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Carlos B Hirschberg - One of the best experts on this subject based on the ideXlab platform.
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a Nucleotide Sugar transporter involved in glycosylation of the toxoplasma tissue cyst wall is required for efficient persistence of bradyzoites
PLOS Pathogens, 2013Co-Authors: Carolina E Caffaro, Carlos B Hirschberg, Li Liu, Anita A Koshy, Gusti M Zeiner, John C BoothroydAbstract:Toxoplasma gondii is an intracellular parasite that transitions from acute infection to a chronic infective state in its intermediate host via encystation, which enables the parasite to evade immune detection and clearance. It is widely accepted that the tissue cyst perimeter is highly and specifically decorated with glycan modifications; however, the role of these modifications in the establishment and persistence of chronic infection has not been investigated. Here we identify and biochemically and biologically characterize a Toxoplasma Nucleotide-Sugar transporter (TgNST1) that is required for cyst wall glycosylation. Toxoplasma strains deleted for the TgNST1 gene (Δnst1) form cyst-like structures in vitro but no longer interact with lectins, suggesting that Δnst1 strains are deficient in the transport and use of Sugars for the biosynthesis of cyst-wall structures. In vivo infection experiments demonstrate that the lack of TgNST1 activity does not detectably impact the acute (tachyzoite) stages of an infection or tropism of the parasite for the brain but that Δnst1 parasites are severely defective in persistence during the chronic stages of the infection. These results demonstrate for the first time the critical role of parasite glycoconjugates in the persistence of Toxoplasma tissue cysts.
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inhibition of Nucleotide Sugar transport in trypanosoma brucei alters surface glycosylation
Journal of Biological Chemistry, 2013Co-Authors: Yuxin Xu, Kacey L Caradonna, Emilia K Kruzel, Barbara A Burleigh, James D Bangs, Carlos B HirschbergAbstract:Abstract Nucleotide Sugar transporters (NSTs) are indispensible for the biosynthesis of glycoproteins by providing the Nucleotide Sugars needed for glycosylation in the lumen of the Golgi apparatus. Mutations in NST genes cause human and cattle diseases and impaired cell walls of yeast and fungi. Information regarding their function in the protozoan parasite, Trypanosoma brucei, a causative agent of African trypanosomiasis, is unknown. Here, we characterized the substrate specificities of four NSTs, TbNST1-4, which are expressed in both the insect procyclic form (PCF) and the mammalian bloodstream form (BSF) stages. TbNST1/2 transport UDP-Gal/UDP-GlcNAc, TbNST3 transports GDP-Man, and TbNST4 transports UDP-GlcNAc, UDP-GalNAc and GDP-Man. TbNST4 is the first NST shown to transport both pyrimidine and purine Nucleotide Sugars and is demonstrated here to be localized at the Golgi apparatus. RNAi-mediated silencing of TbNST4 in PCF caused underglycosylated surface glycoprotein EP-procyclin. Similarly, defective glycosylation of the variant surface glycoprotein (VSG221) as well as the lysosomal membrane protein, p67 was observed in ΔTbNST4 BSF T. brucei. Relative infectivity analysis showed that defects in glycosylation of the surface coat resulting from TbNST4 deletion were insufficient to impact the ability of this parasite to infect mice. Notably, the fact that inactivation of a single NST gene results in measurable defects in surface glycoproteins in different life cycle stages of the parasite, highlights the essential role of NST(s) in glycosylation of T. brucei. Thus, results presented in this study provide a framework for conducting functional analyses of other NSTs identified in T. brucei.
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Developmental diseases caused by impaired Nucleotide Sugar transporters
Glycoconjugate Journal, 2013Co-Authors: Carlos B HirschbergAbstract:Nucleotide Sugar transporters play critical roles in glycosylation of proteins, lipids and proteoglycans, which are essential for organogenesis, development, mammalian cellular immunity and pathogenicity of human pathogenic agents. Functional deficiencies of these transporters result in global defects of glycoconjugates, which in turn lead to a diversity of biochemical, physiological and pathological phenotypes. In this short review, we will highlight human and bovine diseases caused by mutations of these transporters.
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the role of Nucleotide Sugar transporters in development of eukaryotes
Seminars in Cell & Developmental Biology, 2010Co-Authors: Li Liu, Carlos B HirschbergAbstract:The Golgi apparatus membrane of all eukaryotes has Nucleotide Sugar transporters which play essential roles in the glycosylation of glycoproteins, proteoglycans and glycolipids. Mutations of these transporters have broad developmental phenotypes across many species including diseases in humans and cattle.
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functional redundancy between two caenorhabditis elegans Nucleotide Sugar transporters with a novel transport mechanism
Journal of Biological Chemistry, 2007Co-Authors: Carolina E Caffaro, Carlos B Hirschberg, Patricia M BerninsoneAbstract:Transporters of Nucleotide Sugars regulate the availability of these substrates required for glycosylation reactions in the lumen of the Golgi apparatus and play an important role in the development of multicellular organisms. Caenorhabditis elegans has seven different Sugars in its glycoconjugates, although 18 putative Nucleotide Sugar transporters are encoded in the genome. Among these, SQV-7, SRF-3, and CO3H5.2 exhibit partially overlapping substrate specificity and expression patterns. We now report evidence of functional redundancy between transporters CO3H5.2 and SRF-3. Reducing the activity of the CO3H5.2 gene product by RNA interference (RNAi) in SRF-3 mutants results in oocyte accumulation and abnormal gonad morphology, whereas comparable RNAi treatment of wild type or RNAi hypersensitive C. elegans strains does not cause detectable defects. We hypothesize this genetic enhancement to be a mechanism to ensure adequate glycoconjugate biosynthesis required for normal tissue development in multicellular organisms. Furthermore, we show that transporters SRF-3 and CO3H5.2, which are closely related in the phylogenetic tree, share a simultaneous and independent substrate transport mechanism that is different from the competitive one previously demonstrated for transporter SQV-7, which shares a lower amino acid sequence identity with CO3H5.2 and SRF-3. Therefore, different mechanisms for transporting multiple Nucleotide Sugars may have evolved parallel to transporter amino acid divergence.
Patricia M Berninsone - One of the best experts on this subject based on the ideXlab platform.
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functional redundancy between two caenorhabditis elegans Nucleotide Sugar transporters with a novel transport mechanism
Journal of Biological Chemistry, 2007Co-Authors: Carolina E Caffaro, Carlos B Hirschberg, Patricia M BerninsoneAbstract:Transporters of Nucleotide Sugars regulate the availability of these substrates required for glycosylation reactions in the lumen of the Golgi apparatus and play an important role in the development of multicellular organisms. Caenorhabditis elegans has seven different Sugars in its glycoconjugates, although 18 putative Nucleotide Sugar transporters are encoded in the genome. Among these, SQV-7, SRF-3, and CO3H5.2 exhibit partially overlapping substrate specificity and expression patterns. We now report evidence of functional redundancy between transporters CO3H5.2 and SRF-3. Reducing the activity of the CO3H5.2 gene product by RNA interference (RNAi) in SRF-3 mutants results in oocyte accumulation and abnormal gonad morphology, whereas comparable RNAi treatment of wild type or RNAi hypersensitive C. elegans strains does not cause detectable defects. We hypothesize this genetic enhancement to be a mechanism to ensure adequate glycoconjugate biosynthesis required for normal tissue development in multicellular organisms. Furthermore, we show that transporters SRF-3 and CO3H5.2, which are closely related in the phylogenetic tree, share a simultaneous and independent substrate transport mechanism that is different from the competitive one previously demonstrated for transporter SQV-7, which shares a lower amino acid sequence identity with CO3H5.2 and SRF-3. Therefore, different mechanisms for transporting multiple Nucleotide Sugars may have evolved parallel to transporter amino acid divergence.
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independent and simultaneous translocation of two substrates by a Nucleotide Sugar transporter
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Carolina E Caffaro, Carlos B Hirschberg, Patricia M BerninsoneAbstract:Nucleotide Sugar transporters play an essential role in protein and lipid glycosylation, and mutations can result in developmental phenotypes. We have characterized a transporter of UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine encoded by the Caenorhabditis elegans gene C03H5.2. Surprisingly, translocation of these substrates occurs in an independent and simultaneous manner that is neither a competitive nor a symport transport. Incubations of Golgi apparatus vesicles of Saccharomyces cerevisiae expressing C03H5.2 protein with these Nucleotide Sugars labeled with 3H and 14C in their Sugars showed that both substrates enter the lumen to the same extent, whether or not they are incubated alone or in the presence of a 10-fold excess of the other Nucleotide Sugar. Vesicles containing a deletion mutant of the C03H5.2 protein transport UDP-N-acetylglucosamine at rates comparable with that of wild-type transporter, whereas transport of UDP-N-acetylgalactosamine was decreased by 85–90%, resulting in an asymmetrical loss of substrate transport.
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loss of srf 3 encoded Nucleotide Sugar transporter activity in caenorhabditis elegans alters surface antigenicity and prevents bacterial adherence
Journal of Biological Chemistry, 2004Co-Authors: Jorg Hoflich, Carlos B Hirschberg, Patricia M Berninsone, Christine Gobel, Maria J Gravatonobre, Brian J Libby, Creg Darby, Samuel M Politz, Jonathan Hodgkin, Ralf BaumeisterAbstract:During the establishment of a bacterial infection, the surface molecules of the host organism are of particular importance, since they mediate the first contact with the pathogen. In Caenorhabditis elegans, mutations in the srf-3 locus confer resistance to infection by Microbacterium nematophilum, and they also prevent biofilm formation by Yersinia pseudotuberculosis, a close relative of the bubonic plague agent Yersinia pestis. We cloned srf-3 and found that it encodes a multitransmembrane hydrophobic protein resembling Nucleotide Sugar transporters of the Golgi apparatus membrane. srf-3 is exclusively expressed in secretory cells, consistent with its proposed function in cuticle/surface modification. We demonstrate that SRF-3 can function as a Nucleotide Sugar transporter in heterologous in vitro and in vivo systems. UDP-galactose and UDP-N-acetylglucosamine are substrates for SRF-3. We propose that the inability of Yersinia biofilms and M. nematophilum to adhere to the nematode cuticle is due to an altered glycoconjugate surface composition of the srf-3 mutant.
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sqv 7 a protein involved in caenorhabditis elegans epithelial invagination and early embryogenesis transports udp glucuronic acid udp n acetylgalactosamine and udp galactose
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Patricia M Berninsone, Ho Yon Hwang, Robert H Horvitz, Irina Zemtseva, Carlos B HirschbergAbstract:Caenorhabditis elegans sqv mutants are defective in vulval epithelial invagination and have a severe reduction in hermaphrodite fertility. The gene sqv-7 encodes a multitransmembrane hydrophobic protein resembling Nucleotide Sugar transporters of the Golgi membrane. A Golgi vesicle enriched fraction of Saccharomyces cerevisiae expressing SQV-7 transported UDP-glucuronic acid, UDP-N-acetylgalactosamine, and UDP-galactose (Gal) in a temperature-dependent and saturable manner. These Nucleotide Sugars are competitive, alternate, noncooperative substrates. The two mutant sqv-7 missense alleles resulted in a severe reduction of these three transport activities. SQV-7 did not transport CMP-sialic acid, GDP-fucose, UDP-N-acetylglucosamine, UDP-glucose, or GDP-mannose. SQV-7 is able to transport UDP-Gal in vivo, as shown by its ability to complement the phenotype of Madin-Darby canine kidney ricin resistant cells, a mammalian cell line deficient in UDP-Gal transport into the Golgi. These results demonstrate that unlike most Nucleotide Sugar transporters, SQV-7 can transport multiple distinct Nucleotide Sugars. We propose that SQV-7 translocates multiple Nucleotide Sugars into the Golgi lumen for the biosynthesis of glycoconjugates that play a pivotal role in development.
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Nucleotide Sugar transporters of the golgi apparatus
Current Opinion in Structural Biology, 2000Co-Authors: Patricia M Berninsone, Carlos B HirschbergAbstract:Glycosylation, sulfation and phosphorylation of proteins, proteoglycans and lipids occur in the lumen of the Golgi apparatus. The Nucleotide substrates of these reactions must be first transported from the cytosol into the Golgi lumen by specific transporters. The topology and structure of these hydrophobic, multi-transmembrane-spanning proteins are beginning to be understood.
Mariusz Olczak - One of the best experts on this subject based on the ideXlab platform.
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biosynthesis of glcnac rich n and o glycans in the golgi apparatus does not require the Nucleotide Sugar transporter slc35a3
Journal of Biological Chemistry, 2020Co-Authors: Bozena Szulc, Paulina Sosicka, Dorota Maszczakseneczko, Teresa Olczak, Edyta Skurska, Auhen Shauchuk, Hudson H Freeze, Mariusz OlczakAbstract:Nucleotide Sugar transporters, encoded by the SLC35 gene family, deliver Nucleotide Sugars throughout the cell for various glycosyltransferase-catalyzed glycosylation reactions. GlcNAc, in the form of UDP-GlcNAc, and galactose, as UDP-Gal, are delivered into the Golgi apparatus by SLC35A3 and SLC35A2 transporters, respectively. However, although the UDP-Gal transporting activity of SLC35A2 has been clearly demonstrated, UDP-GlcNAc delivery by SLC35A3 is not fully understood. Therefore, we analyzed a panel of CHO, HEK293T, and HepG2 cell lines including WT cells, SLC35A2 knockouts, SLC35A3 knockouts, and double-knockout cells. Cells lacking SLC35A2 displayed significant changes in N- and O-glycan synthesis. However, in SLC35A3-knockout CHO cells, only limited changes were observed; GlcNAc was still incorporated into N-glycans, but complex type N-glycan branching was impaired, although UDP-GlcNAc transport into Golgi vesicles was not decreased. In SLC35A3-knockout HEK293T cells, UDP-GlcNAc transport was significantly decreased but not completely abolished. However, N-glycan branching was not impaired in these cells. In CHO and HEK293T cells, the effect of SLC35A3 deficiency on N-glycan branching was potentiated in the absence of SLC35A2. Moreover, in SLC35A3-knockout HEK293T and HepG2 cells, GlcNAc was still incorporated into O-glycans. However, in the case of HepG2 cells, no qualitative changes in N-glycans between WT and SLC35A3 knockout cells nor between SLC35A2 knockout and double-knockout cells were observed. These findings suggest that SLC35A3 may not be the primary UDP-GlcNAc transporter and/or different mechanisms of UDP-GlcNAc transport into the Golgi apparatus may exist.
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biosynthesis of glcnac rich n and o glycans in the golgi apparatus does not require the Nucleotide Sugar transporter slc35a3
Journal of Biological Chemistry, 2020Co-Authors: Bozena Szulc, Paulina Sosicka, Dorota Maszczakseneczko, Teresa Olczak, Edyta Skurska, Auhen Shauchuk, Hudson H Freeze, Mariusz OlczakAbstract:Nucleotide Sugar transporters, encoded by the SLC35 gene family, deliver Nucleotide Sugars throughout the cell for various glycosyltransferase-catalyzed glycosylation reactions. N-acetylglucosamine, in the form of UDP-GlcNAc, and galactose, as UDP-Gal, are delivered into the Golgi apparatus by SLC35A3 and SLC35A2 transporters, respectively. However, although the UDP-Gal transporting activity of SLC35A2 has been clearly demonstrated, UDP-GlcNAc delivery by SLC35A3 is not fully understood. Therefore, we analyzed a panel of CHO, HEK293T and HepG2 cell lines including wild type cells, SLC35A2 knockouts, SLC35A3 knockouts, and double knock-out cells. Cells lacking SLC35A2 displayed significant changes in N- and O-glycan synthesis. However, in SLC35A3-knock-out CHO cells, only limited changes were observed - GlcNAc was still incorporated into N-glycans but complex type N-glycan branching was impaired, although UDP-GlcNAc transport into Golgi vesicles was not decreased. In SLC35A3-knock-out HEK293T cells, UDP-GlcNAc transport was significantly decreased, but not completely abolished. However, N-glycan branching was not impaired in these cells. In CHO and HEK293T cells the effect of SLC35A3 deficiency on N-glycan branching was potentiated in the absence of SLC35A2. Moreover, in SLC35A3-knock-out HEK293T and HepG2 cells GlcNAc was still incorporated into O-glycans. However, in the case of HepG2 cells, no qualitative changes in N-glycans between wild type and SLC35A3 knock-out cells, as well as between SLC35A2 knock-out and double knock-out cells were observed. These findings suggest that SLC35A3 may not be the primary UDP-GlcNAc transporter and/or different mechanisms of UDP-GlcNAc transport into the Golgi apparatus may exist.
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n acetylglucosaminyltransferases and Nucleotide Sugar transporters form multi enzyme multi transporter assemblies in golgi membranes in vivo
Cellular and Molecular Life Sciences, 2019Co-Authors: Fawzi Khoderagha, Paulina Sosicka, Mariusz Olczak, Maria Escriva Conde, Antti Hassinen, Tuomo Glumoff, Sakari KellokumpuAbstract:Branching and processing of N-glycans in the medial-Golgi rely both on the transport of the donor UDP-N-acetylglucosamine (UDP-GlcNAc) to the Golgi lumen by the SLC35A3 Nucleotide Sugar transporter (NST) as well as on the addition of the GlcNAc residue to terminal mannoses in nascent N-glycans by several linkage-specific N-acetyl-glucosaminyltransferases (MGAT1-MGAT5). Previous data indicate that the MGATs and NSTs both form higher order assemblies in the Golgi membranes. Here, we investigate their specific and mutual interactions using high-throughput FRET- and BiFC-based interaction screens. We show that MGAT1, MGAT2, MGAT3, MGAT4B (but not MGAT5) and Golgi alpha-mannosidase IIX (MAN2A2) form several distinct molecular assemblies with each other and that the MAN2A2 acts as a central hub for the interactions. Similar assemblies were also detected between the NSTs SLC35A2, SLC35A3, and SLC35A4. Using in vivo BiFC-based FRET interaction screens, we also identified novel ternary complexes between the MGATs themselves or between the MGATs and the NSTs. These findings suggest that the MGATs and the NSTs self-assemble into multi-enzyme/multi-transporter complexes in the Golgi membranes in vivo to facilitate efficient synthesis of complex N-glycans.
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SLC35A5 Protein—A Golgi Complex Member with Putative Nucleotide Sugar Transport Activity
International journal of molecular sciences, 2019Co-Authors: Paulina Sosicka, Yauhen Shauchuk, Bożena Bazan, Dorota Maszczak-seneczko, Teresa Olczak, Mariusz OlczakAbstract:Solute carrier family 35 member A5 (SLC35A5) is a member of the SLC35A protein subfamily comprising Nucleotide Sugar transporters. However, the function of SLC35A5 is yet to be experimentally determined. In this study, we inactivated the SLC35A5 gene in the HepG2 cell line to study a potential role of this protein in glycosylation. Introduced modification affected neither N- nor O-glycans. There was also no influence of the gene knock-out on glycolipid synthesis. However, inactivation of the SLC35A5 gene caused a slight increase in the level of chondroitin sulfate proteoglycans. Moreover, inactivation of the SLC35A5 gene resulted in the decrease of the uridine diphosphate (UDP)-glucuronic acid, UDP-N-acetylglucosamine, and UDP-N-acetylgalactosamine Golgi uptake, with no influence on the UDP-galactose transport activity. Further studies demonstrated that SLC35A5 localized exclusively to the Golgi apparatus. Careful insight into the protein sequence revealed that the C-terminus of this protein is extremely acidic and contains distinctive motifs, namely DXEE, DXD, and DXXD. Our studies show that the C-terminus is directed toward the cytosol. We also demonstrated that SLC35A5 formed homomers, as well as heteromers with other members of the SLC35A protein subfamily. In conclusion, the SLC35A5 protein might be a Golgi-resident multiprotein complex member engaged in Nucleotide Sugar transport.
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slc35a5 protein a golgi complex member with putative Nucleotide Sugar transport activity
International Journal of Molecular Sciences, 2019Co-Authors: Paulina Sosicka, Dorota Maszczakseneczko, Bozena Bazan, Yauhen Shauchuk, Teresa Olczak, Mariusz OlczakAbstract:Solute carrier family 35 member A5 (SLC35A5) is a member of the SLC35A protein subfamily comprising Nucleotide Sugar transporters. However, the function of SLC35A5 is yet to be experimentally determined. In this study, we inactivated the SLC35A5 gene in the HepG2 cell line to study a potential role of this protein in glycosylation. Introduced modification affected neither N- nor O-glycans. There was also no influence of the gene knock-out on glycolipid synthesis. However, inactivation of the SLC35A5 gene caused a slight increase in the level of chondroitin sulfate proteoglycans. Moreover, inactivation of the SLC35A5 gene resulted in the decrease of the uridine diphosphate (UDP)-glucuronic acid, UDP-N-acetylglucosamine, and UDP-N-acetylgalactosamine Golgi uptake, with no influence on the UDP-galactose transport activity. Further studies demonstrated that SLC35A5 localized exclusively to the Golgi apparatus. Careful insight into the protein sequence revealed that the C-terminus of this protein is extremely acidic and contains distinctive motifs, namely DXEE, DXD, and DXXD. Our studies show that the C-terminus is directed toward the cytosol. We also demonstrated that SLC35A5 formed homomers, as well as heteromers with other members of the SLC35A protein subfamily. In conclusion, the SLC35A5 protein might be a Golgi-resident multiprotein complex member engaged in Nucleotide Sugar transport.
Nobuhiro Ishida - One of the best experts on this subject based on the ideXlab platform.
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variety of Nucleotide Sugar transporters with respect to the interaction with nucleoside mono and diphosphates
Journal of Biological Chemistry, 2007Co-Authors: Masatoshi Muraoka, Nobuhiro Ishida, Toshiaki Miki, Takahiko Hara, Masao KawakitaAbstract:Nucleotide Sugar transporters have long been assumed to be antiporters that exclusively use nucleoside monophosphates as antiport substrates. Here we present evidence indicating that two other types of Nucleotide Sugar transporters exist that differ in their antiport substrate specificity. Biochemical studies using microsomes derived from Saccharomyces cerevisiae cells expressing either human (h) UGTrel7 or the Drosophila (d) FRC (Fringe connection) transporter revealed that (i) efflux of preloaded UDP-glucuronic acid from the yeast microsomes expressing hUGTrel7 was strongly enhanced by UDP-GlcNAc added in the external medium, but not by UMP or UDP, suggesting that hUGTrel7 may be described as a UDP-Sugar/UDP-Sugar antiporter, and (ii) addition of UDP-Sugars, UDP, or UMP in the external medium stimulated the efflux of preloaded UDP-GlcNAc from the yeast microsomes expressing dFRC to a comparable extent, suggesting that UDP, as well as UMP, may serve as an antiport substrate of dFRC. Antiport of UDP-Sugars with these specific substrates was reproduced and definitively confirmed using proteoliposomes reconstituted from solubilized and purified transporters. Possible physiological implications of these observations are discussed.
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identification and characterization of human golgi Nucleotide Sugar transporter slc35d2 a novel member of the slc35 Nucleotide Sugar transporter family
Genomics, 2005Co-Authors: Nobuhiro Ishida, Kazuhisa Aoki, Masao Kawakita, Toshiyasu Kuba, Shoichiro Miyatake, Yutaka SanaiAbstract:We report the molecular cloning of SLC35D2, a novel member of the SLC35 Nucleotide Sugar transporter family. The gene SLC35D2 maps to chromosome 9q22.33. SLC35D2 cDNA codes for a hydrophobic protein consisting of 337 amino acid residues with 10 putative transmembrane helices. Northern blot analysis revealed the SLC35D2 mRNA as a single major band corresponding to 2.0 kb in length. SLC35D2 was localized in the Golgi membrane and exhibited around 50% similarity with three Nucleotide Sugar transporters: human SLC35D1 (UDP-glucuronic acid/UDP-N-acetylgalactosamine transporter), fruitfly fringe connection (frc) transporter, and nematode SQV-7 transporter, the latter two being involved in developmental and organogenetic processes. Heterologous expression of SLC35D2 protein in yeast indicated that UDP-N-acetylglucosamine is a candidate for the substrate(s) of the transporter. The sequence similarity, subcellular localization, and transporting substrate suggest that SLC35D2 is a good candidate for the ortholog of frc transporter, which is involved in the Notch signaling system by providing the fringe N-acetylglucosaminyltransferase with the substrate. We also describe the identification and categorization of the human SLC35 gene family.
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molecular physiology and pathology of the Nucleotide Sugar transporter family slc35
Pflügers Archiv: European Journal of Physiology, 2004Co-Authors: Nobuhiro Ishida, Masao KawakitaAbstract:The solute carrier family SLC35 consists of at least 17 molecular species in humans. The family members so far characterized encode Nucleotide Sugar transporters localizing at the Golgi apparatus and/or the endoplasmic reticulum (ER). These transporters transport Nucleotide Sugars pooled in the cytosol into the lumen of these organelles, where most glycoconjugate synthesis occurs. Pathological analyses and developmental studies of small, multicellular organisms deficient in Nucleotide Sugar transporters have shown these transporters to be involved in tumour metastasis, cellular immunity, organogenesis and morphogenesis. Leukocyte adhesion deficiency type II (LAD II) or the congenital disorder of glycosylation type IIc (CDG IIc) are the sole human congenital disorders known to date that are caused by a defect of GDP-fucose transport. Along with LAD II, the possible involvement of Nucleotide Sugar transporters in disorders of connective tissues and muscles is also discussed.
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molecular characterization of human udp glucuronic acid udp n acetylgalactosamine transporter a novel Nucleotide Sugar transporter with dual substrate specificity
FEBS Letters, 2001Co-Authors: Masatoshi Muraoka, Masao Kawakita, Nobuhiro IshidaAbstract:Abstract A novel human Nucleotide Sugar transporter (NST) which transports both UDP-glucuronic acid (UDP-GlcA) and UDP-N-acetylgalactosamine (UDP-GalNAc) has been identified, cloned and characterized. The strategy for the identification of the novel NST involved a search of the expressed sequence tags database for genes related to the human UDP-galactose transporter-related isozyme 1, followed by heterologous expression of a candidate gene (hUGTrel7) in Saccharomyces cerevisiae and biochemical analyses. Significantly more UDP-GlcA and UDP-GalNAc were translocated from the reaction medium into the lumen of microsomes prepared from the hUGTrel7-expressing yeast cells than into the control microsomes from cells not expressing hUGTrel7. The possibility that this transporter participates in glucuronidation and/or chondroitin sulfate biosynthesis is discussed.
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Nucleotide Sugar transporters elucidation of their molecular identity and its implication for future studies
Journal of Biochemistry, 1998Co-Authors: Masao Kawakita, Nobuhiro Ishida, Nobuhiko Miura, Gehong Sunwada, Shigemi YoshiokaAbstract:Nucleotide Sugar transporters are mainly located in the Golgi membranes and carry Nucleotide Sugars, that are produced outside the Golgi apparatus, into the organelle, where they serve as substrates for the elongation of carbohydrate chains by glycosyltransferases. They are thus indispensable for cellular glycoconjugate synthesis and, moreover, may have regulatory roles in producing the structural variety of cellular glycoconjugates. Their occurrence has long been well recognized, but studies on the molecular bases of their strict substrate specificities and modes of action have been hampered by the lack of information on their precise molecular structures. Complementary DNAs encoding several of these transporters were cloned recently, which represented a substantial step forward as to the above mentioned issues. The products of these cDNAs are mutually related hydrophobic proteins consisting of 320-400 amino acid residues with multiple putative transmembrane helix domains, and are located in the Golgi apparatus. This review briefly summarizes the present status of the field of Nucleotide Sugar transporter research, and also presents an outlook of the study in this field.
Miranda G S Yap - One of the best experts on this subject based on the ideXlab platform.
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an investigation of intracellular glycosylation activities in cho cells effects of Nucleotide Sugar precursor feeding
Biotechnology and Bioengineering, 2010Co-Authors: Niki S C Wong, Lydia Wati, Peter Morin Nissom, Huatao Feng, May May Lee, Miranda G S YapAbstract:Controlling glycosylation of recombinant proteins produced by CHO cells is highly desired as it can be directed towards maintaining or increasing product quality. To further our understanding of the different factors influencing glycosylation, a glycosylation sub-array of 79 genes and a capillary electrophoresis method which simultaneously analyzes 12 Nucleotides and 7 Nucleotide Sugars; were used to generate intracellular N-glycosylation profiles. Specifically, the effects of Nucleotide Sugar precursor feeding on intracellular glycosylation activities were analyzed in CHO cells producing recombinant human interferon-γ (IFN-γ). Galactose (±uridine), glucosamine (±uridine), and N-acetylmannosamine (ManNAc) (±cytidine) feeding resulted in 12%, 28%, and 32% increase in IFN-γ sialylation as compared to the untreated control cultures. This could be directly attributed to increases in Nucleotide Sugar substrates, UDP-Hex (∼20-fold), UDP-HexNAc (6- to 15-fold) and CMP-sialic acid (30- to 120-fold), respectively. Up-regulation of B4gal and St3gal could also have enhanced glycan addition onto the proteins, leading to more complete glycosylation (sialylation). Combined feeding of glucosamine + uridine and ManNAc + cytidine increased UDP-HexNAc and CMP-sialic acid by another two- to fourfold as compared to feeding Sugar precursors alone. However, it did not lead to a synergistic increase in IFN-γ sialylation. Other factors such as glycosyltransferase or glycan substrate levels could have become limiting. In addition, uridine feeding increased the levels of uridine- and cytidine-activated Nucleotide Sugars simultaneously, which could imply that uridine is one of the limiting substrates for Nucleotide Sugar synthesis in the study. Hence, the characterization of intracellular glycosylation activities has increased our understanding of how Nucleotide Sugar precursor feeding influence glycosylation of recombinant proteins produced in CHO cells. It has also led to the optimization of more effective strategies for manipulating glycan quality. Biotechnol. Bioeng. 2010;107: 321–336. © 2010 Wiley Periodicals, Inc.
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an investigation of intracellular glycosylation activities in cho cells effects of Nucleotide Sugar precursor feeding
Biotechnology and Bioengineering, 2010Co-Authors: Niki S C Wong, Lydia Wati, Peter Morin Nissom, Huatao Feng, May May Lee, Miranda G S YapAbstract:Controlling glycosylation of recombinant proteins produced by CHO cells is highly desired as it can be directed towards maintaining or increasing product quality. To further our understanding of the different factors influencing glycosylation, a glycosylation sub-array of 79 genes and a capillary electrophoresis method which simultaneously analyzes 12 Nucleotides and 7 Nucleotide Sugars; were used to generate intracellular N-glycosylation profiles. Specifically, the effects of Nucleotide Sugar precursor feeding on intracellular glycosylation activities were analyzed in CHO cells producing recombinant human interferon-gamma (IFN-gamma). Galactose (+/-uridine), glucosamine (+/-uridine), and N-acetylmannosamine (ManNAc) (+/-cytidine) feeding resulted in 12%, 28%, and 32% increase in IFN-gamma sialylation as compared to the untreated control cultures. This could be directly attributed to increases in Nucleotide Sugar substrates, UDP-Hex ( approximately 20-fold), UDP-HexNAc (6- to 15-fold) and CMP-sialic acid (30- to 120-fold), respectively. Up-regulation of B4gal and St3gal could also have enhanced glycan addition onto the proteins, leading to more complete glycosylation (sialylation). Combined feeding of glucosamine + uridine and ManNAc + cytidine increased UDP-HexNAc and CMP-sialic acid by another two- to fourfold as compared to feeding Sugar precursors alone. However, it did not lead to a synergistic increase in IFN-gamma sialylation. Other factors such as glycosyltransferase or glycan substrate levels could have become limiting. In addition, uridine feeding increased the levels of uridine- and cytidine-activated Nucleotide Sugars simultaneously, which could imply that uridine is one of the limiting substrates for Nucleotide Sugar synthesis in the study. Hence, the characterization of intracellular glycosylation activities has increased our understanding of how Nucleotide Sugar precursor feeding influence glycosylation of recombinant proteins produced in CHO cells. It has also led to the optimization of more effective strategies for manipulating glycan quality.
