title: Novel enzymatic synthesis of spacer-linked P(k) trisaccharide targeting for neutralization of Shiga toxin.
abstract: A novel alkyl spacer-conjugated derivative of P(k) trisaccharide (P(k)), one of the active receptors of Shiga toxins (Stxs; Stx1 and Stx2) produced by pathogenic Escherichia coli (STEC), was designed and synthesized by a combination of cellulase-mediated condensation from Trichoderma reesei and α1,4-galactosyltransferase (LgtC) from Neisseria gonorrhoeae. The specific activity of N. gonorrhoeae LgtC was 66U/mg, which was 13-fold higher than that from N. meningitidis expressed in E. coli. 5-trifluoroacetamidopentyl-β-P(k) (TFAP-P(k)) was synthesized (yield of 86%, based on the amount of TFAP-lactose added) and its binding to Stx1a-B and Stx2a-B was evaluated. The dissociation constants (KDs) of Stx1a-B and Stx2a-B to the spacer-linked P(k), immobilized on a CM5 sensor chip, were 6.8×10(-6) M (kon=4.1×10(1)M(-1)S(-1), koff=2.8×10(-4)S(-1)) and 2.2×10(-5)M (kon=3.9×10(2)M(-1)S(-1), koff=8.6×10(-3)S(-1)), respectively. This result suggests that the monovalent P(k)-derivative, conjugated to a pentylamino group, represents a promising Stx-neutralizing agent. This cellulase-mediated condensation using cellulase and glycosyltransferase is a valuable tool for the synthesis of spacer-linked oligosaccharide.
authors.affiliation: Laboratory of Biotechnology, Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan; Laboratory of Biotechnology, Green Chemistry Research Division, Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya Suruga-ku, Shizuoka 422-8529, Japan; Laboratory of Biotechnology, Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Kato
authors.affiliation: Laboratory of Biotechnology, Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Oizumi
authors.affiliation: Department of Chemistry and Biochemistry, Fukushima National College of Technology, 30 Nagao, Iwaki, Fukushima 970-8034 Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Ogata
authors.affiliation: Laboratory of Biotechnology, Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan.
authors.forename: Akiko
authors.initials: A
authors.lastname: Murakawa
authors.affiliation: Laboratory of Biotechnology, Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan.
authors.forename: Taichi
authors.initials: T
authors.lastname: Usui
authors.affiliation: Laboratory of Biotechnology, Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan; Laboratory of Biotechnology, Green Chemistry Research Division, Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya Suruga-ku, Shizuoka 422-8529, Japan; Laboratory of Biotechnology, Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan. Electronic address: park.enoch@shizuoka.ac.jp.
authors.forename: Enoch Y
authors.initials: EY
authors.lastname: Park
mesh_terms: D002480:Cellulase / Q000378:metabolism*; D005700:Galactosyltransferases / Q000378:metabolism*; D009344:Neisseria gonorrhoeae / Q000201:enzymology; D022603:Shiga Toxins / Q000037:antagonists & inhibitors*; D054323:Shiga-Toxigenic Escherichia coli / Q000378:metabolism; D014242:Trichoderma / Q000201:enzymology; D014312:Trisaccharides / Q000096:biosynthesis* / Q000494:pharmacology
chemical_list: D022603:Shiga Toxins; D014312:Trisaccharides; D005700:Galactosyltransferases; D002480:Cellulase
keywords: Cellulase-mediated condensation; Globotriose; Neisseria gonorrhoeae; Shiga toxin; α1,4-Galactosyltransferase