Acceptor specificity in the transglycosylation reaction using Endo-M

To determine the structural specificity of the glycosyl acceptor of the transglycosylation reaction using endo-β-N-acetylglucosaminidase (ENGase) (EC 3.2.1.96) from Mucor hiemalis (Endo-M), several acceptor derivatives were designed and synthesized. The narrow regions of the 1,3-diol structure from the 4- to 6-hydroxy functions of GlcNAc were found to be essential for the transglycosylation reaction using Endo-M. Furthermore, it was determined that Endo-M strictly recognizes a 1,3-diol structure consisting of primary and secondary hydroxyl groups.     

Molecular cloning of Mucor hiemalis endo-beta-N-acetylglucosaminidase and some properties of the recombinant enzyme

Endo-M, endo-beta-N-acetylglucosaminidase from Mucor hiemalis, is known as a useful enzyme for the synthesis of neoglycopeptides due to its transglycosylation activity. We cloned the Endo-M gene encoding a putative 744 amino acids, which shows high identity to glycoside hydrolase family 85 endo-beta-N-acetylglucosaminidases. The gene encoding Endo-M was expressed in protease-deficient Candida boidinii with a molecular mass of 85 kDa as a monomeric form. Recombinant Endo-M could liberate both high-mannose type and biantennary complex type oligosaccharides from glycopeptides, which was same as the native enzyme. The Km and Kcat values for DNS-Man6GlcNAc2Asn were 0.51 mM and 8.25 s(-1), respectively. Recombinant Endo-M also exhibited transglycosylation activity toward high-mannose type and biantennary complex type oligosaccharides, which were transferred to alcohols, monosaccharides, oligosaccharides, and glycosides. To investigate about the catalytically essential amino acids of Endo-M, site-directed mutagenesis was performed, and it was found that mutants E177G and E177Q completely abolished the hydrolytic activity and W228R partially abolished the transglycosylation activity.     

High efficiency of transferring a native sugar chain from a glycopeptide by a microbial endoglycosidase in organic solvents

We examined the transglycosylation reaction by the recombinant endo-beta-N-acetylglucosaminidase from Mucor hiemalis (Endo-M) expressed in Candida boidinii in media containing organic solvents. The recombinant Endo-M could transglycosylate a disialo biantennary complex-type oligosaccharide from hen egg yolk glycopeptide to p-nitrophenyl N-acetyl-beta-D-glucosaminide even in the presence of 30% acetone, dimethyl sulfoxide, or methanol. The yield of the transglycosylation product reached 21-34% of the total amount of acceptor, while the yield was only about 14% in aqueous solution.     

1-O-Acetyl-beta-D-galactopyranose: a novel substrate for the transglycosylation reaction catalyzed by the beta-galactosidase from Penicillium sp

1-O-Acetyl-beta-D-galactopyranose (AcGal), a new substrate for beta-galactosidase, was synthesized in a stereoselective manner by the trichloroacetimidate procedure. Kinetic parameters (K(M) and k(cat)) for the hydrolysis of 1-O-acetyl-beta-D-galactopyranose catalyzed by the beta-D-galactosidase from Penicillium sp. were compared with similar characteristics for a number of natural and synthetic substrates. The value for k(cat) in the hydrolysis of AcGal was three orders of magnitude greater than for other known substrates. The beta-galactosidase hydrolyzes AcGal with retention of anomeric configuration. The transglycosylation activity of the beta-D-galactosidase in the reaction of AcGal and methyl beta-D-galactopyranoside (1) as substrates was investigated by 1H NMR spectroscopy and HPLC techniques. The transglycosylation product using AcGal as a substrate was beta-D-galactopyranosyl-(1-->6)-1-O-acetyl-beta-D-galactopyranose (with a yield of approximately 70%). In the case of 1 as a substrate, the main transglycosylation product was methyl beta-D-galactopyranosyl-(1-->6)-beta-D-galactopyranoside. Methyl beta-D-galactopyranosyl-(1-->3)-beta-D-galactopyranoside was found to be minor product in the latter reaction.     

A gel diffusion assay for visualization and quantification of chitinase activity

Higher plants, bacteria, fungi, insects, and crustaceans all produce chitinases. Chitinase genes in many organisms are currently under investigation. Chitinase activity is usually assayed with radiolabeled or fluorogenic substrates. We developed a simple, inexpensive, nonradioactive gel-diffusion assay for chitinase that can be used to screen large numbers of samples. In this assay, chitinase diffuses from a small circular well cut in an agarose or agar gel containing the substrate glycol chitin, a soluble, modified form of chitin. Chitinase catalyzes the cleavage of glycol chitin as it diffuses through the gel, leaving a dark, unstained circular zone around the well, because the fluorescent dye calcofluor binds only to undigested chitin. Sample activities can be determined from linear regression of logstandard enzyme concentration versus the zone diameter of internal standards on each Petri dish used for a diffusion assay.     

Transglycosylation reaction of Mucor hiemalis endo-beta-N-acetylglucosaminidase using sugar derivatives modified at C-1 or C-2 as oligosaccharide acceptors

We investigated the transglycosylation reaction of the recombinant endo-beta-N-acetylglucosaminidase from Mucor hiemalis (Endo-M) expressed in Candida boidinii using such sugar derivatives as N-acylated d-glucosamines, C-glucosyl derivatives, and a 2-O-glycosylated disaccharide as acceptors. We found that a variety of sugar derivatives modified at C-1 or C-2 could be used as acceptors for transglycosylation by Endo-M to create novel oligosaccharides.     

Fungal beta-N-acetylhexosaminidases with high beta-N-acetylgalactosaminidase activity and their use for synthesis of beta-GalNAc-containing oligosaccharides

About 60 fungal strains were tested for production of extracellular beta-N-acetylhexosaminidases. A unique beta-N-acetylhexosaminidase with the beta-GalNAc-ase/beta-GlcNAc-ase ratio of 2.3-2.8 was found in the culture filtrates of some strains of Penicillium oxalicum. Addition of 20% (w/v) MgSO(4) increased the beta-GalNAc-ase/beta-GlcNAc-ase ratio to the value of 3.35. Cultivation conditions influence this ratio as well. beta-N-Acetylhexosaminidases from P. oxalicum CCF 2430 and Aspergillus oryzae CCF 1066 considerably differing in the GalNAc-ase activity were used for the synthesis of the following structures beta-D-GalpNAc-(1-->4)-D-GlcpNAc, beta-D-GalpNAc-(1-->6)-D-GlcpNAc, beta-D-GalpNAc-(1-->6)-D-GalpNAc, beta-D-GalpNAc-(1-->4)-alpha-D-GlcpNAcOAll and beta-D-GalpNAc-(1-->6)-beta-D-Galp-(1-->4)-alpha-D-GlcpNAcOAll to demonstrate the application of these new enzymes.     

A novel fluorometric assay for ADP-ribose pyrophosphatase activity

ADP-ribose pyrophosphatase (ADPRase) hydrolyzes ADP-ribose to ribose-5-phosphate and AMP. The ADPRase activity have been assessed by coupling the reaction to alkaline phosphatase and colorimetrically measuring the amount of inorganic phosphate released from AMP that is one of the products of ADPRase. Another but less sensitive colorimetric method has been employed: the reaction mixture was treated with charcoal to adsorb the adenine-containing compounds such as AMP and ADPR and subsequently remaining ribose-5-phosphate was measured colorimetrically. However, the measurement of inorganic phosphate cannot be feasible to assay ADPRase in phosphate-containing samples and the determination of ribose-5-phosphate also is less sensitive. Here we develop a fluorescent assay for ADPRase that utilizes 1, N(6)-etheno ADP-ribose, a fluorescent analogue of ADP-ribose. This method measures fluorescent 1, N(6)-etheno adenosine that is produced by coupling the hydrolysis of 1, N(6)-etheno ADP-ribose to dephosphorylation with alkaline phosphatase. The fluorometric assay is comparable in sensitivity and useful for ADPRase assay in phosphate-containing samples.     

Development of an enzyme-linked immunosorbent assay for measurement of activity of myristoyl-coenzyme A:protein N-myristoyltransferase

Myristoyl-coenzyme A (CoA):protein N-myristoyltransferase (NMT) catalyzes the covalent attachment of myristate to the N-terminal glycine residue of various proteins. To develop a high-throughput assay for NMT, the principle of enzyme-linked immunosorbent assay (ELISA) is used, in which anti-N-myristoylglycine (anti-N-Myr-Gly) monoclonal antibody is utilized for the detection of the N-myristoylglycine moiety of the product of NMT catalysis. Enzyme-catalyzed reaction was performed using recombinant NMT expressed in Escherichia coli, myristoyl-CoA, and an octapeptide substrate that is biotinylated at its C terminus. The mixture of the products of the reaction was added to immunoplate wells precoated with anti-N-Myr-Gly monoclonal antibody. Then, the N-myristoyl-biotinylated octapeptide product was specifically captured by the antibody and stained with streptavidin-biotinylated peroxidase and tetramethylbenzidine substrate. This was followed by absorbance measurement (lambda(450)-lambda(630)). In this ELISA, the calibration curve showed a strong correlation between the concentration of the synthetic N-myristoyl-biotinylated octapeptide and the absorbance, indicating that this system may be useful for enzyme kinetics studies. Using this ELISA system, we assayed for serinal derivatives to determine their NMT inhibitory activity and found that serinal bisulfite inhibits yeast NMT activity. This is the first report of the measurement of NMT activity by the ELISA system.     

A general fluorescence-based coupled assay for S-adenosylmethionine-dependent methyltransferases

We have developed a simple and sensitive fluorescence-based two-step coupled enzyme assay to report the activity of S-adenosylmethionine-dependent methyltransferases. This assay relies on a fluorescein-cystamine-methyl red (FL-S-S-MR) reporter molecule that can be activated by thiols. In the absence of thiols, fluorescence from the reporter is quenched through fluorescence resonance energy transfer between the two chromophores. In this report, we use catechol-O-methyltransferase with the addition of S-adenosylhomocysteine hydrolase to produce the thiol homocysteine. The presence of homocysteine leads to disulfide bond cleavage in the cystamine tether and fluorescence dequenching as the uncoupled chromophores are diluted into the surrounding media. The sensitivity and specificity of FL-S-S-MR to thiols enabled detection of 相似文献   

Cell-based assay for beta-secretase activity

The cerebral deposition of amyloid beta-peptide (Abeta) is a major factor in the etiology of Alzheimer's disease. beta-Secretase (BACE) initiates the generation of Abeta by cleaving the amyloid precursor protein at the beta-site and is therefore a prime target for therapeutic intervention. Here we report a cell-based method suitable for monitoring BACE activity and the efficacy of protease inhibitors. A fusion protein containing the amino-terminal transmembrane domain of Golgi alpha-mannosidase II, a Drosophila Golgi integral membrane protein, linked to human alkaline phosphatase (AP) by a short beta-site sequence, was expressed in Drosophila S2 cells. While the uncleaved fusion protein was retained in the Golgi apparatus, cleavage of the beta-site by BACE resulted in the release of AP to the culture medium, where it was easily detected and quantified. Three peptidomimetic inhibitors (LB83190, LB83192, LB83202) were tested for their efficacy with this cell-based assay. While LB83190 and LB83192 effectively blocked BACE activity, LB83202, a carboxylated derivative of LB83192, did not. This is consistent with the inability of LB83202 to permeate the cell membrane. The present cell-based assay could provide a convenient tool for high-throughput screening of substances that can interfere with BACE in living cells.     

A fluorescence-based assay for 2-oxoglutarate-dependent oxygenases

A widely used generic assay for 2-oxoglutarate-dependent oxygenases relies upon monitoring the release of 14CO2 from labeled [1-14C]-2-oxoglutarate. We report an alternative assay in which depletion of 2-oxoglutarate is monitored by its postincubation derivatization with o-phenylenediamine to form a product amenable to fluorescence analysis. The utility of the procedure is demonstrated by assays with hypoxia-inducible factor hydroxylases where it was shown to give results similar to those reported with the radioactive assay, but it is more efficient and readily adapted to a multiwell format. The process should be amenable to the assay of other 2-oxoglutarate-consuming enzymes and to the discovery of inhibitors.     

Mucor hiemalis endo-beta-N-acetylglucosaminidase can transglycosylate a bisecting hybrid-type oligosaccharide from an ovalbumin glycopeptide

We found that the recombinant endo-beta-N-acetylglucosaminidase of Mucor hiemalis (Endo-M) expressed in Candida boidinii had the transglycosylation activity of transferring a bisecting hybrid-type oligosaccharide from an ovalbumin glycopeptide to the acceptor (p-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside) in a good yield of 43%.     

Enzymatic syntheses of T antigen-containing glycolipid mimicry using the transglycosylation activity of endo-α-N-acetylgalactosaminidase

Thomsen–Friedenreich antigen (T antigen) disaccharide, β- -galactose-(1→3)-α-N-acetyl- -galactosamine (β- -Gal-(1→3)-α- -GalNAc), containing glycolipid mimicry was synthesized using the transglycosylation activity of endo-α-N-acetylgalactosaminidase from Bacillus sp. This enzyme could transfer the disaccharide from a p-nitrophenyl substrate to water-soluble 1-alkanols and other alcohols at a transfer ratio of 70% or more. Although the transfer ratios were lower for water-insoluble than water-soluble alcohols, they were shown to increase by adding sodium cholate to the reaction mixtures. The enzyme also transferred the disaccharide directly from asialofetuin to 1-alkanols. The anomeric bond between the disaccharide and 1-alkanols of the transglycosylation product is in the α configuration as determined by sequential digestion of jack bean β-galactosidase and Acremonium α-N-acetylgalactosaminidase. Since the transglycosylation product, β- -Gal-(1→3)-α- -GalNAc-(1→O)-hexyl, efficiently inhibits the binding of anti-T antigen monoclonal antibody to asialofetuin, it has potential as an agent for blocking T antigen-mediated cancer metastasis.     

Fluorescence-based assay for polyprenyl phosphate-GlcNAc-1-phosphate transferase (WecA) and identification of novel antimycobacterial WecA inhibitors

Polyprenyl phosphate-GlcNAc-1-phosphate transferase (WecA) is an essential enzyme for the growth of Mycobacterium tuberculosis (Mtb) and some other bacteria. Mtb WecA catalyzes the transformation from UDP-GlcNAc to decaprenyl-P-P-GlcNAc, the first membrane-anchored glycophospholipid that is responsible for the biosynthesis of mycolylarabinogalactan in Mtb. Inhibition of WecA will block the entire biosynthesis of essential cell wall components of Mtb in both replicating and non-replicating states, making this enzyme a target for development of novel drugs. Here, we report a fluorescence-based method for the assay of WecA using a modified UDP-GlcNAc, UDP-Glucosamine-C₆-FITC (1), a membrane fraction prepared from an M. smegmatis strain, and the E. coli B21WecA. Under the optimized conditions, UDP-Glucosamine-C₆-FITC (1) can be converted to the corresponding decaprenyl-P-P-Glucosamine-C₆-FITC (3) in 61.5% yield. Decaprenyl-P-P-Glucosamine-C₆-FITC is readily extracted with n-butanol and can be quantified by ultraviolet–visible (UV–vis) spectrometry. Screening of the compound libraries designed for bacterial phosphotransferases resulted in the discovery of a selective WecA inhibitor, UT-01320 (12) that kills both replicating and non-replicating Mtb at low concentration. UT-01320 (12) also kills the intracellular Mtb in macrophages. We conclude that the WecA assay reported here is amenable to medium- and high-throughput screening, thus facilitating the discovery of novel WecA inhibitors.     

A 96-well dot-blot assay for carbohydrate sulfotransferases

Here we describe an efficient dot-blot assay for high-throughput screening of two enzymes, heparan sulfate N-deacetylase/N-sulfotransferase (NDST-1) and high-endothelial cell GlcNAc-6-sulfotransferase (HEC-GlcNAc-6-ST). The assay proceeds by transfer of 35S-labeled sulfate from [35S]-3(')-phosphoadenosine-5(')-phosphosulfate (PAPS) to the free amino groups of de-N-sulfated heparin (NDST-1), or the 6-hydroxyl groups of N-acetylglucosamine residues linked to a polyacrylamide scaffold (HEC-GlcNAc-6-ST). The 35S-labeled products are then captured on an appropriate membrane, taking advantage of their polymeric architecture. In one step, 35S-labeled by-products are then eluted from the membrane, leaving spatially separated 35S-labeled product "dots" for subsequent quantification. This assay allows for direct product detection on the membrane, obviating excessive washing and elution steps endemic to other assays. The assay was validated by measuring K(M) values for PAPS and K(I) values for PAP, the product of sulfuryl transfer. The assay method should be useful for inhibitor screens for both enzymes. In addition, the general assay architecture should be readily applicable to high-throughput screens of other carbohydrate sulfotransferases.     

The effects of N-glycosylation sites and the N-terminal region on the biological function of beta1,3-N-acetylglucosaminyltransferase 2 and its secretion

Human beta1,3-N-acetylglucosaminyltransferase 2 (beta3GnT2) is thought to be an enzyme that extends the polylactosamine acceptor chains, but its function and structure analysis are unknown. To obtain insight into the structure of beta3GnT2, the effects of N-glycosylation on its biological function were evaluated using the addition of inhibitors, site-directed mutagenesis of potential N-glycosylation sites, and deletion of its N-terminal region using a fusion protein with GFP(uv) in a baculovirus expression system. Four of five potential N-glycosylation sites were found to be occupied, and their biological function and secretion were inhibited with the treatment of N-glycosylation inhibitor, tunicamycin. The N-glycosylation at Asn219 was necessary for the beta3GnT activity; moreover, N-glycosylation at Asn127 and Asn219 was critical for efficient protein secretion. When Ser221 was replaced with Thr, fusion protein was expressed as a single band, indicating that the double band of the expressed fusion protein was due to the heterogeneity of the glycosylation at Asn219. The truncated protein consisting of amino acids 82-397 (GFP(uv)-beta3GnT2Delta83), which lacked both one N-glycosylation site at Asn79 and the stem region of glycosyltransferase, was expressed as only a small form and showed no beta3GnT activity. These results suggest that the N-glycosylation site at Asn219, which is conserved throughout the beta1,3-glycosyltransferase family, is indispensable not only with regard to its biological function, but also to its secretion. The N-terminal region, which belongs to a stem region of glycosyltransferase, might also be important to the active protein structure.     

A colorimetric assay for the determination of 4-diphosphocytidyl-2-C-methyl-D-erythritol 4-phosphate synthase activity

A new method for the determination of the activity of 4-diphosphocytidyl-2-C-methyl-D-erythritol 4-phosphate synthase, the enzyme catalyzing the third reaction of the 2-C-methyl-D-erythritol 4-phosphate pathway for biosynthesis of isoprenoids, is described. This is an end-point assay based on the transformation of inorganic pyrophosphate, one of the products of the reaction, to phosphate by using inorganic pyrophosphatase as auxiliary enzyme. The phosphate formed is reacted then with the dye malachite green to yield a colored product which can be determined spectrophotometrically. The method is easy to perform, sensitive, and robust and can be used in automated high-throughput screening analyses for the search of inhibitors of the enzyme.     

A new route for the synthesis of Streptococcus pneumoniae 19F and 19A capsular polysaccharide fragments avoiding the β-mannosamine glycosylation step

The recently described [Attolino, E.; Bonaccorsi, F.; Catelani, G.; D’Andrea, F. Carbohydr. Res. 2008, 343, 2545–2556.] β-d-MaNAcp-(1→4)-β-d-Glcp thiophenyl glycosyl donor 3 was used in α-glycosylation reactions of OH-2 and OH-3 of the suitably protected p-MeO-benzyl α-l-rhamnopyranoside acceptors 7 and 8. Glycosylation of the axial OH-2 of 7 took place in high yield (76%) and with acceptable stereoselectivity (α/β = 3.4) leading to the protected trisaccharide α-11, corresponding to the repeating unit of Streptococcus pneumoniae 19F. The same reaction on equatorial OH-3 of acceptor 8 gave the trisaccharide α-15, a constituent of the repeating unit of S. pneumoniae 19A, but in lower yield (41%) and without stereoselection (α/β = 1:1.3). Utilizing the introduced orthogonal protection of OH-1 and OH-4″, the trisaccharide α-11 was transformed into a trisaccharide building block suitable for the synthesis of its phosphorylated oligomers.     

Convenient preparation and characterization of a monoclonal antibody for the N-linked sugar chain of a glycoprotein using a microbial endoglycosidase

We attempted to obtain the monoclonal antibody specific for the N-linked complex-type sialo-oligosaccharide in glycoproteins. We first synthesized a chimeric immunoantigen having an N-linked complex-type of oligosaccharide of glycopeptide, which was bound to a p-formylphenyl compound and conjugated with phosphatidylethanolamine dimylistoyl using the transglycosylation activity of a microbial endoglycosidase (Endo-M) and a reductive amination reaction. This preparative method was convenient and provided a good yield. By immunizing mice with this chimeric neoglycolipid, the monoclonal antibody for the complex-type of sialo-oligosaccharide was obtained in the culture fluid of the cell line even though it was relatively unstable. The monoclonal antibody reacted with various glycoproteins having complex-type sialo-oligosaccharides, but not with those having complex-type asialo-oligosaccharides and high mannose types of oligosaccharides, or with any glycosphingolipids. One of epitopes of this monoclonal antibody seemed to be an α-2,6-linked sialic acid at the non-reducing end of the sialo-oligosaccharide of the glycoprotein.