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:2.4.99.6 (sialyltransferase)
1,546 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Enzymatic carbohydrate synthesis using glycosyltransferases is highly regio- and stereospecific and does not require extensive protecting group designs. Naturally occurring carbohydrates have been prepared by this biomimetic pathway successfully. As more and more transferases are isolated and get cloned and overexpressed, non-natural substrates were probed with these biocatalysts. Key-polar groups and non-essential residues of the substrates have been determined. Consequently, this technique was employed to generate natural and non-natural carbohydrate libraries for pharmaceutical purposes. The synthesis of sialyl-Lewis(a)- and sialyl-Lewis(x) libraries and non-natural Linear-B derivatives applying glycosyltransferases is presented in this article. The respective transferases investigated are alpha(1-3)galactosyltransferase, beta(1-3)galactosyltransferase, beta(1-4)galactosyltransferase, alpha(2-3)sialyltransferase, alpha(1- 3)fucosyltransferase III and alpha(1-3)fucosyltransferase VI. With respect to the natural acceptors, the aglycon part and the N-acetyl group of the glucosamide have been varied. All enzymes tolerate an unexpected wide range of non-natural acceptors, which is not yet exploited in its full scale. In addition, fucosyltransferase III and VI can be employed to convert also non-natural donors with non-natural acceptors at the same time. Thus sialyl- Lewis(a)- and sialyl-Lewis(x)-libraries which differ in three positions compared to the natural tetrasaccharides are generated very efficiently. Also a library of linear-B trisaccharides, a reactive xenoantigen, has been prepared enzymatically. The aglycon part and the natural N-acetyl group of the glucosamine which is part of the acceptor substrate have been altered widely. This convenient methodology is compared with the evolving solid-phase carbohydrate synthesis using conventional chemistry. The potential use of transferases in solid-phase carbohydrate chemistry is discussed together with the possibility to use these biocatalysts to synthesize carbohydrate mimetics. The presented findings may also be useful to design potential glycosyltransferase inhibitors.
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PMID:Carbohydrates and derivatives as potential drug candidates with emphasis on the selectin and linear-B area. 1236 62

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

Aberrant glycosylation of membrane components due to specific alterations of glycosyltransferase activity is a common feature of carcinoma cells and is usually associated with invasion and metastasis. In a prospective study, the enzyme activity of the sialyltransferases ST6GAL-I and ST3GAL-III was studied in gastric cancer and normal mucosa in 55 patients by a radiometric assay. Cellular localization of sialyltransferase ST6GAL-I mRNA expression was studied by in situ hybridization. Sialyltransferase ST6GAL-I mRNA expression was mainly localized to epithelial cells. ST6GAL-I enzyme activity was enhanced within the tumor tissue. Significant correlations were found between the presence of signet ring cells and enhanced ST6GAL-I activity in the tumor tissue (p = 0.047) or in the mucosa (p = 0.024), and between signet ring cells and ST3GAL-III activity in the mucosa (p < 0.001). Multivariate Cox analysis demonstrated that only lymph node metastases (p = 0.044) had a significant influence on tumor-related survival. ST3GAL-III and ST6GAL-I activity showed no independent prognostic relevance in multivariate analysis, but high levels of ST3GAL-III and ST6GAL-I in the tumor tissue correlated with secondary local tumor recurrence (p = 0.005; p = 0.012). Interestingly, also the nonmalignant and uninvolved mucosa of tumor patients was altered on the molecular level and in some cases showed enhanced sialyltransferase levels indicative of the alteration of glycosylation very early during tumorigenesis.
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PMID:Clinical relevance of sialyltransferases ST6GAL-I and ST3GAL-III in gastric cancer. 1293 Oct 20

Solid-phase assays for measuring the activity of four different glycosyltransferase enzymes that utilize N-acetyllactosamine as an acceptor are reported. These enzymes are alpha1,3-galactosyltransferase (E.C. 2.4.1.151), alpha1,3-fucosyltransferase (E.C. 2.4.1.65), alpha2,6-(N)-sialyltransferase (E.C. 2.4.99.1), and alpha2,3-(N)-sialyltransferase (E.C. 2.4.99.5). The acceptor is immobilized on a cellulose membrane in two different ways, through either an amine-cleavable linker or a photolinker. Incubation with a glycosyltransferase and nucleotide donor sugar resulted in the transfer of a monosaccharide from the donor to immobilized N-acetyllactosamine. For galactosyltransferase, transfer was confirmed by mass spectrometry of the products cleaved from the membrane surface after amine treatment or irradiation. When radioactive donors were utilized, the transfer of radioactive sugars could be monitored by autoradiography. Alternatively the transfer of radioactive sugar onto the membranes could be measured by scintillation counting of the products after cleavage from the membrane. Cytidine 5(')-monophosphate-sialic acid carrying a fluorescent tag in the saccharide was also successfully utilized in this assay system. Fluorescent product on the membrane surface was detected by imaging. Glycosyltransferase assays with these versatile membranes have the potential to be adapted for high-throughput screening.
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PMID:Glycosyltransferase assays utilizing N-acetyllactosamine acceptor immobilized on a cellulose membrane. 1462 51

Reduction of pig cell-surface alpha-galactosyl (Gal) epitope, Galalpha1, 3Galbeta1, 4GlcNAc-R, by the introduction of glycosyltransferase genes is effective in suppressing hyperacute rejection (HAR) in pig-to-human xenotransplantation. The transmission of porcine endogenous retroviruses (PERVs) has been recognized as a potential risk factor associated with xenotransplantation. In this study, effects of the introduction of glycosyltransferase genes to pig cells on the sensitivity of gammaretroviruses to human serum were investigated. Pig endothelial cells (PEC), PEC transduced with alpha1,2 fucosyltransferase (FT), alpha2,3 sialyltransferase (ST), or N-acetylglucosaminyltransferase III (GnT-III), and human embryonic kidney (HEK) 293 cells were transduced with the LacZ gene with the packaging signal of murine leukemia virus (MuLV) under the control of the long terminal repeat of MuLV by a pseudotype infection. Then, the cells were further infected with PERV subtype B (PERV-B) or feline leukemia virus subgroup B (FeLV-B). Culture supernatants of the infected cells were mixed with human serum (HS) and then inoculated to HEK293 cells. The inoculated cells were histochemically stained and lacZ-positive blue foci were counted. Glycosyltransferase activity, xenoantigenicity, and alpha-Gal epitope density in the cells were measured at the time of the infection experiments. PERV-B or FeLV-B particles from the parental PEC were efficiently neutralized by HS, while those from PEC transduced with alpha1,2FT, alpha2,3ST or GnT-III were less sensitive to HS. The transduced PEC exhibited high levels of activity of the introduced glycotransferases, and expressed fewer xenoantigens and cell-surface alpha-Gal epitopes. Our results suggest that gammaretroviruses including PERVs produced by transgenic pigs, that are generally modified to reduce the cell-surface alpha-Gal epitope to overcome the HAR in xenotransplantation, are less sensitive to HS.
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PMID:Sensitivity to human serum of gammaretroviruses produced from pig endothelial cells transduced with glycosyltransferase genes. 1470 22

Polysialoglycoprotein (PSGP) is a major cortical alveolus glycoprotein of rainbow trout eggs that is characterized by the attachment of a polysialic acid structure to its O-glycan chains. It has been demonstrated that the polysialic acid structure is synthesized by at least three sialyltransferases mostly localized in cortical alveoli. Here we have cloned a cDNA encoding the sialyltransferase, designated rtST6GalNAc, responsible for the transfer of the first sialic acid residue onto the O-glycan chain of PSGP. This enzyme belongs to the vertebrate ST6GalNAc II family, and is strongly expressed in ovaries. Of those O-glycoproteins tested as substrates, asialo-PSGP is the best substrate. These results indicate that rtST6GalNAc is the enzyme responsible for the sialylation of PSGP during oogenesis. Furthermore, the rtST6GalNAc mRNA is expressed throughout oogenesis, is down-regulated at the late yolk vesicle stage (May), and then up-regulated during vitellogenesis (until August). This developmental profile is highly similar to that of STL2, a cortical alveolus lectin, while it is quite different from that of PSGP, which is extensively expressed at the yolk vesicle stage and down-regulated at later stages. Thus, not all cortical alveolus components are transcribed concomitantly. This is the first description of a developmental change in the transcription of a glycosyltransferase during oogenesis.
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PMID:Developmental expression of a sialyltransferase responsible for sialylation of cortical alveolus glycoprotein during oogenesis in rainbow trout (Oncorhynchus mykiss). 1549 90

Sialyltransferases catalyze reactions that transfer a sialic acid from CMP-sialic acid to an acceptor (a structure terminated with galactose, N-acetylgalactosamine, or sialic acid). They are key enzymes that catalyze the synthesis of sialic acid-containing oligosaccharides, polysaccharides, and glycoconjugates that play pivotal roles in many critical physiological and pathological processes. The structures of a truncated multifunctional Pasteurella multocida sialyltransferase (Delta24PmST1), in the absence and presence of CMP, have been determined by X-ray crystallography at 1.65 and 2.0 A resolutions, respectively. The Delta24PmST1 exists as a monomer in solution and in crystals. Different from the reported crystal structure of a bifunctional sialyltransferase CstII that has only one Rossmann domain, the overall structure of the Delta24PmST1 consists of two separate Rossmann nucleotide-binding domains. The Delta24PmST1 structure, thus, represents the first sialyltransferase structure that belongs to the glycosyltransferase-B (GT-B) structural group. Unlike all other known GT-B structures, however, there is no C-terminal extension that interacts with the N-terminal domain in the Delta24PmST1 structure. The CMP binding site is located in the deep cleft between the two Rossmann domains. Nevertheless, the CMP only forms interactions with residues in the C-terminal domain. The binding of CMP to the protein causes a large closure movement of the N-terminal Rossmann domain toward the C-terminal nucleotide-binding domain. Ser 143 of the N-terminal domain moves up to hydrogen-bond to Tyr 388 of the C-terminal domain. Both Ser 143 and Tyr 388 form hydrogen bonds to a water molecule, which in turn hydrogen-bonds to the terminal phosphate oxygen of CMP. These interactions may trigger the closure between the two domains. Additionally, a short helix near the active site seen in the apo structure becomes disordered upon binding to CMP. This helix may swing down upon binding to donor CMP-sialic acid to form the binding pocket for an acceptor.
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PMID:Cytidine 5'-monophosphate (CMP)-induced structural changes in a multifunctional sialyltransferase from Pasteurella multocida. 1647 3

Carbohydrate chains of cancer glycoprotein antigens contain major outer changes dictated by tissue-specific regulation of glycosyltransferase genes, the availability of sugar nucleotides, and competition between enzymes for acceptor intermediates during glycan elongation. However, it is evident from recent studies with recombinant mucin probes that the final glycosylation profiles of mucin glycoproteins are mainly determined by the cellular repertoire of glycosyltransferases. Hence, we examined various cancer cell lines for the levels of fucosyl-, beta-galactosyl, beta-N-acetylgalactosaminyl-, sialyl-, and sulfotransferase activities that generate the outer ends of the oligosaccharide chains. We have identified glycosyltransferases activities at the levels that would give rise to O-glycan chains as reported by others in breast cancer cell lines, T47D, ZR75-1, MCF-7, and MDA-MB-231. Most breast cancer cells express Gal-3-O-sulfotransferase specific for T-hapten Gal beta1-->3GalNAc alpha-, whereas the enzyme from colon cancer cells exhibits a vast preference for the Gal beta1,4GlcNAc terminal unit in O-glycans. We also studied ovarian cancer cells SW626 and PA-1 and hepatic cancer cells HepG2. Our studies show that alpha1,2-L-fucosyl-T, alpha(2,3) sialyl-T, and 3-O-Sulfo-T capable of acting on the mucin core 2 tetrasaccharide, Gal beta1,4GlcNAc beta1,6(Gal beta1,3)GalNAc alpha-, can also act on the Globo H antigen backbone, Gal beta1,3GalNAc beta1,3Gal alpha-, suggesting the existence of unique carbohydrate moieties in certain cancer-associated glycolipids. Briefly, our study indicates the following: (i) 3'-Sulfo-T-hapten has an apparent relationship to the tumorigenic potential of breast cancer cells; (ii) the 3'-sulfo Lewis(x), the 3-O-sulfo-Globo unit, and the 3-fucosylchitobiose core could be uniquely associated with colon cancer cells; (iii) synthesis of a polylactosamine chain and T-hapten are favorable in ovarian cancer cells due to negligible sialyltransferase activities; and (iv) a 6'-sialyl LacNAc unit and 3'-sialyl T-hapten appear to be prevalent structures in hepatic cancer cell glycans. Thus, it is apparent that different cancer cells are expressing unique glycan epitopes, which could be novel targets for cancer diagnosis and treatment.
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PMID:The pattern of glycosyl- and sulfotransferase activities in cancer cell lines: a predictor of individual cancer-associated distinct carbohydrate structures for the structural identification of signature glycans. 1654 47

A MUC1-related glycopeptide having five core-2 hexasaccharide branches (C330H527N46O207, MW = 8450.9) was synthesized by a new strategy using a combination of microwave-assisted solid-phase synthesis (MA-SPGS) and enzymatic sugar elongation. Synthesis of a key glycopeptide intermediate was best achieved in a combination of PEGA [poly(ethylene glycol)-poly-(N,N-dimethylacrylamide) copolymer] resin and MA-SPGS using glycosylated amino acid building blocks with high speed and high purity. Deprotection of the glycopeptide intermediate and subsequent glycosyltransferase-catalyzed sugar elongations were performed for generation of the additional diversities with the sugar moieties of glycopeptides using beta1,4-galactosyltransferase (beta1,4-GalT) and two kinds of alpha2,3-sialyltransferases [ST3Gal III; alpha2,3-(N)-SiaT and ST3Gal II; alpha2,3-(O)-SiaT]. These reactions proceeded successfully in the presence of 0.2% Triton X-100 to convert the chemically synthesized trisaccharide glycans to disialylated hexasaccharide.
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PMID:Construction of highly glycosylated mucin-type glycopeptides based on microwave-assisted solid-phase syntheses and enzymatic modifications. 1659 99

Ganglioside glycosyltransferases organize as multienzyme complexes that localize in different sub-Golgi compartments. Here we studied whether in CHO-K1 cells lacking CMP-NeuAc: GM3 sialyltransferase (SialT2), the sub-Golgi localization of UDP-Gal:glucosylceramide beta-1,4-galactosyltransferase (GalT1) and CMP-NeuAc:lactosylceramide sialyltransferase (SialT1) complex is affected when SialT2, another member of this complex, is coexpressed. GalT1 and SialT1 sub-Golgi localization was determined by studying the effect of brefeldin A (BFA) and monensin on the synthesis of glycolipids and on the sub-Golgi localization of GalT1(1-52)-CFP (cyan fluorescent protein) and SialT1(1-54)-YFP (yellow fluorescent protein) chimeras by single cell fluorescence microscopy and by isopycnic subfractionation. We found that BFA, and also monensin, impair the synthesis of glycolipids beyond GM3 ganglioside in wild type (WT) cells but beyond GlcCer in SialT2(+) cells. Although BFA redistributed GalT1-CFP and SialT1-YFP to the endoplasmic reticulum in WT cells, a fraction of these chimeras remained associated with a distal Golgi compartment, enriched in trans Golgi network, and recycling endosome markers in SialT2(+) cells. In BFA-treated cells, the percentage of GalT1-CFP and SialT1-YFP associated with Golgi-like membrane fractions separated by isopycnic subfractionation was higher in SialT2(+) cells than in WT cells. These effects were reverted by knocking down the expression of SialT2 with specific siRNA. Results indicate that sub-Golgi localization of glycosyltransferase complexes may change according to the relative levels of the expression of participating enzymes and reveal a capacity of the organelle to adapt the topology of the glycolipid synthesis machinery to functional states of the cell.
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PMID:Modulation of GalT1 and SialT1 sub-Golgi localization by SialT2 expression reveals an organellar level of glycolipid synthesis control. 1695 Jul 84


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