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The binding properties of the rat sublingual glycoprotein (RSL) and its asialo product with lectins were characterized by quantitative precipitin (QPA) and precipitin inhibition (QPIA) assays. Among twenty lectins tested for QPA, native RSL reacted well only with Artocarpus integrifolia (jacalin) but weakly or not at all with the other lectins. However, its asialo product (asialo-RSL) reacted strongly with many Gal and GalNAc specific lectins- it bound best to three of the GalNAcalpha1?Ser/Thr (II) and/or Galbeta1?4GlcNAc active lectins [jacalin, Wistaria floribunda and Ricinus communis agglutinins] and completely precipitated each of these three lectins. Asialo-RSL also reacted well with Abrus precatorius, Glycine max Bauhinia purpurea alba, and Maclura pomifera agglutinins and abrin-a, but not with Arachis hypogeae and Dolichos biflorus agglutinins. The interaction between asialo-RSL and lectins were inhibited by either Galbeta1?4GlcNAc, p-NO2-phenylalpha-GalNAc or both. The mapping of the precipitation and inhibition profiles leads to the conclusion that the asialo rat sublingual glycoprotein provides important ligands for II(Galbeta1?4GlcNAcbeta1?) and Tn(GalNAcalpha1 ?Ser/Thr) active lectins.

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A novel lectin was purified to homogeneity from winter buds of Lysichiton camtschatcensis (L.) Schott of the Araceae family. It was a tetramer composed of two non-covalently associated polypeptides with small subunits (11 kDa) and large subunits (12 kDa). Sequencing of both subunits yielded unique N-terminal sequences. A cDNA encoding the lectin was cloned. The isolated cDNA contained an open reading frame that encoded 267 amino acids. It encoded both subunits, indicating that the lectin is synthesized as a single precursor protein that is post-translationally processed into two different subunits with 45% sequence identity. Each subunit contained a mannose-binding motif known to be conserved in monocot mannose-binding lectins, but its activity was not inhibited by monosaccharides, including methyl alpha-mannoside. Asialofetuin and yeast invertase were potent inhibitors. Lectin activity was detected in the buds formed during the winter season but not in the expanded leaves.

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The lipid carrier specificity of the protein N-glycosylation enzyme C. jejuni PglB was tested using a logical, synthetic array of natural and unnatural C10, C20, C30, and C40 polyisoprenol sugar pyrophosphates, including those bearing repeating cis-prenyl units. Unusual, short, synthetically accessible C20 prenols (nerylnerol 1d and geranylnerol 1e) were shown to be effective lipid carriers for PglB sugar substrates. Kinetic analyses for PglB revealed clear KM-only modulation with lipid chain length, thereby implicating successful in vitro application at appropriate concentrations. This was confirmed by optimized, efficient in vitro synthesis allowing >90% of Asn-linked beta-N-GlcNAc-ylated peptide and proteins. This reveals a simple, flexible biocatalytic method for glycoconjugate synthesis using PglB N-glycosylation machinery and varied chemically synthesized glycosylation donor precursors.

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A novel methodology for the enzymatic preparation from suitably derivatized oligosaccharides of N-linked neoglycopeptides using the microbial glutaminyl-peptide gamma-glutamyl transferase, transglutaminase (TGase), is described. N-Allyl glycosides of various oligosaccharides were photochemically coupled with cysteamine to yield amino-terminated thioether spacers, which were accepted by transglutaminase to transamidate the side-chain gamma-carboxamide group in the dipeptide Z-Gln-Gly.

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(Chemical Presented) Strengthening the bond: The introduction of a thiol tag in combination with chemoselective ligation to form a disulfide-linked bioconjugate is a selective and useful method for site-selective protein glycosylation. The phosphine-mediated desulfurization of such glycoconjugates to their reductant-resistant thioether-linked counterparts completes a convergent, site-selective synthesis of thioether-linked glycoproteins (see scheme).

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Glycosynthases are promising enzyme catalysts for glycoside synthesis. Derived from glycoside hydrolases by mechanistic repurposing of their active site, glycosynthases utilize suitably activated glycosyl donors for glycosylation, yet they are unable to hydrolyze the products thus formed. Although primed for synthetic application by their design, glycosynthases have yet to see actual use in carbohydrate production. To challenge limitations on glycosynthase applicability perceived from the process chemistry point of view, here we developed a glycosynthase (D746E variant) from Bifidobacterium bifidum beta-N-acetylhexosaminidase that is highly active synthetically (?100 mumol min-1 mg-1) and fully chemo- and regioselective when using N-acetyl-d-glucosamine 1,2-oxazoline for beta-1,3-glycosylation of lactose. We thus established a chemoenzymatic process technology for production of lacto-N-triose II, a core structural unit of human milk oligosaccharides. Using equivalent amounts of oxazoline (prepared chemically in 40% yield from N-acetyl-d-glucosamine) and lactose, we obtained lacto-N-triose II (515 mM; 281 mg mL-1 90% yield; ?1 h reaction time) immediately recoverable from the reaction in 85% purity. These metrics of process efficiency reveal the prodigious potential of the glycosynthase for trisaccharide production.

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This work is a structure-activity relationship study that investigates the influence of the nature and amount of negative charge in carbohydrate substrates on the affinity of beta-N-acetylhexosaminidases, and on the stimulation of natural killer cells. It describes synthetic procedures yielding novel glycosides that are useful in immunoactivation. Specifically, we present a thorough study on the ability of six C-6 modified beta-N-acetylhexosaminides (aldehyde, uronate, 6-O-sulfate, 6-O-phosphate) to serve as substrates for cleavage and glycosylation by a library of beta-N-acetylhexosaminidases from various sources. Four novel disaccharides with one or two (negatively) charged groups were prepared in synthetic reactions in good yields. Surprisingly, the 6-O-phosphorylated substrate, although cleaved by a number of enzymes from the series, worked neither as a donor nor as an acceptor in transglycosylation reactions. The results of wet experiments were supported by molecular modeling of substrates in the active site of two representative enzymes from the screening. All ten prepared compounds were examined in terms of their immunoactivity, namely as ligands of two activation receptors of natural killer (NK) cells, NKR-P1 and CD69, both with isolated proteins and whole cells. Sulfated disaccharides in particular acted as very efficient protectants of NK cells against activation-induced apoptosis, and as stimulants of the natural killing of resistant tumor cells, which makes them good candidates for potential clinical use in cancer treatment. Copyright

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[Problem] To provide a method for obtaining an alkyl-N-acetylglucosaminide with a low content of N-acetylglucosamine, which is an impurity, inexpensively, easily, and efficiently. [Solution] This method for producing an alkyl-N-acetylglucosaminide comprises a step for reducing the content of N-acetylglucosamine by bringing a mixture of an alkyl-N-acetylglucosaminide and N-acetylglucosamine into contact with a strongly basic anion-exchange resin.

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As a unique and unappreciated protein posttranslational modification, arginine N-glycosylation was recently discovered to play an important role in the process that bacteria counteract host defenses. To provide chemical tools for further proteomic and biochemical studies on arginine N-glycosylation, we report the first general strategy for a rapid and costeffective synthesis of glycopeptides carrying single or multiple arginine N-GlcNAcyl groups. These glycopeptides were successfully utilized to generate the first antibodies that can specifically recognize arginine N-GlcNAcylated peptides or proteins in a sequence-independent manner.

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The chemoenzymic synthesis of the sialic acid Neu5Boc from the commercially-available carbohydrate N-acetyl-D-glucosamine is presented. A basic resin-catalysed epimerisation of N-acetyl-D-glucosamine to N-acetyl-D-mannosamine is also discussed.

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