Mass Production of Bacterial α2,6-Sialyltransferase and Enzymatic Syntheses of Sialyloligosaccharides

Yamamoto, Takeshi; Nagae, Hideki; Kajihara, Yasuhiro; Terada, Ichiro

doi:10.1271/bbb.62.210

Cited by 26 publications

(19 citation statements)

References 13 publications

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“…First, it has been revealed that P. damselae α2,6-sialyltransferase transfers Neu5Ac to both glycoproteins including asialo- N -linked and asialo- O -linked glycoproteins and glycolipids [46,47]. However, rat liver α2,6-sialyltransferase has a K m value approximately 33-times higher for lactose than for N -acetyllactosaminide, P. damselae α2,6-sialyltransferase recognizes lactose and N -acetyllactosaminide as acceptor substrates with almost equal K m values [48].…”

Section: Enzymatic Properties Of Marine Bacterial Sialyltransferasesmentioning

confidence: 99%

Marine Bacterial Sialyltransferases

Yamamoto

2010

Marine Drugs

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Sialyltransferases transfer N-acetylneuraminic acid (Neu5Ac) from the common donor substrate of these enzymes, cytidine 5′-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac), to acceptor substrates. The enzymatic reaction products including sialyl-glycoproteins, sialyl-glycolipids and sialyl-oligosaccharides are important molecules in various biological and physiological processes, such as cell-cell recognition, cancer metastasis, and virus infection. Thus, sialyltransferases are thought to be important enzymes in the field of glycobiology. To date, many sialyltransferases and the genes encoding them have been obtained from various sources including mammalian, bacterial and viral sources. During the course of our research, we have detected over 20 bacteria that produce sialyltransferases. Many of the bacteria we isolated from marine environments are classified in the genus Photobacterium or the closely related genus Vibrio. The paper reviews the sialyltransferases obtained mainly from marine bacteria.

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Section: Enzymatic Properties Of Marine Bacterial Sialyltransferasesmentioning

confidence: 99%

Marine Bacterial Sialyltransferases

Yamamoto

2010

Marine Drugs

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“…The flexible donor and acceptor substrate specificities of the enzyme were described and the enzyme was applied in the synthesis of α2,6-linked sialosides and glycopeptides (Yamamoto et al 1998a;Yu et al 2006;Yamamoto et al 1998b;Kajihara et al 1996;Teo et al 2005;Yamamoto et al 1996;Endo et al 2001). Pd2,6ST shares amino acid sequence homology with a multifunctional Pasteurella multocida sialyltransferase PmST1 encoded by gene Pm0188 (GenBank accession numbers: AAY89061, AAK02272) (Yu et al 2005), a Haemophilus ducreyi α2,3-sialyltransferase encoded by gene Hd0053 (GenBank accession number: AAP95068) (Li et al 2007), a Photobacterium phosphoreum α2,3-sialyltransferase encoded by gene pst3-467 (GenBank accession number: BAF63530) , and a Photobacterium leiognathi α2,6-sialyltransferase ).…”

Section: Introductionmentioning

confidence: 99%

N-Terminal 112 amino acid residues are not required for the sialyltransferase activity of Photobacterium damsela α2,6-sialyltransferase

Sun

Chokhawala

et al. 2007

Biotechnol Lett

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Photobacterium damsela-α2,6-sialyltransferase was cloned as N-and C-His-tagged fusion proteins with different lengths (16-497 aa or 113-497 aa). Expression and activity assays indicated that the N-terminal 112 amino acid residues of the protein were not required for its α2,6-sialyltransferase activity. Among four truncated forms tested, N-His-tagged Δ15Pd2,6ST(N) containing 16-497 amino acid residues had the highest expression level. Similar to the Δ15Pd2,6ST (N), the shorter Δ112Pd2,6ST(N) was active in a wide pH range of 7.5-10.0. A divalent metal ion was not required for the sialyltransferase activity, and the addition of EDTA and dithiothreitol did not affect the activity significantly.

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“…2), which was higher than that obtained from other expression systems. For example, the expression level of secreted rat recombinant ST6Gal1 using Sf9 insect cells was reported as 0.03 U/ml [23], mammalian ST6Gal1 using yeast Pichia pastoris was reported as 0.3 × 10 -3 U/ml [24] and bacterial ST6Gal1 using Photobacterium damsela JT0160 cell was reported at 0.55 U/ml [25]. In addition, we produced various N -terminal-tagged ST6Gal1 using our silkworm expression system.…”

Section: Discussionmentioning

confidence: 99%

Synthesis of sialoglycopolypeptide for potentially blocking influenza virus infection using a rat α2,6-sialyltransferase expressed in BmNPV bacmid-injected silkworm larvae

et al. 2009

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BackgroundSialic 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.ResultsFLAG-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.ConclusionThe 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.

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Mass Production of Bacterial α2,6-Sialyltransferase and Enzymatic Syntheses of Sialyloligosaccharides

Cited by 26 publications

References 13 publications

Marine Bacterial Sialyltransferases

Marine Bacterial Sialyltransferases

N-Terminal 112 amino acid residues are not required for the sialyltransferase activity of Photobacterium damsela α2,6-sialyltransferase

Synthesis of sialoglycopolypeptide for potentially blocking influenza virus infection using a rat α2,6-sialyltransferase expressed in BmNPV bacmid-injected silkworm larvae

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