Publications

• Characterization of the second type of human beta-galactoside alpha2,6-sialyltransferase (ST6Gal II) that sialylates Galbeta1,4GlcNAc structures on oligosaccharides preferentially.

  Takashima S., Tsuji S., Tsujimoto M.

  A novel member of the human beta-galactoside alpha2,6-sialyltransferase (ST6Gal) family, designated ST6Gal II, was identified by BLAST analysis of expressed sequence tags and genomic sequences. The sequence of ST6Gal II encoded a protein of 529 amino acids, and it showed 48.9% amino acid sequence ... >> More

  A novel member of the human beta-galactoside alpha2,6-sialyltransferase (ST6Gal) family, designated ST6Gal II, was identified by BLAST analysis of expressed sequence tags and genomic sequences. The sequence of ST6Gal II encoded a protein of 529 amino acids, and it showed 48.9% amino acid sequence identity with human ST6Gal I. Recombinant ST6Gal II exhibited alpha2,6-sialyltransferase activity toward oligosaccharides that have the Galbeta1,4GlcNAc sequence at the nonreducing end of their carbohydrate groups, but it exhibited relatively low and no activities toward some glycoproteins and glycolipids, respectively. It is concluded that ST6Gal II is an oligosaccharide-specific enzyme compared with ST6Gal I, which exhibits broad substrate specificities, and is mainly involved in the synthesis of sialyloligosaccharides. The expression of the ST6Gal II gene was significantly detected by reverse transcription PCR in small intestine, colon, and fetal brain, whereas the ST6Gal I gene was ubiquitously expressed, and its expression levels were much higher than those of the ST6Gal II gene. The ST6Gal I gene was also expressed in all tumors examined, but no expression was observed for the ST6Gal II gene in these tumors. The ST6Gal II gene is located on chromosome 2 (2q11.2-q12.1), and it spans over 85 kb of human genomic DNA consisting of at least eight exons and shares a similar genomic structure with the ST6Gal I gene. In this paper, we have shown that ST6Gal I, which has been known as the sole member of the ST6Gal family, also has the counterpart enzyme (ST6Gal II) like other sialyltransferases. << Less

  J. Biol. Chem. 277:45719-45728(2002) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• The sialyltransferase "sialylmotif" participates in binding the donor substrate CMP-NeuAc.

  Datta A.K., Paulson J.C.

  All members of the sialyltransferase gene family cloned to date contain a conserved region, the "sialylmotif," consisting of 48-49 amino acids in the center of the coding sequence. To investigate the function of this motif, mutant constructs of the Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase we ... >> More

  All members of the sialyltransferase gene family cloned to date contain a conserved region, the "sialylmotif," consisting of 48-49 amino acids in the center of the coding sequence. To investigate the function of this motif, mutant constructs of the Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase were designed by site-directed mutagenesis, replacing 11 individual conserved amino acids with alanine. Each of the mutants was expressed in COS-1 cells, and eight of these retained sialyltransferase activity, allowing comparison of their enzymatic properties with that of the wild type enzyme. Kinetic analysis showed that six of eight mutants had a 3-12-fold higher Km for the donor substrate CMP-NeuAc relative to the wild type enzyme, while the Km values for the acceptor substrate were within 0.5-1.2-fold of the wild type for all eight mutants evaluated. The Ki of the donor substrate analog CDP was also evaluated for the recombinant sialyltransferase with the Val to Ala mutation at residue 220, which produced a 6-fold increase in Km of CMP-NeuAc. A corresponding increase in Ki of 3.4-fold was observed for CDP, indicating a decreased affinity for the cytidine nucleotide. Taken together, these results suggest that the conserved sialylmotif in the sialyltransferase gene family participates in the binding of the common donor substrate, CMP-NeuAc. << Less

  J Biol Chem 270:1497-1500(1995) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Physical and chemical studies on ceruloplasmin. 8. Preparation of N-acetylneuraminic acid-1-14C-labeled ceruloplasmin.

  Hickman J., Ashwell G., Morell A.G., van den Hamer C.J., Scheinberg I.H.

  J Biol Chem 245:759-766(1970) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Purification and characterization of an endogenous inhibitor of the sialyltransferase CMP-N-acetylneuraminate: lactosylceramide alpha 2,6-N-acetylneuraminyltransferase (EC 2.4.99.-).

  Albarracin I., Lassaga F.E., Caputto R.

  The presence in the 100,000 g supernatant of rat brain homogenate of an inhibitor of the sialyltransferase has been confirmed. It is also present in chicken and bovine brain and in other rat and bovine organs. The inhibitor has been purified, a preparation with a specific activity 130-fold higher ... >> More

  The presence in the 100,000 g supernatant of rat brain homogenate of an inhibitor of the sialyltransferase has been confirmed. It is also present in chicken and bovine brain and in other rat and bovine organs. The inhibitor has been purified, a preparation with a specific activity 130-fold higher than that of the original 100,000 g supernatant of brain being obtained. It runs as a single peak in polyacrylamide-gel electrophoresis; when run in the presence of SDS, two components appeared. The apparent Mr of the components were 14,800 and 22,400. The inhibitor has been characterized as a heat-stable protein of acidic nature. It has effect on the glycolipid and the glycoprotein sialyltransferase activities but has no effect on the galactosaminyltransferase activity. << Less

  Biochem J 254:559-565(1988) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• The sialic acids. XV. Transfer of sialic acid to glycoproteins by a sialyltransferase from colostrum.

  Bartholomew B.A., Jourdian G.W., Roseman S.

  J Biol Chem 248:5751-5762(1973) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Glycoprotein biosynthesis: studies on thyroglobulin. Thyroid sialyltransferase.

  Spiro M.J., Spiro R.G.

  J Biol Chem 243:6520-6528(1968) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Primary structure of beta-galactoside alpha 2,6-sialyltransferase. Conversion of membrane-bound enzyme to soluble forms by cleavage of the NH2-terminal signal anchor.

  Weinstein J., Lee E.U., McEntee K., Lai P.-H., Paulson J.C.

  This report describes the primary structure of a rat liver beta-galactoside alpha 2,6-sialyltransferase (EC 2.4.99.1), a Golgi apparatus enzyme involved in the terminal sialylation of N-linked carbohydrate groups of glycoproteins. The complete amino acid sequence was deduced from the nucleotide se ... >> More

  This report describes the primary structure of a rat liver beta-galactoside alpha 2,6-sialyltransferase (EC 2.4.99.1), a Golgi apparatus enzyme involved in the terminal sialylation of N-linked carbohydrate groups of glycoproteins. The complete amino acid sequence was deduced from the nucleotide sequence of cDNA clones of the enzyme. The primary structure suggests that the topology of the enzyme in the Golgi apparatus consists of a short NH2-terminal cytoplasmic domain, a 17-residue hydrophobic sequence which serves as the membrane anchor and signal sequence, and a large lumenal, catalytic domain. NH2-terminal sequence analysis of a truncated form of the enzyme, obtained by purification from tissue homogenates, reveals that it is missing a 63-residue NH2-terminal peptide which includes the membrane binding domain. These and supporting results show that soluble forms of the sialyltransferase can be generated by proteolytic cleavage between the NH2-terminal signal-anchor and the catalytic domain. << Less

  J. Biol. Chem. 262:17735-17743(1987) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Identification and functional expression of a second human beta-galactoside alpha2,6-sialyltransferase, ST6Gal II.

  Krzewinski-Recchi M.-A., Julien S., Juliant S., Teintenier-Lelievre M., Samyn-Petit B., Montiel M.-D., Mir A.-M., Cerutti M., Harduin-Lepers A., Delannoy P.

  BLAST analysis of the human and mouse genome sequence databases using the sequence of the human CMP-sialic acid:beta-galactoside alpha-2,6-sialyltransferase cDNA (hST6Gal I, EC2.4.99.1) as a probe allowed us to identify a putative sialyltransferase gene on chromosome 2. The sequence of the corresp ... >> More

  BLAST analysis of the human and mouse genome sequence databases using the sequence of the human CMP-sialic acid:beta-galactoside alpha-2,6-sialyltransferase cDNA (hST6Gal I, EC2.4.99.1) as a probe allowed us to identify a putative sialyltransferase gene on chromosome 2. The sequence of the corresponding cDNA was also found as an expressed sequence tag of human brain. This gene contained a 1590 bp open reading frame divided in five exons and the deduced amino-acid sequence didn't correspond to any sialyltransferase already known in other species. Multiple sequence alignment and subsequent phylogenic analysis showed that this new enzyme belonged to the ST6Gal subfamily and shared 48% identity with hST6Gal-I. Consequently, we named this new sialyltransferase ST6Gal II. A construction in pFlag vector transfected in COS-7 cells gave raise to a soluble active form of ST6Gal II. Enzymatic assays indicate that the best acceptor substrate of ST6Gal II was the free disaccharide Galbeta1-4GlcNAc structure whereas ST6Gal I preferred Galbeta1-4GlcNAc-R disaccharide sequence linked to a protein. The alpha2,6-linkage was confirmed by the increase of Sambucus nigra agglutinin-lectin binding to the cell surface of CHO transfected with the cDNA encoding ST6Gal II and by specific sialidases treatment. In addition, the ST6Gal II gene showed a very tissue specific pattern of expression because it was found essentially in brain whereas ST6Gal I gene is ubiquitously expressed. << Less

  Eur. J. Biochem. 270:950-961(2003) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Universal phosphatase-coupled glycosyltransferase assay.

  Wu Z.L., Ethen C.M., Prather B., Machacek M., Jiang W.

  A nonradioactive glycosyltransferase assay is described here. This method takes advantage of specific phosphatases that can be added into glycosyltransferase reactions to quantitatively release inorganic phosphate from the leaving groups of glycosyltransferase reactions. The released phosphate gro ... >> More

  A nonradioactive glycosyltransferase assay is described here. This method takes advantage of specific phosphatases that can be added into glycosyltransferase reactions to quantitatively release inorganic phosphate from the leaving groups of glycosyltransferase reactions. The released phosphate group is then detected using colorimetric malachite-based reagents. Because the amount of phosphate released is directly proportional to the sugar molecule transferred in a glycosyltransferase reaction, this method can be used to obtain accurate kinetic parameters of the glycosyltransferase. The assay can be performed in multiwell plates and quantitated by a plate reader, thus making it amenable to high-throughput screening. It has been successfully applied to all glycosyltransferases available to us, including glucosyltransferases, N-acetylglucosaminyltransferases, N-acetylgalactosyltransferases, galactosyltransferases, fucosyltransferases and sialyltransferases. As examples, we first assayed Clostridium difficile toxin B, a protein O-glucosyltransferase that specifically monoglucosylates and inactivates Rho family small GTPases; we then showed that human KTELC1, a homolog of Rumi from Drosophila, was able to hydrolyze UDP-Glc; and finally, we measured the kinetic parameters of human sialyltransferase ST6GAL1. << Less

  Glycobiology 21:727-733(2011) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Probing the substrate specificity of four different sialyltransferases using synthetic beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->O) (CH(2))7CH3 analogues general activating effect of replacing N-acetylglucosamine by N-propionylglucosamine.

  Rohfritsch P.F., Joosten J.A.F., Krzewinski-Recchi M.-A., Harduin-Lepers A., Laporte B., Juliant S., Cerutti M., Delannoy P., Vliegenthart J.F.G., Kamerling J.P.

  The acceptor specificities of ST3Gal III, ST3Gal IV, ST6Gal I and ST6Gal II were investigated using a panel of beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->O)(CH(2))(7)CH(3) analogues. Modifications introduced at either C2, C3, C4, C5, or C6 of terminal D-Gal, as well as N-propiony ... >> More

  The acceptor specificities of ST3Gal III, ST3Gal IV, ST6Gal I and ST6Gal II were investigated using a panel of beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->O)(CH(2))(7)CH(3) analogues. Modifications introduced at either C2, C3, C4, C5, or C6 of terminal D-Gal, as well as N-propionylation instead of N-acetylation of subterminal D-GlcN were tested for their influence on the alpha-2,3- and alpha-2,6-sialyltransferase acceptor activities. Both ST3Gal enzymes displayed the same narrow acceptor specificity, and only accept reduction of the Gal C2 hydroxyl function. The ST6Gal enzymes, however, do not have the same acceptor specificity. ST6Gal II seems less tolerant towards modifications at Gal C3 and C4 than ST6Gal I, and prefers beta-D-GalpNAc-(1-->4)-beta-D-GlcpNAc (LacdiNAc) as an acceptor substrate, as shown by replacing the Gal C2 hydroxyl group with an N-acetyl function. Finally, a particularly striking feature of all tested sialyltransferases is the activating effect of replacing the N-acetyl function of subterminal GlcNAc by an N-propionyl function. << Less

  Biochim. Biophys. Acta 1760:685-692(2006) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Enzymatic properties of beta-D-galactoside alpha2 leads to 6 sialytransferase from bovine colostrum.

  Paulson J.C., Rearick J.I., Hill R.L.

  The substrate specificity and kinetic properties of a pure sialyltransferase from bovine colostrum have been examined. The transferase appears to incorporate sialic acid into the sequence, NeuAcalpha2 leads to 6Galbeta1 leads to 4GlcNAc, which is commonly found in glycoproteins. It has a strict su ... >> More

  The substrate specificity and kinetic properties of a pure sialyltransferase from bovine colostrum have been examined. The transferase appears to incorporate sialic acid into the sequence, NeuAcalpha2 leads to 6Galbeta1 leads to 4GlcNAc, which is commonly found in glycoproteins. It has a strict substrate specificity for CMP-NeuAc and forms only the alpha2 leads to 6 sialyl linkage with beta-D-galactosides. N-Acetyllactosamine (Galbeta1 leads to 4GlcNAc) and asialo-glycoproteins containing the N-acetyllactosaminyl linkage at the nonreducing ends of the oligosaccharides prosthetic groups are the best acceptor substrates. Isomers of N-acetyllactosamine with beta1 leads to 3 or beta1 leads to 6 glycosidic linkages are less than 1% as effective as acceptor substates as the beta1 leads to 4-linked isomer. Lactose (Galbeta1 leads to 4Glc) is also a poor acceptor, indicating the importance of the 2-acetamido group in the N-acetylglucosaminyl residues. The unnatural substrate beta-methyl-L-arabinopyrano-side, a five-carbon analog of beta-methyl-D-galactoside which contains no 6-hydroxyl, also acts as a poor acceptor of the transferase and the sialylated product has been partially characterized. Kinetic properties of the enzyme in the presence and absence of inhibitors suggest that the transferase has an equilibrium random order mechanism. << Less

  J Biol Chem 252:2363-2371(1977) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• The structure of human alpha-2,6-sialyltransferase reveals the binding mode of complex glycans.

  Kuhn B., Benz J., Greif M., Engel A.M., Sobek H., Rudolph M.G.

  Human β-galactoside α-2,6-sialyltransferase I (ST6Gal-I) establishes the final glycosylation pattern of many glycoproteins by transferring a sialyl moiety to a terminal galactose. Complete sialylation of therapeutic immunoglobulins is essential for their anti-inflammatory activity and protein stab ... >> More

  Human β-galactoside α-2,6-sialyltransferase I (ST6Gal-I) establishes the final glycosylation pattern of many glycoproteins by transferring a sialyl moiety to a terminal galactose. Complete sialylation of therapeutic immunoglobulins is essential for their anti-inflammatory activity and protein stability, but is difficult to achieve in vitro owing to the limited activity of ST6Gal-I towards some galactose acceptors. No structural information on ST6Gal-I that could help to improve the enzymatic properties of ST6Gal-I for biotechnological purposes is currently available. Here, the crystal structures of human ST6Gal-I in complex with the product cytidine 5'-monophosphate and in complex with cytidine and phosphate are described. These complexes allow the rationalization of the inhibitory activity of cytosine-based nucleotides. ST6Gal-I adopts a variant of the canonical glycosyltransferase A fold and differs from related sialyltransferases by several large insertions and deletions that determine its regiospecificity and substrate specificity. A large glycan from a symmetry mate localizes to the active site of ST6Gal-I in an orientation compatible with catalysis. The glycan binding mode can be generalized to any glycoprotein that is a substrate of ST6Gal-I. Comparison with a bacterial sialyltransferase in complex with a modified sialyl donor lends insight into the Michaelis complex. The results support an SN2 mechanism with inversion of configuration at the sialyl residue and suggest substrate-assisted catalysis with a charge-relay mechanism that bears a conceptual similarity to serine proteases. << Less

  Acta Crystallogr. D 69:1826-1838(2013) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Comparison of the enzymatic properties of mouse beta-galactoside alpha2,6-sialyltransferases, ST6Gal I and II.

  Takashima S., Tsuji S., Tsujimoto M.

  The cDNA encoding a second type of mouse beta-galactoside alpha2,6-sialyltransferase (ST6Gal II) was cloned and characterized. The sequence of mouse ST6Gal II encoded a protein of 524 amino acids and showed 77.1% amino acid sequence identity with human ST6Gal II. Recombinant ST6Gal II exhibited al ... >> More

  The cDNA encoding a second type of mouse beta-galactoside alpha2,6-sialyltransferase (ST6Gal II) was cloned and characterized. The sequence of mouse ST6Gal II encoded a protein of 524 amino acids and showed 77.1% amino acid sequence identity with human ST6Gal II. Recombinant ST6Gal II exhibited alpha2,6-sialyltransferase activity toward oligosaccharides that have the Galbeta1,4GlcNAc sequence at the nonreducing end of their carbohydrate groups, but it exhibited relatively low and no activity toward some glycoproteins and glycolipids, respectively. On the other hand, ST6Gal I, which has been known as the sole member of the ST6Gal-family for more than ten years, exhibited broad substrate specificity toward oligosaccharides, glycoproteins, and a glycolipid, paragloboside. The ST6Gal II gene was mainly expressed in brain and embryo, whereas the ST6Gal I gene was ubiquitously expressed, and its expression levels were higher than those of the ST6Gal II gene. The ST6Gal II gene is located on chromosome 17 and spans over 70 kb of mouse genomic DNA consisting of at least 6 exons. The ST6Gal II gene has a similar genomic structure to the ST6Gal I gene. In this paper, we have shown that ST6Gal II is a counterpart of ST6Gal I. << Less

  J. Biochem. 134:287-296(2003) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Purification of a sialyltransferase from bovine colostrum by affinity chromatography on CDP-agarose.

  Paulson J.C., Beranek W.E., Hill R.L.

  CDP-hexanolamine agarose was used as an affinity adsorbent to purify a CMP-N-acetylneuraminate: beta-D-galactosyl-glycoprotein N-acetylneuraminyltransferase (EC 2.4.99.1) from bovine colostrum. Upon binding of the enzyme to the adsorbent, elution is achieved either nonspecifically, with 0.5 to 1.0 ... >> More

  CDP-hexanolamine agarose was used as an affinity adsorbent to purify a CMP-N-acetylneuraminate: beta-D-galactosyl-glycoprotein N-acetylneuraminyltransferase (EC 2.4.99.1) from bovine colostrum. Upon binding of the enzyme to the adsorbent, elution is achieved either nonspecifically, with 0.5 to 1.0 M sodium chloride, or specifically, with CDP. A highly purified sialyltransferase is obtained with a specific activity 440,000 times that of whole colostrum. Fractionation of the purified enzyme by gel filtration gives two species with different molecular weights but equal specific activities toward asialo-alpha1-acid glycoprotein (26.0 to 28.0 micronmol/min/mg of enzyme). The molecular weights of these two forms are about 56,000 and 43,000 as judged by sodium doedcyl sulfate-gel electrophoresis, sedimentation equilibrium, and gel filtration. The catalytic properties of both forms have been examined (Paulson, J. C., Rearick, J. I., and Hill, R. L. (1977) J. Biol. Chem. 252, 2363-2371). It is concluded that the lower molecular weight form may be a partially degraded species of the enzyme of higher molecular weight. << Less

  J Biol Chem 252:2356-2362(1977) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Glycosyltransferases involved in elongation of N-glycosidically linked oligosaccharides of the complex or N-acetyllactosamine type.

  Schachter H., Narasimhan S., Gleeson P., Vella G.

  Methods Enzymol 98:98-134(1983) [PubMed] [EuropePMC]

  This publication is cited by 9 other entries.