Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.4.99.7 (sialyltransferase)
1,534 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The extracellular polysaccharide capsule is an essential virulence factor of Neisseria meningitidis, a leading cause of severe bacterial meningitis and sepsis. Serogroup B strains, the primary disease causing isolates in Europe and America, are encapsulated in alpha-2,8 polysialic acid (polySia). The capsular polymer is synthesized from activated sialic acid by action of a membrane-associated polysialyltransferase (NmB-polyST). Here we present a comprehensive characterization of NmB-polyST. Different from earlier studies, we show that membrane association is not essential for enzyme functionality. Recombinant NmB-polyST was expressed, purified and shown to synthesize long polySia chains in a non-processive manner in vitro. Subsequent structure-function analyses of NmB-polyST based on refined sequence alignments allowed the identification of two functional motifs in bacterial sialyltransferases. Both (D/E-D/E-G and HP motif) are highly conserved among different sialyltransferase families with otherwise little or no sequence identity. Their functional importance for enzyme catalysis and CMP-Neu5Ac binding was demonstrated by mutational analysis of NmB-polyST and is emphasized by structural data available for the Pasteurella multocida sialyltransferase PmST1. Together our data are the first description of conserved functional elements in the highly diverse families of bacterial (poly)sialyltransferases and thus provide an advanced basis for understanding structure-function relations and for phylogenetic sorting of these important enzymes.
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PMID:Biochemical characterization of a Neisseria meningitidis polysialyltransferase reveals novel functional motifs in bacterial sialyltransferases. 1766 40

Glycoconjugates containing polysialic acid have many biological activities and represent target molecules for therapeutic interventions. Enzymatic synthesis of these glycoconjugates should give access to these important molecules to evaluate their potential. The polysialyltransferases from both Neisseria meningitidis and Escherichia coli were cloned and expressed as recombinant proteins in E. coli. We have used synthetic acceptors to probe the acceptor requirement of these enzymes and to examine the basic enzymology. The minimum number of sialic acid residues (Neu5Ac) on the acceptor for activity in vitro was shown to be 2 for both enzymes, but a large increase in activity was seen if the acceptor had three Neu5Ac residues. The polysialyltransferase from N. meningitidis generated longer reaction products than the enzyme from E. coli on FCHASE acceptors. Examination of the products showed them to be a heterogeneous mixture, but products with >50 Neu5Ac residues could be seen using capillary zone electrophoresis analyses. In addition we made fusion proteins of these polysialyltransferase enzymes with the bifunctional alpha-2,3/alpha-2,8-sialyltransferase from Campylobacter jejuni to create self priming polysialyltransferases. These bifunctional sialyltransferases utilized various synthetic disaccharide acceptors with a terminal galactose, and we demonstrate here that the PST enzyme from N. meningitidis and its fusion protein with the C. jejuni sialyltransferase can be used to create polysialic acid on O-linked glycopeptides.
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PMID:Characterization of the alpha-2,8-polysialyltransferase from Neisseria meningitidis with synthetic acceptors, and the development of a self-priming polysialyltransferase fusion enzyme. 1800 29

A chemo-enzymatic synthesis of [(5-acetamido-9-O-acetyl-3,5-dideoxy-D-glycero-alpha-D-galacto-2-nonulopyranosylonic acid)-(2-->3)-O-(beta-D-galactopyranosyl)-(1-->3)-O-(2-acetamido-2-deoxy-alpha-D-galactopyranosyl)]-l-serine acetate (1) has been accomplished by a regioselective chemical acetylation of Neu5Ac (2) to give 9-O-acetylated sialic acid 3, which was enzymatically converted into CMP-Neu5,9Ac(2) (4) employing a recombinant CMP-sialic acid synthetase from Neisseria meningitis [EC 2.7.7.43]. The resulting compound was then employed for the enzymatic glycosylation of the C-3' hydroxyl of chemically prepared glycosylated amino acid 10 using recombinant rat alpha-(2-->3)-O-sialyltransferase expressed in Spodooptera frugiperda [EC 2.4.99.4] to give, after deprotection of the N(alpha)-benzyloxycarbonyl (CBz)-protecting group of serine, target compound 1. The N(alpha)-CBz-protected intermediate 11 can be employed for the synthesis of glycolipopeptides for immunization purposes.
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PMID:Chemo-enzymatic synthesis of C-9 acetylated sialosides. 1850 39

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.
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PMID:Multifunctionality of Campylobacter jejuni sialyltransferase CstII: characterization of GD3/GT3 oligosaccharide synthase, GD3 oligosaccharide sialidase, and trans-sialidase activities. 1850 8

Sialic acids are abundant nine-carbon sugars expressed terminally on glycoconjugates of eukaryotic cells and are crucial for a variety of cell biological functions such as cell-cell adhesion, intracellular signaling, and in regulation of glycoproteins stability. In bacteria, N-acetylneuraminic acid (Neu5Ac) polymers are important virulence factors. Cytidine 5'-monophosphate (CMP)-N-acetylneuraminic acid synthetase (CSS; EC 2.7.7.43), the key enzyme that synthesizes CMP-N-acetylneuraminic acid, the donor molecule for numerous sialyltransferase reactions, is present in both prokaryotes and eukaryotic systems. Herein, we emphasize the source, function, and biotechnological applications of CSS enzymes from bacterial sources. To date, only a few CSS from pathogenic bacterial species such as Neisseria meningitidis, Escherichia coli, group B streptococci, Haemophilus ducreyi, and Pasteurella hemolytica and an enzyme from nonpathogenic bacterium, Clostridium thermocellum, have been described. Overall, the enzymes from both Gram-positive and Gram-negative bacteria share common catalytic properties such as their dependency on divalent cation, temperature and pH profiles, and catalytic mechanisms. The enzymes, however, can be categorized as smaller and larger enzymes depending on their molecular weight. The larger enzymes in some cases are bifunctional; they have exhibited acetylhydrolase activity in addition to their sugar nucleotidyltransferase activity. The CSSs are important enzymes for the chemoenzymatic synthesis of various sialooligosaccharides of significance in biotechnology.
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PMID:Bacterial CMP-sialic acid synthetases: production, properties, and applications. 1871 69

Sialic acid content in FSH is modulated by GnRH and sexual steroids. Galbeta1,3GlcNAcalpha2,3-sialyltransferase (ST3Gal III) and Galbeta1,4GlcNAcalpha2,6-sialyltransferase (ST6Gal I) incorporate sialic acid residues into FSH oligosaccharides. The aim of the present study was to assess pituitary FSH molecular microheterogeneity and ST3Gal III/ST6Gal I expression during sexual development and after castration in male rats. Preparative isoelectric focusing and lectin chromatography were used to isolate FSH glycosylation variants according to charge and complexity of their oligosaccharides; RT-PCR and immunohistochemistry were employed to analyse sialyltransferase expression. Sexual development was associated with a progressive shift towards more acidic/sialylated FSH glycoforms concomitantly with an increment in ST6Gal I gene and protein expression. After castration, a transient decrease followed by a marked increase in ST6Gal I expression were observed. Less acidic/sialylated FSH glycoforms bearing incomplete oligosaccharides increased after castration, despite high ST6Gal I expression. ST3Gal III expression remained unchanged in all the experimental conditions examined. These results show that the synthesis of FSH isoforms possessing alpha2,6-linked sialic acid is hormonally regulated in male rats.
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PMID:Hormonal regulation of pituitary FSH sialylation in male rats. 1946 43

Cell surface glycans are often terminated by sialic acid, which is incorporated onto sugar acceptors by sialyltransferases. The crystal structure of the GT family 42 Campylobacter jejuni alpha-2,3/2,8-sialyltransferase (CstII) provides key insights into the sialyl-transfer mechanism, including tentative identification of His188 as the catalytic base. In support of this hypothesis, the CstII-H188A mutant is able to catalyze sialyl transfer from CMP-Neu5Ac to added anions such as azide and formate but not to its natural sugar acceptor lactose. Complementing this work, NMR spectroscopy was used to investigate the structure and dynamics of CstII and to measure the intrinsic pK(a) value of His188 for comparison with the pK(a) determined from the pH-dependent k(cat)/K(M) of the enzyme. By systematically introducing point mutations at the subunit interfaces, two active monomeric variants, CstII-F121D and CstII-Y125Q, were obtained and characterized. In contrast to the wild-type tetramer, the monomeric CstII variants yielded good quality (1)H/(15)N-HSQC and (1)H/(13)C-methyl-TROSY NMR spectra. However, the absence of signals from approximately one-half of the amides in the (1)H/(15)N-HSQC spectra of both monomeric forms suggests that the enzyme undergoes substantial conformational exchange on a millisecond to microsecond time scale. The histidine pK(a) values of CstII-F121D in its apo form were measured by monitoring the pH-dependent chemical shifts of [(13)C(epsilon1)]histidine, biosynthetically incorporated into the otherwise uniformly deuterated protein. Consistent with its proposed catalytic role, the site-specific pK(a) value approximately 6.6 of His188 matches the apparent pK(a) value approximately 6.5 governing the pH dependence of k(cat)/K(M) for CstII toward CMP-Neu5Ac in the presence of saturating acceptor substrate.
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PMID:NMR spectroscopic characterization of the sialyltransferase CstII from Campylobacter jejuni: histidine 188 is the general base. 1982 95

In order to understand the biological importance of naturally occurring sialic acid variations on disialyl structures in nature, we developed an efficient two-step multienzyme approach for the synthesis of a series of GD3 ganglioside oligosaccharides and other disialyl glycans containing a terminal Siaalpha2-8Sia component with different natural and non-natural sialic acids. In the first step, alpha2-3- or alpha2-6-linked monosialylated oligosaccharides were obtained using a one-pot three-enzyme approach. These compounds were then used as acceptors for the alpha2-8-sialyltransferase activity of a recombinant truncated multifunctional Campylobacter jejuni sialyltransferase CstII mutant, CstIIDelta32(I53S), to produce disialyl oligosaccharides. The alpha2-8-sialyltransferase activity of CstIIDelta32(I53S) has promiscuous donor substrate specificity and can tolerate various substitutions at C-5 or C-9 of the sialic acid in CMP-sialic acid, while its acceptor substrate specificity is relatively restricted. The terminal sialic acid residues in the acceptable monosialylated oligosaccharide acceptors are restricted to Neu5Ac, Neu5Gc, KDN, and some of their C-9-modified forms but not their C-5 derivatives. The disialyl oligosaccharides obtained are valuable probes for their biological studies.
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PMID:Chemoenzymatic synthesis of GD3 oligosaccharides and other disialyl glycans containing natural and non-natural sialic acids. 1994 30

Alterations of the receptor-binding properties of swine influenza A viruses (SIVs) during their isolation in embryonated chicken eggs have not been well studied. In this study, the receptor-binding properties of classical H1 SIVs isolated solely in eggs or Madin-Darby canine kidney (MDCK) cells were examined. Sequencing analysis revealed substitutions of D190V/N or D225G in the haemagglutinin (HA) proteins in egg isolates, whereas MDCK isolates retained HA genes identical to those of the original viruses present in the clinical samples. Egg isolates with substitution of either D190V/N or D225G had increased haemagglutinating activity for mouse and sheep erythrocytes, but reduced activity for rabbit erythrocytes. Additionally, egg isolates with D225G had increased haemagglutination activity for chicken erythrocytes. A direct binding assay using a sialyl glycopolymer that possessed either a 5-N-acetylneuraminic acid (Neu5Ac) alpha2,6galactose (Gal) or a Neu5Acalpha2,3Gal linkage revealed that the egg isolates used in this study showed higher binding activity to the Neu5Acalpha2,3Gal receptor than MDCK isolates. Increased binding activity of the egg isolates to the Neu5Acalpha2,3Gal receptor was also confirmed by haemagglutination assay with resialylated chicken erythrocytes by Galbeta1,3/4GlcNAcalpha2,3-sialyltransferase. These observations were reinforced by flow-cytometric and N-glycan analyses of the erythrocytes. The alpha2,3-linked sialic acids were expressed predominantly on the surface of mouse and sheep erythrocytes. Chicken erythrocytes expressed Neu5Acalpha2,3Gal more abundantly than Neu5Acalpha2,6Gal, and rabbit erythrocytes expressed both 5-N-glycolylneuraminic acid (Neu5Gc) alpha2,6Gal and Neu5Acalpha2,6Gal. Our results demonstrate clearly that classical H1 SIVs undergo alterations in receptor-binding activity associated with an amino acid substitution in the HA protein during isolation and propagation in embryonated chicken eggs.
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PMID:Alterations in receptor-binding properties of swine influenza viruses of the H1 subtype after isolation in embryonated chicken eggs. 2000 53

Human monocyte-derived dendritic cells (mo-DCs) express highly sialylated structures, with recognized but poorly understood function in maturation, immunogenicity and endocytosis capacity. We have previously shown that mo-DCs surface sialylation is changeable upon different stimuli, which led us to hypothesise the existence of cell surface (non-intracellular) sialyltransferases, rapidly restoring or altering mo-DC surface sialylation, thus modulating specific functions. Here, we demonstrate that, in the presence of exogenous CMP-Neu5Ac, mo-DCs incorporate considerable amounts of sialic acids into cell surface, predominantly when mo-DCs were previously desialylated or matured. This is a genuine sialyltransferase activity, confirmed by specific inhibition assays, which is not influenced by secreted enzymes. Functionally, the ecto-sialyltransferase activity causes a significant down-regulation of mo-DCs endocytic capacity, without affecting the maturation state. These findings suggest that ecto-sialyltransferases participate in a dynamic control of mo-DC sialylation, with functional repercussions. This activity is possibly related with specific physiological and pathological conditions, as inflammation and infection, contributing to protection and homeostasis regulation.
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PMID:Human dendritic cells contain cell surface sialyltransferase activity. 2020 7


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