[show abstract][hide abstract] ABSTRACT: trans-Sialidases constitute a special group of the sialidase family. They occur in some trypanosome species and, in a unique reversible reaction, transfer sialic acids from one glycosidic linkage with galactose (donor) to another galactose (acceptor), to form (α2-3)-sialyl linkages. Trypanosomes cause such devastating human diseases as Chagas disease in South America (Trypanosoma cruzi) or sleeping sickness in Africa (Trypanosoma brucei). The trans-sialidases strongly contribute to the pathogenicity of the trypanosomes by scavenging sialic acids from the host or blood meal to coat the parasite surface; this aids their survival strategy in the insect's intestine, and in the blood circulation or cells of the host, and serves to compromise the immune system of the human or animal host. American and African trypanosomes express trans-sialidases at different stages of their vector/host development. They are transmitted to humans by insect vectors (tsetse fly or other insect "bug" species). trans-Sialidase activity with varying linkage specificity has also been found in a few bacteria species and in human serum. trans-Sialidases are of increasing practical importance for the chemo-enzymatic synthesis of sialylated glycans. The search for appropriate inhibitors of trans-sialidases and vaccination strategies is intensifying, as less toxic medicaments for the treatment of these widespread and often chronic tropical diseases are required.
[show abstract][hide abstract] ABSTRACT: Enhanced expression of 9-O-acetylated sialoglycoproteins (Neu5,9Ac(2)-GPs) and 9-O-acetylated disialoganglioside (9-OAcGD3) was observed on lymphoblasts of childhood acute lymphoblastic leukemia (ALL). Sialate-O-acetyltransferase (SOAT) and sialate-O-acetylesterase (SIAE) are the two main enzymes responsible for the quantity of the O-acetyl ester groups on sialic acids (Sias). We have earlier shown an enhanced level of SOAT activity, capable of transferring acetyl groups to Sias of glycoconjugates in the microsomes of lymphoblasts of these children. We further observed a decreased SIAE activity in both lysosomal and cytosolic fractions of ALL cell lines and primary cells from bone marrow of patients compared with peripheral blood mononuclear cells from healthy donors, which preferentially hydrolyze O-acetyl groups at C-9 of Sia. The level of O-acetylated Sias in the cytosolic and the lysosomal fractions showed a good correlation with SIAE activity in the corresponding fractions. The apparent K(M) values for SIAE in the lysosomal and the cytosolic fractions from lymphoblasts of ALL patients are 0.38 and 0.39 mM, respectively. These studies demonstrate that both SIAE and SOAT activities seem to be responsible for the enhanced level of Neu5,9Ac(2) in lymphoblasts, which is a hallmark in ALL. This was subsequently confirmed by using an enzyme-linked immunosorbent assay that also demonstrated a steady decline in SOAT activities even in cell lysates of lymphoblasts during successful chemotherapy, like radioactive methods have shown earlier.
[show abstract][hide abstract] ABSTRACT: Sialic acids are important sugars at the reducing end of glycoproteins and glycolipids. They are among many other functions involved in cell-cell interactions, host-pathogen recognition and the regulation of serum half-life of glycoproteins. An important modification of sialic acids is O-acetylation, which can alter or mask the biological properties of the parent sialic acid molecule. The nature of mammalian sialate-O-acetyltransferases (EC 126.96.36.199) involved in their biosynthesis is still unknown. We have identified the human CasD1 (capsule structure1 domain containing 1) gene as a candidate to encode the elusive enzyme. The human CasD1 gene encodes a protein with a serine-glycine-asparagine-histidine hydrolase domain and a hydrophobic transmembrane domain. Expression of the Cas1 protein tagged with enhanced green fluorescent protein in mammalian and insect cells directed the protein to the medial and trans-cisternae of the Golgi. Overexpression of the Cas1 protein in combination with α-N-acetyl-neuraminide α-2,8-sialyltransferase 1 (GD3 synthase) resulted in an up to 40% increased biosynthesis of 7-O-acetylated ganglioside GD3. By quantitative real-time polymerase chain reaction, we found up to 5-fold increase in CasD1 mRNA in tumor cells overexpressing O-Ac-GD3. CasD1-specific small interfering RNA reduced O-acetylation in tumor cells. These results suggest that the human Cas1 protein is directly involved in O-acetylation of α2-8-linked sialic acids.
[show abstract][hide abstract] ABSTRACT: GD3 (CD60a) and its 9-O-acetylated variant (CD60b) are intracellular regulators of apoptosis in T lymphocytes. Surface expressed 9-O-acetyl- and 7-O-acetyl-GD3 (CD60b and CD60c) may have a functional impact on activated T and B cells. In order to investigate the balance between surface and intracellular expression and synthesis and degradation of these glycosphingolipids in human lymphocytes of various differentiation stages, we analyzed (i) expression of GD3 molecules on native T and B cells and thymocytes by flow cytometry and (ii) activity and regulation of possible key enzymes for CD60a,b,c synthesis and degradation at the transcriptional level. Both, surface and cytoplasmic expression of CD60a and CD60c was highest in tonsillar T cells. In thymocytes, CD60c outweighs the other CD60 variants and was mainly found in the cytoplasm. All lymphocyte preparations contained sialate O-acetyltransferase activity producing 7-O-acetyl-GD3. Sialidase activity was highest in peripheral blood lymphocytes followed by thymocytes and tonsillar T and B cells. Transcription of GD3 synthase (ST8SiaI), the key enzyme for GD3 synthesis, was highest in tonsillar T cells, whereas transcriptional levels of sialidase NEU3 and O-acetylesterase H-Lse were lowest in activated T cells. This balance between enzymes of sialic acid metabolism may explain the strong overall staining intensity for all GD3 forms in T cells. Both CASD1, presumably encoding a sialic acid-specific O-acetyltransferase, and H-Lse showed highest transcription in peripheral B lymphocytes corresponding to the low expression of CD60b and c in these cells. Our data point to regulatory functions of these anabolic and catabolic key enzymes for the expression of GD3 and its O-acetylated variants in lymphocytes at a given differentiation stage.
[show abstract][hide abstract] ABSTRACT: The Publisher regrets that this article is an accidental duplication of an article that has already been published, doi: 10.1016/j.carres.2010.01.003. The duplicate article has therefore been withdrawn.
Carbohydrate research 02/2010; · 2.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: The wide occurrence of sialic acids (Sia) in various chemical forms linked as monomers or polymers in an outstanding position in a multitude of complex carbohydrates of animals and microorganisms renders them as most versatile function modulators in cell biology and pathology. A survey is presented of recent advances in the study of the influences that Sias have as bulky hydrophilic and electronegatively charged monosaccharides on animal cells and on their interaction with microorganisms. Some highlights are: sialylation leads to increased anti-inflammatory activity of IgG antibodies, facilitates the escape of microorganisms from the host's immune system, and in polymeric form is involved in the regulation of embryogenesis and neuronal growth and function. The role of siglecs in immunoregulation, the dynamics of lymphocyte binding to selectins and the interactions of toxins, viruses, and other microorganisms with the host's Sia are now better understood. N-Glycolylneuraminic acid from food is antigenic in man and seems to have pathogenic potential. Sia O-acetylation mediated by various eukaryotic and prokaryotic O-acetyltransferases modulates the affinity of these monosaccharides to mammalian and microbial receptors and hinders apoptosis. The functionally versatile O-acetylated ganglioside GD3 is an onco-fetal antigen.
Current Opinion in Structural Biology 09/2009; 19(5):507-14. · 8.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: The affinity to sialic acid-containing oligosaccharides of the small-animal lectin SHL-I isolated from the venom of the Chinese bird-hunting spider Selenocosmia huwena is here described for the first time. By a strategic combination of NMR techniques, molecular modeling, and data mining tools it was possible to identify the crucial amino acid residues that are responsible for SHL-I's ability to bind sialic acid residues in a specific way. Furthermore, we are able to discuss the role of the functional groups of sialic acid when bound to SHL-I. Also the impact of Pro31 in its cis- or trans-form on SHL-I's ligand affinity is of special interest, since it answers the question if Trp32 is a crucial amino acid for stabilizing complexes between SHL-I and sialic acid. SHL-I can be considered as a proper model system that provides further insights into the binding mechanisms of small-animal lectins to sialic acid on a sub-molecular level.
Carbohydrate research 07/2009; 344(12):1515-25. · 2.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: The sialic acids of the platypus, birds, and reptiles were investigated with regard to the occurrence of N-glycolylneuraminic (Neu5Gc) acid. They were released from tissues, eggs, or salivary mucin samples by acid hydrolysis, and purified and analyzed by thin-layer chromatography, high-performance liquid chromatography, and mass spectrometry. In muscle and liver of the platypus only N-acetylneuraminic (Neu5Ac) acid was found. The nine bird species studied also did not express N-glycolylneuraminic acid with the exception of an egg, but not tissues, from the budgerigar and traces in poultry. Among nine reptiles, including one turtle, N-glycolylneuraminic acid was only found in the egg and an adult basilisk, but not in a freshly hatched animal. BLAST analysis of the genomes of the platypus, the chicken, and zebra finch against the CMP-N-acetylneuraminic acid hydroxylase did not reveal the existence of a similar protein structure. Apparently monotremes (platypus) and sauropsids (birds and reptiles) cannot synthesize Neu5Gc. The few animals where Neu5Gc was found, especially in eggs, may have acquired this from the diet or by an alternative pathway. Since Neu5Gc is antigenic to man, the observation that this monosaccharide does not or at least only rarely occur in birds and reptiles, may be of nutritional and clinical significance.
Carbohydrate research 06/2009; 344(12):1494-500. · 2.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: Zusammenfassung. Sialidase (EC 188.8.131.52) ist ein Pathogenitätsfaktor vieler Mikroorganismen, und könnte auch bei der Anheftung der Hautpilze an die Epithelien ihrer Wirte eine Rolle spielen, indem sie die terminalen, negativ geladenen Sialinsäuren von Glykokonjugaten der Zelloberflächen hydrolytisch abspaltet, und subterminale Zucker für eine Bindung durch Pilzlektine freilegt. Daher wurden 116 Stämme von sieben Species der Dermatophyten auf Sialidasebildung untersucht. Zwei empfindliche, quantitative Sialidase-Tests wurden auf Zellhomogenate und Kulturüberstän-de aus sieben verschiedenen Nährlösungen dieser Pilze angewandt, ergaben aber stets negative Ergebnisse für Sialidase. Dagegen war Sialidase für Ophiostoma stenoceras als Positivkontrolle unter gleichen Bedingungen stets nachweisbar, wobei sich das Enzym durch sialylierte Muzine als induzierbar erwies. Ein Pathomechanismus über Sialidase ist für Dermatophyten nach den vorliegenden Ergebnissen wenig wahrscheinlich.Summary. Sialidase (EC 184.108.40.206) is a pathogenicity factor of many microorganisms, and may also play a role in adhesion of dermatophytes to the epithelia of their hosts by the hydrolytical cleavage of terminal, negatively charged sialic acids of glycoconjugates on the cell surfaces, thus allowing fungal lectins to bind to the subterminal sugars. Therefore, 116 strains of seven species of dermatophytes were investigated for sialidase production. Two highly sensitive, quantitative sialidase assays were applied to cell homogenates and culture supernatants from seven different media of the fungi, but were always negative for sialidase activity. However, sialidase activity was always detected in Ophiostoma stenoceras used as a positive control cultivated in parallel; the enzyme was inducable by sialylated mucins. A sialidase-dependent pathomechanism for dermatophytes appears unlikely based on the results presented.
[show abstract][hide abstract] ABSTRACT: Previous studies had established an over-expression of 9-O-acetylated sialoglycoproteins (Neu5,9Ac(2)-GPs) on lymphoblasts of childhood acute lymphoblastic leukaemia (ALL). Here, we report the discovery and characterization of sialate-O-acetyltransferase enzyme in ALL-cell lines and lymphoblasts from bone marrow of children diagnosed with B- and T-ALL. We observed a positive correlation between the enhanced sialate-O-acetyltransferase activity and the enhanced expression of Neu5,9Ac(2)-GPs in these lymphoblasts. Sialate-O-acetyltransferase activity in cell lysates or microsomal fractions of lymphoblasts of patients was always higher than that in healthy donors reaching up to 22-fold in microsomes. Additionally, the V (max) of this enzymatic reaction with AcCoA was over threefold higher in microsomal fractions of lymphoblasts. The enzyme bound to the microsomal fractions showed high activity with CMP-N-acetylneuraminic acid, ganglioside GD3 and endogenous sialic acid as substrates. N-acetyl-7-O-acetylneuraminic acid was the main reaction product, as detected by radio-thin-layer chromatography and fluorimetrically coupled radio-high-performance liquid chromatography. CMP and coenzyme A inhibited the microsomal enzyme. Sialate-O-acetyltransferase activity increased at the diagnosis of leukaemia, decreased with clinical remission and sharply increased again in relapsed patients as determined by radiometric-assay. A newly-developed non-radioactive ELISA can quickly detect sialate-O-acetyltransferase, and thus, may become a suitable tool for ALL-monitoring in larger scale. This is the first report on sialate-O-acetyltransferase in ALL being one of the few descriptions of an enzyme of this type in human.
[show abstract][hide abstract] ABSTRACT: The O-acetylation of sialic acids is one of the most frequent modifications of these monosaccharides and modulates many cell biological and pathological events. Sialic acid-specific O-acetyltransferases and O-acetylesterases are responsible for the metabolism of esterified sialic acids. Assays were developed for the analysis of the activities and specificities of these enzymes. The methods had to be varied in dependence on the substrate assayed, the kind of biological source, and the state of enzyme purity. With the new techniques the primary site of O-acetyl incorporation at C-7, catalyzed by the animal sialate-O-acetyltransferases studied, was ascertained. Correspondingly, this enzyme, for example from bovine submandibular gland, can be denominated as AcCoA:sialate-7-O-acetyltransferase (EC 220.127.116.11). Methods for assaying the activity of esterases de-O-acetylating sialic acids and their metabolic cooperation with the O-acetyltransferases are presented.
[show abstract][hide abstract] ABSTRACT: Sialate-O-acetylesterase was purified almost 900-fold from particle-free supernatants of horse liver by gel filtration, ion-exchange chromatography and isoelectric focussing. The native enzyme on gel filtration exhibits a molecular weight of 54,000 Da. It was separated by isoelectric focussing into two forms with pI values of 4.8 and 5.7, respectively. The esterase with a lower pI hydrolyses only 9-O-acetyl groups from sialic acids (K(M) 1.1 mM), while that with the higher pI esterifies both 4- and 9-O-acetylated monosaccharides at similar rates (K(M) 0.3 M and 1.3 mM, respectively). Both forms are inactive with 7-O-acetylated N-acetylneuraminic acid. Enzyme assays were carried out at the pH optimum (pH 8.4-8.6) using free O-acetylated sialic acids followed by direct analysis of the reaction products by isocratic anion-exchange HPLC. Glycosidically bound sialic acids can also be de-O-acetylated. Horse liver esterase seems to be an essential enzyme for the catabolism of 4-O-acetylated sialoglycoconjugates, since sialidase from this tissue cannot act on 4-O-acetylated sialic acids.
[show abstract][hide abstract] ABSTRACT: The O-acetylation of sialic acids in various positions is a frequent modification of these residues in glycoproteins and glycolipids of higher animals and some bacteria. Sialic acid O-acetylation is involved in the regulation of many cell biological and pathophysiological events. Since the properties and the structural and molecular genetic aspects of the eukaryotic sialate O-acetyltransferases are not yet known, we attempted to isolate the enzyme from bovine submandibular glands. O-Acetyltransferase was solubilised from its microsomal location with a zwitterionic detergent and enriched by approximately 50-fold in three steps, including affinity chromatography on coenzyme A. It exhibits a molecular mass of 150-160 kDa. Evidence was obtained for the putative existence of a low-molecular-mass, dialysable enzyme activator. The enzyme showed best activity with CMP-N-acetylneuraminic acid (CMP-Neu5Ac), followed by N-acetylneuraminic acid (Neu5Ac). These compounds, as well as AcCoA, have high affinity for both the microsome-bound and the partially purified O-acetyltransferase. CoA is a strong inhibitor. N-Acetyl-9-O-acetylneuraminic acid was found to be the main reaction product. No evidence was obtained for the involvement of an isomerase that might be responsible for the migration of O-acetyl groups within the sialic acid side chain.
[show abstract][hide abstract] ABSTRACT: Infections by mouse hepatitis viruses result in disease of the liver, the gastrointestinal tract, respiratory tract, and the central nervous system. Coronaviruses related to mouse hepatitis virus express a hemagglutinin-esterase surface glycoprotein, which specifically hydrolyses either 5-N-acetyl-4-O-acetyl neuraminic acid (Neu4,5Ac(2)) or 5-N-acetyl-9-O-acetyl neuraminic acid (Neu5,9Ac(2)). Moreover, these sialic acids represent potential cellular receptor determinants for murine coronaviruses. Until now, the distribution of these sialic acids in mouse brain was not thoroughly investigated. Particularly Neu4,5Ac(2) was not yet found in mouse brain. Using a sensitive method of gas chromatography coupled to mass spectrometry in the electron impact mode of ionization this manuscript demonstrates the occurrence of 13 different sialic acids varying in their alkyl and acyl substituents in mouse tissues including 5-N-acetyl-4-O-acetyl-9-O-lactyl-neuraminic acid (Neu4,5Ac(2)9Lt), 5-N-acetyl-9-O-lactyl-neuraminic acid (Neu5Ac9Lt), 5-N-acetyl-8-O-methyl-neuraminic acid (Neu5Ac8Me) and the 1,7-lactone (Neu5Ac1,7L) of neuraminic acid. Neu4,5Ac(2), relatively abundant in the gut, was present as a minor compound in all tissues, including liver, olfactory lobe, telencephalon, metencephalon and hippocampus. Neu5,9Ac(2) was also found in these tissues, except in the liver. It is suggested that these sialic acids represent the endogenous substrate and receptor determinants for murine coronaviruses.
[show abstract][hide abstract] ABSTRACT: A previous study (Bergwerff et al., Biochimie 74 (1992) 25-37) reported that sialic acids present in Asterias rubens gonads were essentially composed of 8-methyl-N-glycolylneuraminic acid (Neu5Gc8Me), a large part of it being acetylated in position 9. Using GC/MS of heptafluorobutyrate derivatives (Zanetta et al., Glycobiology 11 (2001) 663-676) on the chloroform/methanol soluble and insoluble fractions, we showed that most sialic acids were found in the latter and demonstrated that all sialic acids were derived from N-glycolylneuraminic acid, most of them being 8-methylated, but that the majority were also acetylated in position 4 or 7 (or both positions). GC/MS analyses of the constituents liberated using acid-catalysed methanolysis verified that major glycoprotein-bound glycans were N-linked and of the gluco-oligomannosidic type. Major fatty acids were poly-unsaturated (especially C20:4) and long-chain bases were C22:1 phytosphingosine and C22:2 6-hydroxysphingenine. Major monosaccharides found in the chloroform/methanol extract (quinovose and fucose) were derived from steroidal saponins.
[show abstract][hide abstract] ABSTRACT: Trans-sialidase (TS; E.C. 18.104.22.168) catalyzes the transfer of preferably alpha2,3-linked sialic acid to another glycan or glycoconjugate, forming a new alpha2,3-linkage to galactose or N-acetylgalactosamine. In the absence of an appropriate acceptor, TS acts as a sialidase, hydrolytically releasing glycosidically linked sialic acid. Interest in TS has increased rapidly in recent years owing to its great relevance to the pathogenicity of trypanosomes and its possible application in the regiospecific synthesis of sialylated carbohydrates and glycoconjugates. Recently, the authors described a newly developed nonradioactive screening test for monitoring TS activity (1). In this highly sensitive and specific assay, 4-methylumbelliferyl-beta-D-galactoside is used as acceptor substrate and sialyllactose as donor to fluorimetrically detect enzyme activity in the low mU range (approximately 0.1-1 mU/mL possible). The test can be applied to screen a large number of samples quickly and reliably during enzyme purification, for testing inhibitors, and for monitoring TS activity during the production of monoclonal antibodies (2). This chapter focuses on the main steps of this assay and gives detailed instructions for performing a nonradioactive TS 96-well-plate fluorescence test. In addition, it describes the controls necessary when starting to monitor an unknown TS and facts to be considered when testing new substrates and inhibitors.
Methods in molecular biology (Clifton, N.J.) 02/2006; 347:93-107.