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Jeffrey D Esko - One of the best experts on this subject based on the ideXlab platform.
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functional udp xylose transport across the endoplasmic reticulum golgi membrane in a chinese hamster ovary Cell Mutant defective in udp xylose synthase
Journal of Biological Chemistry, 2009Co-Authors: Hans Bakker, Xiaomei Bai, Takuji Oka, Angel Ashikov, Ajit Yadav, Monika Berger, Nadia A Rana, Yoshifumi Jigami, Robert S Haltiwanger, Jeffrey D EskoAbstract:Abstract In mammals, xylose is found as the first sugar residue of the tetrasaccharide GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser, initiating the formation of the glycosaminoglycans heparin/heparan sulfate and chondroitin/dermatan sulfate. It is also found in the trisaccharide Xylα1-3Xylα1-3Glcβ1-O-Ser on epidermal growth factor repeats of proteins, such as Notch. UDP-xylose synthase (UXS), which catalyzes the formation of the UDP-xylose substrate for the different xylosyltransferases through decarboxylation of UDP-glucuronic acid, resides in the endoplasmic reticulum and/or Golgi lumen. Since xylosylation takes place in these organelles, no obvious requirement exists for membrane transport of UDP-xylose. However, UDP-xylose transport across isolated Golgi membranes has been documented, and we recently succeeded with the cloning of a human UDP-xylose transporter (SLC25B4). Here we provide new evidence for a functional role of UDP-xylose transport by characterization of a new Chinese hamster ovary Cell Mutant, designated pgsI-208, that lacks UXS activity. The Mutant fails to initiate glycosaminoglycan synthesis and is not capable of xylosylating Notch. Complementation was achieved by expression of a cytoplasmic variant of UXS, which proves the existence of a functional Golgi UDP-xylose transporter. A ∼200 fold increase of UDP-glucuronic acid occurred in pgsI-208 Cells, demonstrating a lack of UDP-xylose-mediated control of the cytoplasmically localized UDP-glucose dehydrogenase in the Mutant. The data presented in this study suggest the bidirectional transport of UDP-xylose across endoplasmic reticulum/Golgi membranes and its role in controlling homeostasis of UDP-glucuronic acid and UDP-xylose production.
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enzyme interactions in heparan sulfate biosynthesis uronosyl 5 epimerase and 2 o sulfotransferase interact in vivo
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Maria A S Pinhal, Koji Kimata, Brian J Smith, Sara K Olson, Junichi Aikawa, Jeffrey D EskoAbstract:The formation of heparan sulfate occurs within the lumen of the endoplasmic reticulum–Golgi complex–trans-Golgi network by the concerted action of several glycosyltransferases, an epimerase, and multiple sulfotransferases. In this report, we have examined the location and interaction of tagged forms of five of the biosynthetic enzymes: galactosyltransferase I and glucuronosyltransferase I, required for the formation of the linkage region, and GlcNAc N-deacetylase/N-sulfotransferase 1, uronosyl 5-epimerase, and uronosyl 2-O-sulfotransferase, the first three enzymes involved in the modification of the chains. All of the enzymes colocalized with the medial-Golgi marker α-mannosidase II. To study whether any of these enzymes interacted with each other, they were relocated to the endoplasmic reticulum (ER) by replacing their cytoplasmic N-terminal tails with an ER retention signal derived from the cytoplasmic domain of human invariant chain (p33). Relocating either galactosyltransferase I or glucuronosyltransferase I had no effect on the other's location or activity. However, relocating the epimerase to the ER caused a parallel redistribution of the 2-O-sulfotransferase. Transfected epimerase was also located in the ER in a Cell Mutant lacking the 2-O-sulfotransferase, but moved to the Golgi when the Cells were transfected with 2-O-sulfotransferase cDNA. Epimerase activity was depressed in the Mutant, but increased upon restoration of 2-O-sulfotransferase, suggesting that their physical association was required for both epimerase stability and translocation to the Golgi. These findings provide in vivo evidence for the formation of complexes among enzymes involved in heparan sulfate biosynthesis. The functional significance of these complexes may relate to the rapidity of heparan sulfate formation.
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enhanced 3 o sulfation of galactose in asn linked glycans and maackia amurenesis lectin binding in a new chinese hamster ovary Cell line
Glycobiology, 2001Co-Authors: Xiaomei Bai, Jillian R Brown, Ajit Varki, Jeffrey D EskoAbstract:We report the characterization of two Chinese hamster ovary Cell lines that produce large amounts of sulfated N-linked oligosaccharides. Clones 26 and 489 were derived by stable transfection of the glycosaminoglycan-deficient Cell Mutant pgsA-745 with a cDNA library prepared from wild-type Cells. Peptide:N-glycanase F released nearly all of the sulfate label, indicating that sulfation had occurred selectively on the Asn-linked glycans. Hydrazinolysis followed by nitrous acid treatment at pH 4 and borohydride reduction yielded reduced sulfated disaccharides that comigrated with standard Gal3SO4beta1-4anhydromannitol. The disaccharides were resistant to periodate oxidation but became sensitive after the sulfate group was removed by methanolysis, indicating that the sulfate was located at C3 of the galactose residues. Maackia amurensis lectin bound to the sulfated glycopeptides on the Cell surface and in free form, even after sialidase treatment. This finding indicates that the lectin requires only a charged group at C3 of the galactose unit and not an intact sialic acid. Growth of Cells with chlorate restored sialidase sensitivity to lectin binding, indicating that sulfation and sialylation occurred largely at the same sites. The enhanced sulfation was due to elevated sulfotransferase activity that catalyzed transfer of sulfate from phosphoadenosine-5'-phosphosulfate to Galbeta1-4(3)GlcNAcbeta-O-naphthalenemethanol.
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an animal Cell Mutant defective in heparan sulfate hexuronic acid 2 o sulfation
Journal of Biological Chemistry, 1996Co-Authors: Xiaomei Bai, Jeffrey D EskoAbstract:Abstract The interaction of heparan sulfate with protein ligands depends on unique oligosaccharide sequences containing iduronic acid (IdUA), N-sulfated glucosamine residues, and O-sulfated sugars. To study the role of O-sulfation in greater detail, we isolated a Chinese hamster ovary Cell Mutant defective in 2-O-sulfation of iduronic acid. The Mutant, pgsF-17, was identified by a colony blotting assay in which colonies of mutagen-treated Cells were replica plated to two disks of polyester cloth. One disk was blotted with 125I-labeled basic fibroblast growth factor (bFGF) to measure binding to Cell surface proteoglycans. The other disk was incubated with 35SO4 to measure proteoglycan biosynthesis. Autoradiography revealed a colony that did not bind 125I-bFGF, but incorporated 35SO4 normally (Mutant pgsF-17). Complete deaminative cleavage of heparan sulfate revealed that material from pgsF-17 lacked IdUA(2OSO3)-GlcNSO3 and IdUA(2OSO3)-GlcNSO3(6OSO3), but contained a higher proportion of glucuronic acid GlcUA-GlcNSO3(6OSO3) and IdUA-GlcNSO3(6OSO3). Assay of the 2-O-sulfotransferase that acts on IdUA residues showed that Mutant 17 lacked enzyme activity. Interestingly, the alteration resulted in accumulation of GlcNSO3 groups, suggesting that under normal conditions 2-O-sulfation decreases GlcNAc N-deacetylation/N-sulfation, and that the reactions occur simultaneously. The formation of IdUA and 6-O-sulfated glucosaminyl residues appears to be independent of 2-O-sulfation. pgsF-17 also lacks 2-O-sulfated GlcUA residues, suggesting that the same enzyme is responsible for 2-O-sulfation of IdUA and GlcUA residues. Mutant 17 provides a useful tool for studying the regulation of heparan sulfate biosynthesis and the relationship of heparan sulfate fine structure to its biological function.
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a single mutation affects both n acetylglucosaminyltransferase and glucuronosyltransferase activities in a chinese hamster ovary Cell Mutant defective in heparan sulfate biosynthesis
Proceedings of the National Academy of Sciences of the United States of America, 1992Co-Authors: Kerstin Lidholt, Fulgentius N. Lugemwa, Julie L Weinke, Cheryl S Kiser, Karen J Bame, Sela Cheifetz, Joan Massague, Ulf Lindahl, Jeffrey D EskoAbstract:Abstract Mutants of Chinese hamster ovary Cells have been found that no longer produce heparan sulfate. Characterization of one of the Mutants, pgsD-677, showed that it lacks both N-acetylglucosaminyl- and glucuronosyltransferase, enzymes required for the polymerization of heparan sulfate chains. pgsD-677 also accumulates 3- to 4-fold more chondroitin sulfate than the wild type. Cell hybrids derived from pgsD-677 and wild type regained both transferase activities and the capacity to synthesize heparan sulfate. Two segregants from one of the hybrids reexpressed the dual enzyme deficiency, the lack of heparan sulfate synthesis, and the enhanced accumulation of chondroitin sulfate, suggesting that all of the traits were genetically linked. These findings indicate that the pgsD locus may represent a gene involved in the coordinate control of glycosaminoglycan formation.
Kentaro Hanada - One of the best experts on this subject based on the ideXlab platform.
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specificity of inhibitors of serine palmitoyltransferase spt a key enzyme in sphingolipid biosynthesis in intact Cells a novel evaluation system using an spt defective mammalian Cell Mutant
Biochemical Pharmacology, 2000Co-Authors: Kentaro Hanada, Masahiro Nishijima, Tetsuro Fujita, Shū KobayashiAbstract:Abstract In the present study, we demonstrate a model Cell system for evaluating the specificity of inhibitors of serine palmitoyltransferase (SPT), the enzyme that catalyzes the first step of sphingolipid biosynthesis. The LY-B strain is a Chinese hamster ovary (CHO) Cell Mutant defective in SPT, and the LY-B/cLCB1 strain is a genetically corrected revertant of the Mutant. Although LY-B Cells grew only slightly in sphingolipid-deficient medium, their growth was restored to the level of LY-B/cLCB1 Cells under sphingosine-supplied conditions, indicating that, in CHO Cells, the growth inhibition caused by SPT inactivation was rescued almost fully by the metabolic complementation of sphingolipids. Cultivation of LY-B/cLCB1 Cells in sphingolipid-deficient medium in the presence of 10 μM sphingofungin B and ISP-1 (myriocin, thermozymocidin), potent inhibitors of SPT activity, caused severe growth inhibition with ∼95% inhibition of de novo sphingolipid synthesis. The growth inhibition by sphingofungin B and ISP-1 was rescued substantially by exogenous sphingosine, whereas the cytotoxicity of two other types of SPT inhibitor, l- cycloserine and β-chloro- l- alanine, was hardly rescued. Similar cytotoxic patterns of these inhibitors also were observed on the growth of SPT-defective LY-B Cells cultured under sphingosine-supplied conditions. The SPT inhibitors did not affect metabolic conversion of exogenous [ 3 H]sphingosine to complex sphingolipids. Thus, the cytotoxicity of sphingofungin B and ISP-1, but not l -cycloserine or β-chloro- l -alanine, is due largely to inhibition of sphingolipid synthesis by inhibiting the SPT activity.
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purification of the serine palmitoyltransferase complex responsible for sphingoid base synthesis by using affinity peptide chromatography techniques
Journal of Biological Chemistry, 2000Co-Authors: Kentaro Hanada, Tomoko Hara, Masahiro NishijimaAbstract:Abstract Serine palmitoyltransferase (SPT), a membrane-bound enzyme of the endoplasmic reticulum, catalyzes the condensation of palmitoyl coenzyme A (CoA) andl-serine to produce 3-ketodihydrosphingosine. This enzyme contains at least two different subunits, named the LCB1 and LCB2 proteins. In the present study, we expressed a FLAG- and His6 peptide-tagged version of the hamster LCB1 protein in a Chinese hamster ovary Cell Mutant strain lacking the endogenous LCB1 subunit and purified SPT from the Cells near to homogeneity by affinity peptide chromatography. The endogenous LCB2 protein was co-purified with the tagged LCB1 protein in purification of SPT. In various aspects, including optimum pH, acyl-CoA specificity, and sphingofungin sensitivity, the activity of purified SPT was consistent with the activity detected in lysates of wild-type Chinese hamster ovary Cells. The optimum concentration of palmitoyl-CoA for 3-ketodihydrosphingosine formation by purified SPT was ∼25 μm, and the apparentK m of l-serine was 0.28 mm. Competition analysis of the SPT reaction with various serine analogs showed that all of the amino, carboxyl, and hydroxyl groups ofl-serine were responsible for the substrate recognition of the enzyme. SDS-polyacrylamide gel electrophoretic analysis of purified SPT, together with immunoprecipitation analysis of metabolically labeled LCB proteins, strongly suggested that the SPT enzyme consisted of the LCB1 and LCB2 proteins with a stoichiometry of 1:1.
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genetic evidence for atp dependent endoplasmic reticulum to golgi apparatus trafficking of ceramide for sphingomyelin synthesis in chinese hamster ovary Cells
Journal of Cell Biology, 1999Co-Authors: Masayoshi Fukasawa, Masahiro Nishijima, Kentaro HanadaAbstract:LY-A strain is a Chinese hamster ovary Cell Mutant resistant to sphingomyelin (SM)-directed cytolysin and has a defect in de novo SM synthesis. Metabolic labeling experiments with radioactive serine, sphingosine, and choline showed that LY-A Cells were defective in synthesis of SM from these precursors, but not syntheses of ceramide (Cer), glycosphingolipids, or phosphatidylcholine, indicating a specific defect in the conversion of Cer to SM in LY-A Cells. In vitro experiments showed that the specific defect of SM formation in LY-A Cells was not due to alterations in enzymatic activities responsible for SM synthesis or degradation. When Cells were treated with brefeldin A, which causes fusion of the Golgi apparatus with the endoplasmic reticulum (ER), de novo SM synthesis in LY-A Cells was restored to the wild-type level. Pulse–chase experiments with a fluorescent Cer analogue, N -(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza- s -indacene-3-pentanoyl)-d- erythro -sphingosine (C 5 -DMB-Cer), revealed that in wild-type Cells C 5 -DMB-Cer was redistributed from intraCellular membranes to the Golgi apparatus in an intraCellular ATP-dependent manner, and that LY-A Cells were defective in the energy-dependent redistribution of C 5 -DMB-Cer. Under ATP-depleted conditions, conversion of C 5 -DMB-Cer to C 5 -DMB-SM and of [ 3 H]sphingosine to [ 3 H]SM in wild-type Cells decreased to the levels in LY-A Cells, which were not affected by ATP depletion. ER-to-Golgi apparatus trafficking of glycosylphosphatidylinositol-anchored or membrane-spanning proteins in LY-A Cells appeared to be normal. These results indicate that the predominant pathway of ER-to-Golgi apparatus trafficking of Cer for de novo SM synthesis is ATP dependent and that this pathway is almost completely impaired in LY-A Cells. In addition, the specific defect of SM synthesis in LY-A Cells suggests different pathways of Cer transport for glycosphingolipids versus SM synthesis.
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sphingolipids are essential for the growth of chinese hamster ovary Cells restoration of the growth of a Mutant defective in sphingoid base biosynthesis by exogenous sphingolipids
Journal of Biological Chemistry, 1992Co-Authors: Kentaro Hanada, Masahiro Nishijima, M Kiso, A Hasegawa, S Fujita, T Ogawa, Yuzuru AkamatsuAbstract:Abstract We previously isolated a temperature-sensitive Chinese hamster ovary Cell Mutant (strain SPB-1) with thermolabile serine palmitoyltransferase, which is involved in the first step of sphingolipid synthesis (Hanada, K., Nishijima, M., and Akamatsu, Y. (1990) J. Biol. Chem. 265, 22137-22142). In this study, sphingolipid-deficient culture medium was used to examine the effect of exogenous sphingolipids on the Cell growth of SPB-1. When cultivated in the sphingolipid-deficient medium, SPB-1 Cells ceased growing at non-permissive temperatures. Under these conditions, de novo sphingolipid synthesis ceased in the SPB-1 Cells, resulting in a decrease in levels of sphingomyelin and ganglioside sialyl lactosylceramide (GM3), whereas the parental CHO-K1 Cells grew logarithmically with normal sphingolipid synthesis. Exogenous sphingosine restored the contents of both sphingomyelin and GM3 in the SPB-1 Cells near to the parental levels through metabolic utilization and allowed the Mutant Cells to grow even at the non-permissive temperature. Similarly, exogenous sphingomyelin restored the sphingomyelin levels and only partly the GM3 levels and also suppressed the temperature-sensitivity of the SPB-1 Cell growth. In contrast, exogenous glucosylceramide, which restored the GM3 levels but not the sphingomyelin levels, failed to suppress the temperature sensitivity of the SPB-1 Cell growth. Combination of exogenous sphingomyelin with ceramide, glucosylceramide, GM3, or sphingoid bases did not show any synergistic or additive effect on the SPB-1 Cell growth enhancement, compared with sphingomyelin alone. The results indicated that the temperature sensitivity of the SPB-1 Cell growth was due to the lack of Cellular sphingolipids, possibly that of sphingomyelin.
Monty Krieger - One of the best experts on this subject based on the ideXlab platform.
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a single point mutation in cop results in temperature sensitive lethal defects in membrane transport in a chinese hamster ovary Cell Mutant
Journal of Biological Chemistry, 1996Co-Authors: Marsha Penman, Bernardo L Trigatti, Monty KriegerAbstract:Abstract At the nonpermissive temperature of 39.5°C, the Chinese hamster ovary Cell conditionally lethal, temperature-sensitive (ts) Mutant ldlF exhibits the following defects: rapid degradation of low density lipoprotein receptors, disruption of ER-through-Golgi transport, and disintegration of the Golgi apparatus. All of these are corrected by transfection with an expression vector for wild-type -COP, a subunit of coatomers (Guo, Q., Vasile, E., and Krieger, M.(1994) J. Cell Biol. 125, 1213-1224). We now report the identification in ldlF Cells of a point mutation in the -COP gene, Glu to Lys, which prevents the corresponding cDNA from correcting the defects in transfected ldlF Cells and the immunochemical analysis of the synthesis, structure, and stability of -COP. At the permissive temperature (34°C), the steady state level of ts--COP in ldlF Cells was about half that of -COP in wild-type Chinese hamster ovary Cells and the isoelectric point of ts--COP was 0.14 pH units higher than that of the wild-type protein. The stability but not the biosynthesis of ts--COP was temperature-sensitive (t > 6 h at 34°C and 1-2 h at 39.5°C), and this accounts for the virtual absence of detectable ts--COP protein in ldlF Cells after incubation at 39.5°C for >6 h. The steady state levels in ldlF Cells of another coatomer subunit, β-COP, and the peripheral Golgi protein ldlCp were not temperature-sensitive. Thus, a mutation in -COP that causes instability at 39.5°C is responsible for all of the temperature-sensitive defects in ldlF Cells, and the stability of β-COP is not linked directly to that of -COP. ldlF Cells should be useful for the future analysis of the structure and function of -COP, the assembly of COPs into coatomers, and the participation of coatomers in intraCellular membrane transport.
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a single point mutation in cop results in temperature sensitive lethal defects in membrane transport in a chinese hamster ovary Cell Mutant
Journal of Biological Chemistry, 1996Co-Authors: Qiu Guo, Marsha Penman, Bernardo L Trigatti, Monty KriegerAbstract:At the nonpermissive temperature of 39.5 degrees C, the Chinese hamster ovary Cell conditionally lethal, temperature-sensitive (ts) Mutant ldlF exhibits the following defects: rapid degradation of low density lipoprotein receptors, disruption of ER-through Golgi transport, and disintegration of the Golgi apparatus. All of these are corrected by transfection with an expression vector for wild-type epsilon-COP, a subunit of coatomers (Guo, Q., Vasile, E., and Krieger, M. (1994) J. Cell Biol. 125, 1213-1224). We now report the identification in ldlF Cells of a point mutation in the epsilon-COP gene, Glu251 to Lys251, which prevents the corresponding cDNA from correcting the defects in transfected ldlF Cells and the immunochemical analysis of the synthesis, structure, and stability of epsilon-COP. At the permissive temperature (34 degrees C), the steady state level of ts-epsilon-COP in ldlF Cells was about half that of epsilon-COP in wild-type Chinese hamster ovary Cells and the isoelectric point of ts-epsilon-COP was 0.14 pH units higher than that of the wild-type protein. The stability but not the biosynthesis of ts-epsilon-COP was temperature-sensitive (t1/2 > 6 h at 34 degrees C and approximately 1-2 h at 39.5 degrees C), and this accounts for the virtual absence of detectable ts-epsilon-COP protein in ldlF Cells after incubation at 39.5 degrees C for > 6h. The steady state levels in ldlF Cells of another coatomer subunit, beta-COP, and the peripheral Golgi protein ldlCp were not temperature-sensitive. Thus, a mutation in epsilon-COP that causes instability at 39.5 degrees C is responsible for all of the temperature-sensitive defects in ldlF Cells, and the stability of beta-COP is not linked directly to that of epsilon-COP. ldlF Cells should be useful for the future analysis of the structure and function of epsilon-COP, the assembly of COPs into coatomers, and the participation of coatomers in intraCellular membrane transport.
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disruptions in golgi structure and membrane traffic in a conditional lethal mammalian Cell Mutant are corrected by epsilon cop
Journal of Cell Biology, 1994Co-Authors: Eliza Vasile, Monty KriegerAbstract:The CHO Cell temperature-sensitive Mutant ldlF exhibits two defects in membrane traffic at the nonpermissive temperature (39.5 degrees C): rapid degradation of LDL receptors, possibly caused by endocytic missorting, and disruption of ER-through-Golgi transport. Here, we show that at 39.5 degrees C, the Golgi in ldlF Cells dissociated into vesicles and tubules. This dissociation was inhibited by AlF4-, suggesting trimeric G proteins are involved in the dissociation mechanism. This resembled the effects of brefeldin A on wild-type Cells. We isolated a hamster cDNA that specifically corrected the ts defects of ldlF Cells, but not those of other similar ts Mutants (ldlE, ldlG, ldlH, and End4). Its predicted protein sequence is conserved in humans, rice, Arabidopsis, and Caenorhabditis elegans, and is virtually identical to that of bovine epsilon-COP, a component of the coatomer complex implicated in membrane transport. This provides the first genetic evidence that coatomers in animal Cells can play a role both in maintaining Golgi structure and in mediating ER-through-Golgi transport, and can influence normal endocytic recycling of LDL receptors. Thus, along with biochemical and yeast genetics methods, mammalian somatic Cell Mutants can provide powerful tools for the elucidation of the mechanisms underlying intraCellular membrane traffic.
Xiaomei Bai - One of the best experts on this subject based on the ideXlab platform.
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functional udp xylose transport across the endoplasmic reticulum golgi membrane in a chinese hamster ovary Cell Mutant defective in udp xylose synthase
Journal of Biological Chemistry, 2009Co-Authors: Hans Bakker, Xiaomei Bai, Takuji Oka, Angel Ashikov, Ajit Yadav, Monika Berger, Nadia A Rana, Yoshifumi Jigami, Robert S Haltiwanger, Jeffrey D EskoAbstract:Abstract In mammals, xylose is found as the first sugar residue of the tetrasaccharide GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser, initiating the formation of the glycosaminoglycans heparin/heparan sulfate and chondroitin/dermatan sulfate. It is also found in the trisaccharide Xylα1-3Xylα1-3Glcβ1-O-Ser on epidermal growth factor repeats of proteins, such as Notch. UDP-xylose synthase (UXS), which catalyzes the formation of the UDP-xylose substrate for the different xylosyltransferases through decarboxylation of UDP-glucuronic acid, resides in the endoplasmic reticulum and/or Golgi lumen. Since xylosylation takes place in these organelles, no obvious requirement exists for membrane transport of UDP-xylose. However, UDP-xylose transport across isolated Golgi membranes has been documented, and we recently succeeded with the cloning of a human UDP-xylose transporter (SLC25B4). Here we provide new evidence for a functional role of UDP-xylose transport by characterization of a new Chinese hamster ovary Cell Mutant, designated pgsI-208, that lacks UXS activity. The Mutant fails to initiate glycosaminoglycan synthesis and is not capable of xylosylating Notch. Complementation was achieved by expression of a cytoplasmic variant of UXS, which proves the existence of a functional Golgi UDP-xylose transporter. A ∼200 fold increase of UDP-glucuronic acid occurred in pgsI-208 Cells, demonstrating a lack of UDP-xylose-mediated control of the cytoplasmically localized UDP-glucose dehydrogenase in the Mutant. The data presented in this study suggest the bidirectional transport of UDP-xylose across endoplasmic reticulum/Golgi membranes and its role in controlling homeostasis of UDP-glucuronic acid and UDP-xylose production.
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temperature sensitive glycosaminoglycan biosynthesis in a chinese hamster ovary Cell Mutant containing a point mutation in glucuronyltransferase i
Journal of Biological Chemistry, 2004Co-Authors: Ge Wei, Xiaomei Bai, Jeffrey D EskoAbstract:Abstract In previous studies, we reported the isolation and characterization of a Chinese hamster ovary Cell Mutant (pgsG) defective in glucuronyltransferase I (GlcATI). This enzyme adds the terminal GlcA residue in the core protein-linkage tetrasaccharide (GlcAβ1,3Galβ1,3Galβ1, 4Xylβ-O-) on which glycosaminoglycan assembly occurs (Bai, X. M., Wei, G., Sinha, A., and Esko, J. D. (1999) J. Biol. Chem. 274, 13017–13024; Wei, G., Bai, X. M., Sarkar, A. K., and Esko, J. D. (1999) J. Biol. Chem. 274, 7857–7864). Here we show that incorporation of 35SO4 into glycosaminoglycans in the Mutant is temperature-sensitive, with greater synthesis occurring at 33 °C compared with 37 °C. Wild-type Cells show the opposite thermal dependence. Rabbit antiserum to hamster GlcATI failed to detect cross-reactive material in pgsG Cells by immunofluorescence and Western blotting. Furthermore, expression of chimeric proteins composed of Mutant GlcATI fused to IgG binding domain of protein A or to green fluorescent protein did not yield the proteins at the expected mass. The green fluorescent protein-tagged version appeared as a truncated protein, and immunofluorescence showed large perinuclear bodies at 30 °C. At 37 °C, the fusion protein was not readily detectable. Sequencing cDNAs from Mutant and wild-type Cells revealed a single base transition (G331A) in the open reading frame in pgsG Cells, which resulted in a Val-111 → Met substitution. These data suggest that pgsG Cells contain a labile form of GlcATI that causes conditional expression of glycosaminoglycans dependent on temperature.
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enhanced 3 o sulfation of galactose in asn linked glycans and maackia amurenesis lectin binding in a new chinese hamster ovary Cell line
Glycobiology, 2001Co-Authors: Xiaomei Bai, Jillian R Brown, Ajit Varki, Jeffrey D EskoAbstract:We report the characterization of two Chinese hamster ovary Cell lines that produce large amounts of sulfated N-linked oligosaccharides. Clones 26 and 489 were derived by stable transfection of the glycosaminoglycan-deficient Cell Mutant pgsA-745 with a cDNA library prepared from wild-type Cells. Peptide:N-glycanase F released nearly all of the sulfate label, indicating that sulfation had occurred selectively on the Asn-linked glycans. Hydrazinolysis followed by nitrous acid treatment at pH 4 and borohydride reduction yielded reduced sulfated disaccharides that comigrated with standard Gal3SO4beta1-4anhydromannitol. The disaccharides were resistant to periodate oxidation but became sensitive after the sulfate group was removed by methanolysis, indicating that the sulfate was located at C3 of the galactose residues. Maackia amurensis lectin bound to the sulfated glycopeptides on the Cell surface and in free form, even after sialidase treatment. This finding indicates that the lectin requires only a charged group at C3 of the galactose unit and not an intact sialic acid. Growth of Cells with chlorate restored sialidase sensitivity to lectin binding, indicating that sulfation and sialylation occurred largely at the same sites. The enhanced sulfation was due to elevated sulfotransferase activity that catalyzed transfer of sulfate from phosphoadenosine-5'-phosphosulfate to Galbeta1-4(3)GlcNAcbeta-O-naphthalenemethanol.
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an animal Cell Mutant defective in heparan sulfate hexuronic acid 2 o sulfation
Journal of Biological Chemistry, 1996Co-Authors: Xiaomei Bai, Jeffrey D EskoAbstract:Abstract The interaction of heparan sulfate with protein ligands depends on unique oligosaccharide sequences containing iduronic acid (IdUA), N-sulfated glucosamine residues, and O-sulfated sugars. To study the role of O-sulfation in greater detail, we isolated a Chinese hamster ovary Cell Mutant defective in 2-O-sulfation of iduronic acid. The Mutant, pgsF-17, was identified by a colony blotting assay in which colonies of mutagen-treated Cells were replica plated to two disks of polyester cloth. One disk was blotted with 125I-labeled basic fibroblast growth factor (bFGF) to measure binding to Cell surface proteoglycans. The other disk was incubated with 35SO4 to measure proteoglycan biosynthesis. Autoradiography revealed a colony that did not bind 125I-bFGF, but incorporated 35SO4 normally (Mutant pgsF-17). Complete deaminative cleavage of heparan sulfate revealed that material from pgsF-17 lacked IdUA(2OSO3)-GlcNSO3 and IdUA(2OSO3)-GlcNSO3(6OSO3), but contained a higher proportion of glucuronic acid GlcUA-GlcNSO3(6OSO3) and IdUA-GlcNSO3(6OSO3). Assay of the 2-O-sulfotransferase that acts on IdUA residues showed that Mutant 17 lacked enzyme activity. Interestingly, the alteration resulted in accumulation of GlcNSO3 groups, suggesting that under normal conditions 2-O-sulfation decreases GlcNAc N-deacetylation/N-sulfation, and that the reactions occur simultaneously. The formation of IdUA and 6-O-sulfated glucosaminyl residues appears to be independent of 2-O-sulfation. pgsF-17 also lacks 2-O-sulfated GlcUA residues, suggesting that the same enzyme is responsible for 2-O-sulfation of IdUA and GlcUA residues. Mutant 17 provides a useful tool for studying the regulation of heparan sulfate biosynthesis and the relationship of heparan sulfate fine structure to its biological function.
Masahiro Nishijima - One of the best experts on this subject based on the ideXlab platform.
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specificity of inhibitors of serine palmitoyltransferase spt a key enzyme in sphingolipid biosynthesis in intact Cells a novel evaluation system using an spt defective mammalian Cell Mutant
Biochemical Pharmacology, 2000Co-Authors: Kentaro Hanada, Masahiro Nishijima, Tetsuro Fujita, Shū KobayashiAbstract:Abstract In the present study, we demonstrate a model Cell system for evaluating the specificity of inhibitors of serine palmitoyltransferase (SPT), the enzyme that catalyzes the first step of sphingolipid biosynthesis. The LY-B strain is a Chinese hamster ovary (CHO) Cell Mutant defective in SPT, and the LY-B/cLCB1 strain is a genetically corrected revertant of the Mutant. Although LY-B Cells grew only slightly in sphingolipid-deficient medium, their growth was restored to the level of LY-B/cLCB1 Cells under sphingosine-supplied conditions, indicating that, in CHO Cells, the growth inhibition caused by SPT inactivation was rescued almost fully by the metabolic complementation of sphingolipids. Cultivation of LY-B/cLCB1 Cells in sphingolipid-deficient medium in the presence of 10 μM sphingofungin B and ISP-1 (myriocin, thermozymocidin), potent inhibitors of SPT activity, caused severe growth inhibition with ∼95% inhibition of de novo sphingolipid synthesis. The growth inhibition by sphingofungin B and ISP-1 was rescued substantially by exogenous sphingosine, whereas the cytotoxicity of two other types of SPT inhibitor, l- cycloserine and β-chloro- l- alanine, was hardly rescued. Similar cytotoxic patterns of these inhibitors also were observed on the growth of SPT-defective LY-B Cells cultured under sphingosine-supplied conditions. The SPT inhibitors did not affect metabolic conversion of exogenous [ 3 H]sphingosine to complex sphingolipids. Thus, the cytotoxicity of sphingofungin B and ISP-1, but not l -cycloserine or β-chloro- l -alanine, is due largely to inhibition of sphingolipid synthesis by inhibiting the SPT activity.
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purification of the serine palmitoyltransferase complex responsible for sphingoid base synthesis by using affinity peptide chromatography techniques
Journal of Biological Chemistry, 2000Co-Authors: Kentaro Hanada, Tomoko Hara, Masahiro NishijimaAbstract:Abstract Serine palmitoyltransferase (SPT), a membrane-bound enzyme of the endoplasmic reticulum, catalyzes the condensation of palmitoyl coenzyme A (CoA) andl-serine to produce 3-ketodihydrosphingosine. This enzyme contains at least two different subunits, named the LCB1 and LCB2 proteins. In the present study, we expressed a FLAG- and His6 peptide-tagged version of the hamster LCB1 protein in a Chinese hamster ovary Cell Mutant strain lacking the endogenous LCB1 subunit and purified SPT from the Cells near to homogeneity by affinity peptide chromatography. The endogenous LCB2 protein was co-purified with the tagged LCB1 protein in purification of SPT. In various aspects, including optimum pH, acyl-CoA specificity, and sphingofungin sensitivity, the activity of purified SPT was consistent with the activity detected in lysates of wild-type Chinese hamster ovary Cells. The optimum concentration of palmitoyl-CoA for 3-ketodihydrosphingosine formation by purified SPT was ∼25 μm, and the apparentK m of l-serine was 0.28 mm. Competition analysis of the SPT reaction with various serine analogs showed that all of the amino, carboxyl, and hydroxyl groups ofl-serine were responsible for the substrate recognition of the enzyme. SDS-polyacrylamide gel electrophoretic analysis of purified SPT, together with immunoprecipitation analysis of metabolically labeled LCB proteins, strongly suggested that the SPT enzyme consisted of the LCB1 and LCB2 proteins with a stoichiometry of 1:1.
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genetic evidence for atp dependent endoplasmic reticulum to golgi apparatus trafficking of ceramide for sphingomyelin synthesis in chinese hamster ovary Cells
Journal of Cell Biology, 1999Co-Authors: Masayoshi Fukasawa, Masahiro Nishijima, Kentaro HanadaAbstract:LY-A strain is a Chinese hamster ovary Cell Mutant resistant to sphingomyelin (SM)-directed cytolysin and has a defect in de novo SM synthesis. Metabolic labeling experiments with radioactive serine, sphingosine, and choline showed that LY-A Cells were defective in synthesis of SM from these precursors, but not syntheses of ceramide (Cer), glycosphingolipids, or phosphatidylcholine, indicating a specific defect in the conversion of Cer to SM in LY-A Cells. In vitro experiments showed that the specific defect of SM formation in LY-A Cells was not due to alterations in enzymatic activities responsible for SM synthesis or degradation. When Cells were treated with brefeldin A, which causes fusion of the Golgi apparatus with the endoplasmic reticulum (ER), de novo SM synthesis in LY-A Cells was restored to the wild-type level. Pulse–chase experiments with a fluorescent Cer analogue, N -(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza- s -indacene-3-pentanoyl)-d- erythro -sphingosine (C 5 -DMB-Cer), revealed that in wild-type Cells C 5 -DMB-Cer was redistributed from intraCellular membranes to the Golgi apparatus in an intraCellular ATP-dependent manner, and that LY-A Cells were defective in the energy-dependent redistribution of C 5 -DMB-Cer. Under ATP-depleted conditions, conversion of C 5 -DMB-Cer to C 5 -DMB-SM and of [ 3 H]sphingosine to [ 3 H]SM in wild-type Cells decreased to the levels in LY-A Cells, which were not affected by ATP depletion. ER-to-Golgi apparatus trafficking of glycosylphosphatidylinositol-anchored or membrane-spanning proteins in LY-A Cells appeared to be normal. These results indicate that the predominant pathway of ER-to-Golgi apparatus trafficking of Cer for de novo SM synthesis is ATP dependent and that this pathway is almost completely impaired in LY-A Cells. In addition, the specific defect of SM synthesis in LY-A Cells suggests different pathways of Cer transport for glycosphingolipids versus SM synthesis.
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sphingolipids are essential for the growth of chinese hamster ovary Cells restoration of the growth of a Mutant defective in sphingoid base biosynthesis by exogenous sphingolipids
Journal of Biological Chemistry, 1992Co-Authors: Kentaro Hanada, Masahiro Nishijima, M Kiso, A Hasegawa, S Fujita, T Ogawa, Yuzuru AkamatsuAbstract:Abstract We previously isolated a temperature-sensitive Chinese hamster ovary Cell Mutant (strain SPB-1) with thermolabile serine palmitoyltransferase, which is involved in the first step of sphingolipid synthesis (Hanada, K., Nishijima, M., and Akamatsu, Y. (1990) J. Biol. Chem. 265, 22137-22142). In this study, sphingolipid-deficient culture medium was used to examine the effect of exogenous sphingolipids on the Cell growth of SPB-1. When cultivated in the sphingolipid-deficient medium, SPB-1 Cells ceased growing at non-permissive temperatures. Under these conditions, de novo sphingolipid synthesis ceased in the SPB-1 Cells, resulting in a decrease in levels of sphingomyelin and ganglioside sialyl lactosylceramide (GM3), whereas the parental CHO-K1 Cells grew logarithmically with normal sphingolipid synthesis. Exogenous sphingosine restored the contents of both sphingomyelin and GM3 in the SPB-1 Cells near to the parental levels through metabolic utilization and allowed the Mutant Cells to grow even at the non-permissive temperature. Similarly, exogenous sphingomyelin restored the sphingomyelin levels and only partly the GM3 levels and also suppressed the temperature-sensitivity of the SPB-1 Cell growth. In contrast, exogenous glucosylceramide, which restored the GM3 levels but not the sphingomyelin levels, failed to suppress the temperature sensitivity of the SPB-1 Cell growth. Combination of exogenous sphingomyelin with ceramide, glucosylceramide, GM3, or sphingoid bases did not show any synergistic or additive effect on the SPB-1 Cell growth enhancement, compared with sphingomyelin alone. The results indicated that the temperature sensitivity of the SPB-1 Cell growth was due to the lack of Cellular sphingolipids, possibly that of sphingomyelin.