Development of a microbioreactor for glycoconjugate synthesis.

A microbioreactor immobilized with a synthase-type mutant enzyme, Endo-M-N175Q (glycosynthase) of endo-ß-N-acetylglucosaminidase derived from Mucor hiemalis (Endo-M), was constructed and used for glycoconjugate synthesis. The transglycosylation was performed with a reaction mixture containing an oxazoline derivative of sialo complex-type glycoside (SG), which was prepared from a sialo complex-type glycopeptide SGP derived from hen egg yolk, as a glycosyl donor and N-Fmoc-N-acetylglucosaminyl-l-asparagine [Fmoc-Asn(GlcNAc)-OH] as an acceptor. The reaction mixture was injected into a glycosynthase microbioreactor at a constant flow rate. Highly efficient and nearly stoichiometric transglycosylation occurred in the microbioreactor, and the transglycosylation product was eluted from the other end of the reactor. The glycosynthase microbioreactor was stable and could be used repeatedly for a long time.

Chemo-enzymatic synthesis of a glycosylated peptide containing a complex N-glycan based on unprotected oligosaccharides by using DMT-MM and Endo-M.

For chemo-enzymatic synthesis of a glycosylated peptide, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) was used for the synthesis of a N-acetylglucosaminyl peptide and a pseudoglycopeptide by solid-phase peptide synthesis without the requirement of protecting groups on the carbohydrate. We also performed transglycosylation of an N-glycan to the N-acetylglucosaminyl peptide using endo-ß-N-acetylglucosaminidase from Mucor hiemalis (Endo-M) to synthesize a glycopeptide containing a complex N-glycan.

Synthesis, structure, and evaluation of a ß-cyclodextrin-artificial carbohydrate conjugate for use as a doxorubicin-carrying molecule.

This paper describes the synthesis of a ß-cyclodextrin (ß-CyD) derivative conjugated with a C,C-glucopyranoside containing a benzene unit. Its doxorubicin-inclusion ability and structure are also discussed. SPR analysis revealed that the ß-CyD conjugate had a high inclusion association value of 3.8×10(6)M(-1) for immobilized doxorubicin. NMR structural analysis suggested that its high doxorubicin-inclusion ability was due to the formation of the inclusion complex as a result of the π-π stacking interaction between the benzene ring of the conjugate and the A ring of doxorubicin.

The effect of glycosylation on the antibody recognition of a MUC2 mucin epitope.

MUC2 glycoprotein, produced by the epithelium of the colon and built up mainly of repeat units of (1) PTTTPITTTTTVTPTPTPTGTQT(23) , can be overexpressed or underglycosylated in gastrointestinal diseases, e.g. in case of colon carcinoma. We have been studying the epitope structure of the MUC2 by focusing on the repeat unit with the mucin peptide specific MAb 996 monoclonal antibody. This antibody recognizes the (18) PTGTQ(22) sequence as minimal, and (16) PTPTGTQ(22) as optimal epitope within the underglycosylated glycoprotein. In this article, we aim to clarify the effect of glycosylation of the epitope on MAb 996 antibody binding including its correlation with the secondary structure of the modified peptides: glycosylation in the epitope core and in the flank. For this we have prepared the (16) PTPTGTQ(22) peptide glycosylated with N-acetylgalactoseamine (Tn antigen) in position 17, 19, 21, or on all three threonines. The MAb 996 antibody binding properties of the peptides were characterized in competitive ELISA experiments, and their solution secondary structure was studied by circular dichroism spectroscopy in water and in the ordered structure promoting trifluoroethanol. Our results show that glycosylation in position 19 (peptide (16) PTPT(GalNAcα)GTQ(22) ) resulted in enhanced antibody recognition and significantly altered secondary structure, while glycosylation in position 21 completely demolished the binding. These findings could be useful in determining the nature of antigen-antibody interaction, and perhaps designing synthetic peptide vaccines for tumor therapy.

Sensor-on-Microtips: Design and Development of Hydrothermally Grown ZnO on Micropipette Tips as a Modified Working Electrode for Detection of Glucose.

Miniaturization of electrochemical components has become less common in the last decade, with the focus predominantly being the design and development of state-of-the-art microelectrodes for achieving small volume analysis of samples. However, such microelectrodes involve cumbersome processing procedures to convert the base material for the required application. A potential paradigm shift in such miniaturization could be achieved by using cheaper alternatives such as plastics to build electrochemical components, such as micropipette tips made of polypropylene, which are commercially available at ease. Hence, this work presents the design of an electrochemical working electrode based upon a micropipette tip, involving minimal processing procedures. Furthermore, such a working electrode was realized by sputtering silver onto a bare micropipette tip using a radio-frequency sputtering technique, to obtain electrical contacts on the tip, followed by hydrothermal growth of ZnO, which acted as the active electrode material. The ZnO nanostructures grown on the micropipette tip were characterized for their morphology and surface properties using a scanning electron microscope (SEM), laser microscope, Raman spectrometer, and X-ray photoelectron spectrometer (XPS). The developed micropipette tip-based electrode was then used as the working electrode in a three-electrode system, wherein its electrochemical stability and properties were analyzed using cyclic voltammetry (CV). Furthermore, the above system was used to detect glucose concentrations of 10-200 µM, to evaluate its sensing properties using amperometry. The developed working electrode exhibited a sensitivity of 69.02 µA/µM cm-2 and limit of detection of 67.5 µM, indicating the potential for using such modified micropipette tips as low-cost miniaturized sensors to detect various bio-analytes in sample solutions.

Effects of length and amino acid sequence of O-mannosyl peptides on substrate specificity of protein O-linked mannose ß1,2-N-acetylglucosaminyltransferase 1 (POMGnT1).

Protein O-linked mannose ß1,2-N-acetylglucosaminyltransferase 1 (POMGnT1) catalyzes the transfer of GlcNAc to O-mannose of glycoproteins. Mutations in the POMGnT1 gene cause muscle-eye-brain disease (MEB). POMGnT1 is a typical type II membrane protein, which is localized in the Golgi apparatus. However, details of the catalytic and reaction mechanism of POMGnT1 are not understood. To develop a better understanding of POMGnT1, we examined the substrate specificity of POMGnT1 using a series of synthetic O-mannosyl peptides based on the human α-dystroglycan (α-DG) sequence as substrates. O-Mannosyl peptides consisting of three to 20 amino acids are recognized as substrates. Enzyme kinetics improved with increasing peptide length up to a length of 8 amino acids but the kinetics of peptides longer than 8 amino acids were similar to those of octapeptides. Our results also show that the amino acid sequence affects POMGnT1 activity. These data suggest that both length and amino acid sequence of mannosyl peptides are determinants of POMGnT1 substrate specificity.

Chemo-enzymatic synthesis of glycosylated insulin using a GlcNAc tag.

Artificial insulin with an N-linked oligosaccharide was synthesized by a chemo-enzymatic method using endo-beta-N-acetylglucosaminidase from Mucor hiemalis (Endo-M). GlcNAc-modified insulin was prepared by the reaction of the carboxymethyl glycoside of GlcNAc and 3 amino groups of bovine insulin using a dimethylphosphinothioic mixed anhydride (Mpt-MA) method. A transglycosylation reaction of the GlcNAc-modified insulin using Endo-M gave mono-transglycosylated insulin predominantly. We determined the transglycosylation site of the mono-transglycosylated insulin.

Protein O-mannosyltransferase activities in lymphoblasts from patients with alpha-dystroglycanopathies.

Defects in O-mannosylation of alpha-dystroglycan cause some forms of congenital muscular dystrophy (CMD), the so-called alpha-dystroglycanopathies. Six genes are responsible for these diseases with overlapping phenotypes. We investigated the usefulness of a biochemical approach for the diagnosis and investigation of the alpha-dystroglycanopathies using immortalized lymphoblasts prepared from genetically diagnosed and undiagnosed CMD patients and from control subjects. We measured the activities of protein O-mannose beta1,2-N-acetylglucosaminyltransferase 1 (POMGnT1) and protein O-mannosyltransferase (POMT). Lymphoblasts from patients harbouring known mutations in either POMGNT1 or POMT1 showed a marked decrease in POMGnT1 or POMT activity, respectively, compared to controls. Furthermore, we identified pathogenic mutations in POMGNT1, POMT1 or POMT2 in six previously genetically uncharacterised patients who had very low enzyme activity. In conclusion, the lymphoblast-based enzymatic assay is a sensitive and useful method (i) to select patients harbouring POMGNT1, POMT1 or POMT2 mutations; (ii) to assess the pathogenicity of new or already described mutations.

Chemo-enzymatic synthesis of eel calcitonin glycosylated at two sites with the same and different carbohydrate structures.

Naturally occurring glycopeptides and glycoproteins usually contain more than one glycosylation site, and the structure of the carbohydrate attached is often different from site to site. Therefore, synthetic methods for preparing peptides and proteins that are glycosylated at multiple sites, possibly with different carbohydrate structures, are needed. Here, we report a chemo-enzymatic approach for accomplishing this. Complex-type oligosaccharides were introduced to the calcitonin derivatives that contained two N-acetyl-D-glucosamine (GlcNAc) residues at different sites by treatment with Mucor hiemalis endo-beta-N-acetylglucosaminidase. Using this enzymatic transglycosylation reaction, three glycopeptides were produced, a calcitonin derivative with the same complex-type carbohydrate at two sites, and two calcitonin derivatives each with one complex-type carbohydrate and one GlcNAc. Starting from the derivatives with one complex-type carbohydrate and one GlcNAc, a high-mannose-type oligosaccharide was successfully transferred to the remaining GlcNAc using another endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae. Thus, we were able to obtain glycopeptides containing not only two complex-type carbohydrates, but also both complex and high-mannose-type oligosaccharides in a single molecule. Using the resultant glycosylated calcitonin derivatives, the effects of di-N-glycosylation on the structure and the activity of calcitonin were studied. The effect appeared to be predictable from the results of mono-N-glycosylated calcitonin derivatives.

Complete NMR assignment of a bisecting hybrid-type oligosaccharide transferred by Mucor hiemalis endo-ß-N-acetylglucosaminidase.

This study describes the complete nuclear magnetic resonance (NMR) spectral assignment of a bisecting hybrid-type oligosaccharide 1, transferred by Mucor hiemalis endo-ß-N-acetylglucosaminidase (Endo-M). Through (1)H- and (13)C-NMR, DQF-COSY, HSQC, HMBC, TOCSY, and NOESY experiments, we determine the structure of the glycoside linkage formed by the Endo-M transglycosylation, i.e., the connection between GlcNAc and GlcNAc in oligosaccharide 1.

A novel peptide synthesis using fluorous chemistry.

Three new fluorous supports for peptide synthesis, i.e., the trialkoxybenzhydryl-type (6), the Wang-type (7) and the tert-butyl-type support (8), were prepared. A bioactive peptide TRH was easily synthesized by an Fmoc strategy using the benzhydryl-type fluorous support with fluorous chemistry.

Enhancement of bioactivity of Saccharomyces cerevisiae alpha-mating factor by attachment of sugar moiety to glutamine residue.

We prepared yeast Saccharomyces cerevisiae alpha-mating factor, a 13-amino acid pheromone produced by haploid alpha-cells, bound with glucose or N-acetylglucosamine at the fifth glutamine residue from the N-terminal by the chemical method of peptide synthesis. It was found that the bioactivity of glucosyl alpha-mating factor was higher than that of native alpha-mating factor. However, it was slightly lower than that of N-acetylglucosaminyl alpha-mating factor. This suggested that the N-acetylamino residue might play some important role in the enhancement of the bioactivity of alpha-mating factor. However, CD spectra analysis of alpha-mating factor and its derivatives demonstrated that their structures were almost identical. On the other hand, we attached a sialo complex type oligosaccharide to N-acetylglucosamine or its glucose residue by means of the transglycosylation activity of endo-beta-N-acetylglucosaminidase from Mucor hiemalis (Endo-M). The attachment of the oligosaccharide to both alpha-mating factors reduced their activities. However, enzymatical trimming of the sialo complex type oligosaccharide recovered its activity.

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%.

Acceptor specificity in the transglycosylation reaction using Endo-M.

To determine the structural specificity of the glycosyl acceptor of the transglycosylation reaction using endo-ß-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.

Self-aggregation of synthetic zinc chlorophyll derivatives possessing multi-perfluoroalkyl chains in perfluorinated solvents.

Zinc 3(1)-hydroxy-13(1)-oxo-chlorins possessing two, three, four and six perfluorooctyl chains were synthesized from naturally occurring chlorophyll-a. Only the synthetic zinc chlorin possessing six perfluorooctyl chains was directly dissolved in perfluorinated solvents due to its high fluorine content in molecular weight (over 50%). In this solution, visible absorption spectra gave a red-shifted Q(y) band at 723 nm (compared to 648 nm in THF) and showed the formation of well-ordered self-aggregates. No monomeric form was observed in the solution from any fluorescence emission spectra from visible absorption spectra. In the aggregate solution, no precipitation occurred during either standing for a long period or heating at 70 degrees C. This showed that the supramolecular structure was stabilized by F-F interactions on its surface among the perfluorooctyl chains on the 17-position and perfluorinated solvents. The core part of the supramolecular structure was constructed by a special intramolecular bond of Zn ... O3(2)-H ... O=C13(1), which was confirmed from resonance Raman spectral analysis.

Neutralizing activity of polyvalent Gb3, Gb2 and galacto-trehalose models against Shiga toxins.

Shiga toxin (Stx) is one of the most critical factors in the development of hemolytic uremic syndrome and other systemic complications following enterohemorrhagic Escherichia coli (EHEC) infection. Substances neutralizing Stx by interfering with toxin-receptor binding have been explored as therapeutic candidates for EHEC infection. In this study, we examined globotriaosyl (Gb3), galabiosyl (Gb2) and galacto-trehalose, each of which was synthetically conjugated with a polyacrylamide backbone, for Stxneutralizing activity. Galacto-trehalose was designed as a Gb2 mimicking, unnatural Stx-ligand that was expected to show tolerance to enzymatic degradation in vivo. Galacto-trehalose copolymer showed neutralizing activity against Stx-1 but not Stx-2 in a HeLa cell cytotoxicity assay. It was thought that galactotrehalose copolymer could be a lead compound for the treatment of Stx-mediated diseases, although it requires modification to show neutralizing activity to Stx-2. The Gb3 copolymer with high sugar unit density showed stronger neutralizing activity against Stx-2 than those with lower density. However, the density-dependency of the neutralizing activity was less obvious against Stx-1. Intravenous administration of the Gb3 copolymer prevented death in mice lethally infected with Stx-1- and Stx-2-producing E. coli O157:H7. Thus, we demonstrated that the artificial Gb3 copolymer could neutralize Stx-1 and the more clinically relevant Stx-2 in vitro and effectively inhibit Stx toxicity in vivo.

Regulation of mammalian protein O-mannosylation: preferential amino acid sequence for O-mannose modification.

O-mannosyl glycans are important in muscle and brain development. Protein O-mannosyltransferase (POMT) catalyzes the initial step of O-mannosyl glycan biosynthesis. To understand which serine (Ser) and threonine (Thr) residues POMT recognizes for mannosylation, we prepared a series of synthetic peptides based on a mucin-like domain in alpha-dystroglycan (alpha-DG), one of the best known O-mannosylated proteins in mammals. In alpha-DG, the mucin-like domain spans amino acid residues 316 to 489. Two similar peptide sequences, corresponding to residues 401-420 and 336-355, respectively, were strongly mannosylated by POMT, whereas other peptides from alpha-DG and peptides of various mucin tandem repeat regions were poorly mannosylated. Peptides 401-420 and 336-355 contained four and six Ser and Thr residues, respectively. Substitution of Ala residues for the Ser or Thr residues showed that Thr-414 of peptide 401-420 and Thr-351 of peptide 336-355 were prominently modified by O-mannosylation. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry and Edman degradation analysis of the mannosylated peptide 401-420 indicated that Thr-414 was the Thr residue that was most prominently modified by O-mannosylation and that O-mannosylation occurred sequentially rather than at random. Based on these results, we propose a preferred amino acid sequence for mammalian O-mannose modification.