Purification to homogeneity of a β-galactoside α2→3 sialyltransferase and partial purification of an α-N-acetylgalactosaminide α2→6 sialyltransferase from porcine submaxillary glands (original) (raw)
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Enzymatic characterization of β-D-galactoside 2–3 sialyltransferase from porcine submaxillary gland
Journal of Biological Chemistry
The substrate requirements, linkage specificity, and kinetic mechanism of a pure sialyltransferase from porcine submaxillary glands have been examined. The enzyme transfers sialic acid from the donor nucleotide, CMP-NeuAc, into the sequence NeuAccw2 + 3Galfil+ 3GalNAc, which is found in both glycoproteins and gangliosides. It forms only the rw2 + 3 linkage with the disaccharide Gal/31 + 3GalNAc or antifreeze glycoprotein, which, along with asialoglycoproteins containing the sequence Galbl + 3GalNAccwl + 0-Thr/Ser, are the best acceptor substrates. Low molecular weight galactosides linked /31+ 3 to glycose residues other than Nacetylgalactosamine are poor acceptors with relatively high K,,, values, while those in fil+ 4 or /31+ 6 linkages have both high K,,, and low V,,,,. With glycoprotein and ganglioside acceptors this substrate specificity appears to be even more strict, with the sequence Gal/?1 --+ 3GalNAc serving as the exclusive acceptor. Thus the present enzyme is not responsible either for the sequence, NeuAccx2 + 3Galpl + 4GlcNAc, found in the asparagine-linked chains of certain glycoproteins, or for the synthesis of hematoside, NeuAccr2 + 3Galpl+ 4GlcPl + 1Cer. Initial rate kinetic studies, with and without inhibitors, suggest that the transferase has an equilibrium random order mechanism.
The Journal of biological chemistry, 1979
By means of affinity chromatography on CDP-hexanolamine-agarose, a CMP-N-acetylneuraminate: alpha-N-acetylgalactosaminide alpha 2 leads to 6 sialyltransferase (EC 2.4.99.1) has been purified 117,000-fold to homogeneity from Triton X-100 extracts of porcine submaxillary glands. The enzyme consists of several electrophoretic forms that can be partially resolved by chromatography on Sephadex G-200, the largest of which has a molecular weight of approximately 160,000 as estimated by sodium dodecyl sulfate-gel electrophoresis. Periodate oxidation studies show that the linkage formed by this enzyme with ovine submaxillary asialo-mucin as the acceptor substrate is NeuAc alpha 2 leads to 6GalNAc alpha 1 leads to O-Thr/Ser. On the basis of initial rate studies and the patterns of inhibition observed with alternate acceptor substrates, the transferase is proposed to have either a random equilibrium kinetic mechanism or an ordered steady state mechanism with the acceptor substrate binding firs...
A sialidase from horse liver was co-purified with b-galactosidase and carboxypeptodase A
20th International Carbohydrate Symposium, 2000
Sialidase (EC 3.2.1.18) is a hydrolytic enzyme that releases sialic acids, which are usually bound through α-2,3, α-2,6, or α-2,8 linkages to oligosaccharides, glycoproteins or glycolipids. In mammals sialidases have been found in various cellular locations, either as cytosolic or membrane-bound enzymes (e.g. lysosomes, Golgi, plasma membrane and nuclear membrane) [1]. The study of membrane-bound sialidases is fraught with difficulties, due to their low stability and their occurrence in a complex with other enzymes such as acid b-galactosidase and carboxypeptidase A [2, 3, 4, 5]. Sialic acids comprise a family of about 40 different derivatives of the 9-carbon sugar neuraminic acid. Sialic acids with a 4-O-acetyl group occur in a number of mammals, including horse, donkey, guinea pig as well as in the monotreme Echidna [1]. Sialic acids containing this modification are resistant to most sialidases [6]. Although the catabolism of 4-O-acetylated neuraminic acid derivatives in horse liver involves the action of an esterase [7], studies on the sialidase from this tissue may provide further insight into the degradation of sialylated glycoconjugates and enhance our understanding of eukaryotic sialidases. Here we show that the sialidase from horse liver is a membrane-bound enzyme. The solubilized enzyme was isolated using Fractogel TMAE (650) followed by affinity chromatography on the b-galactosidasespecific medium p-aminophenylthio-b-D-galactopyranoside-agarose and chromatofocusing on PBE 94. The enzyme was found to occur in a complex with b-galactosidase and carboxypeptidase A. Using sialyl-methylumbelliferyl a-glycoside as substrate the isolated sialidase exhibits temperature and pH optima of 42-46°C and 4.5 respectively, while the b-galactosidase is optimally active at 50°C and pH 4.0 with galactosyl-methylumbelliferyl b-glycoside. On the other hand, the carboxypeptidase A shows temperature and pH optima of 42°C and 5.8, respectively, with N-Carbobenzyloxy-Phe-Leu. [1] C. Traving and R. Schauer (1998) Cell. Mol. Life Sci. 54, 1330-1349 [2] F. W. Verheijen, S. Palmeri, A. T. Hoogeven and H. Galjaard (1985) Eur. J. Biochem. 149, 315-321 [3] M. Hiraiwa M. Saitoh, Y. Uda, N. Azuma, B. M. Martin, Y. Kishimoto and J. S. O’Brien (1996) Comp. Biochem. Physiol. 115B, 541-546 [4] M. Hiraiwa, M. Saitoh, N. Arai, T. Shirashi, S. Odani, Y. Uda, T. Ono and J. S. O’Brien (1997) Biochim. Biophys. Acta 1314, 189-199 [5] M. Nagaoka, M. Saitoh, T. Shirashi, H. Nagaoka, N. Irimaya, K. Furuhata and Y. Uda (1998) Bio. Pharm. Bull. 21, 682-687 [6] R. Schauer (1997) Trends Glycosci. Glycotechnol. 9, 315-330 [7] R. Schauer, G. Reuter and S. Stoll (1998) Biochimie 70, 1511-1519
Glycoconjugate Journal, 2008
Sialyltransferases are a family of enzymes catalyzing the transfer of sialic acid residues to terminal non-reducing positions of oligosaccharide chains of glycoproteins and glycolipids. Although expression of sialic acid is well documented in animals of the deuterostomian lineage, sialyltransferases have been predominantly described for relatively recent vertebrate lineages such as birds and mammals. This study outlines the characterization of the only sialyltransferase gene found in the tunicate Ciona intestinalis, the first such report of a non-vertebrate deuterostomian sialyltransferase, which has been discussed as a possible orthologue of the common ancestor of galactose α2,3sialyltransferases. We also report for the first time the characterization of a ST3Gal II gene from the bony fish Takifugu rubripes. We demonstrate that both genes encode functional α2,3-sialyltransferases that are structurally and functionally related to the ST3Gal family of mammalian sialyltransferases. However, characterization of the recombinant, purified forms of both enzymes reveal novel acceptor substrate specificities, with sialylation of the disaccharide Galβ1-3GalNAc and asialofetuin, but not GM1 or GD1b observed. This is in contrast to the mammalian ST3Gal II that predominantly sialylates gangliosides. Taken together the ceramide binding/recognition site previously proposed for the mouse ST3Gal II might represent a unique feature of mammalian ST3Gal II that is missing in the evolutionary more distant fish and tunicate species reported here. This suggests that during the evolution of the ST3Gal II, probably following the separation of the teleosts, a significant shift in substrate specificity enabling the sialylation of gangliosides took place.
Purification and characterization of ?(2-6)-sialyltransferase from human liver
Glycoconjugate Journal, 1991
A Galβ1-4GlcNAc α(2-6)-sialyltransferase from human liver was purified 34 340-fold with 18% yield by dye chromatography on Cibacron Blue F3GA and cation exchange FPLC. The enzyme preparation was free of other sialyltransferases. It did not contain CMP-NeuAc hydrolase, protease, or sialidase activity, and was stable at −20°C for at least eight months. The donor substrate specificity was examined with CMP-NeuAc analogues modified at C-5 or C-9 of theN-acetylneuraminic acid moiety. Affinity of the human enzyme for parent CMP-NeuAc and each CMP-NeuAc analogue was substantially higher than the corresponding Galβ1-4GlcNAc α(2-6)-sialyltransferase from rat liver.
1996
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, Neu5Ac␣2,3Gal1,3GalNAc and glycoconjugates carrying the same sequence such as G M1b and fetuin. The disialylated tetrasaccharide formed by reacting the sialyltransferase with the aforementioned sialoside was analyzed by one-and two-dimensional 1 H and 13 C NMR spectroscopy and was shown to be the Neu5Ac␣2,3-Gal1,3(Neu5Ac␣2,6)GalNAc sialoside. This indicates that the enzyme is a GalNAc ␣2,6-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 D1␣ from G M1b .