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At least 19 recordsLinked to original sources
PubMed1995

Release of sialyltransferases from rat liver Golgi membranes by a cathepsin D-like proteinase: comparison of the release of Gal beta 1-4GlcNAc alpha 2-6 sialyltransferase, Gal beta 1-3(4)GlcNAc alpha 2-3 sialyltransferase and lactosylceramide alpha 2-3 sialyltransferase (SAT-1).

The activities of Gal beta 1-3(4)GlcNAc alpha 2-3 sialyltransferase and SAT-1 were measured in rat liver Golgi in inflammation; both enzymes decreased by about 50%. This compares with increases of about 3-fold for the Gal beta 1-4GlcNAc alpha 2-6 sialyltransferase. All three sialyltransferases were released from disrupted Golgi membranes by incubation at reduced pH which activates an endogenous cathepsin D which is believed to be the lysosomal enzyme. Pepstatin A was found to block the release of all three sialyltransferases providing support for the role of cathepsin D as the proteinase that clips the catalytic portions of the enzymes from their membrane anchor and stem regions.

Animals↗
PubMed1996

Characterization of two glycolipid: alpha 2-3sialyltransferases, SAT-3 (CMP-NeuAc:nLcOse4Cer alpha 2-3sialyltransferase) and SAT-4 (CMP-NeuAc:GgOse4Cer alpha 2-3sialyltransferase), from human colon carcinoma (Colo 205) cell line.

Sialyltransferase activities, SAT-3 (CMP-NeuAc:nLcOse4Cer alpha 2-3sialyltransferase) and SAT-4 (CMP-NeuAc:GgOse4Cer alpha 2-3sialyltransferase), in Colo 205 cells catalyze the transfer of sialic acid to the terminal galactose of GlcNc-- and GalNAc-containing glycolipid substrates, respectively. Competition kinetic studies with nLcOse4Cer and GM1 as substrates in a sialyltransferase assay show that these two activities are catalyzed by two different catalytic entities. The two enzymes were co-solubilized with taurochlorate and resolved by DEAE--Cibacron Blue--Sepharose column chromatography into two elution peaks. The column eluent with SAT-3 activity failed to transfer sialic acid to asialo alpha(1)-acid glycoprotein, indicating that this enzyme is different from the sialyltransferase (ST3N) that synthesizes NeuAc alpha 2-3Gal linkage in asparagine-linked oligosaccharides of glycoprotein. However, SAT-3 activity can be immunoprecipitated with a polyclonal antibody produced against a protein expressed in Escherichia coli as GST-fusion protein from an ECB cDNA homolog of an alpha 2-3 sialyltransferase SAT-3 or STZ) the has been cloned from human melanoma cell and human placenta. Thus a concentration-dependent decrease in the residual SAT-3 activity relative to SAT-4 activity was observed in the supernatant after precipitation of the immune complex. Expression of SAT-3 (STZ) cDNA was also detected in Colo 205 cell by RT-PCR, followed by sequence analysis of the RT-PCR product. Characterization of the catalytic reaction products of SAT-3 and SAT-4 with thin-layer chromatography, sialidase treatment, and binding to specific antibodies indicates that both SAT-3 and SAT-4 catalyze the formation of alpha 2-3 linkage between sialic acid and terminal galactose of glycolipid substrates.

Base Sequence↗
PubMed1982

Purification of a Gal beta 1 to 4GlcNAc alpha 2 to 6 sialyltransferase and a Gal beta 1 to 3(4)GlcNAc alpha 2 to 3 sialyltransferase to homogeneity from rat liver.

A Gal beta 1 to 4GlcNAc alpha 2 to 6 sialyltransferse and a Gal beta 1 to 3(4)GlcNAc alpha 2 to 3 sialyltransferase have been purified 23,000- and 860,000-fold to homogeneity from Triton CF-54 extracts of rat liver membranes. The two enzymes were concentrated by affinity chromatography on CDP-hexanolamine-agarose and resolved by NaCl gradient elution from the same adsorbent. Final purification of the Gal beta 1 to 4GlcNAc alpha 2 to 6 sialytransferase, the most abundant enzyme, was achieved by specific elution from CDP-agarose with CDP. The Gal beta 1 to 3(4)GlcNAc alpha 2 to 3 sialyltransferase was also purified further by CDP elution from CDP-agarose, but final purification required affinity chromatography on an adsorbent prepared by coupling asialoprothrombin to cyanogen bromide-activated agarose. Asialoprothrombin contains the terminal sequence Gal beta 1 to 3GlcNAc on N-linked oligosaccharides and is the best acceptor substrate of the enzyme (Km congruent to 6 microM). The Gal beta 1 to 3(4)GlcNAc alpha 2 to 3 sialyltransferase was found to bind to asialoprothrombin-agarose in the presence of CDP and could be eluted with a solution containing 0.2 M lactose and no CDP. Sodium dodecyl sulfate-gel electrophoresis of the Gal beta 1 to 4GlcNAc alpha 2 to 6 and Gal beta 1 to 3(4)GlcNAc alpha 2 to 3 sialyltransferases revealed a single major protein band for each enzyme with apparent molecular weights of 40,500 and 44,000, respectively. Rabbit antibodies raised to the Gal beta 1 to 4GlcNAc alpha 2 to 6 sialyltransferase inhibit its enzymatic activity greater than 99% but caused little or no inhibition of Gal beta 1 to 3(4)GlcNAc alpha 2 to 3 sialytransferase. Moreover, the Gal beta 1 to 4GlcNAc alpha 2 to 6 sialyltransferase quantitatively bound to a column containing antibody adsorbed to Protein A-agarose, while the Gal beta 1 to 3(4) GlcNAc alpha 2 to 3 sialyltransferase did not bind. This demonstrated that the two sialyltransferases are antigenically unrelated and formed the basis for removal of contaminating Gal beta 1 to 4GlcNAc alpha 2 to 6 sialyltransferase from solutions of the Gal beta 1 to 3(4)GlcNAc alpha 2 to 3 sialyltransferase. Enzymatic characterization of the two sialyltransferases suggests that their major biological roles are in the terminal glycosylation of N-linked oligosaccharides of glycoproteins. (Weinstein, J., de Souza-e-Silva, U., and Paulson J. C. (1982) J. Biol. Chem. 257, 13845-13853. The alpha 2 to 6 sialyltransferase efficiently forms the NeuAc alpha 2 to 6Gal beta 1 to 4GlcNAc sequence, and the alpha 2 to 3 sialyltransferase forms the NeuAc alpha 2 to 3Gal beta 1 to 3GlcNAc and NeuAc alpha 2 to 3Ga; beta 1 to 4GlcNAc sequences.

Animals↗
PubMed1989

Evidence for an O-glycan sialylation system in brain. Characterization of a beta-galactoside alpha 2,3-sialyltransferase from rat brain regulating the expression of an alpha-N-acetylgalactosaminide alpha 2,6-sialyltransferase activity.

We present evidence for the existence in rat brain of several sialyltransferases able to sialylate sequentially asialofetuin. [14C]Sialylated glycans of asialofetuin were analyzed by gel filtration. Three types of [14C]sialylated glycans were synthesized: N-glycans and monosialylated and disialylated O-glycans. The varying effects of N-ethylmaleimide, lysophosphatidylcholine (lysoPtdCho) and trypsin, were helpful in the identification of these different sialyltransferases. One of them, selectively inhibited by N-ethylmaleimide, was identified as the Neu5Ac alpha 2----3Gal beta 1----3GalNAc-R:alpha 2----6 sialyltransferase previously described [Baubichon-Cortay, H., Serres-Guillaumond, M., Louisot, P. and Broquet, P. (1986) Carbohydr. Res. 149, 209-223]. This enzyme was responsible for the synthesis of disialylated O-glycans. LysoPtdCho and trypsin selectively inhibited the enzyme responsible for the synthesis of monosialylated O-glycan. N-ethylmaleimide, lysoPtdCho and trypsin did not inhibit Neu5Ac transfer onto N-glycans, giving evidence for three different molecular species. To identify the enzyme responsible for monosialylated O-glycan synthesis, we used another substrate: Gal beta 1----3GalNAc--protein obtained after galactosylation of desialylated ovine mucin by a GalNAc-R:beta 1----3 galactosyltransferase from porcine submaxillary gland. This acceptor was devoid of N-glycans and of NeuAc in alpha 2----3 linkages on the galactose residue. When using N-ethylmaleimide we obtained the synthesis of only one product, a monosialylated structure. After structural analysis by HPLC on SAX and SiNH2 columns, we identified this product as Neu5Ac alpha 2----3Gal beta 1----3GalNAc. The enzyme leading to synthesis of this monosialylated O-glycan was identified as a Gal beta 1----3GalNAc-R:alpha 2----3 sialyltransferase. When using lysoPtdCho and trypsin, sialylation was completely abolished, although the Neu5Ac alpha 2----3Gal beta 1----3GalNAc-R:alpha 2----6 sialyltransferase was not inhibited. We provided thus evidence for the interpendence between the two enzymes, the alpha 2----3 sialyltransferase regulates the alpha 2----6 sialyltransferase activity since it synthesizes the alpha 2----6 sialyltransferase substrate.

Animals↗
PubMed1989

Different reactivity of two brain sialyltransferases towards sulfhydryl reagents. Evidence for a thiol group involved in the nucleotide-sugar binding site of the NeuAc alpha 2-3Gal beta 1-3GalNAc alpha(2-6)sialyltransferase.

We have studied the amino-acid residues involved in the catalytic activity of two distinct brain sialyltransferases acting on fetuin and asialofetuin. These two enzymes were strongly inhibited by N-bromosuccinimide, a specific blocking reagent for tryptophan residues. This result suggests the involvement of such residues in the catalytic process of the two sialyltransferases. Furthermore, chemical modifications by various sulfhydryl reagents led to a strong inhibition of the fetuin sialyltransferase while the asialofetuin sialyltransferase was only slightly inhibited. For a more thorough understanding of the thiol inactivation mechanism of the fetuin sialyltransferase, we studied in more detail the reactivity of this enzyme with NEM (N-ethylmaleimide), an irreversible reagent. The time-dependent inactivation followed first-order kinetics and these kinetic data afforded presumptive evidence for the binding of 1 mol NEM per mol of enzyme. Only CMP-NeuAc protected the enzyme against NEM inactivation effectively. MnCl2 did not enhance the protective effect of CMP-NeuAc. The modifications of the fetuin sialyltransferase kinetic parameters by NEM showed a competitive mechanism between NEM and CMP-NeuAc. The results suggest the involvement of a sulfhydryl residue in or near the nucleotide-sugar binding site of the fetuin sialyltransferase (but we could not excluded that CMP-NeuAc binding may induce a change in conformation of the protein, leading to a decreased accessibility of this thiol group located near the nucleotide-sugar binding site). This SH group is essential to the enzyme activity, which is not the case for the asialofetuin sialyltransferase.

Animals↗
PubMed2001

Overexpression of sialyltransferase CMP-sialic acid:Galbeta1,3GalNAc-R alpha6-Sialyltransferase is related to poor patient survival in human colorectal carcinomas.

Thomsen-Friedenreich (TF)-related blood group antigens, such as TF, Tn, and their sialylated variants, belong to a family of tumor-associated carbohydrates. The aim of the present study was to examine tumor-associated alterations of glycosyltransferases involved in the biosynthesis of the TF glycotope in colorectal carcinomas. To this end, glycosyltransferase expression was examined in 40 cases of colorectal carcinoma specimens classified according to the WHO/Union International Contre Cancer guidelines and in "normal" mucosa of the same patients. Occurrence of TF glycotope was examined by immunohistochemistry with the monoclonal antibody A78-G/A7. Expression of sialyltransferases CMP-sialic acid:Galbeta1,3GalNAc-R alpha3-sialyltransferase I and II (ST3Gal-I and ST3Gal-II) and CMP-sialic acid:Galbeta1,3GalNAc-R alpha6-sialyltransferase (ST6GalNAc-II) and of core 2 beta1,6-N-acetylglucosaminyltransferase was determined by reverse transcription-PCR in the same cryostat sections used for immunohistochemistry. Additionally, alpha2,3-sialyltransferase enzyme activity was studied in each of these tissues. The TF glycotope was detected in 7% of the normal mucosa, but in 57% of the carcinoma samples. Expression of alpha2,3-sialyltransferases ST3Gal-I, ST3Gal-II, and enzyme activity of alpha2,3-sialyltransferase was significantly increased (P < 0.001) in carcinoma specimens compared with normal mucosa. ST3Gal-I mRNA expression was significantly increased (P = 0.05) in cases showing invasion of lymph vessels. Expression of ST6GalNAc-II was significantly increased (P = 0.04) in cases with metastases to lymph nodes along the vascular trunk. Moreover, ST6GalNAc-II expression provides an prognostic factor for patient survival (log rank, P = 0.02). In an attempt to study the functional relevance of the glycosyltransferases for TF biosynthesis, SW480 colorectal cells were transfected with each of the enzymes, and cell surface expression of the TF glycotope was examined by flow cytometry. The presence of TF was not altered by transfection of the cells with either sialyltransferase ST3Gal-I or ST3Gal-II. However, successful transfection with core 2 beta1,6-N-acetylglucosaminyltransferase led to reduced expression of TF. In contrast, increased cell surface expression of TF was found after ST6GalNAc-II transfection. Thus, expression of TF on the cell surface of SW480 colorectal carcinoma cells depends on the ratio of core 2 beta1,6-N-acetylglucosaminyltransferase and ST6GalNAc-II. Earlier immunohistological studies demonstrated that TF is a prognostic factor for patient survival. Our results suggest that sialyltransferase ST6GalNAc-II is of crucial relevance for the prognostic significance of TF.

Adult↗
PubMed1992

Primary structure of Gal beta 1,3(4)GlcNAc alpha 2,3-sialyltransferase determined by mass spectrometry sequence analysis and molecular cloning. Evidence for a protein motif in the sialyltransferase gene family.

The Gal beta 1,3(4)GlcNAc alpha 2,3-sialyltransferase forms the NeuAc alpha 2,3Gal beta 1,3(4)GlcNAc sequences found in terminal carbohydrate groups of glycoproteins and glycolipids. High energy collision-induced dissociation analysis of tryptic peptides from only 300 pmol of the purified Gal beta 1,3(4)GlcNAc alpha 2,3-sialyltransferase provided 25% of the total amino acid sequence and led to the successful cloning of this enzyme. The peptide sequence information was used to design short degenerate primers for use in the polymerase chain reaction. A long specific cDNA fragment was amplified which was used to isolate a clone from a rat liver cDNA library. The cloned cDNA encodes a 374-amino acid protein containing an amino-terminal signal-anchor sequence characteristic of all cloned glycosyltransferases and produced sialyltransferase activity when transiently expressed in COS-1 cells. When compared with two other cloned sialyltransferases, the primary structure of Gal beta 1,3(4)GlcNAc alpha 2,3-sialyltransferase revealed a homologous region in all three enzymes consisting of a stretch of 55 amino acids located in their catalytic domains. This feature together with lack of homology in the remaining 85% of the sequence of the three sialyltransferases defines a pattern of sequence homology not found in cloned cDNAs of other glycosyltransferase families.

Amino Acid Sequence↗
PubMed1995

Donor substrate specificities of Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase and Gal beta 1,3GalNAc alpha 2,3-sialyltransferase: comparison of N-acetyl and N-glycolylneuraminic acids.

Using cloned sialyltransferases, Gal beta 1,3GalNAc alpha 2,3-sialyltransferase (ST3Gal I) and Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase (ST6Gal I) from both chicken and mouse, CMP-NeuAc and CMP-NeuGc were compared as donor substrates with pyridylamino-oligo-saccharides as acceptors. ST6Gal I showed 4-7-times higher activity toward CMP-NeuGc than CMP-NeuAc, while for ST3Gal I there was no significant difference between them, irrespective of the origin of the enzymes. Also, the difference in donor substrate (i.e., NeuAc and NeuGc) had little effect on the preference to acceptor substrates of these enzymes. Thus, the results showed that the cloned sialyltransferases can utilize both CMP-NeuAc and CMP-NeuGc as donor substrates, and that the preference difference between the sialyltransferases to CMP-NeuGc and CMP-NeuAc could, at least partly, explain the discrepancy in the ratio of NeuAc and NeuGc in glycolipids and glycoproteins in individual tissues.

Animals↗
PubMed2005

The animal sialyltransferases and sialyltransferase-related genes: a phylogenetic approach.

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.

Amino Acid Sequence↗
PubMed1993

Sialyltransferase activity in FR3T3 cells transformed with ras oncogene: decreased CMP-Neu5Ac:Gal beta 1-3GalNAc alpha-2,3-sialyltransferase.

We have investigated the activity of CMP-Neu5Ac:Gal beta 1-3GalNAc alpha-2,3-sialyltransferase (EC 2.4.99.4) in FR3T3 cells transformed by the Ha-ras oncogene in which we have previously demonstrated the higher expression of the beta-galactosidase alpha-2,6-sialyltransferase (EC 2.4.99.1) [21]. We demonstrate that the presence of the activated ras gene decreases the activity of this specific alpha-2,3-sialyltransferase fourfold. According to the kinetic parameters and to mixing experiments, we can assume that this decreased enzymatic activity reflects a decrease in the number of active O-glycan alpha-2,3-sialyltransferase polypeptides in ras-transformed cells. However, no change in the binding of Peanut agglutinin was observed on the cell surface of ras-transformed FR3T3 suggesting that no change in the sialylation of O-glycan core 1 appeared in these cells, although the activity of the alpha-2,3-sialyltransferase was decreased.

Animals↗
PubMed1995

Enzymatic activity of a developmentally regulated member of the sialyltransferase family (STX): evidence for alpha 2,8-sialyltransferase activity toward N-linked oligosaccharides.

We have detected sialyltransferase activity of recombinant mouse STX, which was cloned from rat brain as a new member of the sialyltransferase family, but sialyltransferase activity of which had not been detected previously [Livingston and Paulson, J. Biol. Chem. (1993) 268, 11504-11507]. The activity of mouse STX was specific toward sialylated glycoproteins. N-Glycanase treatment and linkage-specific sialidase treatment of glycoproteins revealed that STX transfers sialic acids through alpha 2,8-linkages to only N-linked oligosaccharides of glycoproteins. However, polymerase activity for polysialic acid synthesis was not detected for this sialyltransferase. Since this alpha 2,8-sialyltransferase gene is highly restricted in fetal and newborn brain, it may be involved in the polysialylation of glycoproteins, especially of N-CAM.

Animals↗
PubMed1979

Purification to homogeneity of a beta-galactoside alpha2 leads to 3 sialyltransferase and partial purification of an alpha-N-acetylgalactosaminide alpha2 leads to 6 sialyltransferase from porcine submaxillary glands.

Two different sialyltransferases (EC 2.4.99.1) have been resolved from Triton X-100 extracts of porcine submaxillary glands by affinity chromatography on CDP-hexanolamine agarose. The predominant sialyltransferase of this tissue, a CMP-N-acetylneuraminate: alpha-D-N-acetylgalactosaminide alpha2 leads to 6 sialyltransferase, has been obtained in a partially purified and stable form. A less abundant but highly active enzyme, a CMP-N-acetylneuraminate: beta-D-galactoside alpha2 leads to 3 sialyltransferase, was purified over 90,000-fold to homogeneity. Chromatography of the latter enzyme on Sephadex G-200 separated two noninterconverting forms, designated A and B, with Stokes radii of 51 A and 31 A, respectively. Both forms have equal specific activity toward lactose and contain a single polypeptide with a molecular weight of about 50,000 as estimated by gel electrophoresis. Form A appears to bind 1.18 g of Triton X-100 per g of protein, or nearly an entire detergent micelle per polypeptide, while Form B binds little or no detergent. The enzymatic properties of both forms are similar (Rearick, J.I., Sadler, J.E., Paulson, J.C., and Hill, R.L. (1979) J. Biol. Chem. 254, 4444-4451) supporting the conclusion that Form A may represent the native sialyltransferase with an intact membrane-binding site, and Form B may be a large proteolytic fragment of Form A.

Acetylgalactosamine↗
PubMed1982

Sialyltransferase activity as a marker for the differentiation of lymphocytes. Increase in sialyltransferase activity of blasts from acute lymphoblastic leukaemia cases by 12-o-tetradecanoylphorbol-13-acetate (TPA).

The level of sialyltransferase activity in leukaemic blasts from acute lymphoblastic leukaemia (ALL) cases was significantly lower (3.29 +/- 2.09 pmoles/5 X 10(7) cells or 1.77 +/- 1.16 pmoles/mg protein) than those (18.80 +/- 4.91 pmoles/5 X 10(7) cells or 7.72 +/- 1.75 pmoles/mg protein) of mature lymphocytes from normal volunteers (T less than 0.001). An inverse relationship between the level of sialyltransferase activity and the level of terminal transferase (TdT)activity was seen in blasts from eight TdT-positive ALL cases. No significant difference was observed in the level of sialyltransferase activity between ALL and cells of chronic myelogenous leukemia (CML) in blast crisis. Short Term culture of ALL blast cells with 12-0-tetradecanoylphorbol-13-acetate (TPA) at the concentration of 10-(6)M to 10-(9)M caused a marked increase in sialyltransferase activity. In one of these three ALL cases the population of TdT-positive cells and the TdT activity of the blasts decreased significantly after culture with TPA. These results suggest that biochemical differentiation of leukaemic lymphoblasts has been induced by the addition of TPA, although morphological changes were not observed. Sialyltransferase activity may be a useful indicator for the analysis of differentiation of lymphocytes.

Cell Differentiation↗
PubMed1982

Discrimination between activity of (alpha 2-3)-sialyltransferase and (alpha 2-6)-sialyltransferase in human platelets using p-nitrophenyl-beta-D-galactoside as acceptor.

Exogenous asialo-glycoproteins and endogenous acceptors are both sialylated by incubating cytidine 5'-monophosphate N-[14C]acetylneuraminic acid (CMP [14C]NeuAc) with a lysate of human platelets but their respective incorporation levels vary with the divalent cation concentration. P-Nitrophenyl-beta-D-galactoside has also been demonstrated to be an acceptor of sialyl residues, and two different sialyl derivatives are synthesized according to the concentration of divalent cations. P-Nitrophenyl-beta-D-[6-3H]galactoside has been prepared by reduction with tritiated borohydride of the compound previously oxidized by galactose oxidase. Using this labelled p-nitrophenyl-beta-D-galactoside as acceptor and unlabelled CMP-NeuAc as donor, the two sialyl derivatives have been identified by methylation analysis as alpha-sialosyl-(2-3)-p-nitrophenyl-beta-D-galactoside and alpha-sialosyl-(2-6)-p-nitrophenyl-beta-D-galactoside. In addition to their different responses to divalent cation requirements, the sialyltransferase activities responsible for the synthesis of the two sialylgalactoside isomers have been clearly distinguished by their temperature and pH optimal values. They also exhibit different susceptibilities to dithioerythritol and different stabilities. These results demonstrate the presence in human platelets of two sialyltransferases: a CMP-NeuAc: galactoside (alpha 2-3)-sialyltransferase and a CMP-NeuAc: galactoside (alpha 2-6)-sialyltransferase.

Blood Platelets↗
PubMed1993

Determination of the specificities of rat liver Gal(beta 1-4)GlcNAc alpha 2,6-sialyltransferase and Gal(beta 1-3/4)GlcNAc alpha 2,3-sialyltransferase using synthetic modified acceptors.

Apparent kinetic parameters have been measured for the transfer of N-acetyl-D-neuraminic acid (Neu5Ac) from CMP-Neu5Ac to analogues of the Gal(beta 1-4)GlcNAc (type II) and Gal(beta 1-3)GlcNAc (type I) substrates by the rat liver Gal(beta 1-4)GlcNAc alpha 2,6-sialyltransferase and the Gal(beta 1-3/4)GlcNAc alpha 2,3-sialyltransferase. In these acceptor analogues, the substituents of the pyranose rings were modified, particularly by deoxygenation, to identify (i) the key polar groups required for efficient transfer and (ii) the substituents that can be removed or modified. A topography including the 6-hydroxyl of the beta Gal and the 2-acetamido of the GlcNAc unit is required for transfer to a terminal type II disaccharide by the alpha 2,6-sialyltransferase. The other hydroxyls can be replaced by hydrogen without a substantial decrease in activity. The alpha 2,3-sialyltransferase requires the 3-, 4-, and 6-hydroxyls of the terminal beta Gal and some contribution from the subterminal sugar. This may explain the cross-reactivity of this enzyme for the type I and type II acceptors. For both enzymes, an influence of the hydrophobic nature of the aglycone is noticed. The results allow an evaluation of the efficiency of the transfer of Neu5Ac to modified substrates.

Animals↗
PubMed1998

New sialyltransferase inhibitors based on CMP-quinic acid: development of a new sialyltransferase assay.

Quinic acid (4) was transformed into phosphitamides 6, 14, and 15, which could be readily linked to 5'-O-unprotected cytidine derivative 7; ensuing oxidation of the obtained phosphite triesters with tert-butylhydroperoxide furnished the corresponding phosphate triesters 8, 16, and 17, respectively. Hydrogenolytic debenzylation of the phosphate moiety, base catalysed removal of acetyl protective groups, and basic hydrolysis of the methylester of the quinic acid moiety furnished CMP-Neu5Ac analogues 1-3. In order to measure their inhibition of sialyltransferases, a nonradioactive sialyltransferase assay [employed for alpha(2-6)-sialyltransferase from rat liver (EC 2.4.99.1)] based on reversed-phase HPLC separation of UV-labelled acceptor 20 (p-nitrophenyl glycoside of N-acetyllactosamine) from the UV-labelled product 21 (p-nitrophenyl glycoside of sialyl alpha(2-6')-N-acetyllactosamine) and p-nitrophenylalanine as internal standard was developed. The assay reproduced the reported K(M) values for CMP-Neu5Ac and N-acetyllactosamine and the Ki values for CDP. 1 and 2 turned out to be potent sialyltransferase inhibitors.

Animals↗
PubMed2002

The Hexapeptide inhibitor of Galbeta 1,3GalNAc-specific alpha 2,3-sialyltransferase as a generic inhibitor of sialyltransferases.

The mammalian Galbeta1,3GalNAc-specific alpha2,3-sialyltransferase (ST3Gal I) was expressed as a secreted glycoprotein in High Five (Trichoplusia ni) cells. Using this recombinant ST3Gal I, we screened the synthetic hexapeptide combinatorial library to explore a sialyltransferase inhibitor. We found that the hexapeptide, NH(2)-GNWWWW, exhibited the most strong inhibition of ST3Gal I among five different hexapeptides that were finally selected. The kinetic analysis of ST3Gal I inhibition demonstrated that this hexapeptide could act as a competitive inhibitor (K(i) = 1.1 microm) on CMP-NeuAc binding to the enzyme. Moreover, the hexapeptide was shown to strongly inhibit both N-glycan-specific alpha2,3- and alpha2,6-sialyltranferase in vitro, suggesting that this peptide may inhibit the broad range of sialyltransferases regardless of their linkage specificity. The inhibitory activity in vivo was investigated by RCA-I lectin blot analyses and by metabolic d-[6-(3)H]GlcNH(2) radiolabeling analyses of N- and O-linked oligosaccharides in Chines hamster ovary cells. Our results demonstrate that the hexapeptide can act as a generic inhibitor of the N- and O-glycan-specific sialyltransferases in mammalian cells, which results in the significantly reduced NeuAc expression on cellular glycoproteins in vivo.

Amino Acid Sequence↗
PubMed1983

Studies on the effect of inflammation on rat liver and serum sialyltransferase. Evidence that inflammation causes release of Gal beta 1 leads to 4GlcNAc alpha 2 leads to 6 sialyltransferase from liver.

Turpentine induced inflammation has been shown to elevate liver sialyl- and galactosyltransferase activities (Turchen, B., Jamieson, J.C., Huebner, E., and van Caeseele, L. (1977) Can. J. Zool. 55, 1567-1571; Lombart, C., Sturgess, J., and Schachter, H. (1980) Biochem. Biophys. Acta 629, 1-12). We now report that serum sialyl-, but not galactosyltransferase activities are significantly elevated in turpentine inflammation. A liver slice system is used to demonstrate that liver releases large amounts of sialyltransferase activity into medium after inflammation, whereas only a low level of galactosyltransferase activity is released. Studies with rat and human asialo-alpha 1-acid glycoprotein as acceptors, coupled with the use of lactose to confirm the nature of the linkages formed, showed that Gal beta 1 leads to 4GlcNAc alpha 2 leads to 6 sialyltransferase is released from liver in turpentine inflammation and is mainly responsible for the elevated sialyltransferase activity found in serum. The alpha 2 leads to 6 sialyltransferase is exhibiting the properties of a typical acute phase reactant.

Animals↗