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Toshiyuki Inazu

Publications and source records attributed to Toshiyuki Inazu.

14 recordsLinked to original sources
PubMed2006

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

A chemical synthesis of uridine 5'-diphospho-N-acetyllactosamine (Galbeta(1-->4)GlcNAc-UDP; UDP-LacNAc) and Galbeta(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.

Hexosyltransferases↗
PubMed2005

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.

Amino Acid Sequence↗
PubMed2005

Combinatorial synthesis of MUC1 glycopeptides: polymer blotting facilitates chemical and enzymatic synthesis of highly complicated mucin glycopeptides.

The chemoselective polymer blotting method allows for rapid and efficient synthesis of glycopeptides based on a "catch and release" strategy between solid-phase and water-soluble polymer supports. We have developed a heterobifunctional linker sensitive to glutamic acid specific protease (BLase). The general procedure consists of five steps, namely (i) the solid-phase synthesis of glycopeptide containing BLase sensitive linker, (ii) subsequent deprotections and the release of the glycopeptide from the resin, (iii) chemoselective blotting of the glycopeptide intermediates in the presence of water-soluble polymers with oxylamino functional groups, (iv) sugar elongations using glycosyltransferases, and (v) the release of target glycopeptides from the polymer platform by selective BLase promoted hydrolysis. The combined use of the solid-phase chemical syntheses of peptides and the enzymatic syntheses of carbohydrates on water-soluble polymers would greatly contribute to the production of complicated glycopeptide libraries, thereby enhancing applicative research. We report here a high-throughput synthetic system for the various types of MUC1 glycopeptides exhibiting a variety of sugar moieties. It is our belief that this concept will become part of the entrenched repertoire for the synthesis of biologically important glycopeptides on the basis of glycosyltransferase reactions in automated and combinatorial syntheses.

Carbohydrate Sequence↗
PubMed2004

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.

Acetylglucosamine↗
PubMed2004

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%.

Acetylglucosamine↗
PubMed2004

Rapid oligosaccharide synthesis using a fluorous protective group.

The Bfp-OH, a novel fluorous protecting reagent, was able to be easily prepared. The Bfp group was readily introduced to a carbohydrate, removed in high yield, and recyclable after cleavage. The use of the Bfp group made it possible to synthesize a pentasaccharide by minimal column chromatography purification. Each synthetic intermediate was able to be easily purified only by simple fluorous-organic solvent extraction and monitored by TLC, NMR, and MS.

Catalysis↗
PubMed2004

Interaction assay of oligosaccharide with lectins using glycosylasparagine.

Glycosyl amino acids having natural glycan were useful for the interaction assay of oligosaccharides. A glycochip containing the whole structure of an oligosaccharide was easily prepared by the immobilization of the glycosyl amino acid. Furthermore, fluorescence probes were introduced into the glycosyl amino acid while maintaining the whole structure of oligosaccharide. By using these labeled oligosaccharides, fluorescence polarization (FP) and fluorescence correlation spectroscopy (FCS) analyzed the carbohydrate-lectin interaction in a solution assay system.

Animals↗
PubMed2004

Chemo-enzymatic synthesis and structure-activity study of artificially N-glycosylated eel calcitonin derivatives with a complex type oligosaccharide.

Starting from N-glycosylated eel calcitonin derivatives that contain an N-acetyl-D-glucosamine residue specifically at the 3rd, 14th, 20th or 26th amino acid residue, corresponding glycopeptides with a complex-type oligosaccharide attached to the respective amino acid residue were synthesized by means of a transglycosylation reaction catalyzed by an endo-beta-N-acetylglucosaminidase from Mucor hiemalis . The use of a recombinant enzyme and an excess of a glycosyl donor led to a yield in excess of 60%. Calcitonin derivatives containing truncated oligosaccharides were also prepared via digestion of the complex-type N-glycan with exoglycosidases. Using these N-glycosylated calcitonin derivatives, the effect of carbohydrate structure and glycosylation site on the three-dimensional structure and the biological activity of the peptide were studied. The conformation of the peptide backbone did not change irrespective of the carbohydrate structure or the glycosylation site. However, hypocalcemic activity, calcitonin-receptor binding activity and the biodistribution of the derivatives were affected by the glycosylation and were dependent on both the carbohydrate structure and the glycosylation site. Although the larger oligosaccharides tended to hinder receptor binding, the biodistribution altered by N-glycosylation appeared to enhance the hypocalcemic activity in some cases, and the magnitude of the effect was dependent on the site of glycosylation.

Animals↗
PubMed2003

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.

Amino Acid Sequence↗
PubMed2002

Chemo-enzymatic synthesis of the glycosylated alpha-mating factor of Saccharomyces cerevisiae and analysis of its biological activity.

The effect of glycosylation on a bioactive peptide was studied using yeast Saccharomyces cerevisiae alpha-mating factor, which is composed of 13 amino acids. In this study, we prepared glycosylated alpha-mating factor by chemo-enzymatic synthesis. At first, N-acetylglucosaminyl alpha-mating factor (Trp-His-Trp-Leu-Gln(GlcNAc)-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr) was chemically synthesized by the solid-phase method. Then, using the transglycosylation activity of Mucor hiemalis endo-beta-N-acetylglucosaminidase, we synthesized glycosylated alpha-mating factor with a glutamine-linked sialo complex type oligosaccharide. The biological activity of alpha-mating factor derivatives was examined by means of a growth arrest assay using secreted-protease-defective a cells of S. cerevisiae. The results showed that the bioactivity of glycosylated alpha-mating factor was lower than that of native alpha-mating factor. However, when sialic acid was removed from the complex type sugar chain of glycosylated alpha-mating factor, its bioactivity was recovered. Glycosylated alpha-mating factor exhibited higher resistance against proteolysis than native alpha-mating factor. It was found that the bioactivity of N-acetylglucosaminyl alpha-mating factor was higher than that of alpha-mating factor. Circular dichroism studies indicated that a slight change in the structure of alpha-mating factor may influence its activity.

Acetylglucosamine↗
PubMed2002

Molecular cloning and expression of a sixth type of alpha 2,8-sialyltransferase (ST8Sia VI) that sialylates O-glycans.

A novel member of the mouse alpha2,8-sialyltransferase (ST8Sia) family, designated ST8Sia VI, was identified by BLAST analysis of expressed sequence tags. The sequence of ST8Sia VI encodes a protein of 398 amino acids and shows 42.0 and 38.3% amino acid sequence identities to mouse alpha2,8-sialyltransferases ST8Sia I (GD3 synthase) and ST8Sia V (GD1c, GT1a, GQ1b, and GT3 synthases), respectively. The recombinant soluble form of ST8Sia VI expressed in COS-7 cells exhibited alpha2,8-sialyltransferase activity toward both glycolipids and glycoproteins that have the NeuAcalpha2,3(6)Gal sequence at the nonreducing end of their carbohydrate groups. This enzyme formed NeuAcalpha2,8NeuAc structures, but not oligosialic or polysialic acid structures. Analysis of the fetuin sialylated by ST8Sia VI indicated that ST8Sia VI prefers O-glycans to N-glycans as acceptor substrates. Substrate specificities and kinetic properties also showed that ST8Sia VI prefers O-glycans to glycolipids as acceptor substrates. ST8Sia VI also exhibited activity toward oligosaccharides such as sialyllactose and sialyllactosamine, and the structure of the minimal acceptor substrate for ST8Sia VI was determined as the NeuAcalpha2,3(6)Gal sequence. The expression of the ST8Sia VI gene was ubiquitous, and the highest expression was observed in kidney, with three major transcripts of 8.2, 3.8, and 2.7 kb. This is the first report of a mammalian alpha2,8-sialyltransferase that sialylates O-glycans preferentially.

Amino Acid Sequence↗