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1.
Glycans (i.e. oligosaccharide chains attached to cellular proteins and lipids) are crucial for nearly all aspects of life, including the development of multicellular organisms. They come in multiple forms, and much of this diversity between molecules, cells, and tissues is generated by Golgi-resident glycosidases and glycosyltransferases. However, their exact mode of functioning in glycan processing is currently unclear. Here we investigate the supramolecular organization of the N-glycosylation pathway in live cells by utilizing the bimolecular fluorescence complementation approach. We show that all four N-glycosylation enzymes tested (β-1,2-N-acetylglucosaminyltransferase I, β-1,2-N-acetylglucosaminyltransferase II, 1,4-galactosyltransferase I, and α-2,6-sialyltransferase I) form Golgi-localized homodimers. Intriguingly, the same enzymes also formed two distinct and functionally relevant heterodimers between the medial Golgi enzymes β-1,2-N-acetylglucosaminyltransferase I and β-1,2-N-acetylglucosaminyltransferase II and the trans-Golgi enzymes 1,4-galactosyltransferase I and α-2,6-sialyltransferase I. Given their strict Golgi localization and sequential order of function, the two heterodimeric complexes are probably responsible for the processing and maturation of N-glycans in live cells.  相似文献   

2.
The α- and β-N-acetylglucosaminidase activity of the limpet Patella vulgata (L.) is due to two enzymes. One of these enzymes hydrolyses both α- and β-N-acetylglucosaminidases and is referred to α,β-N-acetylglucosaminidase. The other is a β-N-acetylglucosaminidase (EC 3.2.1.30). Both enzymes have been isolated and characterized as glycoproteins containing 12% hexose, mainly galactose. The amino acid, neutral sugar and amino sugar content of the two enzymes is very similar, and the main difference lies in the presence of 9% sialic acid in β-N-acetylglucosaminidase. The molecular weight of α,β-N-acetylglucosaminidase is 217 000 and that of β-N-acetylglucosaminidase is 136 000. Evidence has been obtained for the presence of an additional sub-unit in the α,β-enzyme.  相似文献   

3.
The specificities of glycosyltransferases make them useful for the synthesis of biologically active oligosaccharides, but also restrict their range of products. In substrate engineering, substrate promiscuity is enhanced by attaching removable interactive groups to weak substrates. Thus, the attachment of β p-nitrophenyl converts galactose from a poor into a good substrate of α-1,3-galactosyltransferase. The crystallographic structure of a complex of α3GT containing p-nitrophenyl-β-galactoside shows that the p-nitrophenyl binds similarly to the N-acetylglucosamine of the substrate, N-acetyllactosamine, interacting with the indole of Trp249. p-Nitrophenyl, unlike N-acetylglucosamine, makes no H-bonds but has more non-polar interactions, making it an effective monosaccharide mimetic.  相似文献   

4.
Enzymatic synthesis of GlcNAc-terminated poly-N-acetyllactosamine β-glycosides GlcNAcβ1,3(Galβ1,4GlcNAcβ1,3)nGalβ1,4GlcNAcβ-pNP (n=1–4) was demonstrated using a transglycosylation reaction of Escherichia freundii endo-β-galactosidase. The enzyme catalyzed a transglycosylation reaction on GlcNAcβ1,3Galβ1,4GlcNAcβ-pNP (1), which served both as a donor and an acceptor, and converted 1 into p-nitrophenyl β-glycosides GlcNAcβ1,3(Galβ1,4GlcNAcβ1,3)1Galβ1,4GlcNAcβ-pNP (2), GlcNAcβ1,3(Galβ1,4GlcNAcβ1,3)2Galβ1,4GlcNAcβ-pNP (3), GlcNAcβ1,3(Galβ1,4GlcNAcβ1,3)3Galβ1,4GlcNAcβ-pNP (4) and GlcNAcβ1,3(Galβ1,4GlcNAcβ1,3)4Galβ1,4GlcNAcβ-pNP (5). When 2 was used as an initial substrate, it led to the preferential synthesis of nonasaccharide β-glycoside 4 to heptasaccharide β-glycoside 3. This suggests that 4 is directly synthesized by transferring the tetrasaccharide unit GlcNAcβ1,3Galβ1,4GlcNAcβ1,3Gal to nonreducing end GlcNAc residue of 2 itself. The efficiency of production of poly-N-acetyllactosamines by E. freundii endo-β-galactosidase was significantly enhanced by the addition of BSA and by a low-temperature condition. Resulting 2 and 3 were shown to be useful for studying endo-β-galactosidase-catalyzed hydrolytic and transglycosylation reactions.  相似文献   

5.
Bacteroides fragilis is a clinically important anaerobic pathogen present in the human gastrointestinal tract and is involved in a high number of anaerobic peritoneal infections. The complete genome sequence of B. fragilis NCTC 9343 revealed the presence of several putative fucosyltransferase gene homologues known as alpha-1,3-fucosyltransferases (α-1,3-FucTs). However, their expression and functional activities have not been studied. Here, we report the molecular cloning, functional expression, and characterization of the alpha-1,3-fucosyltransferase 3 (α-1,3-FucT3) enzyme from B. fragilis NCTC 9343. The polymerase chain reaction (PCR)-based approach was used to clone the 331 amino acid long (MW, ~39 kDa) PCR product encoding fucosyltransferase enzyme. The enzyme had low identity of 30–40% with other known α-1,3-FucTs from Azospirillum sp, Rickettsia bellii, and different strains of Helicobacter pylori. An in vitro enzyme reaction analysis showed the ability of the enzyme to transfer the fucose moiety from guanosine-5′-diphosphate β-l-fucose to the N-acetyllactosamine to produce Lewis X. The reaction product, Lewis X was confirmed by thin layer chromatography, liquid chromatography-mass spectroscopy, and 1H-nuclear magnetic resonance analyses.  相似文献   

6.
Glycoproteins derived from Hansenula polymorpha can not be used for therapeutic purposes due to their high-mannose type asparagine-linked (N-linked) glycans, which result in immune reactions and poor pharmacokinetic behaviors in human body. Previously, we reported that the trimannosyl core N-linked glycans (Man3GlcNAc2) intermediate can be generated in endoplasmic reticulum in HpALG3 and HpALG11 double-mutant H. polymorpha. Here, we describe the further modification of the glycosylation pathway in this double-defect strain to express glycoproteins with complex human-like glycans. After eliminating the impact of HpOCH1, three glycosyltransferases were introduced into this triple-mutant strain. When human β-1,2-N-acetylglucosaminyltransferase I (hGnTI) was efficiently targeted in early Golgi, more than 95 % glycans attached to the glycoproteins were added one N-acetylglucosamine (GlcNAc). With subsequently introduction of rat β-1,2-N-acetylglucosaminyltransferase II (rGnTII) and human β-1,4-galactosyltransferase I (hGalTI), several glycoengineered strains can produce glycoproteins bearing glycans with terminal N-acetylglucosamine or galactose. The expression of glycoproteins with glycan Gal2GlcNAc2Man3GlcNAc2 represents a significant step toward the ability to express fully humanized glycoproteins in H. polymorpha. Furthermore, several shake-flask and bioreactor fermentation experiments indicated that, although the cells do display a reduction in growth rate, the glycoengineered strains are still suitable for high-density fermentation.  相似文献   

7.
Trypanosoma cruzi, an intracellular protozoan etiologic agent of Chagas disease is covered by a dense coat of mucin-type glycoproteins, which is important to promote the parasite entry and persistence in the mammalian host cells. The O-glycosylation of T. cruzi mucins (Tc-mucins) is initiated by enzymatic addition of α-O-N-acetylglucosamine (GlcNAc) to threonine (Thr) by the UDP-GlcNAc:polypeptide α-N-acetylglucosaminyltransferase (pp-α-GlcNAcT) in the Golgi. The Tc-mucin is characterized by the presence of a high structural diversity of O-linked oligosaccharides found among different parasite strains, comprising two O-glycan Cores. In the Core 1, from strains principally associated with the domestic transmission cycle of Chagas disease, the GlcNAc O-4 is substituted with a β-galactopyranose (βGalp) unit, and in the most complex oligosaccharides the GlcNAc O-6 is further processed by the addition of β1?→?2-linked Galp residues creating a short linear Galp-containing chain. In the Core 2 structures, expressed by strains isolated from T. cruzi sylvatic hosts, the GlcNAc O-4 carries a β-galactofuranose (βGalf) unit and the GlcNAc O-6 can carry a branched Galpβ1?→?3[Galpβ1?→?2]Galpβ1?→?6 motif. The O-glycans carrying nonreducing terminal βGalp are available for sialylation by a surface T. cruzi trans-sialidase activity. Based on structural results, this review summarizes available data on the highly conserved process, which adds the GlcNAc unit in α-linkage to Thr residues the basis of the post-translational modification system in T. cruzi mucins. In addition, a mechanism unique employed by the parasite to transfer exogenous sialic acid residues to Tc-mucins is presented.  相似文献   

8.
Abstract: Antibodies specific for α-N-acetyl-β-endorphins have been prepared by injecting into rabbits either α-N-acetyl-β-endorphin(1-31) or [α-N-acetyl, ε-acetyl-Lys9]-β-endorphin(1-9) linked by carbodiimide to bovine thyroglobulin. Both antisera were used to develop specific radioimmunoassays for α-N-acetyl-β-endorphins. The radioimmunoassays were used to measure α-N-acetylated β-endorphins in extracts of pituitary regions from different species. By comparison of the amounts of total β-endorphin and α-N-acetyl-β-endorphin immunoreactivity, a relative ratio of β-endorphin acetylation was obtained. The relative acetylation of β-endorphin was highest in rat posterior-intermediate lobe extracts (>90%). Beef and monkey intermediate lobes had a lower degree of acetylation (53 and 31%, respectively). Anterior lobe extracts from all three species contained low amounts of acetylated β-endorphin. Human pituitary extracts did not contain acetylated β-endorphins. By the use of cation exchange and high performance liquid chromatography, six different acetylated derivatives and fragments of β-endorphin were resolved in extracts of rat posterior-intermediate pituitaries. Two of these peptides corresponded to α-N-acetyl-β-endorphin(1-31) and -(1-27). One acetylated β-endorphin fragment had the same size as α-N-acetyl-β-endorphin(1-27) but was eluted earlier from the cation exchange column. This peptide had full cross-reactivity with antibodies directed against the middle and amino-terminal parts of β-endorphin. Compared with α-N-acetyl-β-endorphin(1-27), it had much less cross-reactivity with antibodies directed against the COOH-terminal part of β-endorphin, suggesting that it was a COOH-terminally modified derivative of β-endorphin(1-27). The remaining N-acetylated β-endorphin derivatives were eluted even earlier from the cation exchange column. The majority of these fragments were slightly larger in size than y-endorphin, i.e., β-endorphin(1-17), but smaller than β-endorphin(1-27). They had full cross-reactivity in an amino-terminally directed β-endorphin radioimmunoassay and a greatly diminished cross-reactivity with antibodies to the middle region of β-endorphin.  相似文献   

9.
β-N-Acetylaminoglucohydrolase (β-2-acetylamino-2-deoxy-D-glucoside acetylaminodeoxyglucohydrolase, EC 3.2.1.30) was extracted from malted barley and purified. The partially purified preparation was free from α-and β-glucosidase, α- and β-galactosidase, α-mannosidase and β-mannosidase. This preparation was free from α-mannosidase only after affinity chromatography with p-amino-N-acetyl-β-D-glucosaminidine coupled to Sepharose. The enzyme was active between pH 3 and 6.5 and had a pH optimum at pH 5. A MW of 92000 was obtained by sodium dodecyl sulfate-acrylamide gel electrophoresis and a sedimentation coefficient of 4.65 was obtained from sedimentation velocity experiments. β-N-Acetylaminoglucohydrolase had a Km of 2.5 × 10?4 M using the p-nitrophenyl N-acetyl β-D-glucosaminidine as the substrate.  相似文献   

10.
A condensation reaction between N-acetyllactosamine and glycerol was directly catalyzed by using a commercially available cellulase preparation from Trichoderma reesei. 1-O-β-N-Acetyllactosaminyl-(R, S)-glycerols (1) were readily synthesized in a 5% yield based on the N-acetyllactosamine added and conveniently isolated by two-step column chromatographies. The use of a partially purified enzyme increased 2.3-fold the yield of 1, compared to that of the crude enzyme containing β-D-galactosidase activity. When various alkanols (n:2-4) were used in the condensation reaction, the corresponding alkyl β-N-acetyllactosaminides were obtained in yields of 0.3-1.1% of the desired compounds.  相似文献   

11.
The α- and β-N-oxalyl derivatives of l-α,β-diaminopropionic acid have been chemically synthesized and also isolated from seed extracts of Lathyrus sativus. Chemical and physical properties of the natural and synthetic isomers were in good agreement. The toxicity of the α-isomer to chicks was evaluated and compared with that of the β-isomer.  相似文献   

12.
SLC35A3 is considered the main UDP-N-acetylglucosamine transporter (NGT) in mammals. Detailed analysis of NGT is restricted because mammalian mutant cells defective in this activity have not been isolated. Therefore, using the siRNA approach, we developed and characterized several NGT-deficient mammalian cell lines. CHO, CHO-Lec8, and HeLa cells deficient in NGT activity displayed a decrease in the amount of highly branched tri- and tetraantennary N-glycans, whereas monoantennary and diantennary ones remained unchanged or even were accumulated. Silencing the expression of NGT in Madin-Darby canine kidney II cells resulted in a dramatic decrease in the keratan sulfate content, whereas no changes in biosynthesis of heparan sulfate were observed. We also demonstrated for the first time close proximity between NGT and mannosyl (α-1,6-)-glycoprotein β-1,6-N-acetylglucosaminyltransferase (Mgat5) in the Golgi membrane. We conclude that NGT may be important for the biosynthesis of highly branched, multiantennary complex N-glycans and keratan sulfate. We hypothesize that NGT may specifically supply β-1,3-N-acetylglucosaminyl-transferase 7 (β3GnT7), Mgat5, and possibly mannosyl (α-1,3-)-glycoprotein β-1,4-N-acetylglucosaminyltransferase (Mgat4) with UDP-GlcNAc.  相似文献   

13.
β1–3-N-Acetylglucosaminyltransferases (β3GlcNAcTs) and β1–4-galactosyltransferases (β4GalTs) have been broadly used in enzymatic synthesis of N-acetyllactosamine (LacNAc)-containing oligosaccharides and glycoconjugates including poly-LacNAc, and lacto-N-neotetraose (LNnT) found in the milk of human and other mammals. In order to explore oligosaccharides and derivatives that can be synthesized by the combination of β3GlcNAcTs and β4GalTs, donor substrate specificity studies of two bacterial β3GlcNAcTs from Helicobacter pylori (Hpβ3GlcNAcT) and Neisseria meningitidis (NmLgtA), respectively, using a library of 39 sugar nucleotides were carried out. The two β3GlcNAcTs have complementary donor substrate promiscuity and 13 different trisaccharides were produced. They were used to investigate the acceptor substrate specificities of three β4GalTs from Neisseria meningitidis (NmLgtB), Helicobacter pylori (Hpβ4GalT), and bovine (Bβ4GalT), respectively. Ten of the 13 trisaccharides were shown to be tolerable acceptors for at least one of these β4GalTs. The application of NmLgtA in one-pot multienzyme (OPME) synthesis of two trisaccharides including GalNAcβ1–3Galβ1–4GlcβProN3 and Galβ1–3Galβ1–4Glc was demonstrated. The study provides important information for using these glycosyltransferases as powerful catalysts in enzymatic and chemoenzymatic syntheses of oligosaccharides and derivatives which can be useful probes and reagents.  相似文献   

14.
It is well documented that glycan synthesis is altered in some pathological processes, including cancer. The most frequently observed alterations during tumourigenesis are extensive expression of β1,6-branched complex type N-glycans, the presence of poly-N-acetyllactosamine structures, and high sialylation of cell surface glycoproteins. This study investigated two integrins, α3β1 and αvβ3, whose expression is closely related to cancer progression. Their oligosaccharide structures in two metastatic melanoma cell lines (WM9, WM239) were analysed with the use of matrix-assisted laser desorption ionisation mass spectrometry. Both examined integrins possessed heavily sialylated and fucosylated glycans, with β1,6-branches and short polylactosamine chains. In WM9 cells, α3β1 integrin was more variously glycosylated than αvβ3; in WM239 cells the situation was the reverse. Functional studies (wound healing and ELISA integrin binding assays) revealed that the N-oligosaccharide component of the tested integrins influenced melanoma cell migration on vitronectin and α3β1 integrin binding to laminin-5. Additionally, more variously glycosylated integrins exerted a stronger influence on these parameters. To the best of our knowledge, this is the first report concerning structural characterisation of αvβ3 integrin glycans in melanoma or in any cancer cells.  相似文献   

15.
Approximately 70% of fucose-labeled glycopeptides from the cell surface and cellular material of rat fibroblasts (3Y1B cells) were hydrolyzed by endo-β-N-acetylglucosaminidase D in the presence of neuraminidase, β-glactosidase and β-N-acetylglucosaminidase. Structure of the suspceptible glycopeptides were found to be very similar to non-membrane glycopeptides of the complex heteropolysaccharide unit, such as the sialylated glycopeptides of thyroglobulin. On the other hand, the resistant glycopeptides were also refractory toward endo-β-N-acethylglucosaminidase H and α-mannosidase, and appeared to be a mixture of glycopeptides with unique structures.  相似文献   

16.
Endo-1,3(4)-β-glucanase (EC 3.2.1.6) from Vigna aconitifolia sprouts was purified to 14.5 fold by gel filtration and ion-exchange chromatography. The enzyme was found to be a glycoprotein, its activity was Ca2+ dependent and specific for β-1,3 linkages in different polysaccharides. The Km value of the enzyme was estimated to be 3.0 mg ml−1 for β-d-glucan as substrate. Circular dichroism studies revealed 8% α-helix, 48% β-pleated and 44% random coil in its secondary structure. Purified β-glucanase was then successfully co-immobilized with glucose oxidase in agarose-chitosan beads, showing better immobilization yield, operational range and stability as compared with the crude β-glucanase beads. The immobilized β-glucanase was successfully used for mini-bioreactor fabrication.  相似文献   

17.
The following new compounds were prepared and characterized: N-benzyl-oxycarbonyl-O-(tetra-O-acetyl-β-D-glucopyranosyl)-N-glycyl-L-serine methyl ester (1) and L-threonine methyl ester (2), N-benzyloxycarbonyl-O-(β-D-glucopyranosyl)-N-glycyl-L-serine amide (3), N-benzyloxycarbonyl-O-(β-D-glucopyranosyl)-N-glycyl-L-threonine methyl ester (4) and L-threonine amide (5), N-benzyloxycarbonyl-O-(tri-O-acetyl-2-deoxy-2-trifluoroacetamido-β-D-glucopyranosyl)-N-glycyl-L-serine methyl ester (6), and N-benzyloxycarbonyl-O-(2-deoxy-2-trifluoroacetamido-β-D-glucopyranosyl)-N-glycyl-L-serine amide (7). Although various modifications of the Koenigs-Knorr synthesis were used, the best, over-all yields of the deacetylated dipeptide derivatives were only 5–10%. Although the products are alkali-labile, deacetylation was accomplished with methanolic ammonia. Of the deacetylated products, the threonine derivatives (4 and 5) were more rapidly hydrolyzed by acids than phenyl β-D-glucopyranoside, which in turn was more rapidly cleaved than the serine derivatives (3 and 7). The stabilities of 3, 4, 5, and 7 to sodium hydroxide and sodium borohydride were similar, and essentially complete β-elimination of the glycosyl residue occurred for the amide derivatives (3, 5, and 7). For the ester derivative 4, pH 9 was optimal; above this pH, ester hydrolysis was more rapid than β-elimination, and the resulting carboxyl derivatives did not undergo β-elimination. Under optimal conditions with sodium borohydride, the β-elimination reaction was complete, but the corresponding alanine and α-aminobutyric acid residues were not formed; presumably reductions to the amino alcohols occurred. A mechanism for the β-elimination is proposed.  相似文献   

18.
Two soluble fucosyltransferases have been demonstrated in human serum. One enzyme transfers l-fucose from GDP-l-fucose to the terminal galactose residues of lactose, N-acetyllactosamine, and sialidase-treated α1-acid glycoprotein, to form the blood group H determinant, α-l-fucosyl-(1 → 2)-β-d-galactosyl-R. The second enzyme transfers fucose to the terminal N-acetylglucosamine residue of sialidase-, β-galactosidase-treated α1-acid glycoprotein. Serum from a donor with the rare “Bombay” Oh blood group (genotype hh) cannot transfer fucose to terminal galactose residues but has normal levels of the enzyme acting on sialidase-, β-galactosidase-treated α1-acid glycoprotein. This observation, as well as mixed substrate experiments, demonstrate that the two fucosyltransferase activities are due to two separate enzymes. The GDP-l-fucose:galactoside fucosyltransferase has a pH optimum of 5.5 and the following Km values: lactose, 31 mm; N-acetyllactosamine, 7.5 mm; sialidase-treated α1-acid glycoprotein, 6.4 mm. The GDP-l-fucose: N-acetylglucosaminide fucosyltransferase has a pH optimum of 5.0 and a Km for sialidase-, β-galactosidase-treated α1-acid glycoprotein of 1.2 mm. The serum GDP-l-fucose: N-acetylglucosaminide fucosyltransferase is distinct from the blood group Lewis-dependent enzyme in milk since the serum enzyme is present in serum from Le (a-b-)donors and since the Le-dependent fucosyltransferase could not be demonstrated in serum from donors carrying the Le gene.  相似文献   

19.
13C-N.m.r. spectra of thirteen xylo-oligosaccharides [a complete series of α- and β-d-xylopyranosyl derivatives of methyl α-d-xylopyranoside, β-d-xylopyranosyl derivatives of methyl 4-O-β-d-xylopyranosyl-d-xylopyranoside, methyl O-α-d-xylopyranosyl-(1→3)-O-β-d-xylopyranosyl-(1→4)-d-xylopyranoside, and a branched methyl β-xylotetraoside] have been interpreted. The data obtained have been used for the carbon signal assignment in the spectra of a number of red-algal xylans. 13C-N.m.r. spectroscopy is shown to be a rapid and convenient method for the structural analysis of xylose-rich polysaccharides.  相似文献   

20.
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