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  1. Article: Molecular Design of Fluorescent Labeled Glycosides as Acceptor Substrates for Sialyltransferases

    OGATA, Makoto / OBARA, Takakiyo / CHUMA, Yasushi / MURATA, Takeomi / PARK, Enoch Y. / USUI, Taichi

    Bioscience, biotechnology, and biochemistry. 2010 Nov. 23, v. 74, no. 11

    2010  

    Abstract: A series of dansyl-labeled glycosides with di-, tetra-, and hexasaccharides carrying the terminal N-acetyllactosamine (LacNAc) sequence were synthesized as acceptor substrates for α2,6- and α2,3-sialyltransferases. As an alternative design, dansyl- ... ...

    Abstract A series of dansyl-labeled glycosides with di-, tetra-, and hexasaccharides carrying the terminal N-acetyllactosamine (LacNAc) sequence were synthesized as acceptor substrates for α2,6- and α2,3-sialyltransferases. As an alternative design, dansyl-labeled LacNAc glycoside carrying a long-spacer linked glycan was engineered by replacement of the LacNAc or lactose units with an alkyl chain. In addition, we designed a dansyl-labeled bi-antennary LacNAc glycoside as an N-linked oligosaccharide mimetic, such as asialo-α₁-acid glycoprotein. The kinetic parameters for the transfer reaction of synthesized dansyl-labeled glycosides by sialyltransferases were determined by the fluorescent HPLC method. The catalytic efficiencies (V ₘₐₓ/K ₘ) of acceptor substrates carrying the terminal LacNAc sequence with various length glycans in the array for α2,6- and α2,3-sialyltransferases decreased in a glycan length-dependent manner. Furthermore, of the acceptor substrates tested, dansyl-labeled bi-antennary LacNAc glycoside displayed the most favorable K ₘ value for α2,6- and α2,3-sialyltransferases.
    Keywords N-acetyllactosamine ; biotechnology ; fluorescence ; fluorescent labeling ; glycoproteins ; glycosides ; glycosyltransferases ; lactose ; polysaccharides
    Language English
    Dates of publication 2010-1123
    Size p. 2287-2292.
    Publishing place Japan Society for Bioscience, Biotechnology, and Agrochemistry
    Document type Article
    Note NAL-light
    ZDB-ID 1106450-x
    ISSN 1347-6947 ; 0916-8451
    ISSN (online) 1347-6947
    ISSN 0916-8451
    DOI 10.1271/bbb.100505
    Database NAL-Catalogue (AGRICOLA)

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    In stock of ZB MED Bonn / Germany

    Z 1783: Show issues

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  2. Article ; Online: Molecular design of fluorescent labeled glycosides as acceptor substrates for sialyltransferases.

    Ogata, Makoto / Obara, Takakiyo / Chuma, Yasushi / Murata, Takeomi / Park, Enoch Y / Usui, Taichi

    Bioscience, biotechnology, and biochemistry

    2010  Volume 74, Issue 11, Page(s) 2287–2292

    Abstract: A series of dansyl-labeled glycosides with di-, tetra-, and hexasaccharides carrying the terminal N-acetyllactosamine (LacNAc) sequence were synthesized as acceptor substrates for α2,6- and α2,3-sialyltransferases. As an alternative design, dansyl- ... ...

    Abstract A series of dansyl-labeled glycosides with di-, tetra-, and hexasaccharides carrying the terminal N-acetyllactosamine (LacNAc) sequence were synthesized as acceptor substrates for α2,6- and α2,3-sialyltransferases. As an alternative design, dansyl-labeled LacNAc glycoside carrying a long-spacer linked glycan was engineered by replacement of the LacNAc or lactose units with an alkyl chain. In addition, we designed a dansyl-labeled bi-antennary LacNAc glycoside as an N-linked oligosaccharide mimetic, such as asialo-α(1)-acid glycoprotein. The kinetic parameters for the transfer reaction of synthesized dansyl-labeled glycosides by sialyltransferases were determined by the fluorescent HPLC method. The catalytic efficiencies (V(max)/K(m)) of acceptor substrates carrying the terminal LacNAc sequence with various length glycans in the array for α2,6- and α2,3-sialyltransferases decreased in a glycan length-dependent manner. Furthermore, of the acceptor substrates tested, dansyl-labeled bi-antennary LacNAc glycoside displayed the most favorable K(m) value for α2,6- and α2,3-sialyltransferases.
    MeSH term(s) Amino Sugars ; Dansyl Compounds ; Fluorescent Dyes/chemistry ; Glycosides/chemistry ; Glycosides/metabolism ; Kinetics ; Oligosaccharides ; Protein Binding ; Sialyltransferases/metabolism
    Chemical Substances Amino Sugars ; Dansyl Compounds ; Fluorescent Dyes ; Glycosides ; Oligosaccharides ; N-acetyllactosamine (3Y5B2K5OOK) ; Sialyltransferases (EC 2.4.99.-)
    Language English
    Publishing date 2010
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 1106450-x
    ISSN 1347-6947 ; 0916-8451
    ISSN (online) 1347-6947
    ISSN 0916-8451
    DOI 10.1271/bbb.100505
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    In stock of ZB MED Cologne/Königswinter

    Zs.A 4647: Show issues Location:
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  3. Article ; Online: Synthesis of sialoglycopolypeptide for potentially blocking influenza virus infection using a rat α2,6-sialyltransferase expressed in BmNPV bacmid-injected silkworm larvae

    Ogata Makoto / Nakajima Makoto / Kato Tatsuya / Obara Takakiyo / Yagi Hirokazu / Kato Koichi / Usui Taichi / Park Enoch Y

    BMC Biotechnology, Vol 9, Iss 1, p

    2009  Volume 54

    Abstract: Abstract Background Sialic acid is a deoxy uronic acid with a skeleton of nine carbons which is mostly found on cell surface in animals. This sialic acid on cell surface performs various biological functions by acting as a receptor for microorganisms, ... ...

    Abstract Abstract Background Sialic acid is a deoxy uronic acid with a skeleton of nine carbons which is mostly found on cell surface in animals. This sialic acid on cell surface performs various biological functions by acting as a receptor for microorganisms, viruses, toxins, and hormones; by masking receptors; and by regulating the immune system. In order to synthesize an artificial sialoglycoprotein, we developed a large-scale production of rat α2,6-sialyltransferase (ST6Gal1). The ST6Gal1 was expressed in fifth instar silkworm larval hemolymph using recombinant both cysteine protease- and chitinase-deficient Bombyx mori nucleopolyhedrovirus (BmNPV- CP - - Chi - ) bacmid. The expressed ST6Gal1 was purified, characterized and used for sialylation of asialoglycopolypeptide. We tested the inhibitory effect of the synthesized α2,6-sialoglycopolypeptide on hemagglutination by Sambucus nigra (SNA) lectin. Results FLAG-tagged recombinant ST6Gal1 was expressed efficiently and purified by precipitation with ammonium sulphate followed by affinity chromatography on an anti-FLAG M2 column, generating 2.2 mg purified fusion protein from only 11 silkworm larvae, with a recovery yield of 64%. The purified ST6Gal1 was characterized and its N -glycan patterns were found to be approximately paucimannosidic type by HPLC mapping method. Fluorescently-labelled N -acetyllactosamine (LacNAc) glycoside containing dansyl group was synthesized chemo-enzymatically as high-sensitivity acceptor substrate for ST6Gal1. The acceptor substrate specificity of the enzyme was similar to that of rat liver ST6Gal1. The fluorescent glycoside is useful as a substrate for a highly sensitive picomole assay of ST6Gal1. Asialoglycopolypeptide was regioselectively and quantitatively sialylated by catalytic reaction at the terminal Gal residue to obtain α2,6-sialoglycopolypeptide using ST6Gal1. The α2,6-sialoglycopolypeptide selectively inhibited hemagglutination induced by Sambucus nigra (SNA) lectin, showing about 780-fold higher affinity than the control fetuin. Asialoglycopolypeptide and γ-polyglutamic acid did not affect SNA lectin-mediated hemagglutination. Conclusion The recombinant ST6Gal1 from a silkworm expression system is useful for the sialylation of asialoglycopeptide. The sialylated glycoprotein is a valuable tool for investigating the molecular mechanisms of biological and physiological events, such as cell-cell recognition and viral entry during infection.
    Keywords Biotechnology ; TP248.13-248.65 ; Chemical technology ; TP1-1185 ; Technology ; T ; DOAJ:Biotechnology ; DOAJ:Life Sciences ; DOAJ:Biology and Life Sciences
    Subject code 540
    Language English
    Publishing date 2009-06-01T00:00:00Z
    Publisher BioMed Central
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  4. Article ; Online: Synthesis of sialoglycopolypeptide for potentially blocking influenza virus infection using a rat alpha2,6-sialyltransferase expressed in BmNPV bacmid-injected silkworm larvae.

    Ogata, Makoto / Nakajima, Makoto / Kato, Tatsuya / Obara, Takakiyo / Yagi, Hirokazu / Kato, Koichi / Usui, Taichi / Park, Enoch Y

    BMC biotechnology

    2009  Volume 9, Page(s) 54

    Abstract: Background: Sialic acid is a deoxy uronic acid with a skeleton of nine carbons which is mostly found on cell surface in animals. This sialic acid on cell surface performs various biological functions by acting as a receptor for microorganisms, viruses, ... ...

    Abstract Background: Sialic acid is a deoxy uronic acid with a skeleton of nine carbons which is mostly found on cell surface in animals. This sialic acid on cell surface performs various biological functions by acting as a receptor for microorganisms, viruses, toxins, and hormones; by masking receptors; and by regulating the immune system. In order to synthesize an artificial sialoglycoprotein, we developed a large-scale production of rat alpha2,6-sialyltransferase (ST6Gal1). The ST6Gal1 was expressed in fifth instar silkworm larval hemolymph using recombinant both cysteine protease- and chitinase-deficient Bombyx mori nucleopolyhedrovirus (BmNPV-CP--Chi-) bacmid. The expressed ST6Gal1 was purified, characterized and used for sialylation of asialoglycopolypeptide. We tested the inhibitory effect of the synthesized alpha2,6-sialoglycopolypeptide on hemagglutination by Sambucus nigra (SNA) lectin.
    Results: FLAG-tagged recombinant ST6Gal1 was expressed efficiently and purified by precipitation with ammonium sulphate followed by affinity chromatography on an anti-FLAG M2 column, generating 2.2 mg purified fusion protein from only 11 silkworm larvae, with a recovery yield of 64%. The purified ST6Gal1 was characterized and its N-glycan patterns were found to be approximately paucimannosidic type by HPLC mapping method. Fluorescently-labelled N-acetyllactosamine (LacNAc) glycoside containing dansyl group was synthesized chemo-enzymatically as high-sensitivity acceptor substrate for ST6Gal1. The acceptor substrate specificity of the enzyme was similar to that of rat liver ST6Gal1. The fluorescent glycoside is useful as a substrate for a highly sensitive picomole assay of ST6Gal1. Asialoglycopolypeptide was regioselectively and quantitatively sialylated by catalytic reaction at the terminal Gal residue to obtain alpha2,6-sialoglycopolypeptide using ST6Gal1. The alpha2,6-sialoglycopolypeptide selectively inhibited hemagglutination induced by Sambucus nigra (SNA) lectin, showing about 780-fold higher affinity than the control fetuin. Asialoglycopolypeptide and gamma-polyglutamic acid did not affect SNA lectin-mediated hemagglutination.
    Conclusion: The recombinant ST6Gal1 from a silkworm expression system is useful for the sialylation of asialoglycopeptide. The sialylated glycoprotein is a valuable tool for investigating the molecular mechanisms of biological and physiological events, such as cell-cell recognition and viral entry during infection.
    MeSH term(s) Animals ; Antiviral Agents/metabolism ; Bombyx/metabolism ; Bombyx/virology ; Cloning, Molecular ; Genetic Vectors ; Hemagglutination Inhibition Tests ; Larva/metabolism ; Larva/virology ; Nucleopolyhedroviruses/genetics ; Orthomyxoviridae/drug effects ; Plant Lectins/metabolism ; Rats ; Recombinant Proteins/biosynthesis ; Recombinant Proteins/genetics ; Recombinant Proteins/isolation & purification ; Ribosome Inactivating Proteins/metabolism ; Sialoglycoproteins/biosynthesis ; Sialoglycoproteins/genetics ; Sialoglycoproteins/isolation & purification ; Sialyltransferases/biosynthesis ; Sialyltransferases/genetics ; Sialyltransferases/isolation & purification
    Chemical Substances Antiviral Agents ; Plant Lectins ; Recombinant Proteins ; Sambucus nigra lectins ; Sialoglycoproteins ; Sialyltransferases (EC 2.4.99.-) ; beta-D-galactoside alpha 2-6-sialyltransferase (EC 2.4.99.1) ; Ribosome Inactivating Proteins (EC 3.2.2.22)
    Language English
    Publishing date 2009-06-05
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ISSN 1472-6750
    ISSN (online) 1472-6750
    DOI 10.1186/1472-6750-9-54
    Database MEDical Literature Analysis and Retrieval System OnLINE

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