Sialylation of lacto-N-neohexaosylceramide by sialyltransferase from embryonic chicken muscle

A sialyltransferase which catalyzes the in vitro biosynthesis of N-acetylneuraminosyllacto-N-neohexaosylceramide from lacto-N-neohexaosylceramide and CMP-NeuAc has been examined in embryonic chicken breast muscle. The maximum enzyme activity was observed in 11-12-day-old embryos. The enzyme has optimum activity at pH 6.8 in the presence of Triton CF-54 and Mg2+. The apparent Km values for lacto-N-neohexaosylceramide and CMP-NeuAc were 0.9 and 0.67 mM, respectively. The enzymic product was characterized by TLC, neuraminidase hydrolysis and permethylation analysis. The structure was identical to authentic N-acetylneuraminosyllacto-N-neohexaosylceramide from chicken muscle. In addition, a disialo derivative has been detected that constitutes 15% of the total radioactivity incorporated. The two sialic acids connected by sialosyl-sialosyl linkage were attached to the terminal galactose residue. To our knowledge, this is the first report of biosynthesis of this disialo compound.     

Relationship between plasmin-trypsin-inhibitory and sialyltransferase activities

Previously we have shown that the measurable soluble sialyltransferase (STase) activity released into the medium during the incubation of rat jejunal slices was dependent upon the presence of a heparin-binding fraction (HBF) from heat-inactivated serum or a trypsin-binding protein (TBP) isolated from HBF. Both HBF and TBP were able to inhibit trypsin and plasmin. The measurement of galactosyltransferase (GTase) activity which was also released in incubations was not dependent on HBF or TBP. The present study is directed towards further exploring the relationship between STase activity and protease inhibitory activity. Heat-inactivated serum from turpentine-treated rats (HTS), had higher plasmin-trypsin-inhibitory (HTS) activities compared to heat-inactivated serum from control rats (HCS). When HTS was used to supplement jejunal incubations, there was a 25–40% increase in the measurable STase activity in the incubation medium compared to similar incubations carried out in buffer alone. In contrast, with HCS the increase was 10–15%. During incubations with hepatocytes, STase activity detected in the incubation medium was increased with the incubation buffer was supplemented with HTS compared to incubations supplemented with HCS. Serum antiproteolytic activity was higher in turpentine rats compared to controls. Incubation of serum at 37°C led to a progressive decrease in plasmin-trypsin-inhibitory and STase activities. TBP a plasmin and trypsin inhibitor was able to prevent the decrease in STase activity. Overall, serum STase activity was higher in the turpentine treated rats. In contrast, GTase activity in serum as well as that detected in the medium during jejunal and hepatocyte incubations was not dependent on protease inhibitory activity. The results show that there is a relationship between soluble STase and plasmin-trypsin-inhibitory activities.     

Sialylation and malignant potential in tumour cell glycosylation mutants

Somatic mutations and drugs that either reduce     

Sialylation in vitro of purified human liver beta-D-N-acetylhexosaminidase

In order to study structure-function relationships of lysosomal enzymes, human liver beta-N-acetylhexosaminidase (2-acetamido-2-deoxy-beta-D-hexoside acetamidodeoxyhexohydrolase, EC 3.2.1.52) has been purified by an extraction/affinity chromatography/ion-exchange procedure. The isoenzymes A and B, native as well as neuraminidase-treated, were incubated with a partially purified preparation of bovine colostrum sialyltransferase (CMP-N-acetylneuraminate: D-galactosyl-glycoprotein N-acetylneuraminyltransferase, EC 2.4.99.1). Native beta-N-acetylhexosaminidases were found to be poor acceptors for the sialyltransferase used. However, incorporation of sialic acid into neuraminidase-treated beta-N-acetylhexosaminidase A and B amounted to a 58 to 72% saturation of the theoretical acceptor sites, respectively. The acceptor specificity of the sialyltransferase suggests that Gal beta(1 leads to 4)-GlcNAc units may be present on at least part of the beta-N-acetylhexosaminidase A and B molecules. However, oligomannosidic-type chains may also occur on the lysosomal enzyme, as shown by sugar composition of the enzyme. The presence and/or amount of sialic acid residues does not appear to affect the kinetic properties of beta-N-acetylhexosaminidase A and B towards 4-methylumbelliferyl glycoside substrate.     

The human sialyltransferase family

The human genome encodes probably more than 20 different sialyltransferases involved in the biosynthesis of sialylated glycoproteins and glycolipids but to date only 15 different human sialyltransferase cDNAs have been cloned and characterized. Each of the sialyltransferase genes is differentially expressed in a tissue-, cell type-, and stage-specific manner to regulate the sialylation pattern of cells. These enzymes differ in their substrate specificity, tissue distribution and various biochemical parameters. However, enzymatic analysis conducted in vitro with recombinant enzyme revealed that one linkage can be synthesized by multiple enzymes. We present here an overview of these human genes and enzymes, the regulation of their occurrence and their involvement in several physiological and pathological processes.     

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 (2-6)-sialyltransferase from rat liver (EC 2.4.99.1)] based on reversed-phase HPLC separation of UV-abelled acceptor 20 (p-nitrophenyl glycoside of N-acetyllactosamine) from the UV-labelled product 21 (p-nitrophenyl glycoside of sialyl (2-6)-N-acetyllactosamine) and p-nitrophenylalanine as internal standard was developed. The assay reproduced the reported KM values for CMP-Neu5Ac and N-acetyllactosamine and the Ki values for CDP. 1 and 2 turned out to be potent sialyltransferase inhibitors. © 1998 Rapid Science Ltd     

Functional characterization of Drosophila sialyltransferase

Sialylation is an important carbohydrate modification of glycoconjugates in the deuterostome lineage of animals. By contrast, the evidence for sialylation in protostomes has been scarce and somewhat controversial. In the present study, we characterize a Drosophila sialyltransferase gene, thus providing experimental evidence for the presence of sialylation in protostomes. This gene encodes a functional alpha2-6-sialyltransferase (SiaT) that is closely related to the vertebrate ST6Gal sialyltransferase family, indicating an ancient evolutionary origin for this family. Characterization of recombinant, purified Drosophila SiaT revealed a novel acceptor specificity as it exhibits highest activity toward GalNAcbeta1-4GlcNAc carbohydrate structures at the non-reducing termini of oligosaccharides and glycoprotein glycans. Oligosaccharides are preferred over glycoproteins as acceptors, and no activity toward glycolipid acceptors was detected. Recombinant Drosophila SiaT expressed in cultured insect cells possesses in vivo and in vitro autosialylation activity toward beta-linked GalNAc termini of its own N-linked glycans, thus representing the first example of a sialylated insect glycoconjugate. In situ hybridization revealed that Drosophila SiaT is expressed during embryonic development in a tissue- and stage-specific fashion, with elevated expression in a subset of cells within the central nervous system. The identification of a SiaT in Drosophila provides a new evolutionary perspective for considering the diverse functions of sialylation and, through the powerful genetic tools available in this system, a means of elucidating functions for sialylation in protostomes.     

Sialylglycoconjugates and sialyltransferase activity in the fungus Cryptococcus neoformans

Cryptococcus neoformans is a fungal pathogen associated with systemic mycoses in up to 10% of AIDS patients. C. neoformans yeasts express sialic acids on the cell wall, where they play an anti-phagocytic role, and may represent a virulence factor at the initial phase of infection. Since the nature of the sialic acid-carrying components is undefined in C. neoformans, our aim in the present work was to identify sialylated molecules in this fungus and study the sialylation process. C. neoformans yeast forms were cultivated in a chemically defined medium free of sialic acids, to search for autologous sialylglycoconjugates. Sialylated glycolipids were not detected. Two glycoproteins with molecular masses of 38 and 67 kDa were recognized by Sambucus nigra agglutinin, an 2,6-sialic acid-specific lectin. The 67 kDa glycoprotein also interacted with Influenza C virus, but not with Limax flavus agglutinin, suggesting the presence of the 9-O-acetylated sialic acid derivative as a constituent of the oligosaccharide chains. A partially purified protein fraction from cryptococcal yeast forms was able to transfer sialic acid from CMP-Neu5Ac to both N-(acetyl-1-¹⁴C)-lactosamine and asialofetuin. Additional evidence for a sialyltransferase in C. neoformans was obtained through the reactivity of fungal proteins with rabbit anti-rat 2,6 sialyltransferase polyclonal antibody. Our results indicate that sialic acids in C. neoformans are linked to glycoproteins, which are sialylated by the action of a fungal sialyltransferase. This is the first demonstration of this biosynthetic step in pathogenic fungi. Published in 2003.     

Plasma sialyltransferase activity in healthy subjects and atherosclerotic patients

Plasma sialyltransferase activity measured by incorporation of cytidine 5;-phospho[14C]acetylneuraminic acid (CMP-NeuAc) into asialofetuin was twofold higher in patients with documented atherosclerosis than in healthy donors. Kinetic studies showed that the enzyme affinity for CMP-NeuAc is the same in donors and patients. Low activity of plasma sialyltransferase in donors may be due to low blood content of this enzyme.     

Structural basis of sialyltransferase activity in trypanosomal sialidases

The intracellular parasite Trypanosoma cruzi, the etiological agent of Chagas disease, sheds a developmentally regulated surface trans-sialidase, which is involved in key aspects of parasite-host cell interactions. Although it shares a common active site architecture with bacterial neuraminidases, the T.cruzi enzyme behaves as a highly efficient sialyltransferase. Here we report the crystal structure of the closely related Trypanosoma rangeli sialidase and its complex with inhibitor. The enzyme folds into two distinct domains: a catalytic beta-propeller fold tightly associated with a lectin-like domain. Comparison with the modeled structure of T.cruzi trans-sialidase and mutagenesis experiments allowed the identification of amino acid substitutions within the active site cleft that modulate sialyltransferase activity and suggest the presence of a distinct binding site for the acceptor carbohydrate. The structures of the Trypanosoma enzymes illustrate how a glycosidase scaffold can achieve efficient glycosyltransferase activity and provide a framework for structure-based drug design.     

3D-QSAR analysis of sialyltransferase inhibitors

A predictive 3D-QSAR model that correlates the biological activities with the chemical structures of a series of sialyltransferase inhibitors, exemplified by the sugar:nucleotide derivatives, was developed by means of comparative molecular field analysis (CoMFA). The resulting cross-validated value (q(2)=0.629), non-cross-validated value (r(2)=0.965) and standard error of estimate (SEE=0.288) indicate that the obtained pharmacophore model indeed mimics the steric and electrostatic environment where inhibitors bind to the enzyme. The developed model also possesses promising predictive ability as discerned by the testing on the external test set, and should be useful to further understand the molecular nature of inhibitor-enzyme interactions and to aid in the design of more potent sialyltransferase inhibitors.     

A new fluorescent assay for sialyltransferase

A new fluorescent assay for the sialyltransferase reaction was established. After incubation of the sialyltransferase reaction, the sialyloligosaccharide obtained was treated by acid hydrolysis, and then the NeuAc that was released was labeled with 1,2-diamino-4,5-methylenedioxibenzene. The fluorescent-labeled NeuAc could be estimated by HPLC (excitation: 373 nm; emission: 448 nm) and a Lineweaver-Burk plot could be plotted with the data from this analysis.     

Glycan microarrays for screening sialyltransferase specificities

Here we demonstrate that glycan microarrays can be used for high-throughput acceptor specificity screening of various recombinant sialyltransferases. Cytidine-5'-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) was biotinylated at position 9 of N-acetylneuraminic acid (Neu5Ac) by chemoenzymatic synthesis generating CMP-9Biot-Neu5Ac. The activated sugar nucleotide was used as donor substrate for various mammalian sialyltranferases which transferred biotinylated sialic acids simultaneously onto glycan acceptors immobilized onto a microarray glass slide. Biotinylated glycans detected with fluorescein-streptavidin conjugate to generate a specificity profile for each enzyme both confirming previously known specificities and reveal additional specificity information. Human alpha2,6sialyltransferase-I (hST6Gal-I) also sialylates chitobiose structures (GlcNAcbeta1-4GlcNAc)(n) including N-glycans, rat alpha2,3sialyltransferase (rST3Gal-III) tolerates fucosylated acceptors such as Lewis(a), human alpha2,3sialyltransferase-IV (hST3Gal-IV) broadly sialylates oligosaccharides of types 1-4 and porcine alpha2,3sialyltransferase-I (pST3Gal-I) sialylates ganglio-oligosaccharides and core 2 O-glycans in our array system. Several of these sialyltransferases perform a substitution reaction and exchange a sialylated acceptor with a biotinylated sialic acid but are restricted to the most specific acceptor substrates. Thus, this method allows for a rapid generation of enzyme specificity information and can be used towards synthesis of new carbohydrate compounds and expand the glycan array compound library.     

Stress causes tissue-specific changes in the sialyltransferase activity

Numerous pathological conditions are associated with specific changes in glycosylation. Recent studies clearly demonstrated a link between stress and the development and course of many diseases. Biochemical mechanisms that link stress and diseases are still not fully understood, but there are some indications that changes in glycosylation are involved in this process. Influence of acute and chronic psychological stress on protein sialylation as well as the activity of sialyltransferases, enzymes that synthesize sialoglycoproteins, has been studied on Fischer rats. Liver, spleen, kidney, skeletal muscle, heart, adrenal gland, serum, cerebellum, hippocampus, medulla oblongata and cortex have been analyzed. Statistically significant tissue- and type of stress-specific changes in total sialyltransferase (ST) activity were observed. Acute stress resulted in 39% increase of ST activity in liver and spleen, while at the same time there was 43% decrease in ST activity in cerebellum. In chronic stress, ST activity increased in spleen (93%) and decreased in liver (17%), cerebellum (38%) and hippocampus (64%). Western-blot analysis using Maackia amurensis and Sambucus nigra lectins did not reveal any difference in protein sialylation. The results of serum corticosterone analysis indicate that showed increase in acute stress and decrease in chronic stress are in good accordance with the hypothesis that corticosterone has a role in the regulation of liver ST activity.     

Identification and characterization of flavonoids as sialyltransferase inhibitors

Sialyltransferases biosynthesize sialyl-glycoconjugates involved in many biological and pathological processes. We investigated and characterized synthetic flavonoid derivatives as sialyltransferase inhibitors. We first examined 54 compounds by solid-phase enzyme assay using β-galactoside α2,6-sialyltransferase 1 (ST6Gal I) and β-galactoside α2,3-sialyltransferase. Several compounds inhibited sialyltransferase enzyme activity regardless of sialyl-linkage reactions. Among them, two compounds showed inhibitory activity against ST6Gal I with IC₅₀ values less than 10 μM. Three characteristic features of flavonoids were determined by structure-inhibitory activity relationships. First, a double bond between C2-C3 linkages is required for the activity. Second, increasing hydrophilic properties on the B-ring markedly augmented the inhibitory effect. Third, a hydrophobic functional group introduced on the hydroxyl groups of the A-ring enhanced the inhibitory activity. Kinetic analysis using human ST6Gal I indicated a mixed inhibition mechanism of the compounds. In conclusion, the flavonoids identified could be applied for control of cellular expression of sialic acid.     

Sialylation of intestinal microvillar membranes in newborn, sucking and weaned pigs

Affinity cytochemistry and biochemistry revealed distinctivetemporal changes in the expression of sialylated and compositionallyrelated membrane glycoconjugates in the pig small intestinebetween birth and weaning. The expression of membrane NeuAc2,6moieties, recognized by Sambucus nigra agglutinin-1, was highin newborn pigs, declined slightly during sucking and was verylow in weaned animals. Conversely, the expression of membraneNeuAc2r3 moieties, recognized by Maackia amurensis agglutinin-2,was low at birth but higher in sucking and weaned animals. Histobloodgroup O- and A-antigen expression was first detected in a minorityof sucking pigs, but was evident in all weaned pigs examined.Lactase glycoforms were isolated from solubilized microvillarmembranes of newborn and weaned pigs. The newborn (predominantly2,6-sialylated) and weaned (predominantly 1,2-fucosylated) glycoformsexhibited similar specific activity, indicating that postnatallactase decline in the pig intestine is unrelated to temporalchanges in membrane sialylation and fucosylation. fucosylation lactase lectins intestine sialylation     

Sialylation enhances the secretion of neurotoxic amyloid-beta peptides

Alzheimer's disease (AD) is characterized by amyloid-beta peptide (Abeta) deposition in the brain. Abeta is produced by sequential cleavage of amyloid precursor protein (APP) by beta-secretase (BACE1: beta-site APP-cleaving enzyme 1) and gamma-secretase. Previously, we demonstrated that BACE1 also cleaves beta-galactoside alpha2,6-sialyltransferase (ST6Gal-I) and down-regulates its transferase activity. Here, we report that overexpression of ST6Gal-I in Neuro2a cells enhanced alpha2,6-sialylation of endogenous APP and increased the extracellular levels of its metabolites [Abeta by two-fold, soluble APPbeta (sAPPbeta) by three-fold and sAPPalpha by 2.5-fold). Sialylation-deficient mutant (Lec-2) cells secreted half as much Abeta as wild-type Chinese hamster ovary (CHO) cells. Furthermore, wild-type CHO cells showed enhanced secretion of the APP metabolites upon ST6Gal-I overexpression, whereas Lec-2 cells did not, indicating that the secretion enhancement requires sialylation of cellular protein(s). Secretion of metabolites from a mutant APP (APP-Asn467,496Ala) that lacked N-glycosylation sites was not enhanced upon ST6Gal-I overexpression, suggesting that the N-glycans on APP itself are required for the enhanced secretion. In the mouse brain, the amount of alpha2,6-sialylated APP appeared to be correlated with the sAPPbeta level. These results suggest that sialylation of APP promotes its metabolic turnover and could affect the pathology of AD.     

Sialylation is modulated through maturation in hemocytes from Macrobrachium rosenbergii

In this work we identified in adult and juvenile freshwater prawn, Macrobrachium rosenbergii, three major type of circulating hemocytes: fusiform; rounded; and large ovoid hemocytes. Rounded and large hemocytes represent the first defense line, since this type of cells exerts phagocytic activity as well as lectin synthesis. Considering that glycosylation plays important roles in cell communication and as a target for pathogenic microorganisms, in this report was also described the main glycosidic modifications that occur in the large and rounded hemocytes from the freshwater prawn during maturation as determined with lectins. Neu5Acalpha2,6Gal, was identified homogeneously distributed in the membrane in 90% of hemocytes from juvenile organisms. Maturation of the freshwater prawn induced a decrease or complete loss of Neu5Acalpha2,6Gal residues that were replaced with Neu5Acalpha2,3 molecules in practically all hemocytes from adult organisms. This change was paralleled by a diminution in 9-O-acetyl-neuraminic acid (Neu5,9Ac(2)) expression. T and Tn antigens (Galbetal,3 GalNAcalpha1-0-Ser/Thr or GalNAcalpha1-0-Ser/Thr, respectively), as well as N-glycosidically linked glycans, seem to be highly conserved throughout maturation. Our results show that sialylation of freshwater prawn hemocytes is modulated throughout the maturation process.