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Xi Chen - One of the best experts on this subject based on the ideXlab platform.
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converting pasteurella multocida α2 3 Sialyltransferase 1 pmst1 to a regioselective α2 6 Sialyltransferase by saturation mutagenesis and regioselective screening
2017Co-Authors: John B Mcarthur, Jie Zeng, Xi ChenAbstract:A microtiter plate-based screening assay capable of determining the activity and regioselectivity of Sialyltransferases was developed. This assay was used to screen two single-site saturation libraries of Pasteurella multocidaα2-3-Sialyltransferase 1 (PmST1) for α2-6-Sialyltransferase activity and total Sialyltransferase activity. PmST1 double mutant P34H/M144L was found to be the most effective α2-6-Sialyltransferase and displayed 50% reduced donor hydrolysis and 50-fold reduced sialidase activity compared to the wild-type PmST1. It retained the donor substrate promiscuity of the wild-type enzyme and was used in an efficient one-pot multienzyme (OPME) system to selectively catalyze the sialylation of the terminal galactose residue in a multigalactose-containing tetrasaccharide lacto-N-neotetraoside.
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decreasing the sialidase activity of multifunctional pasteurella multocida α2 3 Sialyltransferase 1 pmst1 by site directed mutagenesis
2011Co-Authors: Go Sugiarto, Hai Yu, Yanhong Li, Zahra Khedri, Diem Thuy Le, Xi ChenAbstract:Pasteurella multocida α2–3-Sialyltransferase 1 (PmST1) is a multifunctional enzyme which has α2–6-Sialyltransferase, α2–3-sialidase, and α2–3-trans-sialidase activities in addition to its major α2–3-Sialyltransferase activity. The presence of the α2–3-sialidase activity of PmST1 complicates its application in enzymatic synthesis of α2–3-linked sialosides as the product formed can be hydrolyzed by the enzyme. Herein we show that the α2–3-sialidase activity of PmST1 can be significantly decreased by protein crystal structure-based site-directed mutagenesis. A PmST1 double mutant E271F/R313Y showed a significantly (6333-fold) decreased sialidase activity without affecting its α2–3-Sialyltransferase activity. The double mutant E271F/R313Y, therefore, is a superior enzyme for enzymatic synthesis of α2–3-linked sialosides.
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decreasing the sialidase activity of multifunctional pasteurella multocida α2 3 Sialyltransferase 1 pmst1 by site directed mutagenesis
2011Co-Authors: Go Sugiarto, Kam Lau, Zahra Khedri, Xi ChenAbstract:Pasteurella multocida α2–3-Sialyltransferase 1 (PmST1) is a multifunctional enzyme which has α2–6-Sialyltransferase, α2–3-sialidase, and α2–3-trans-sialidase activities in addition to its major α2–3-Sialyltransferase activity. The presence of the α2–3-sialidase activity of PmST1 complicates its application in enzymatic synthesis of α2–3-linked sialosides as the product formed can be hydrolyzed by the enzyme. Herein we show that the α2–3-sialidase activity of PmST1 can be significantly decreased by protein crystal structure-based site-directed mutagenesis. A PmST1 double mutant E271F/R313Y showed a significantly (6333-fold) decreased sialidase activity without affecting its α2–3-Sialyltransferase activity. The double mutant E271F/R313Y, therefore, is a superior enzyme for enzymatic synthesis of α2–3-linked sialosides.
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trans sialidase activity of photobacterium damsela α2 6 Sialyltransferase and its application in the synthesis of sialosides
2010Co-Authors: Jiansong Cheng, Shengshu Huang, Kam Lau, Xi ChenAbstract:Trans-sialidases catalyze the transfer of a sialic acid from one sialoside to an acceptor to form a new sialoside. alpha2,3-Trans-sialidase activity was initially discovered in the parasitic protozoan Trypanosoma cruzi, and more recently was found in a multifunctional Pasteurella multocida Sialyltransferase PmST1. alpha2,8-Trans-sialidase activity was also described for a multifunctional Campylobacter jejuni Sialyltransferase CstII. We report here the discovery of the alpha2,6-trans-sialidase activity of a previously reported recombinant truncated bacterial alpha2,6-Sialyltransferase from Photobacterium damsela (Delta15Pd2,6ST). This is the first time that the alpha2,6-trans-sialidase activity has ever been identified. Kinetic studies indicate that Delta15Pd2,6ST-catalyzed trans-sialidase reaction follows a ping-pong bi-bi reaction mechanism. Cytidine 5'-monophosphate, the product of Sialyltransferase reactions, is not required by the trans-sialidase activity of the enzyme but enhances the trans-sialidase activity modestly as a non-essential activator. Using chemically synthesized Neu5AcalphapNP and LacbetaMU, alpha2,6-linked sialoside Neu5Acalpha2,6LacbetaMU has been obtained in one-step in high yield using the trans-sialidase activity of Delta15Pd2,6ST. In addition to the alpha2,6-trans-sialidase activity, Delta15Pd2,6ST also has alpha2,6-sialidase activity. The multifunctionality is thus a common feature of many bacterial Sialyltransferases.
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multifunctionality of campylobacter jejuni Sialyltransferase cstii characterization of gd3 gt3 oligosaccharide synthase gd3 oligosaccharide sialidase and trans sialidase activities
2008Co-Authors: Jiansong Cheng, Harshal A Chokhawala, Shengshu Huang, Kam Lau, Vinod K Tiwari, Xi ChenAbstract:CstII from bacterium Campylobacter jejuni strain OH4384 has been previously characterized as a bifunctional Sialyltransferase having both alpha2,3-Sialyltransferase (GM3 oligosaccharide synthase) and alpha2,8-Sialyltransferase (GD3 oligosaccharide synthase) activities which catalyze the transfer of N-acetylneuraminic acid (Neu5Ac) from cytidine 5'-monophosphate (CMP)-Neu5Ac to C-3' of the galactose in lactose and to C-8 of the Neu5Ac in 3'-sialyllactose, respectively (Gilbert M, Karwaski MF, Bernatchez S, Young NM, Taboada E, Michniewicz J, Cunningham AM, Wakarchuk WW. 2002. The genetic bases for the variation in the lipo-oligosaccharide of the mucosal pathogen, Campylobacter jejuni. Biosynthesis of sialylated ganglioside mimics in the core oligosaccharide. J Biol Chem. 277:327-337). We report here the characterization of a truncated CstII mutant (CstIIDelta32(I53S)) cloned from a synthetic gene whose codons are optimized for an Escherichia coli expression system. In addition to the alpha2,3- and alpha2,8-Sialyltransferase activities reported before for the synthesis of GM3- and GD3-type oligosaccharides, respectively, the CstIIDelta32(I53S) has alpha2,8-Sialyltransferase (GT3 oligosaccharide synthase) activity for the synthesis of GT3 oligosaccharide. It also has alpha2,8-sialidase (GD3 oligosaccharide sialidase) activity that catalyzes the specific cleavage of the alpha2,8-sialyl linkage of GD3-type oligosaccharides and alpha2,8-trans-sialidase (GD3 oligosaccharide trans-sialidase) activity that catalyzes the transfer of a sialic acid from a GD3 oligosaccharide to a different GM3 oligosaccharide (3'-sialyllactoside). The donor substrate specificity study of the CstIIDelta32(I53S) GD3 oligosaccharide synthase activity indicates that the enzyme is flexible in using different CMP-activated sialic acids and their analogs for the synthesis of GD3 oligosaccharides containing natural and nonnatural modifications at the terminal sialic acid.
Takeshi Yamamoto - One of the best experts on this subject based on the ideXlab platform.
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loss of function mutation in bi functional marine bacterial Sialyltransferase
2012Co-Authors: Hitomi Kajiwara, Toshiki Mine, Sakurako Katayama, Yoshimitsu Kakuta, Nozomu Okino, Makoto Ito, Takeshi YamamotoAbstract:An α2,3-Sialyltransferase produced by Photobacterium phosphoreum JT-ISH-467 is a bi-functional enzyme showing both α2,3-Sialyltransferase and α2,3-linkage specific sialidase activity. To date, the crystal structures of several Sialyltransferases have been solved, but the roles of amino acid residues around the catalytic site have not been completely clarified. Hence we performed a mutational study using α2,3-Sialyltransferase cloned from P. phosphoreum JT-ISH-467 as a model enzyme to study the role of the amino acid residues around the substrate-binding site. It was found that a mutation of the glutamic acid at position 342 in the Sialyltransferase resulted in a loss of sialidase activity, although the mutant showed no decrease in Sialyltransferase activity. Based on this result, it is strongly expected that the Glu342 of the enzyme is an important amino acid residue for sialidase activity.
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efficient synthesis of 6 sialyllactose 6 6 disialyllactose and 6 kdo lactose by metabolically engineered e coli expressing a multifunctional Sialyltransferase from the photobacterium sp jt ish 224
2010Co-Authors: Sophie Drouillard, Hitomi Kajiwara, Toshiki Mine, Takeshi Yamamoto, Eric SamainAbstract:We have previously reported the efficient conversion of lactose into 3'-sialyllactose by high cell density cultures of a genetically engineered Escherichia coli strain expressing the Neisseria meningitidis gene for alpha-(2-->3)-Sialyltransferase [Fierfort, N.; Samain, E. J. Biotechnol. 2008, 134, 261-265.]. First attempts to use a similar strategy to produce 6'-sialyllactose with a strain expressing alpha-(2-->6)-Sialyltransferase from the Photobacterium sp. JT-ISH-224 led to the production of a trisaccharide that was identified as KDO-lactose (2-keto-3-deoxy-manno-octonyllactose). This result showed that alpha-(2-->6)-Sialyltransferase was able to use CMP-KDO as sugar donor and preferentially used CMP-KDO over CMP-Neu5Ac. By reducing the expression level of the Sialyltransferase gene and increasing that of the neuABC genes, we have been able to favour the formation of 6'-sialyllactose and to prevent the formation of KDO-lactose. However, in this case, a third lactose derivative, which was identified as 6,6'-disialyllactose, was also produced. Formation of 6,6'-disialyllactose was mainly observed under conditions of lactose shortage. On the other hand, when the culture was continuously fed with an excess of lactose, 6'-sialyllactose was almost the only product detected and its final concentration was higher than 30g/L of culture medium.
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An α2,6-Sialyltransferase cloned from Photobacterium leiognathi strain JT-SHIZ-119 shows both Sialyltransferase and neuraminidase activity
2009Co-Authors: Toshiki Mine, Hitomi Kajiwara, Sakurako Katayama, Masako Tsunashima, Hiroshi Tsukamoto, Yoshimitsu Takakura, Takeshi YamamotoAbstract:We cloned, expressed, and characterized a novel beta-galactoside alpha2,6-Sialyltransferase from Photobacterium leiognathi strain JT-SHIZ-119. The protein showed 56-96% identity to the marine bacterial alpha2,6-Sialyltransferases classified into glycosyltransferase family 80. The Sialyltransferase activity of the N-terminal truncated form of the recombinant enzyme was 1477 U/L of Escherichia coli culture. The truncated recombinant enzyme was purified as a single band by sodium dodecyl sulfate polyacrylamide gel electrophoresis through 3 column chromatography steps. The enzyme had distinct activity compared with known marine bacterial alpha2,6-Sialyltransferases. Although alpha2,6-Sialyltransferases cloned from marine bacteria, such as Photobacterium damselae strain JT0160, P. leiognathi strain JT-SHIZ-145, and Photobacterium sp. strain JT-ISH-224, show only alpha2,6-Sialyltransferase activity, the recombinant enzyme cloned from P. leiognathi strain JT-SHIZ-119 showed both alpha2,6-Sialyltransferase and alpha2,6-linkage-specific neuraminidase activity. Our results provide important information toward a comprehensive understanding of the bacterial Sialyltransferases belonging to the group 80 glycosyltransferase family in the CAZy database.
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Toshiki Mine, Sakurako Katayama, Hitomi Kajiwara,
2009Co-Authors: Masako Tsunashima, Hiroshi Tsukamoto, Takeshi YamamotoAbstract:An α2,6-Sialyltransferase cloned from Photobacterium leiognathi strain JT-SHIZ-119 shows both Sialyltransferase and neuraminidase activit
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Photobacterium sp. JT-ISH-224 Produces Two Sialyltransferases, α-/β-Galactoside α2,3-Sialyltransferase and β-Galactoside α2,6-Sialyltransferase
2007Co-Authors: Hiroshi Tsukamoto, Toshiki Mine, Yoshimitsu Takakura, Takeshi YamamotoAbstract:A novel bacterium, Photobacterium sp. JT-ISH-224, that produces alpha-/beta-galactoside alpha2,3-Sialyltransferase and beta-galactoside alpha2,6-Sialyltransferase, was isolated from the gut of a Japanese barracuda. The genes that encode the enzymes were cloned from the genomic library of the bacterium using the genes encoding alpha-/beta-galactoside alpha2,3-Sialyltransferase from P. phosphoreum and beta-galactoside alpha2,6-Sialyltransferase from P. damselae as probes. The nucleotide sequences were determined, and open reading frames of 1,230 and 1,545 bp for encoding an alpha2,3-Sialyltransferase and an alpha2,6-Sialyltransferase of 409- and 514-amino acid residues, respectively, were identified. The alpha2,3-Sialyltransferase had 92% amino acid sequence identity with the P. phosphoreum alpha2,3-Sialyltransferase, whereas the alpha2,6-Sialyltransferase had 54% amino acid sequence identity with the P. damselae alpha2,6-Sialyltransferase. For both enzymes, the DNA fragments that encoded the full-length protein and its truncated form lacking the putative signal peptide sequence were amplified by a polymerase chain reaction and cloned into an expression vector. Each gene was expressed in Escherichia coli, and the lysate from each strain had enzymatic activity. The alpha2,3-Sialyltransferase catalysed the transfer of N-acetylneuraminic acid (NeuAc) from CMP-NeuAc to lactose, alpha-methyl-galactopyranoside and beta-methyl-galactopyranoside with low apparent K(m) and the alpha2,6-Sialyltransferase catalysed the transfer of NeuAc from CMP-NeuAc to lactose with low apparent K(m).
James C. Paulson - One of the best experts on this subject based on the ideXlab platform.
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systemic blockade of sialylation in mice with a global inhibitor of Sialyltransferases
2014Co-Authors: Matthew S Macauley, Britni M Arlian, Cory D Rillahan, Pohchoo Pang, Nikki Bortell, Maria Cecilia G Marcondes, Stuart M Haslam, Anne Dell, James C. PaulsonAbstract:Sialic acid terminates glycans of glycoproteins and glycolipids that play numerous biological roles in health and disease. Although genetic tools are available for interrogating the effects of decreased or abolished sialoside expression in mice, pharmacological inhibition of the Sialyltransferase family has, to date, not been possible. We have recently shown that a sialic acid analog, 2,4,7,8,9-pentaacetyl-3Fax-Neu5Ac-CO2Me (3F-NeuAc), added to the media of cultured cells shuts down sialylation by a mechanism involving its intracellular conversion to CMP-3F-NeuAc, a competitive inhibitor of all Sialyltransferases. Here we show that administering 3F-NeuAc to mice dramatically decreases sialylated glycans in cells of all tissues tested, including blood, spleen, liver, brain, lung, heart, kidney, and testes. A single dose results in greatly decreased sialoside expression for over 7 weeks in some tissues. Although blockade of sialylation with 3F-NeuAc does not affect viability of cultured cells, its use in vivo has a deleterious “on target” effect on liver and kidney function. After administration of 3F-NeuAc, liver enzymes in the blood are dramatically altered, and mice develop proteinuria concomitant with dramatic loss of sialic acid in the glomeruli within 4 days, leading to irreversible kidney dysfunction and failure to thrive. These results confirm a critical role for sialosides in liver and kidney function and document the feasibility of pharmacological inhibition of Sialyltransferases for in vivo modulation of sialoside expression.
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Spatiotemporal expression patterns of sialoglycoconjugates during nephron morphogenesis and their regional and cell type-specific distribution in adult rat kidney
2003Co-Authors: Christian Zuber, James C. Paulson, Valeriu Toma, Harry C. Winter, Irwin J. Goldstein, Jürgen RothAbstract:The expression of α2,6- and α2,3-linked sialic acids on N -glycans was studied in embryonic, postnatal, and adult rat kidney. Histochemistry and blotting using Polyporus squamosus and Sambucus nigra lectins for α2,6-linked sialic acids and the Maackia amurensis lectin for α2,3-linked sialic acids were performed and Sialyltransferase activity was assayed. N -glycans with α2,6- and α2,3-linked sialic acid were differently expressed in the two embryonic anlagen and early stages of nephron. Metanephrogenic mesenchyme was positive for α2,3-linked sialic acid but not for the α2,6-linked one, which became detectable initially in the proximal part of S-shaped bodies. Collecting ducts were positive for α2,6-linked sialic acid, whereas α2,3-linked sialic acid was restricted to their ampullae. Although positive in embryonic kidney, S1 and S2 of proximal tubules became unreactive for α2,3-linked sialic acid in postnatal and adult kidneys. In adult kidney, intercalated but not principal cells of collecting ducts were reactive for α2,3-linked sialic acid. In contrast, α2,6-linked sialic acids were detected in all cells of adult kidney nephron. Blot analysis revealed a different but steady pattern of bands reactive for α2,6- and α2,3-linked sialic acid in embryonic, postnatal, and adult kidney. Activity of α2,6 and α2,3 Sialyltransferases was highest in embryonic kidney and decreased over postnatal to adult kidney with the activity of α2,6 Sialyltransferase always being three to fourfold that of α2,3 Sialyltransferase. Thus, α2,6- and α2,3-linked sialic acids are differently expressed in embryonic anlagen and mesenchyme-derived early stages of nephron and show regional and cell type-specific differences in adult kidney.
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mutation of the Sialyltransferase s sialylmotif alters the kinetics of the donor and acceptor substrates
1998Co-Authors: Arun K Datta, Abhishek Sinha, James C. PaulsonAbstract:Protein sequence analysis of the cloned Sialyltransferase gene family has revealed the presence of two conserved protein motifs in the middle of the lumenal catalytic domain, termed L-sialylmotif and S-sialylmotif. In our previous study (Datta, A. K., and Paulson, J. C. (1995) J. Biol. Chem.270, 1497–1500) the larger L-sialylmotif of ST6Gal I was analyzed by site-directed mutagenesis, which provided evidence that it participates in the binding of the CMP-NeuAc, a common donor substrate for all the Sialyltransferases. However, none of the mutants tested in this motif had any significant effect on their binding affinities toward the acceptor substrate asialo α1-acid glycoprotein. In this study, we have investigated the role of the S-sialylmotif of the same enzyme ST6Gal I. In total, nine mutants have been constructed by changing the conserved amino acids of this motif to mostly alanine by site-directed mutagenesis. Kinetic analysis for the mutants which retained Sialyltransferase activity showed that the mutations in the S-sialylmotif caused a change ofKm values for both the donor and the acceptor substrates. Our results indicated that this motif participates in the binding of both the substrates. A sequence homology search also supported this finding, which showed that the downstream amino acid sequence of the S-sialylmotif is conserved for each subgroup of this enzyme family, indicating its association with the acceptor substrate.
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molecular cloning of a developmentally regulated n acetylgalactosamine 2 6 Sialyltransferase specific for sialylated glycoconjugates
1996Co-Authors: Eric R Sjoberg, Hiroshi Kitagawa, John Glushka, Herman Van Halbeek, James C. PaulsonAbstract:Abstract A cDNA encoding a novel Sialyltransferase has been isolated employing the polymerase chain reaction using degenerate primers to conserved regions of the sialylmotif that is present in all eukaryotic members of the Sialyltransferase gene family examined to date. The cDNA sequence revealed an open reading frame coding for 305 amino acids, making it the shortest Sialyltransferase cloned to date. This open reading frame predicts all the characteristic structural features of other Sialyltransferases including a type II membrane protein topology and both sialylmotifs, one centrally located and the second in the carboxyl-terminal portion of the cDNA. When compared with all other Sialyltransferase cDNAs, the predicted amino acid sequence displays the lowest homology in the Sialyltransferase gene family. Northern analysis shows this Sialyltransferase to be developmentally regulated in brain with expression persisting through adulthood in spleen, kidney, and lung. Stable transfection of the full-length cDNA in the human kidney carcinoma cel line 293 produced an active Sialyltransferase with marked specificity for the sialoside, Neu5Acα2,3Galβ1,3GalNAc and glycoconjugates carrying the same sequence such as G and fetuin. The disialylated tetrasaccharide formed by reacting the Sialyltransferase with the aforementioned sialoside was analyzed by one- and two-dimensional 1H and C NMR spectroscopy and was shown to be the Neu5Acα2,3Galβ1,3(Neu5Acα2,6)GalNAc sialoside. This indicates that the enzyme is a GalNAc α2,6-Sialyltransferase. Since two other ST6GalNAc Sialyltransferase cDNAs have been isolated, this Sialyltransferase has been designated ST6GalNAc III. Of these three, ST6GalNAc III displays the most restricted acceptor specificity and is the only Sialyltransferase cloned to date capable of forming the developmentally regulated ganglioside G from G.
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large scale expression of recombinant Sialyltransferases and comparison of their kinetic properties with native enzymes
1995Co-Authors: Mark A Williams, James C. Paulson, Hiroshi Kitagawa, Arun K Datta, James C JamiesonAbstract:Values ofKm were determined for three purified Sialyltransferases and the corresponding recombinant enzymes. The enzymes were Galβ1-4GlcNAc α2-6Sialyltransferase and Galβ1-3(4)GlcNAc α2-3Sialyltransferase from rat liver; these enzymes are responsible for the attachment of sialic acid to N-linked oligosaccharide chains; and the Galβ1-3GalNAc α2-3Sialyltransferase from porcine submaxillary gland that is responsible for the attachment of sialic acid to O-linked glycoproteins and glycolipids. A procedure for the large scale expression of active Sialyltransferases from recombinant baculovirus-infected insect cells is described. For the liver enzymes values ofKm were determined using rat and human asialoα1 acid glycoprotein andN-acetyllactosamine as variable substrates; lacto-N-tetraose was also used with the Galβ1-3(4)GlcNAc α2-3Sialyltransferase. Antifreeze glycorprotein was used as the macromolecular acceptor for the porcine enzyme. Values forKm were also determined using CMP-NeuAc as the variable substrate.
Philippe Delannoy - One of the best experts on this subject based on the ideXlab platform.
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Novel Zebrafish Mono-α2,8-Sialyltransferase (ST8Sia VIII): An Evolutionary Perspective of α2,8-Sialylation
2019Co-Authors: Lan-yi Chang, Maxence Noel, Pierreandre Gilormini, Virginie Cogez, Cedric Lion, Christophe Biot, Elin Teppa, Mathieu Decloquement, Anne-marie Mir, Philippe DelannoyAbstract:The mammalian mono-α2,8-Sialyltransferase ST8Sia VI has been shown to catalyze the transfer of a unique sialic acid residues onto core 1 O-glycans leading to the formation of di-sialylated O-glycosylproteins and to a lesser extent to diSia motifs onto glycolipids like GD1a. Previous studies also reported the identification of an orthologue of the ST8SIA6 gene in the zebrafish genome. Trying to get insights into the biosynthesis and function of the oligo-sialylated glycoproteins during zebrafish development, we cloned and studied this fish α2,8-Sialyltransferase homologue. In situ hybridization experiments demonstrate that expression of this gene is always detectable during zebrafish development both in the central nervous system and in non-neuronal tissues. Intriguingly, using biochemical approaches and the newly developed in vitro MicroPlate Sialyltransferase Assay (MPSA), we found that the zebrafish recombinant enzyme does not synthetize diSia motifs on glycoproteins or glycolipids as the human homologue does. Using comparative genomics and molecular phylogeny approaches, we show in this work that the human ST8Sia VI orthologue has disappeared in the ray-finned fish and that the homologue described in fish correspond to a new subfamily of α2,8-Sialyltransferase named ST8Sia VIII that was not maintained in Chondrichtyes and Sarcopterygii.
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il 6 and il 8 increase the expression of glycosyltransferases and sulfotransferases involved in the biosynthesis of sialylated and or sulfated lewisx epitopes in the human bronchial mucosa
2008Co-Authors: Sophie Grouxdegroote, Marieange Krzewinskirecchi, Aurelie Cazet, Audrey Vincent, Sylvain Lehoux, Jeanjacques Lafitte, Isabelle Van Seuningen, Philippe DelannoyAbstract:Bronchial mucins from patients suffering from CF (cystic fibrosis) exhibit glycosylation alterations, especially increased amounts of the sialyl-Lewis x (NeuAcα2-3Galβ1-4[Fucα1-3]GlcNAc-R) and 6-sulfo-sialyl-Lewis x (NeuAcα2-3Galβ1-4[Fucα1-3][SO 3 H-6]GlcNAc-R) terminal structures. These epitopes are preferential receptors for Pseudomonas aeruginosa , the bacteria responsible for the chronicity of airway infection and involved in the morbidity and early death of CF patients. However, these glycosylation changes cannot be directly linked to defects in CFTR (CF transmembrane conductance regulator) gene expression since cells that secrete airway mucins express no or very low amounts of the protein. Several studies have shown that inflammation may affect glycosylation and sulfation of various glycoproteins, including mucins. In the present study, we show that incubation of macroscopically healthy fragments of human bronchial mucosa with IL-6 (interleukin-6) or IL-8 results in a significant increase in the expression of α1,3/4-fucosyltransferases [ FUT11 (fucosyltransferase 11 gene) and FUT3 ], α2-6- and α2,3-Sialyltransferases [ ST3GAL6 (α2,3-Sialyltransferase 6 gene) and ST6GAL2 (α2,6-Sialyltransferase 2 gene)] and GlcNAc-6- O -sulfotransferases [ CHST4 (carbohydrate sulfotransferase 4 gene) and CHST6 ] mRNA. In parallel, the amounts of sialyl-Lewis x and 6-sulfo-sialyl-Lewis x epitopes at the periphery of high-molecular-mass proteins, including MUC4, were also increased. In conclusion, our results indicate that IL-6 and -8 may contribute to the increased levels of sialyl-Lewis x and 6-sulfo-sialyl-Lewis x epitopes on human airway mucins from patients with CF.
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The animal Sialyltransferases and Sialyltransferase-related genes: a phylogenetic approach.
2005Co-Authors: Anne Harduin-lepers, Philippe Delannoy, Rosella Mollicone, Rafael OriolAbstract:The animal Sialyltransferases are Golgi type II transmembrane glycosyltransferases. Twenty distinct Sialyltransferases have been identified in both human and murine genomes. These enzymes catalyze transfer of sialic acid from CMP-Neu5Ac to the glycan moiety of glycoconjugates. Despite low overall identities, they share four conserved peptide motifs [L (large), S (small), motif III, and motif VS (very small)] that are hallmarks for Sialyltransferase identification. We have identified 155 new putative genes in 25 animal species, and we have exploited two lines of evidence: (1) sequence comparisons and (2) exon-intron organization of the genes. An ortholog to the ancestor present before the split of ST6Gal I and II subfamilies was detected in arthropods. An ortholog to the ancestor present before the split of ST6GalNAc III, IV, V, and VI subfamilies was detected in sea urchin. An ortholog to the ancestor present before the split of ST3Gal I and II subfamilies was detected in ciona, and an ortholog to the ancestor of all the ST8Sia was detected in amphioxus. Therefore, single examples of the four families (ST3Gal, ST6Gal, ST6GalNAc, and ST8Sia) have appeared in invertebrates, earlier than previously thought, whereas the four families were all detected in bony fishes, amphibians, birds, and mammals. As previously hypothesized, sequence similarities among Sialyltransferases suggest a common genetic origin, by successive duplications of an ancestral gene, followed by divergent evolution. Finally, we propose predictions on these invertebrates Sialyltransferase-related activities that have not previously been demonstrated and that will ultimately need to be substantiated by protein expression and enzymatic activity assays.
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the human Sialyltransferase family
2001Co-Authors: Anne Harduinlepers, Veronica Vallejoruiz, Marieange Krzewinskirecchi, Benedicte Samynpetit, Sylvain Julien, Philippe DelannoyAbstract:The human genome encodes probably more than 20 different Sialyltransferases involved in the biosynthesis of sialylated glycoproteins and glycolipids but to date only 15 different human Sialyltransferase cDNAs have been cloned and characterized. Each of the Sialyltransferase genes is differentially expressed in a tissue-, cell type-, and stage-specific manner to regulate the sialylation pattern of cells. These enzymes differ in their substrate specificity, tissue distribution and various biochemical parameters. However, enzymatic analysis conducted in vitro with recombinant enzyme revealed that one linkage can be synthesized by multiple enzymes. We present here an overview of these human genes and enzymes, the regulation of their occurrence and their involvement in several physiological and pathological processes.
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multiplex reverse transcription polymerase chain reaction assessment of Sialyltransferase expression in human breast cancer
1998Co-Authors: Marieange Recchi, Anne Harduinlepers, M Hebbar, Louis Hornez, Jeanphilippe Peyrat, Philippe DelannoyAbstract:Increased sialylation, especially involving the Sialyl-Lewisa and Sialyl-Lewisx determinants, has been reported in breast cancer. A multiplex reverse transcription-PCR method was used here to determine the expression of five Sialyltransferases (ST3Gal III, ST6Gal I, ST3Gal IV, ST3Gal I, and ST3Gal II) in 49 patients surgically treated for locoregional breast cancer. We assessed the relationship between these expressions and clinical, pathological, and biological features. The most expressed Sialyltransferase was ST3Gal 1II, which is involved in Sialyl-Lewisa synthesis. ST3Gal III expression was positively correlated to ST6Gal I and ST3Gal IV expressions, to tumor size, and to the number of involved axillary nodes. Patients with high ST3Gal III expression had a shorter overall survival. High ST6Gal I expression was associated with histoprognostic grade III. ST6Gal I expression was negatively correlated to expression of progesterone receptor. In conclusion, high ST3Gal III and ST6Gal I expressions in human breast tumors are associated with poor prognosis markers.
Yoshimitsu Takakura - One of the best experts on this subject based on the ideXlab platform.
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An α2,6-Sialyltransferase cloned from Photobacterium leiognathi strain JT-SHIZ-119 shows both Sialyltransferase and neuraminidase activity
2009Co-Authors: Toshiki Mine, Hitomi Kajiwara, Sakurako Katayama, Masako Tsunashima, Hiroshi Tsukamoto, Yoshimitsu Takakura, Takeshi YamamotoAbstract:We cloned, expressed, and characterized a novel beta-galactoside alpha2,6-Sialyltransferase from Photobacterium leiognathi strain JT-SHIZ-119. The protein showed 56-96% identity to the marine bacterial alpha2,6-Sialyltransferases classified into glycosyltransferase family 80. The Sialyltransferase activity of the N-terminal truncated form of the recombinant enzyme was 1477 U/L of Escherichia coli culture. The truncated recombinant enzyme was purified as a single band by sodium dodecyl sulfate polyacrylamide gel electrophoresis through 3 column chromatography steps. The enzyme had distinct activity compared with known marine bacterial alpha2,6-Sialyltransferases. Although alpha2,6-Sialyltransferases cloned from marine bacteria, such as Photobacterium damselae strain JT0160, P. leiognathi strain JT-SHIZ-145, and Photobacterium sp. strain JT-ISH-224, show only alpha2,6-Sialyltransferase activity, the recombinant enzyme cloned from P. leiognathi strain JT-SHIZ-119 showed both alpha2,6-Sialyltransferase and alpha2,6-linkage-specific neuraminidase activity. Our results provide important information toward a comprehensive understanding of the bacterial Sialyltransferases belonging to the group 80 glycosyltransferase family in the CAZy database.
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Photobacterium sp. JT-ISH-224 Produces Two Sialyltransferases, α-/β-Galactoside α2,3-Sialyltransferase and β-Galactoside α2,6-Sialyltransferase
2007Co-Authors: Hiroshi Tsukamoto, Toshiki Mine, Yoshimitsu Takakura, Takeshi YamamotoAbstract:A novel bacterium, Photobacterium sp. JT-ISH-224, that produces alpha-/beta-galactoside alpha2,3-Sialyltransferase and beta-galactoside alpha2,6-Sialyltransferase, was isolated from the gut of a Japanese barracuda. The genes that encode the enzymes were cloned from the genomic library of the bacterium using the genes encoding alpha-/beta-galactoside alpha2,3-Sialyltransferase from P. phosphoreum and beta-galactoside alpha2,6-Sialyltransferase from P. damselae as probes. The nucleotide sequences were determined, and open reading frames of 1,230 and 1,545 bp for encoding an alpha2,3-Sialyltransferase and an alpha2,6-Sialyltransferase of 409- and 514-amino acid residues, respectively, were identified. The alpha2,3-Sialyltransferase had 92% amino acid sequence identity with the P. phosphoreum alpha2,3-Sialyltransferase, whereas the alpha2,6-Sialyltransferase had 54% amino acid sequence identity with the P. damselae alpha2,6-Sialyltransferase. For both enzymes, the DNA fragments that encoded the full-length protein and its truncated form lacking the putative signal peptide sequence were amplified by a polymerase chain reaction and cloned into an expression vector. Each gene was expressed in Escherichia coli, and the lysate from each strain had enzymatic activity. The alpha2,3-Sialyltransferase catalysed the transfer of N-acetylneuraminic acid (NeuAc) from CMP-NeuAc to lactose, alpha-methyl-galactopyranoside and beta-methyl-galactopyranoside with low apparent K(m) and the alpha2,6-Sialyltransferase catalysed the transfer of NeuAc from CMP-NeuAc to lactose with low apparent K(m).
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crystal structure of vibrionaceae photobacterium sp jt ish 224 α2 6 Sialyltransferase in a ternary complex with donor product cmp and acceptor substrate lactose catalytic mechanism and substrate recognition
2007Co-Authors: Yoshimitsu Kakuta, Hitomi Kajiwara, Yoshimitsu Takakura, Masako Ichikawa, Nozomu Okino, Makoto Ito, Takeshi YamamotoAbstract:Sialyltransferases are a family of glycosyltransferases that catalyze the transfer of N-acetylneuraminic acid residues from cytidine monophosphate N-acetylneuraminic acid (CMP-NeuAc) as a donor substrate to the carbohydrate groups of glycoproteins and glycolipids as acceptor substrates. We determined the crystal structure of Delta16psp26ST, the N-terminal truncated form of alpha2,6-Sialyltransferase from Vibrionaceae Photobacterium sp. JT-ISH-224, complexed with a donor product CMP and an acceptor substrate lactose. Delta16psp26ST has three structural domains. Domain 1 belongs to the immunoglobulin-like beta-sandwich fold, and domains 2 and 3 form the glycosyltransferase-B structure. The CMP and lactose were bound in the deep cleft between domains 2 and 3. In the structure, only Asp232 was within hydrogen-binding distance of the acceptor O6 carbon of the galactose residue in lactose, and His405 was within hydrogen-binding distance of the phosphate oxygen of CMP. Mutation of these residues greatly decreased the activity of the enzyme. These structural and mutational results indicated that Asp232 might act as a catalytic base for deprotonation of the acceptor substrate, and His405 might act as a catalytic acid for protonation of the donor substrate. These findings are consistent with an in-line-displacement reaction mechanism in which Delta16psp26ST catalyzes the inverting transfer reaction. Unlike the case with multifunctional Sialyltransferase (Delta24PmST1) complexed with CMP and lactose, the crystal structure of which was recently reported, the alpha2,6 reaction specificity of Delta16psp26ST is likely to be determined by His123.
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purification cloning and expression of an α β galactoside α 2 3 Sialyltransferase from a luminous marine bacterium photobacterium phosphoreum
2007Co-Authors: Hiroshi Tsukamoto, Yoshimitsu Takakura, Takeshi YamamotoAbstract:A novel Sialyltransferase, α/β-galactoside α-2,3-Sialyltransferase, was purified from the cell lysate of a luminous marine bacterium, Photobacterium phosphoreum JT-ISH-467, isolated from the Japanese common squid (Todarodes pacificus). The gene encoding the enzyme was cloned from the genomic library of the bacterium using probes derived from the NH2-terminal and internal amino acid sequences. An open reading frame of 409 amino acids was identified, and the sequence had 32% identity with that of β-galactoside α-2,6-sialyltrasferase in Photobacterium damselae JT0160. DNA fragments that encoded the full-length protein and a protein that lacked the sequence between the 2nd and 24th residues at the NH2 terminus were amplified by polymerase chain reactions and cloned into an expression vector. The full-length and truncated proteins were expressed in Escherichia coli, producing active enzymes of 0.25 and 305 milliunits, respectively, per milliliter of the medium in the lysate of E. coli. The truncated enzyme was much more soluble without detergent than the full-length enzyme. The enzyme catalyzed the transfer of N-acetylneuraminic acid from CMP-N-acetylneuraminic acid to disaccharides, such as lactose and N-acetyllactosamine, with low apparent Km and to monosaccharides, such as α-methyl-galactopyranoside and β-methyl-galactopyranoside, with much lower apparent Km. Thus, this Sialyltransferase is unique and should be very useful for achieving high productivity in E. coli with a wide substrate range.
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A β-galactoside α2,6-Sialyltransferase produced by a marine bacterium, Photobacterium leiognathi JT-SHIZ-145, is active at pH 8
2007Co-Authors: Takeshi Yamamoto, Hitomi Kajiwara, Toshiki Mine, Hiroshi Tsukamoto, Yoko Hamada, Masako Ichikawa, Yoshimitsu TakakuraAbstract:A gene encoding a Sialyltransferase produced by Photobacterium leiognathi JT-SHIZ-145 was cloned, sequenced, and expressed in Escherichia coli. The Sialyltransferase gene contained an open reading frame of 1494 base pairs (bp) encoding a predicted protein of 497 amino acid residues. The deduced amino acid sequence of the Sialyltransferase had no significant similarity to mammalian Sialyltransferases and did not contain sialyl motifs, but did show high homology to another marine bacterial Sialyltransferase, a beta-galactoside alpha2,6-Sialyltransferase produced by P. damselae JT0160. The acceptor substrate specificity of the new enzyme was similar to that of the alpha2,6-Sialyltransferase from P. damselae JT0160, but its activity was maximal at pH 8. This property is quite different from the properties of all mammalian and bacterial Sialyltransferases reported previously, which have maximal activity at acidic pH. In general, both sialosides and cytidine-5'-monophospho-N-acetylneuraminic acid, the common donor substrate of Sialyltransferases, are more stable under basic conditions. Therefore, a Sialyltransferase with an optimum pH in the basic range should be useful for the preparation of sialosides and the modification of glycoconjugates, such as asialo-glycoproteins and asialo-glycolipids. Thus, the Sialyltransferase obtained from P. leiognathi JT-SHIZ-145 is a promising tool for the efficient production of sialosides.
