Toshiyuki Inazu

Protein O-mannosyltransferase activities in lymphoblasts from patients with α-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.

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 Stx‐neutralizing 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 galacto‐trehalose 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.

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.

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.

Effects of length and amino acid sequence of O-mannosyl peptides on substrate specificity of protein O-linked mannose β1,2-N-acetylglucosaminyltransferase 1 (POMGnT1)

Protein O-linked mannose β1,2-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.

A chemical synthesis of UDP-LacNAc and its regioisomer for finding 'oligosaccharide transferases'

A chemical synthesis of uridine 5’-diphospho-N-acetyllactosamine (Galβ(1→4)GlcNAc-UDP; UDP-LacNAc) and Galβ(1→3)GlcNAc-UDP is described. Coupling of the disaccharide imidate derivatives with dibenzylphosphate gave the corresponding 1-phosphates, which were condensed with UMP-imidazolate to give the target UDP-oligosaccharides after purification by anion exchange HPLC and gel filtration column chromatography. Using this methodology a variety of oligosaccharide nucleotide analogues can be synthesized. These UDP-oligosaccharides may be useful for finding so-called `oligosaccharide transferases’, the glycosyltransferases which transfer the oligosaccharide moiety onto glycosyl acceptors.

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

This paper describes the synthesis of a β-cyclodextrin (β-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 β-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 1PTTTPITTTTTVTPTPTPTGTQT23, 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 18PTGTQ22 sequence as minimal, and 16PTPTGTQ22 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 16PTPTGTQ22 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 16PTPT(GalNAcα)GTQ22) 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.

Development of a microbioreactor for glycoconjugate synthesis

A microbioreactor immobilized with a synthase-type mutant enzyme, Endo-M-N175Q (glycosynthase) of endo-β-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.