhanced the self-renewal of NSCs. Conversely, suppression of Fut10 expression induced the differentiation of NSCs and embryonic stem cells. In addition, knockdown of Fut10 expression in the cortical ventricular zone of the embryonic brain by in utero electroporation of Fut10-miRNAs impaired the radial migration of neural precursor cells. Our data suggest that Fut10 is involved in a unique α1,3-fucosyltransferase activity with stringent substrate specificity, and that this activity is required to maintain stem cells in an undifferentiated state.

Estrogen induced β-1,4-galactosyltransferase 1 expression regulates proliferation of human breast cancer MCF-7 cells

Beta 1,4-galactosyltransferase 1 (B4GALT1) synthesizes galactose β-1,4-N-acetylglucosamine (Galβ1-4GlcNAc) groups on N-linked sugar chains of glycoproteins, which play important roles in many biological events, including the proliferation and migration of cancer cells. A previous microarray study reported that this gene is expressed by estrogen treatment in breast cancer. In this study, we examined the regulatory mechanisms and biological functions of estrogen-induced B4GALT1 expression. Our data showed that estrogen-induced expression of B4GALT1 is localized in intracellular compartments and in the plasma membrane. In addition, B4GALT1 has an enzyme activity involved in the production of the Galβ1-4GlcNAc structure. The result from a promoter assay and chromatin immunoprecipitation revealed that 3 different estrogen response elements (EREs) in the B4GALT1 promoter are critical for responsiveness to estrogen. In addition, the estrogen antagonists ICI 182,780 and ER-α-ERE binding blocker TPBM inhibit the expression of estrogen-induced B4GALT1. However, the inhibition of signal molecules relating to the extra-nuclear pathway, including the G-protein coupled receptors, Ras, and mitogen-activated protein kinases, had no inhibitory effects on B4GALT1 expression. The knock-down of the B4GALT1 gene and the inhibition of membrane B4GALT1 function resulted in the significant inhibition of estrogen-induced proliferation of MCF-7 cells. Considering these results, we propose that estrogen regulates the expression of B4GALT1 through the direct binding of ER-α to ERE and that the expressed B4GALT1 plays a crucial role in the proliferation of MCF-7 cells through its activity as a membrane receptor.

Jack bean α-mannosidase digestion profile of hybrid-type N-glycans: Effect of reaction pH on substrate preference

Jack bean α-mannosidase (JBM) is a well-studied plant vacuolar α-mannosidase, and is widely used as a tool for the enzymatic analysis of sugar chains of glycoproteins. In this study, the JBM digestion profile of hybrid-type N-glycans was examined using pyridylamino (PA-) sugar chains. The digestion efficiencies of the PA-labeled hybrid-type N-glycans Manα1,6(Manα1,3)Manα1,6(GlcNAcβ1,2Manα1,3)Manβ1,4GlcNAcβ1,4GlcNAc-PA (GNM5-PA) and Manα1,6(Manα1,3)Manα1,6(Galβ1,4GlcNAcβ1,2Manα1,3)Manβ1,4GlcNAcβ1,4GlcNAc-PA (GalGNM5-PA) were significantly lower than that of the oligomannose-type N-glycan Manα1,6(Manα1,3)Manα1,6Manβ1,4GlcNAcβ1,4GlcNAc-PA (M4-PA), and the trimming pathways of GNM5-PA and GalGNM5-PA were different from that of M4-PA, suggesting a steric hindrance to the JBM activity caused by GlcNAcβ1-2Man(α) residues of the hybrid-type N-glycans. We also found that the substrate preference of JBM for the terminal Manα1–6Man(α) and Manα1–3Man(α) linkages in the hybrid-type N-glycans was altered by the change in reaction pH, suggesting a pH-dependent change in the enzyme–substrate interaction.

ChemInform Abstract: Fascination with Natural Glycosides

AbstractReview: 12 refs.

Determination of primary factors with adhesive property of Lactobacillus brevis FSB-1 to rat colonic mucin

To maintain the valid physiological effects of lactic acid bacteria LAB in large intestine, the adhesive properties of LAB to human colonic mucin HCM is essential. In this study, the primary factors associated with the adhesive property of LAB to rat colonic mucin RCM were investigated, which contains sugar chains similar to those in HCM. Specific lectins bound to RCM were isolated by using the reaction between the surface layer Slayer protein of Lactobacillus brevis FSB-1 and RCM-coated membrane. When the isolated specific lectins of L. brevis FSB-1 analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE, 3 bands about 24, 55, and 75 kDa were observed on the electrophoretograms. And the specific sugar chains in glycoprotein of mucin also investigated by using modified colonic mucin-binding assay. The results indicated that Slayer protein of L. brevis FSB-1 bound to sialic acid and GalNAcα1-3Gal. Therefore, it could be concluded that Slayer protein of L. brevis FSB-1 recognized the terminal sugar chains of RCM and bound to it.

ChemInform Abstract: Structures and Functions of the Sugar Chains of Glycoproteins

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Fascination with Natural Glycosides

Recent progress in glycobiology reveals that sugar-chains play a crucial role in cell-cell recognition among immunity, inflammation, and malignant tumor in the living body. To study the mechanism of action requires a sample of sufficient quantity. However, isolating a sugar chain and a glycoside in pure form, difficulty follows sugar chain composition again, and studies have been limited to a few sugar chains. Glycosides are a group of compounds known to be the active principle of a natural drug. We have isolated triterpene glycosides from a Leguminosae plant, which improved liver disorder, and steroid glycosides from a plant of the Solanaceous with cytotoxic activity against human cancer cell lines. A biological activity test suggested an important role of the aglycone part and the sugar chain part of those glycosides. Although many studies of sugar chains of glycoprotein and glycolipid are known, there are few examples of studies of the sugar chain function of a glycoside of a natural drug, and the role of a sugar chain of a glycoside for its pharmacologic action expression is unknown. Therefore, as a biological tool investigating the function of a sugar chain of a natural glycoside, we have begun to synthesize a useful "glycoside" which can be utilized as a lead compound having activity.

N‐glycan based models improve diagnostic efficacies in hepatitis B virus‐related hepatocellular carcinoma

The early diagnosis of hepatocellular carcinoma (HCC) is of great clinical desirable due to lack of specific and sensitive markers. Alterations in the sugar chains of glycoprotein synthesized by the liver contribute to the molecular basis of abnormalities in carcinogenesis. This study aims to construct and assess the diagnostic value of N-glycan based diagnostic model in HCC identification and follow-up. A total of 393 subjects including HBV-related HCC, liver fibrosis and healthy controls were recruited. Follow-up was carried out before and after surgical treatment in HCC. N-glycome of serum glycoprotein was profiled by DNA sequencer-assisted fluorophore-assisted carbohydrate electrophoresis (DSA-FACE). Multiparameters diagnostic models were constructed based on N-glycan markers. The result found that 2 N-glycan structure abundances (NG1A2F, Peak 4; NA3Fb, Peak 9) were useful as N-glycan markers. The diagnostic efficacy of the log ratio [log(p9/4)] was similar to that of AFP in differentiating HCC from fibrosis. The accuracy and sensitivity of the diagnostic model combining AFP and N-glycan markers (Cscore B) were increased 7-10% compared with that of AFP. Log(p9/4) was more efficient in monitoring the progression of HCC with regarding to vascular invasion at improved specificity (16%) and accuracy (8%) compared with that of AFP. The N-glycan markers were found to be changed significantly after surgical resection in HCC follow-up. We conclude that the branching alpha (1,3)-fucosylated triantennary glycan and a biantennary glycan are promising as N-glycan markers. The diagnostic models based on the N-glycan markers and AFP improve the efficacy in HCC diagnosis and progression monitoring.

Akira Kobata: a man who established the structural basis for glycobiology of N-linked sugar chains

Akira Kobata is a pioneer of the glycobiology of N-linked sugar chains. He established the basis of glycobiology by developing a series of reliable methods to analyse the structures of N-linked sugar chains. The sensitive methods established by him greatly contributed to our understanding of the structural characteristics of the sugar chains and the biosynthetic mechanisms responsible for the production of such characteristics. He also provided new aspects that the sugar chains of glycoproteins play an important role in cell-to-cell recognition, and that the structures of sugar chains are altered under physiological and pathological conditions, including many tumours and diseases on a structural basis. In this article, the author would like to sketch out Kobata's main contributions to glycobiology for the sake of young scientists, who are planning to enter this scientific field in the future.

β1,4-Galactosyltransferase V Functions as a Positive Growth Regulator in Glioma

beta1,4-galactosyltransferase V (GalT V; EC 2.4.1.38) can effectively galactosylate the GlcNAcbeta1-->6Man arm of the highly branched N-glycans that are characteristic of glioma. Previously, we have reported that the expression of GalT V is increased in the process of glioma. However, currently little is known about the role of GalT V in this process. In this study, the ectopic expression of GalT V could promote the invasion and survival of glioma cells and transformed astrocytes. Furthermore, decreasing the expression of GalT V in glioma cells promoted apoptosis, inhibited the invasion and migration and the ability of tumor formation in vivo, and reduced the activation of AKT. In addition, the activity of GalT V promoter could be induced by epidermal growth factor, dominant active Ras, ERK1, JNK1, and constitutively active AKT. Taken together, our results suggest that GalT V functioned as a novel glioma growth activator and might represent a novel target in glioma therapy.

Altered glycosylation of proteins produced by malignant cells, and application for the diagnosis and immunotherapy of tumours

Most secretory and membrane-bound proteins produced by mammalian cells contain covalently linked sugar chains. Alterations of the sugar chain structures of glycoproteins have been found to occur in various tumours. Because the sugar chains of glycoproteins are essential for the maintenance of the ordered social behaviour of differentiated cells in multicellular organisms, alterations to the sugar chains are the molecular basis of abnormal social behaviours in tumour cells, such as invasion into the surrounding tissues and metastasis. In this review, the structure and enzymatic basis of typical alterations of the N-linked sugar chains, which are found in various tumours, are introduced. These data are useful for devising diagnostic methods and immunotherapies for the clinical treatment of tumours. Three beta-N-acetylglucosaminyltransferases, GnT-III, -IV and -V, play roles in the structural alteration of the complex-type sugar chains in various tumours. In addition, transcriptional changes in various glycosyltransferases, together with the transporters of sugar nucleotides and sulfate, which are responsible for the formation of the outer chain moieties of complex-type sugar chains, are the keys to inducing the alterations.

Novel bifidobacterial glycosidases acting on sugar chains of mucin glycoproteins

Bifidobacterium bifidum was found to produce a specific 1,2-alpha-L-fucosidase. Its gene (afc A) has been cloned and the DNA sequence was determined. The Afc A protein consisting of 1959 amino acid residues with a predicted molecular mass of 205 kDa can be divided into three domains; the N-terminal function-unknown domain (576 aa), the catalytic domain (898 aa), and the C-terminal bacterial Ig-like domain (485 aa). The recombinant catalytic domain specifically hydrolyzed the terminal alpha-(1-->2)-fucosidic linkages of various oligosaccharides and sugar chains of glycoproteins. The primary structure of the catalytic domain exhibited no similarity to those of any glycoside hydrolases but showed similarity to those of several hypothetical proteins in a database, which resulted in establishment of a novel glycoside hydrolase family (GH family 95). Several bifidobacteria were found to produce a specific endo-alpha-N-acetylgalactosaminidase, which is the endoglycosidase liberating the O-glycosidically linked galactosyl beta1-->3 N-acetylgalactosamine disaccharide from mucin glycoprotein. The molecular cloning of endo-alpha-N-acetylgalactosaminidase was carried out on Bifidobacterium longum based on the information in the database. The gene was found to comprise 1966 amino acid residues with a predicted molecular mass of 210 kDa. The recombinant protein released galactosyl beta1-->3 N-acetylgalactosamine disaccharide from natural glycoproteins. This enzyme of B. longum is believed to be involved in the catabolism of oligosaccharide of intestinal mucin glycoproteins. Both 1,2-alpha-L-fucosidase and endo-alpha-N-acetylgalactosaminidase are novel and specific enzymes acting on oligosaccharides that exist mainly in mucin glycoproteins. Thus, it is reasonable to conclude that bifidobacteria produce these enzymes to preferentially utilize the oligosaccharides present in the intestinal ecosystem.

Thermodynamic Analysis of Interactions between N-Linked Sugar Chains and F-Box Protein Fbs1

Fbs1 is a recently discovered F-box protein that was proposed to recognize high-mannose-type asparagine-linked glycoprotein sugar chains. To reveal the specificity of Fbs1, Manalpha1-->6Manbeta1-->4GlcNAc(2), Manalpha1-->3Manbeta1-->4GlcNAc(2), and Manalpha1-->3(Manalpha1--> 6)Manbeta1-->4GlcNAc(2) were synthesized and their affinities for Fbs1 were evaluated in comparison with previously synthesized Man(9)GlcNAc(2) and Man(8)GlcNAc(2). These analyses revealed that Man(3)GlcNAc(2) had the strongest affinity and the chitobiose and alpha1-->6 linked Man residue are necessary for Fbs1 to recognize a sugar.