Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.4.99.10 (sialyltransferase)
 1,547 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Expression of recombinant full length human alpha 2,6(N)sialyltransferase has been scaled-up in S. cerevisiae in a 150-l bioreactor yielding 47 U at a concentration of 0.31 U/l. The protein specific activity as measured in reconstituted yeast lyophilisate was 0.8 mU/mg protein. The recombinant enzyme exhibited similar Michaelis constants as previously determined for the native rat enzyme. By immunoblotting the enzyme was shown to be heterogeneous by size (44-48 kD) and N-glycosylated. We conclude that recombinant alpha 2,6(N)sialyltransferase expressed in S. cerevisiae is retained in the endoplasmic reticulum as a fully active enzyme.
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PMID:Scaled-up expression of human alpha 2,6(N)sialyltransferase in Saccharomyces cerevisiae. 774 34

Biosynthesis and intracellular transport of recombinant human full-length beta 1,4 galactosyltransferase (GT) and full-length alpha 2,6 sialyltransferase (ST) were investigated in Saccharomyces cerevisiae. Recently, enzymic activity of recombinant GT (rGT) in crude homogenates of S. cerevisiae could successfully be demonstrated [Krezdorn, C., Watzele, G., Kleene, R. B., Ivanov, S. X. & Berger, E. G. (1993) Eur. J. Biochem. 212, 113-120]. In the present work, we show that, in yeast strains transformed with plasmid pDPSIA containing the cDNA coding for human ST, rST enzymic activity using asialo-fetuin or N-acetyllactosamine as acceptor substrates could readily be detected. Analysis by 1H-NMR spectroscopy of the disaccharide product of rGT, as recently reported, and the trisaccharide product of rST demonstrated that only the expected glycosidic linkages were formed. Following mechanical disruption of yeast cells, both enzymes sedimented with a fraction enriched in membranes of the endoplasmic reticulum (ER) and were activated by Triton X-100 3-5-fold. rGT and rST could be immunoprecipitated from their [35S]Met-labelled transformed yeast extracts using polyclonal antibodies raised against fusion proteins consisting of beta-galactosidase-GT or beta-galactosidase-ST, respectively, expressed in Escherichia coli. For rGT a single glycosylated form of apparent molecular mass 48 kDa was reported, but for rST two main bands corresponding to apparent molecular masses of 48 kDa and 44 kDa, respectively, were detected. Immunoprecipitation from either tunicamycin-treated [35S]Met-labelled transformed yeast cells or labelling with radio-active sugars both indicated that the 44-kDa form of rST was non-glycosylated and that the 48-kDa form of rST was core N-glycosylated. In addition, core glycosylation of both recombinant enzymes demonstrated that they were competent for translocation across the ER membranes. However, the 44-kDa form of rST was converted to the 48-kDa glycosylated form only slowly, suggesting a mechanism of posttranslational translocation. Absence of hyperglycosylation of rST and rGT in wild type and lack of the Golgi-specific man-alpha 1,6-man epitope suggest that the recombinant enzymes did not enter the yeast Golgi apparatus. These results indicated that both rGT and rST are retained as enzymically active enzymes in the ER of yeast and suggest a ribonucleoprotein-independent import of rST into the ER.
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PMID:Human beta 1,4 galactosyltransferase and alpha 2,6 sialyltransferase expressed in Saccharomyces cerevisiae are retained as active enzymes in the endoplasmic reticulum. 814 35

Target inactivation analysis was used to measure the functional size of uridine diphosphogalactose: N-acetylglucosamine beta(1,4)galactosyltransferase (galactosyltransferase), cytidine monophospho-N-acetyl-neuraminic acid: beta-galactoside alpha(2,6) sialytransferase (sialyltransferase), and uridine diphosphatase (UDPase) in Golgi membranes isolated from rat liver. The size of nucleoside diphosphatase (NDPase), an enzyme similar to UDPase but localized in rat liver endoplasmic reticulum, was also estimated by target inactivation analysis. The related enzymes, UDPase and NDPase, have target sizes of 96 +/- 4 and 77 +/- 3 kDa, while galactosyltransferase and sialyltransferase have target sizes of 97 +/- 10 and 130 +/- 20 kDa, respectively. The target inactivation sizes of galactosyltransferase and of sialyltransferase are about twice the monomer molecular weights of these enzymes obtained from sedimentation studies of the solubilized membranes as well as those predicted from previously reported cDNA sequences. We conclude from our studies that galactosyltransferase and sialyltransferase probably function as dimers in the Golgi membrane.
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PMID:Target sizes of galactosyltransferase, sialyltransferase, and uridine diphosphatase in Golgi apparatus of rat liver. 838 32

Gal beta-1,4-GlcNAc alpha 2,6-sialyltransferase (CMP-N-acetylneuraminate:beta-galactoside alpha 2,6 sialyltransferase, EC 2.4.99.1) is a glycoprotein containing carbohydrate chains of the complex type (Jamieson, J.C. (1989) Life Sci. 43, 691-697). The carbohydrate chains may be important for controlling the expression of sialyltransferase catalytic activity during transit of the enzyme from the rough endoplasmic reticulum to the Golgi complex where it is active as a membrane bound enzyme anchored to the luminal face. To study the role of the carbohydrate chains of sialyltransferase for enzyme activity, conditions were established in which the native enzyme was deglycosylated with N-Glycanase and endo F. It was found that Glycanase removed the carbohydrate chains from native sialyltransferase, but methanol or ethanol had to be present for rapid and complete deglycosylation. Presence of methanol or ethanol were not essential for removal of carbohydrate chains with endo F. There was a correlation between the loss of catalytic activity of sialyltransferase with increased deglycosylation. After deglycosylation with Glycanase for 18 h catalytic activity was largely eliminated and there was a reduction in molecular mass of about 5 kDa compared to the untreated enzyme when examined by immunoblot analysis; this reduction was identical to that found when the denatured enzyme was deglycosylated with Glycanase. At shorter times of incubation partially deglycosylated forms of the enzyme were detected. Complete deglycosylation of native or denatured sialyltransferase with endo F could not be achieved. However, incubation with endo F for 24 h resulted in a loss of catalytic activity of about 60%. Immunoblot analysis showed the presence of three forms of the enzyme corresponding in molecular mass to the native and deglycosylated enzyme and a third form corresponding to a partially deglycosylated enzyme. Sialyltransferase was also subjected to sequential treatment with exoglycosidases. Removal of NeuAc and Gal had little effect on catalytic activity, but subsequent removal of GlcNAc resulted in a significant loss in catalytic activity suggesting that the presence of the trimannose core with GlcNAc attached is important for the expression of catalytic activity. The presence of organic solvents during deglycosylation with Glycanase may be a useful method that can be applied to other glycoproteins.
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PMID:The role of the carbohydrate chains of Gal beta-1,4-GlcNAc alpha 2,6-sialyltransferase for enzyme activity. 839 96

Rat intoxication with a single dose of 1,2-dichloroethane (DCE) (50 microliters/100 g b.w) is able to induce a significant modification of protein glycosylation in the liver endoplasmic reticulum and Golgi apparatus. HPLC analysis shows that within 5-60 min after DCE-intoxication, the levels of total dolichol, free dolichol and dolichyl phosphate strongly decreased in the microsomes and Golgi apparatus. Particularly in total microsomes, dolichyl phosphate, which is rate-limiting for the biosynthesis of the N-linked oligosaccharide chains, drops to values significantly lower than in the control group 15 min after DCE poisoning. In the Golgi apparatus, the total dolichol, essential to enhance the fluidity and permeability of these membranes, early and significantly decreases already 5 min after DCE poisoning. Moreover, in the Golgi apparatus galactosyl- and sialyltransferase activities, the main enzymatic activities of terminal protein glycosylation, are significantly reduced, as measured 15 min after DCE intoxication. These data suggest that the impairment of glycoprotein synthesis, maturation and secretion may be involved in the pathogenesis of liver injury induced by acute DCE-intoxication.
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PMID:Effects of 1,2-dichloroethane intoxication on dolichol levels and glycosyltransferase activities in rat liver microsomes and Golgi apparatus. 856 May 3

Interactions between selectins and their oligosaccharide-decorated ligands play a crucial role in the initiation of leukocyte extravasation. We have shown that synthetic multivalent sialyl Lewis x glycans inhibit strongly the adhesion of lymphocytes to endothelium at sites of inflammation. However, enzyme-assisted synthesis of these oligosaccharides si hampered by the lack of sufficient amounts of specific glycosyltransferases. We report here the construction of Saccharomyces cerevisiae strains expressing the soluble catalytic ectodomain of rat Gal(beta)1-3/4GlcNac alpha 2,3-sialyltransferase (ST3Ne) fused to the C-terminus of the hsp150 delta-carrier polypeptide. The hsp150 delta-carrier, which is an N-terminal fragmented of a natural secretory protein of yeast, is able to confer secretion-competence to several heterologous proteins, which otherwise remain in the yeast endoplasmic reticulum. The ST3Ne portion of the hsp 150 delta-ST3Ne fusion protein adopted an enzymatically active conformation and was N-glycosylated and disulfide-bonded. Hsp150 delta-ST3Ne was secreted with a half-time of about 7.5 min and remained intercalated in the cell wall, which covers the yeast plasma membrane. About 110 mU of sialyltransferase per litre was produced in 16 h. Whole live yeast cells were able to transfer sialic acid from CMP-NeuNAc to N-acetyllactosamine yielding alpha 2,3-sialyl-N-acetyllactosamine, as evidenced by paper chromatography, cleavage by linkage-specific sialidase, and NMR analysis. Our data suggest that yeast cells externalizing mammalian glycosyltransferases with the aid of the hsp150 delta-carrier could provide a source of enzymes for synthesis of valuable oligosaccharides.
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PMID:Targeting of active rat alpha 2,3-sialyltransferase to the yeast cell wall by the aid of the hsp 150 delta-carrier: toward synthesis of sLe(x)-decorated L-selectin ligands. 902 48

We have addressed the question of whether or not Golgi fragmentation, as exemplified by that occurring during drug-induced microtubule depolymerization, is accompanied by the separation of Golgi subcompartments one from another. Scattering kinetics of Golgi subcompartments during microtubule disassembly and reassembly following reversible nocodazole exposure was inferred from multimarker analysis of protein distribution. Stably expressed alpha-2,6-sialyltransferase and N-acetylglucosaminyltransferase-I (NAGT-I), both C-terminally tagged with the myc epitope, provided markers for the trans-Golgi/trans-Golgi network (TGN) and medial-Golgi, respectively, in Vero cells. Using immunogold labeling, the chimeric proteins were polarized within the Golgi stack. Total cellular distributions of recombinant proteins were assessed by immunofluorescence (anti-myc monoclonal antibody) with respect to the endogenous protein, beta-1,4-galactosyltransferase (GalT, trans-Golgi/TGN, polyclonal antibody). ERGIC-53 served as a marker for the intermediate compartment). In HeLa cells, distribution of endogenous GalT was compared with transfected rat alpha-mannosidase II (medial-Golgi, polyclonal antibody). After a 1-h nocodazole treatment, Vero alpha-2,6-sialyltransferase and GalT were found in scattered cytoplasmic patches that increased in number over time. Initially these structures were often negative for NAGT-I, but over a two- to threefold slower time course, NAGT-I colocalized with alpha-2,6-sialyltransferase and GalT. Scattered Golgi elements were located in proximity to ERGIC-53-positive structures. Similar trans-first scattering kinetics was seen with the HeLa GalT/alpha-mannosidase II pairing. Following nocodazole removal, all cisternal markers accumulated at the same rate in a juxtanuclear Golgi. Accumulation of cisternal proteins in scattered Golgi elements was not blocked by microinjected GTPgammaS at a concentration sufficient to inhibit secretory processes. Redistribution of Golgi proteins from endoplasmic reticulum to scattered structures following brefeldin A removal in the presence of nocodazole was not blocked by GTPgammaS. We conclude that Golgi subcompartments can separate one from the other. We discuss how direct trafficking of Golgi proteins from the TGN/trans-Golgi to endoplasmic reticulum may explain the observed trans-first scattering of Golgi transferases in response to microtubule depolymerization.
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PMID:Scattered Golgi elements during microtubule disruption are initially enriched in trans-Golgi proteins. 943

Sialylation is a biosynthetic process occurring in the trans compartments of the Golgi apparatus. Corresponding evidence is based on localization and biochemical studies of alpha2, 6(N)-sialyltransferase (ST6Gal I) as previously reported. Here we describe generation and characterization of polyclonal antibodies to recombinant rat alpha2,3(N)-sialyltransferase (ST3Gal III) expressed as a soluble enzyme in Sf9 cells or as a beta-galactosidase-human-ST3Gal III fusion-protein from E.coli , respectively. These antibodies were used to localize ST3Gal III by immunofluorescence in various cell lines and rat kidney tissue sections. In transiently transfected COS cells the antibodies directed to soluble sialyltransferase or the sialyltransferase portion of the fusion-protein only recognized the recombinant antigen retained in the endoplasmic reticulum. However, an antibody fraction crossreactive with beta-galactosidase recognized natively expressed ST3Gal III which was found to be colocalized with beta1, 4-galactosyltransferase in the Golgi apparatus of several cultured cell lines. Antibodies affinity purified on the beta-galactosidase-ST3Gal III fusion-protein column derived from both antisera have then been used to localize the enzyme in perfusion-fixed rat kidney sections. We found strong staining of the Golgi apparatus of tubular epithelia and a brush-border-associated staining which colocalized with cytochemical staining of the H+ATPase. This subcellular localization was not observed for ST6Gal I which localized to the Golgi apparatus. These data show colocalization in the Golgi apparatus and different post-Golgi distributions of the two sialyltransferases.
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PMID:Immunocytochemical localization of alpha2,3(N)-sialyltransferase (ST3Gal III) in cell lines and rat kidney tissue sections: evidence for golgi and post-golgi localization. 945 Oct 34

The varicella-zoster virus (VZV) is the etiological agent of two different human pathologies, chickenpox (varicella) and shingles (zoster). This alphaherpesvirus is believed to acquire its lipidic envelope in the trans-Golgi network (TGN). This is consistent with previous data showing that the most abundant VZV envelope glycoprotein gE accumulates at steady-state in this organelle when expressed from cloned cDNA. In the present study, we have investigated the intracellular trafficking of gI, another VZV envelope glycoprotein. In transfected cells, this protein shows a very slow biosynthetic transport to the cell surface where it accumulates. However, upon co-expression of gE, gI experiences a dramatic increase in its exit rate from the endoplasmic reticulum, it accumulates in a sialyltransferase-positive compartment, presumably the TGN, and cycles between this compartment and the cell surface. This differential behavior results from the ability of gE and gI to form a complex in the early stages of the biosynthetic pathway whose intracellular traffic is exclusively determined by the sorting information in the tail of gE. Thus, gI provides the first example of a molecule localized to the TGN by means of its association with another TGN protein. We also show that, during the early stages of VZV infection, both proteins are also found in the TGN of the host cell. This suggests the existence of an intermediate stage during VZV biogenesis in which the envelope glycoproteins, transiently arrested in the TGN, could promote the envelopment of newly synthesized nucleocapsids into this compartment and, therefore, the assembly of infective viruses.
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PMID:Intracellular transport of the glycoproteins gE and gI of the varicella-zoster virus. gE accelerates the maturation of gI and determines its accumulation in the trans-Golgi network. 959 75

In this study we compare intracellular transport and processing of a recombinant glycoprotein in mammalian and insect cells. Detailed analysis of the N-glycosylation of recombinant human IFN-gamma by matrix-assisted laser-desorption mass spectrometry showed that the protein secreted by Chinese hamster ovary and baculovirus-infected insect Sf9 cells was associated with complex sialylated or truncated tri-mannosyl core glycans, respectively. However, the intracellular proteins were predominantly associated with high-mannose type oligosaccharides (Man-6 to Man-9) in both cases, indicating that endoplasmic reticulum to cis-Golgi transport is a predominant rate-limiting step in both expression systems. In CHO cells, although there was a minor intracellular subpopulation of sialylated IFN-gamma glycoforms identical to the secreted product (therefore associated with late-Golgi compartments or secretory vesicles), no other intermediates were evident. Therefore, anterograde transport processes in the Golgi stack do not limit secretion. In Sf9 insect cells, there was no direct evidence of post-ER glycan-processing events other than core fucosylation and de-mannosylation, both of which were glycosylation site-specific. To investigate the influence of nucleotide-sugar availability on cell-specific glycosylation, the cellular content of nucleotide-sugar substrates in both mammalian and insect cells was quantitatively determined by anion-exchange HPLC. In both host cell types, UDP-hexose and UDP-N-acetylhexosamine were in greater abundance relative to other substrates. However, unlike CHO cells, sialyltransferase activity and CMP-NeuAc substrate were not present in uninfected or baculovirus-infected Sf9 cells. Similar data were obtained for other insect cell hosts, Sf21 and Ea4. We conclude that although the limitations on intracellular transport and secretion of recombinant proteins in mammalian and insect cells are similar, N-glycan processing in Sf insect cells is limited, and that genetic modification of N-glycan processing in these insect cell lines will be constrained by substrate availability to terminal galactosylation.
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PMID:Constraints on the transport and glycosylation of recombinant IFN-gamma in Chinese hamster ovary and insect cells. 1039 12


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