Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.4.99.6 (sialyltransferase)
 1,546 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Purified lactotetraosylceramide (Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4Glc1-Cer) was tested for its ability to accept [14C]sialic acid from CMP-[14C]sialic into monosialoganglioside fractions in the presence of membrane fractions purified from human colorectal carcinoma cells (SW1116). Membrane fractions were isolated by three different methods: sucrose density centrifugation, CMP-agarose gel column chromatography, and LcOse4 gel chromatography. We optimized the incubation conditions for detergent dependency (taurocholate), pH (6.3), and acceptor concentration. The sialyltransferase activity was dependent on membrane protein and linear for time up to at least 4 h. The LcOse4 affinity chromatography of the crude microsomal membrane pellet from these cells yielded a membrane fraction that was 136-fold enriched in LcOse4 acceptor specific activity compared to cell homogenates. The apparent Km for the sialyltransferase activity with LcOse4Cer acceptor in the presence of affinity-purified membranes was 20 microM and the Vmax was 7 pmol h-1 (100 micrograms of protein)-1. Acceptor capabilities of other core structures were 5-20-fold lower: LcOse4Cer much greater than GgOse4Cer greater than nLcOse4Cer much greater than GbOse4Cer. The enzymatic activity was purified further (900-fold) by a combination of LcOse4 and CMP affinity gels. SDS-PAGE electrophoresis of this material showed a major set of closely migrating bands of Mr 58,000-54,000 compared to authentic proteins, as well as a minor band at 27,000. We analyzed picomole quantities of the radioactive product by convenient controlled short-term hydrolyses with an endoglycoceramidase and sialidases (from four different sources) in comparison to sialylated tetrasaccharides of known structure.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Purification and characterization of a CMP-sialic:LcOse4Cer sialyltransferase from human colorectal carcinoma cell membranes. 306 17

A sialyltransferase involved in the biosynthesis in vitro of LD1c (NeuAc alpha 2-8NeuGc alpha 2-3Gal beta 1-4Glc-NAc beta 1-3Gal beta 1-4Glc-Cer) has been characterized from 9 to 11-day-old embryonic chicken brains. The CMP-[14C]NeuAc:LM1(alpha 2-8)sialyltransferase (SAT-2) sedimented (75%) at the junction of 0.75 and 1.2 M on a discontinuous sucrose density gradient when still membrane bound. In addition to the biosynthesis of LD1c, the detergent-solubilized (0.4% Nonidet P-40) preparation also catalyzes the transfer of sialic acid to O-8 of sialic acid in GM3 to form GD3 (NeuAc alpha 2-8NeuAc alpha 2 - 3Gal beta 1 - 4Glc - Cer). Substrate inhibition studies indicated that these two reactions are probably catalyzed by the same enzyme, SAT-2. The kinetic parameters of SAT-2 activity were determined. The Km values were 70 and 63 microM with CMP-[14C]NeuAc and LM1, respectively, when the detergent-solubilized supernatant fraction was used as enzyme source. The (alpha 2-8)-linkage between the terminal and penultimate sialic acids was determined using nonradioactive CMP-NeuAc and [Ac-14C]LM1 as substrates (Higashi, H., and Basu, S. (1982) Anal. Biochem. 120, 159-164) for the enzyme, followed by identification of the permethylated [14C]sialic acid of the product by radioautography. At 0.5 mM N-ethylmaleimide, the SAT-2 activity was inhibited 50% whereas SAT-1 and SAT-3 activities (Basu, M., Basu, S., Stoffyn, A., and Stoffyn, P. (1982) J. Biol. Chem. 257, 12765-12769) remained uninhibited.
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PMID:Biosynthesis in vitro of disialosylneolactotetraosylceramide by a solubilized sialyltransferase from embryonic chicken brain. 383 72

A cancer-associated glycolipid antigen defined by monoclonal antibody 19-9 has the structure NeuAc alpha 2-3Gal Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc beta 1-Cer. We have (formula; see text) studied its biosynthesis by testing the capacity of a crude microsomal fraction of SW 1116 cells to catalyze the addition of fucosyl or sialyl residues from GDP-fucose or CMP-sialic acid to glycolipid or oligosaccharide precursors. When the tetrasaccharide NeuAc alpha 2-3Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc (LSTa) is incubated with GDP-[14C]fucose and SW 1116 microsomes, a 14C-labeled oligosaccharide is formed that can be separated from the incubation mixture on an affinity column containing antibody 19-9 bound to protein A-Sepharose. The product migrates slower than LSTa when analyzed by paper or thin-layer chromatography. After treatment with neuraminidase, it co-migrates with the pentasaccharide Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc (formula; see text) (LNF II) in both chromatographic systems. Similar experiments demonstrate that SW 1116 microsomes catalyze the addition of a sialyl residue to the tetrasaccharide Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc to form LSTa. However, when LNF II is incubated with CMP-[14C]sialic acid and SW 1116 microsomes, no 19-9-active product is detected by affinity chromatography or by paper or thin-layer chromatography. Results using glycolipid precursors are consistent with these findings and also demonstrate the presence of the Lewis fucosyltransferase in SW 1116 cells. Thus, the biosynthesis of the sialyl-Lea antigen proceeds by addition of sialic acid to a type 1 precursor chain by a sialyltransferase, followed by addition of fucose by the Lewis fucosyltransferase.
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PMID:Biosynthesis of the cancer-associated sialyl-Lea antigen. 401 78

1. There are more glycolipid acceptor sites for NeuNAc than for glycoproteins in 11--15 day old rat cerebra. 2. The glycolipid acceptors appear to be almost exclusively Cer-Glc-Gal and GM1 ganglioside and each is a substrate for a different sialyltransferase. 3. The sialyltransferase(s) that acted on glycoprotein could be differentiated from the ones that acted on the glycolipids. 4. The apparent Km for CMP-NeuNAc was the same for all four of the sialyltransferase reactions studied. 5. Electron microscopic examination and marker enzyme studies on continuous sucrose gradient fractions found that most of the sialyltransferase activities appeared to be localized in smooth microsomal membrane and the Golgi complex derivatives and not associated with the synaptosomes.
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PMID:Sialyltransferases in young rat brain. 615 54

A sialyltransferase activity present in 7- to 12-day-old embryonic chicken brain catalyzes the transfer of sialic acid from CMP-sialic acid to the terminal galactose residue of [3H]nLcOse4Cer ([3H]Gal(beta 1-4).GlcNAc(beta 1-3)Gal(beta 1-4)Glc-Cer) to form NeuAc(alpha 2-3)-[3H]nLcOse4Cer (LM1 ganglioside). The product is sialidase-labile (96%), and the NeuAc group is linked to O-3 of the terminal galactose residue. The (alpha 2-3) linkage between sialic acid and the terminal galactose was determined on the basis of identification of 2,4,6-tri-O-methyl[3H]galactose obtained after hydrolysis of the permethylated enzymatic product. The CMP-sialic acid:nLcOse4Cer (alpha 2-3)sialyltransferase activity sediments (90%) at the junction of 1.2 M and 1.5 M on a discontinuous sucrose density gradient when still membrane bound (insoluble in 0.2% Triton X-100). The enzyme preparation also catalyzes the transfer of sialic acid from CMP-sialic acid to O-3 of GgOse4Cer (Gal(beta 1-3)GalNAc(beta 1-4)Gal(beta 1-4)Glc-Cer) to form NeuAc (alpha 2-3)GgOse4Cer (GM1b). Substrate inhibition studies indicate that these two reactions are probably catalyzed by the same enzyme.
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PMID:Biosynthesis in vitro of sialyl(alpha 2-3)neolactotetraosylceramide by a sialyltransferase from embryonic chicken brain. 713 Jan 78

The CMP-sialic acid:poly alpha 2,8sialosyl sialyltransferase (polyST) in neurotropic Escherichia coli K1 inner membranes catalyzes synthesis of the alpha 2,8-linked polysialic acid capsule. The capsule is a neurovirulent determinant associated with neonatal meningitis in humans. A functionally similar polyST in human neuroblastomas polysialylates neural cell adhesion molecules. While bacteria do not synthesize glycosphingolipids (GSLs), we report here that the E. coli K1 polyST can selectively polysialylate several structurally related GSLs, when added as exogenous sialyl acceptors. A structural feature common to the preferred sialyl acceptors (GD3 > GT1a > GQ1b = GT1b > GD2 = GD1b = GD1a > GM1) was the disialyl glycotope, Sia alpha 2,8Sia, alpha 2,3-linked to galactose (Sia is sialic acid). A linear tetrasaccharide with a terminal Sia residue (e.g., GD3) was the minimum length oligosaccharide recognized by the polyST. Endo-N-acylneuraminidase was used to confirm the alpha 2,8-specific polysialylation of GSL. Ceramide glycanase was used to release the polysialyllactose chains from the ceramide moiety. Size analysis of these chains showed that 60-80 Sia residues were transferred to the disialyllactose moiety of GD3. The significance of these findings is two-fold. (i) The E. coli K1 polyST can be used as a synthetic reagent to enzymatically engineer the glycosyl moiety of GSL, thus creating oligo- or polysialylated GSLs. Such "designer" GSLs may have potentially important biological and pharmacological properties. (ii) The use of GSLs as exogenous sialyl acceptors increases the sensitivity of detecting polyST activity. The practical advantage of this finding is that polyST activity can be identified and studied in those eukaryotic cells that express low levels of this developmentally regulated enzyme and/or its acceptor.
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PMID:Polysialic acid engineering: synthesis of polysialylated neoglycosphingolipids by using the polysialyltransferase from neuroinvasive Escherichia coli K1. 797 78

Using an expression cloning approach, we have isolated a cDNA encoding GD3 synthase (CMP-NeuAc:NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1'Cer alpha 2,8-sialyltransferase, EC 2.4.99.8), which is a key regulatory enzyme determining the prominence of the ganglioside biosynthesis pathway. The cloned cDNA encodes a 341-amino acid protein containing a single transmembrane domain at its N-terminal region, suggesting that the protein has a type II transmembrane topology. The sequence of alpha 2,8-sialyltransferase showed a high level of similarity with other sialyltransferases at two conserved regions typical in the sialyltransferase family. Transfected cells containing the cloned cDNA expressed GD3 ganglioside on the cell surface, which was detectable with specific anti-GD3 antibody by immunofluorescence and immunostaining after separation of isolated glycolipids on thin-layer chromatography. The cDNA hybridized to a single mRNA species of 2.4 kb in melanoma cells. This sialyltransferase is distinctive in catalyzing the formation of the alpha 2-8 linkage of sialic acids.
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PMID:Expression cloning of a CMP-NeuAc:NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1'Cer alpha 2,8-sialyltransferase (GD3 synthase) from human melanoma cells. 805 40

A human Gal beta(1-3/1-4)GlcNAc alpha 2,3-sialyltransferase, called ST-4, is a sialyltransferase involved in the in vivo biosynthesis of sialyl Lewis X (NeuNAc alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc) determinant. The ST-4 enzyme could utilize nLc4Cer (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1'Cer) containing type 2 sugar chain, Lc4Cer (Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1'Cer) containing type 1 sugar chain, Gg4Cer (Gal beta 1-3GalNAc beta 1-3Gal beta 1-4Glc beta 1-1'Cer), and LacCer as glycolipid acceptor substrates, but not other neutral glycolipids (GalCer, GlcCer, Gb3Cer, Gg3Cer, Gb4Cer) and gangliosides (GM1a, GM2, GM3, GD1a, GD1b, and GT1b) as substrates. The order of sialic acid incorporation into glycolipids for the enzyme was nLc4Cer > Gg4Cer > Lc4Cer > LacCer. The apparent Km values of ST-4 for nLc4Cer and Gg4Cer were 0.47 and 2.5 mM, respectively. Thus, the ST-4 could efficiently utilize both nLc4Cer and Gg4Cer as glycolipid acceptor substrates in vitro, suggesting that the substrate specificity of the enzyme may be similar to that of a glycolipid sialyltransferase (SAT-3), which is defined as the enzyme that uses both nLc4Cer and Gg4Cer as glycolipid acceptor substrates.
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PMID:Glycolipid acceptor specificity of a human Gal beta(1-3/1-4) GlcNAc alpha 2,3-sialyltransferase. 855 8

GD3 synthase (CMP-NeuAc:NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1'Cer alpha 2,8-sialyltransferase) is a member of the sialyltransferase family, whose members are characterized by having the sialyl motif and a key regulatory enzyme that controls the ganglioside biosynthesis pathway. The chromosomal location of the GD3 synthase gene (SIAT8) was determined in human and mouse using fluorescence in situ hybridization and interspecific backcross analysis, respectively. The human GD3 synthase gene was mapped to p12.1-p11.2 of chromosome 12. The mouse homologue was mapped 2.8 cM distal to D6Mit52 and 4.3 cM proximal to D6Mit25; this region is syntenic to the short arm of human chromosome 12.
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PMID:Chromosome mapping of the GD3 synthase gene (SIAT8) in human and mouse. 878 3

Although most glycosphingolipids (GSLs) are thought to be located in the outer leaflet of the plasma membrane, recent evidence indicates that GSLs and their precursor, ceramide, are also associated with intracellular organelles and, particularly, mitochondria. GSL biosynthesis starts with the formation of ceramide in the endoplasmic reticulum (ER), which is transported by controversial mechanisms to the Golgi apparatus, where stepwise addition of monosaccharides on to ceramides takes place. We now report the presence of GSL-biosynthetic enzymes in a subcompartment of the ER previously characterized and termed 'mitochondria-associated membrane' (MAM). MAM is a membrane bridge between the ER and mitochondria that is involved in the biosynthesis and trafficking of phospholipids between the two organelles. Using exogenous acceptors coated on silica gel, we demonstrate the presence of ceramide glucosyltransferase (Cer-Glc-T), glucosylceramide galactosyltransferase and sialyltransferase (SAT) activities in the MAM. Estimation of the marker-enzyme activities showed that glycosyltransferase activities could not be ascribed to cross-contamination of MAM by Golgi membranes. Cer-Glc-T was found to have a marked preference for ceramide bearing phytosphingosine as sphingoid base. SAT activities in MAM led to the synthesis of G(M3) ganglioside and small amounts of G(D3). G(M1) was also synthesized along with G(M3) upon incubation of the fraction with exogenous unlabelled G(M3), underlying the presence of other sphingolipid-specific glycosyltransferases in MAM. On the basis of our results, we propose MAM as a privileged compartment in providing GSLs for mitochondria.
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PMID:The mitochondria-associated endoplasmic-reticulum subcompartment (MAM fraction) of rat liver contains highly active sphingolipid-specific glycosyltransferases. 1257 62


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