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Endo-β-N-acetylglucosaminidase (endo-β-GlcNAc-ase) is a unique endoglycosidase that hydrolytically cleaves the N, N′-diacetylchitobiose moiety of asparagine-linked oligosaccharides of various glycoproteins. A novel endo-β-GlcNAc-ase in the culture fluid of Mucor hiemalis isolated from soil was found to have transglycosylation activity. This endo-β-GlcNAc-ase, Endo-M, couldtransfer the intact complex-type oligosaccharide from glycopeptide to suitable acceptors with an Nacetylglucosamine (GlcNAc) residue during hydrolysis of the glycopeptide. Using Endo-M, the chemo-enzymatic synthesis of a bioactive glycopeptide by chemical synthesis of N-acetylglucosaminyl peptide and enzymatic transfer of oligosaccharide was attempted. Peptide T can block HIV infection of human T cells. We added the sialo complex-type oligosaccharide to chemically synthesized N-acetylglucosaminyl Peptide T using the transglycosylation activity of Endo-M. The glycopeptide T thus produced showed a higher degree of resistance to protease digestion. We also prepared calcitonin glycopeptide by the chemo-enzymatic method described above. Calcitonin is a calcium-regulating hormone which is widely used in therapy for hyper calcemia. This glycopeptide demonstrated sufficient physiological activity. N-Acetylglucosaminyl glutamine was also a good glycoside acceptor of Endo-M. We were able to add oligosaccharides to the glutamine residues of Substance P neuropeptide and a-mating factor of yeast Saccharomyces cerevisiae using Endo-M. The glycosylated Substance P was biologically active and stable against peptidase digestion. On the other hand, the bioactivity of the glycosylated a-mating factor was lower than that of native one, although that of N-acetylglucosaminyl a-mating factor was higher. Using the transglycosylation activity of Endo-M, we prepared alkyl compound having oligosaccharide of glycoprotein. We used it as an antigen for preparing monoclonal antibody against oligosaccharide of glycoprotein.

The chemoenzymatic synthesis of a glycopeptide by chemical synthesis of N-acetylglucosaminyl peptide and enzymatic transfer of an oligosaccharide is described. We synthesized glycosylated Peptide T which blocks infection of human T cells by human immunodeficiency virus. The first step of the chemoenzymatic method is the solid-phase chemical synthesis of N-acetylglucosaminyl Peptide T (Ala-Ser-Thr-Thr-Thr-Asn(GlcNAc)-Tyr-Thr) with an N-acetylglucosamine moiety bound to the asparaginyl residue by a solid-phase method. This product was prepared in high yield by the dimethylphosphinothioic mixed anhydride method without protecting the hydroxyl functions of the sugar moiety using Fmoc-N-acetylglucosaminyl asparagine instead of Fmoc-asparagine. The second step was transglycosylation of complex type oligosaccharide to N-acetylglucosaminyl Peptide T by a microbial endoglycosidase. The endo-beta-N-acetylglucosaminidase of Mucor hiemalis transfer the oligosaccharide of human transferrin glycopeptide to N-acetylglucosaminyl Peptide T. The transglycosylation product was confirmed to be the glycosylated Peptide T with a sialo biantennary complex type oligosaccharide by mass spectrometry. The glycosylated Peptide T was highly stable against proteolysis in comparison to native Peptide T and N-acetylglucosaminyl Peptide T.

Recently, we reported the ‘chemo-enzymatic synthesis’ [1] of complex glycopeptide based on the chemical synthesis of glycopeptide containing N-acetylglucosamine (GlcNAc) attached to the asparagine (Asn) residue of the peptide [2] and the transglycosylation of oligosaccharide to this N -acetylglucosaminyl peptide catalyzed by endo-β-N-acetylglucosaminidase of Mucor hiemalis (Endo-M) [3]. Such complex glycopeptides as glycosylated Peptide T [4] or calcitonin [5, 6] derivatives, in which sialo complex-type asparagine (N)-linked oligosaccharides were attached to the Asn residue of the peptides, were synthesized by this method. Substance P is a neuropeptide consisting of eleven amino acids [H–Arg– Pro–Lys–Pro–Gln–Gln–Phe–Phe–Gly–Leu–Met–NH2 ]. This peptide has no Asn residue but has two glutamine (Gln) residues at the position 5 and 6, to which biological addition of oligosaccharide is impossible. We tried to add N -linked oligosaccharides to the each Gln residue of this peptide by using our chemo-enzymatic method, in order to investigate the effect of sugar or sugar chains on the solubility, activity or biological stability of the peptide.

A lignin-degrading basidiomycete, Ceriporiopsis subvermispora produces a series of alkyl- and alkenylitaconates (ceriporic acids). Previously, two alkylitaconic acids with tetradecyl and hexadecyl side chains were isolated and identified as 1-heptadecene-2,3-dicarboxylic acid (ceriporic acid A) and 1-nonadecene-2,3-dicarboxylic acid (ceriporic acid B). In the present study, one hexadecenylitaconate (ceriporic acid C) was isolated and its chemical structure was analyzed by glycolation and subsequent (1) trimethylsilation, or (2) acetalation with acetone and acetone-d6. Analyses of the isolated metabolite demonstrated that the hexadecenylitaconic acid was (Z)-1,10-nonadecadiene-2,3-dicarboxylic acid. The structure of the side chain in ceriporic acid C was the same as that of hexadecenylcitraconate, chaetomellic acid B. Thus, it was found that ceriporic acids share close structural similarity with alk(en)yl citraconate derivatives, chaetomellic acids and other lichen lactones, protolichesterinic, lichesterinic, and murolic acids.

A bioactive peptide containing a glutamine-linked oligosaccharide was chemo-enzymatically synthesized by use of the solid-phase method of peptide synthesis and the transglycosylation activity of endo-beta-N-acetylglucosaminidase. Substance P, a neuropeptide, is an undecapeptide containing two L-glutamine residues. A substance P derivative with an N-acetyl-D-glucosamine residue attached to the fifth or sixth L-glutamine residue from the N-terminal region was chemically synthesized. A sialo complex-type oligosaccharide derived from a glycopeptide of hen egg yolk was added to the N-acetyl-D-glucosamine moiety of the substance P derivative using the transglycosylation activity of endo-beta-N-acetylglucosaminidase from Mucor hiemalis, and a substance P derivative with a sialo complex-type oligosaccharide attached to the L-glutamine residue was synthesized. This glycosylated substance P was biologically active, although the activity was rather low, and stable against peptidase digestion. The oligosaccharide moiety attached to the L-glutamine residue of the peptide was not liberated by peptide-N(4)-(N-acetyl-beta-D-glucosaminyl) asparagine amidase F.

The chemoenzymatic synthesis of a glycopeptide by chemical synthesis of N-acetylglucosaminyl peptide and enzymatic transfer of an oligosaccharide is described. We synthesized glycosylated Peptide T which blocks infection of human T cells by human immunodeficiency virus. The first step of the chemoenzymatic method is the solid-phase chemical synthesis of N-acetylglucosaminyl Peptide T (Ala-Ser-Thr-Thr-Thr-Asn(GlcNAc)-Tyr-Thr) with an N-acetylglucosamine moiety bound to the asparaginyl residue by a solid-phase method. This product was prepared in high yield by the dimethylphosphinothioic mixed anhydride method without protecting the hydroxyl functions of the sugar moiety using Fmoc-N-acetylglucosaminyl asparagine instead of Fmoc-asparagine. The second step was transglycosylation of complex type oligosaccharide to N-acetylglucosaminyl Peptide T by a microbial endoglycosidase. The endo-beta-N-acetylglucosaminidase of Mucor hiemalis transfer the oligosaccharide of human transferrin glycopeptide to N-acetylglucosaminyl Peptide T. The transglycosylation product was confirmed to be the glycosylated Peptide T with a sialo biantennary complex type oligosaccharide by mass spectrometry. The glycosylated Peptide T was highly stable against proteolysis in comparison to native Peptide T and N-acetylglucosaminyl Peptide T.

Background Mothers of young children are at high-risk for developing adverse mental health effects following a nuclear accident. Using the Japanese pregnancy registration system, the prefecture of Fukushima launched a population-based survey of women who were pregnant at the time of the Fukushima nuclear accident in order to assess their and their newborns’ health. In this paper, we focus on the results of a screen for depressive symptoms among new mothers and its association with geographical region and interruption of obstetrical care after the Fukushima nuclear accident, which occurred after the Great East Japan Earthquake on March 11, 2011. Methods The survey targeted women who lived in Fukushima prefecture and who had registered their pregnancies between August 1, 2010 and July 31, 2011. Among the 16,001 women targeted, 9,321 returned the questionnaires (response proportion = 58.3%) and data from 8,196 women with singleton live births were analyzed. The main outcome measure was a standard two-item depression screen. Regional radiation levels were determined from the prefecture’s periodical reports, and interruption in obstetrical care after the Fukushima nuclear accident was determined from mothers’ individual responses to the questionnaire. Results Among the 8,196 women, 2,262 (28%) screened positive for depressive symptoms. After adjusting for maternal and infant characteristics, both mothers in Soso, the region in which the nuclear power plant is located, and mothers that had changed obstetrical care facilities were significantly more likely to screen positive for depression. In contrast, mothers in Iwaki and Aizu, regions with relatively low radiation levels, were significantly less likely to screen positive for depression. Conclusions Our findings suggest that improving mental health support for mothers with infants should be a high priority in the acute phase of nuclear disaster response. We further recommend that in the strategic provisioning of parental support, close attention should be paid to regional variations in negative mental health consequences, particularly to those who experienced an interruption in their obstetrical care.