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Journal articles on the topic 'CMP-sialic acid transport proteins'
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Hipgrave Ederveen, Agnes L., Noortje de Haan, Melissa Baerenfaenger, Dirk J. Lefeber, and Manfred Wuhrer. "Dissecting Total Plasma and Protein-Specific Glycosylation Profiles in Congenital Disorders of Glycosylation." International Journal of Molecular Sciences 21, no. 20 (October 15, 2020): 7635. http://dx.doi.org/10.3390/ijms21207635.
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Protein N-glycosylation is a multifactorial process involved in many biological processes. A broad range of congenital disorders of glycosylation (CDGs) have been described that feature defects in protein N-glycan biosynthesis. Here, we present insights into the disrupted N-glycosylation of various CDG patients exhibiting defects in the transport of nucleotide sugars, Golgi glycosylation or Golgi trafficking. We studied enzymatically released N-glycans of total plasma proteins and affinity purified immunoglobulin G (IgG) from patients and healthy controls using mass spectrometry (MS). The applied method allowed the differentiation of sialic acid linkage isomers via their derivatization. Furthermore, protein-specific glycan profiles were quantified for transferrin and IgG Fc using electrospray ionization MS of intact proteins and glycopeptides, respectively. Next to the previously described glycomic effects, we report unprecedented sialic linkage-specific effects. Defects in proteins involved in Golgi trafficking (COG5-CDG) and CMP-sialic acid transport (SLC35A1-CDG) resulted in lower levels of sialylated structures on plasma proteins as compared to healthy controls. Findings for these specific CDGs include a more pronounced effect for α2,3-sialylation than for α2,6-sialylation. The diverse abnormalities in glycomic features described in this study reflect the broad range of biological mechanisms that influence protein glycosylation.
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Gangi Setty, Thanuja, Christine Cho, Sowmya Govindappa, Michael A. Apicella, and S. Ramaswamy. "Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site." Acta Crystallographica Section D Biological Crystallography 70, no. 7 (June 24, 2014): 1801–11. http://dx.doi.org/10.1107/s139900471400830x.
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Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteriaFusobacterium nucleatum,Pasteurella multocidaandVibrio choleraeand their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of theHaemophilus influenzaeprotein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states.
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Urbanek, Kelly, Danica M. Sutherland, Robert C. Orchard, Craig B. Wilen, Jonathan J. Knowlton, Pavithra Aravamudhan, Gwen M. Taylor, Herbert W. Virgin, and Terence S. Dermody. "Cytidine Monophosphate N-Acetylneuraminic Acid Synthetase and Solute Carrier Family 35 Member A1 Are Required for Reovirus Binding and Infection." Journal of Virology 95, no. 2 (October 21, 2020): e01571-20. http://dx.doi.org/10.1128/jvi.01571-20.
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ABSTRACTEngagement of cell surface receptors by viruses is a critical determinant of viral tropism and disease. The reovirus attachment protein σ1 binds sialylated glycans and proteinaceous receptors to mediate infection, but the specific requirements for different cell types are not entirely known. To identify host factors required for reovirus-induced cell death, we conducted a CRISPR-knockout screen targeting over 20,000 genes in murine microglial BV2 cells. Candidate genes required for reovirus to cause cell death were highly enriched for sialic acid synthesis and transport. Two of the top candidates identified, CMP N-acetylneuraminic acid synthetase (Cmas) and solute carrier family 35 member A1 (Slc35a1), promote sialic acid expression on the cell surface. Two reovirus strains that differ in the capacity to bind sialic acid, T3SA+ and T3SA−, were used to evaluate Cmas and Slc35a1 as potential host genes required for reovirus infection. Following CRISPR-Cas9 disruption of either gene, cell surface expression of sialic acid was diminished. These results correlated with decreased binding of strain T3SA+, which is capable of engaging sialic acid. Disruption of either gene did not alter the low-level binding of T3SA−, which does not engage sialic acid. Furthermore, infectivity of T3SA+ was diminished to levels similar to those of T3SA− in cells lacking Cmas and Slc35a1 by CRISPR ablation. However, exogenous expression of Cmas and Slc35a1 into the respective null cells restored sialic acid expression and T3SA+ binding and infectivity. These results demonstrate that Cmas and Slc35a1, which mediate cell surface expression of sialic acid, are required in murine microglial cells for efficient reovirus binding and infection.IMPORTANCE Attachment factors and receptors are important determinants of dissemination and tropism during reovirus-induced disease. In a CRISPR cell survival screen, we discovered two genes, Cmas and Slc35a1, which encode proteins required for sialic acid expression on the cell surface and mediate reovirus infection of microglial cells. This work elucidates host genes that render microglial cells susceptible to reovirus infection and expands current understanding of the receptors on microglial cells that are engaged by reovirus. Such knowledge may lead to new strategies to selectively target microglial cells for oncolytic applications.
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Souter, E., M. Pypaert, and G. Warren. "The Golgi stack reassembles during telophase before arrival of proteins transported from the endoplasmic reticulum." Journal of Cell Biology 122, no. 3 (August 1, 1993): 533–40. http://dx.doi.org/10.1083/jcb.122.3.533.
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HeLa cells arrested in prometaphase were pulse-labeled with [35S]methionine and chased in the absence of nocodazole to allow passage through mitosis and into G1. Transport of histocompatibility antigen (HLA) molecules to the medial- and trans-Golgi cisternae was measured by monitoring the resistance to endoglycosidase H and the acquisition of sialic acid residues, respectively. Transport to the plasma membrane was measured using neuraminidase to remove sialic acid residues on surface HLA molecules. The half-time for transport to each of these compartments was about 65-min longer in cells progressing out of mitosis than in G1 cells. This delay was only 5-min longer than the half-time for the fall in histone H1 kinase activity suggesting that inactivation of the mitotic kinase triggers the resumption of protein transport. The half-time for reassembly of the Golgi stack, measured using stereological procedures, was also 65 min, suggesting that both transport and reassembly are triggered at the same time. However, since reassembly was complete within 5 min, whereas HLA took 25 min to reach the medial-cisterna, we can conclude that the Golgi stack has reassembled by the time HLA reaches it.
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Qian, Mengding, and Billy Tsai. "Lipids and Proteins Act in Opposing Manners To Regulate Polyomavirus Infection." Journal of Virology 84, no. 19 (July 28, 2010): 9840–52. http://dx.doi.org/10.1128/jvi.01093-10.
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ABSTRACT How receptors control virus infection is poorly understood. Polyomavirus (Py) binds to the sialic acid-galactose moiety on receptors to gain entry into host cells and cause infection. We previously demonstrated that the sialic acid-galactose-containing glycolipids called gangliosides GD1a and GT1b promote Py infection, in part, by sorting the virus from the endolysosomes to the endoplasmic reticulum (ER), a critical infection route. Whether these glycolipids act as Py entry receptors, however, is not clear. Additionally, as the majority of glycoproteins also harbor terminal sialic acid-galactose residues, their roles in Py infection are also not well established. Using a ganglioside-deficient cell line, we show that GD1a is the functional entry receptor for Py. GD1a binds to Py on the plasma membrane, and the receptor-virus complex is internalized and transported to the late endosomes and then the ER to initiate infection. In contrast, our findings indicate that glycoproteins act as decoy receptors, restricting the ER transport and infection of Py. Thus, glycolipids and glycoproteins, two major constituents of the plasma membrane, execute opposing functions in regulating infection by a defined virus.
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Bensing, Barbara A., José A. López, and Paul M. Sullam. "The Streptococcus gordonii Surface Proteins GspB and Hsa Mediate Binding to Sialylated Carbohydrate Epitopes on the Platelet Membrane Glycoprotein Ibα." Infection and Immunity 72, no. 11 (November 2004): 6528–37. http://dx.doi.org/10.1128/iai.72.11.6528-6537.2004.
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ABSTRACT Platelet binding by Streptococcus gordonii strain M99 is dependent on expression of the cell wall-anchored glycoprotein GspB. This large cell surface protein is exported from the M99 cytoplasm via a dedicated transport system that includes SecA2 and SecY2. GspB is highly similar to Hsa, a protein expressed by S. gordonii Challis that has been characterized as a sialic acid binding hemagglutinin. In this study, we compared the contribution of GspB and Hsa to the adherence of S. gordonii to selected glycoproteins. Our results indicate that GspB can mediate binding to a variety of sialylated glycoproteins. GspB facilitates binding to carbohydrates bearing sialic acid in either α(2-3) or α(2-6) linkages, with a slight preference for α(2-3) linkages. Furthermore, GspB readily mediates binding to sialic acid residues on immobilized glycocalicin, the extracellular portion of the platelet membrane glycoprotein (GP) Ibα (the ligand binding subunit of the platelet von Willebrand factor receptor complex GPIb-IX-V). Although Hsa is required for the binding of S. gordonii Challis to sialic acid, most of the Hsa expressed by Challis is retained in the cytoplasm. The deficiency in export is due, at least in part, to a nonsense mutation in secA2. Hsa export can be enhanced by complementation with secA2 from M99, which also results in significantly greater binding to sialylated glycoproteins, including glycocalicin. The combined results indicate that GspB and Hsa contribute similar binding capabilities to M99 and Challis, respectively, but there may be subtle differences in the preferred epitopes to which these adhesins bind.
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Brigham, Christopher, Ruth Caughlan, Rene Gallegos, Mary Beth Dallas, Veronica G. Godoy, and Michael H. Malamy. "Sialic Acid (N-Acetyl Neuraminic Acid) Utilization by Bacteroides fragilis Requires a Novel N-Acetyl Mannosamine Epimerase." Journal of Bacteriology 191, no. 11 (March 20, 2009): 3629–38. http://dx.doi.org/10.1128/jb.00811-08.
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ABSTRACT We characterized the nanLET operon in Bacteroides fragilis, whose products are required for the utilization of the sialic acid N-acetyl neuraminic acid (NANA) as a carbon and energy source. The first gene of the operon is nanL, which codes for an aldolase that cleaves NANA into N-acetyl mannosamine (manNAc) and pyruvate. The next gene, nanE, codes for a manNAc/N-acetylglucosamine (NAG) epimerase, which, intriguingly, possesses more similarity to eukaryotic renin binding proteins than to other bacterial NanE epimerase proteins. Unphosphorylated manNAc is the substrate of NanE, while ATP is a cofactor in the epimerase reaction. The third gene of the operon is nanT, which shows similarity to the major transporter facilitator superfamily and is most likely to be a NANA transporter. Deletion of any of these genes eliminates the ability of B. fragilis to grow on NANA. Although B. fragilis does not normally grow with manNAc as the sole carbon source, we isolated a B. fragilis mutant strain that can grow on this substrate, likely due to a mutation in a NAG transporter; both manNAc transport and NAG transport are affected in this strain. Deletion of the nanE epimerase gene or the rokA hexokinase gene, whose product phosphorylates NAG, in the manNAc-enabled strain abolishes growth on manNAc. Thus, B. fragilis possesses a new pathway of NANA utilization, which we show is also found in other Bacteroides species.
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Henriquez, Tania, Larissa Wirtz, Dan Su, and Heinrich Jung. "Prokaryotic Solute/Sodium Symporters: Versatile Functions and Mechanisms of a Transporter Family." International Journal of Molecular Sciences 22, no. 4 (February 13, 2021): 1880. http://dx.doi.org/10.3390/ijms22041880.
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The solute/sodium symporter family (SSS family; TC 2.A.21; SLC5) consists of integral membrane proteins that use an existing sodium gradient to drive the uphill transport of various solutes, such as sugars, amino acids, vitamins, or ions across the membrane. This large family has representatives in all three kingdoms of life. The human sodium/iodide symporter (NIS) and the sodium/glucose transporter (SGLT1) are involved in diseases such as iodide transport defect or glucose-galactose malabsorption. Moreover, the bacterial sodium/proline symporter PutP and the sodium/sialic acid symporter SiaT play important roles in bacteria–host interactions. This review focuses on the physiological significance and structural and functional features of prokaryotic members of the SSS family. Special emphasis will be given to the roles and properties of proteins containing an SSS family domain fused to domains typically found in bacterial sensor kinases.
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Baeuerle, P. A., and W. B. Huttner. "Tyrosine sulfation is a trans-Golgi-specific protein modification." Journal of Cell Biology 105, no. 6 (December 1, 1987): 2655–64. http://dx.doi.org/10.1083/jcb.105.6.2655.
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The trans-Golgi has been recognized as having a key role in terminal glycosylation and sorting of proteins. Here we show that tyrosine sulfation, a frequent modification of secretory proteins, occurs specifically in the trans-Golgi. The heavy chain of immunoglobulin M (IgM) produced by hybridoma cells was found to contain tyrosine sulfate. This finding allowed the comparison of the state of sulfation of the heavy chain with the state of processing of its N-linked oligosaccharides. First, the pre-trans-Golgi forms of the IgM heavy chain, which lacked galactose and sialic acid, were unsulfated, whereas the trans-Golgi form, identified by the presence of galactose and sialic acid, and the secreted form of the IgM heavy chain were sulfated. Second, the earliest form of the heavy chain detectable by sulfate labeling, as well as the heavy chain sulfated in a cell-free system in the absence of vesicle transport, already contained galactose and sialic acid. Third, sulfate-labeled IgM moved to the cell surface with kinetics identical to those of galactose-labeled IgM. Lastly, IgM labeled with sulfate at 20 degrees C was not transported to the cell surface at 20 degrees C but reached the cell surface at 37 degrees C. The data suggest that within the trans-Golgi, tyrosine sulfation of IgM occurred at least in part after terminal glycosylation and therefore appeared to be the last modification of this constitutively secreted protein before its exit from this compartment. Furthermore, the results establish the covalent modification of amino acid side chains as a novel function of the trans-Golgi.
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Tiralongo, Joe, Samia Abo, Basil Danylec, Rita Gerardy-Schahn, and Mark von Itzstein. "A High-Throughput Assay for Rat Liver Golgi and Saccharomyces cerevisiae-Expressed Murine CMP-N-Acetylneuraminic Acid Transport Proteins." Analytical Biochemistry 285, no. 1 (October 2000): 21–32. http://dx.doi.org/10.1006/abio.2000.4705.
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Doms, R. W., G. Russ, and J. W. Yewdell. "Brefeldin A redistributes resident and itinerant Golgi proteins to the endoplasmic reticulum." Journal of Cell Biology 109, no. 1 (July 1, 1989): 61–72. http://dx.doi.org/10.1083/jcb.109.1.61.
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Brefeldin A (BFA) has been reported to block protein transport from the ER and cause disassembly of the Golgi complex. We have examined the effects of BFA on the transport and processing of the vesicular stomatitis virus G protein, a model integral membrane protein. Delivery of G protein to the cell surface was reversibly blocked by 6 micrograms/ml BFA. Pulse-label experiments revealed that in the presence of BFA, G protein became completely resistant to endoglycosidase H digestion. Addition of sialic acid, a trans-Golgi event, was not observed. Despite processing by cis- and medial Golgi enzymes, G protein was localized by indirect immunofluorescence to a reticular distribution characteristic of the ER. By preventing transport of G protein from the ER with the metabolic inhibitor carbonyl cyanide m-chlorophenylhydrazone or by use of the temperature-sensitive mutant ts045, which is restricted to the ER at 40 degrees C, we showed that processing of G protein occurred in the ER and was not due to retention of newly synthesized Golgi enzymes. Rather, redistribution of preexisting cis and medial Golgi enzymes to the ER occurred as soon as 2.5 min after addition of BFA, and was complete by 10-15 min. Delivery of Golgi enzymes to the ER was energy dependent and occurred only at temperatures greater than or equal to 20 degrees C. BFA also induced retrograde transport of G protein from the medial Golgi to the ER. Golgi enzymes were completely recovered from the ER 10 min after removal of BFA. These findings demonstrate that BFA induces retrograde transport of both resident and itinerant Golgi proteins to the ER in a fully reversible manner.
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Tisdale, E. J., J. R. Bourne, R. Khosravi-Far, C. J. Der, and W. E. Balch. "GTP-binding mutants of rab1 and rab2 are potent inhibitors of vesicular transport from the endoplasmic reticulum to the Golgi complex." Journal of Cell Biology 119, no. 4 (November 15, 1992): 749–61. http://dx.doi.org/10.1083/jcb.119.4.749.
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We have examined the role of ras-related rab proteins in transport from the ER to the Golgi complex in vivo using a vaccinia recombinant T7 RNA polymerase virus to express site-directed rab mutants. These mutations are within highly conserved domains involved in guanine nucleotide binding and hydrolysis found in ras and all members of the ras superfamily. Substitutions in the GTP-binding domains of rab1a and rab1b (equivalent to the ras 17N and 116I mutants) resulted in proteins which were potent trans dominant inhibitors of vesicular stomatitis virus glycoprotein (VSV-G protein) transport between the ER and cis Golgi complex. Immunofluorescence analysis indicated that expression of rab1b121I prevented delivery of VSV-G protein to the Golgi stack, which resulted in VSV-G protein accumulation in pre-Golgi punctate structures. Mutants in guanine nucleotide exchange or hydrolysis of the rab2 protein were also strong trans dominant transport inhibitors. Analogous mutations in rab3a, rab5, rab6, and H-ras did not inhibit processing of VSV-G to the complex, sialic acid containing form diagnostic of transport to the trans Golgi compartment. We suggest that at least three members of the rab family (rab1a, rab1b, and rab2) use GTP hydrolysis to regulate components of the transport machinery involved in vesicle traffic between early compartments of the secretory pathway.
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Zolotarev, A. S., R. R. Townsend, A. Stuart-Tilley, and S. L. Alper. "HCO3(-)-dependent conformational change in gastric parietal cell AE2, a glycoprotein naturally lacking sialic acid." American Journal of Physiology-Gastrointestinal and Liver Physiology 271, no. 2 (August 1, 1996): G311—G321. http://dx.doi.org/10.1152/ajpgi.1996.271.2.g311.
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Although the AE1 chloride/bicarbonate exchanger of the red blood cell is among the most thoroughly investigated of membrane transport proteins, less is known about the related AE2 polypeptide of parietal cells. We have studied enzymatic deglycosylation of native AE2 polypeptide in gastric mucosal membranes from pig and rabbit. Deglycosylation of AE2 was maximal at low ionic strength. Deglycosylation of AE2 in membranes was preferentially inhibited by bicarbonate compared with other anions. This inhibition was maximal at alkaline pH and was not evident after detergent solubilization of AE2. Deglycosylation of AE2 increased its susceptibility to proteolytic degradation, but the presence of bicarbonate protected against this degradation. Bicarbonate failed to inhibit deglycosylation of the membrane glycoproteins AE1 and gastric H(+)-K(+)-adenosinetriphosphatase beta-subunit or deglycosylation of the soluble glycoproteins fetuin and ribonuclease B. These data suggest that bicarbonate induces a conformational change in AE2 that can protect the polypeptide from deglycosylation and proteolysis. Pig AE2 was purified in sodium dodecyl sulfate, and its monosaccharide composition was determined after blotting onto polyvinylidene fluoride membrane. AE2 was found to be devoid of sialic acid, with a composition suggestive of the presence of lactosamine-type chains.
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Anba-Mondoloni, Jamila, Stéphane Chaillou, Monique Zagorec, and Marie-Christine Champomier-Vergès. "Catabolism ofN-Acetylneuraminic Acid, a Fitness Function of the Food-Borne Lactic Acid Bacterium Lactobacillus sakei, Involves Two Newly Characterized Proteins." Applied and Environmental Microbiology 79, no. 6 (January 18, 2013): 2012–18. http://dx.doi.org/10.1128/aem.03301-12.
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ABSTRACTIn silicoanalysis of the genome sequence of the meat-borne lactic acid bacterium (LAB)Lactobacillus sakei23K has revealed a repertoire of potential functions related to the adaptation of this bacterium to the meat environment. Among these functions, the ability to useN-acetyl-neuraminic acid (NANA) as a carbon source could provide a competitive advantage for growth on meat in which this amino sugar is present. In this work, we proposed to analyze the functionality of a gene cluster encompassingnanTEARandnanK(nanTEAR-nanK). We established that this cluster encoded a pathway allowing transport and early steps of the catabolism of NANA in this genome. We also demonstrated that this cluster was absent from the genome of otherL. sakeistrains that were shown to be unable to grow on NANA. Moreover,L. sakei23KnanA,nanT,nanK, andnanEgenes were able to complementEscherichia colimutants. Construction of different mutants inL. sakei23K ΔnanR, ΔnanT, and ΔnanKand the double mutantL. sakei23K Δ(nanA-nanE) made it possible to show that all were impaired for growth on NANA. In addition, two genes located downstream fromnanK,lsa1644andlsa1645, are involved in the catabolism of sialic acid inL. sakei23K, as aL. sakei23K Δlsa1645mutant was no longer able to grow on NANA. All these results demonstrate that the gene clusternanTEAR-nanK-lsa1644-lsa1645is indeed involved in the use of NANA as an energy source byL. sakei.
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Пыко, К. В., Ю. А. Беспалов, and С. С. Осочук. "The Role of Sialic Acid and Red Blood Cells Zeta Potential in Oxygen Transport from Bloodstream to Vital Organs and Tissues: Review of Current Data." Лабораторная диагностика. Восточная Европа, no. 3 (September 22, 2022): 339–48. http://dx.doi.org/10.34883/pi.2022.11.3.009.
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Транспорт кислорода из русла крови в ткани жизненно важных органов является одним из наиболее важных факторов обеспечения энергетического баланса организма. При нарушении его развиваются изменения метаболизма и физиологического состояния организма, проявляющиеся в диапазоне от ощущения недомогания до летального исхода, что связано прежде всего с недостаточным потреблением тканями организма кислорода как конечного акцептора электрон-транспортной цепи митохондрий. Понимание и углубленное изучение этих механизмов может значительно повлиять на существующие подходы к лечению пациентов, подготовке спортсменов и разработке технологий двойного назначения, в том числе для военизированных подразделений. Одними из недостаточно полно изученных участников транспорта кислорода и энергообеспечения клеток являются сиаловые кислоты и формируемый ими ζ-потенциал эритроцитов.В обзоре рассмотрены и обобщены современные знания о синтезе, биодеградации сиаловых кислот, их роли в посттрансляционной модификации белков, клеточных и субклеточных структур, транспорте кислорода, энергопродукции и развитии в ряде случаев не совместимых с жизнью заболеваний. Представленные данные литературы являются основой для проведения экспериментальных исследований, которые могут помочь уточнить механизмы транспорта кислорода. Oxygen transport from bloodstream to tissues is an important goal in maintaining the energy balance of the body. If this balance undergoes an extreme change, it could lead to number of pathological condition from fatigue to death because tissue has lack of oxygen as the final acceptor of the electron transport chain in the mitochondria. Understanding and researching these mechanisms could bring important changes in modern treatment guidelines, athletic training or military training and invention of dual-use technology. One of the unknown factors that has an impact on oxygen delivery is sialic acid and zeta- potential.This review examines and summarizes the current knowledge about the synthesis, biodegradation of sialic acids and the role in posttranslational modification of proteins, modification of cellular and subcellular structures, oxygen delivery, bioenergetics, organism development and how they influence the mortality rates of some diseases. The data presented are the basis for conducting experimental studies that can help to understand the mechanism of oxygen transport.
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Hobman, T. C., L. Woodward, and M. G. Farquhar. "Targeting of a heterodimeric membrane protein complex to the Golgi: rubella virus E2 glycoprotein contains a transmembrane Golgi retention signal." Molecular Biology of the Cell 6, no. 1 (January 1995): 7–20. http://dx.doi.org/10.1091/mbc.6.1.7.
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Rubella virus (RV) envelope glycoproteins, E2 and E1, form a heterodimeric complex that is targeted to medial/trans-Golgi cisternae. To identify the Golgi targeting signal(s) for the E2/E1 spike complex, we constructed chimeric proteins consisting of domains from RV glycoproteins and vesicular stomatitis virus (VSV) G protein. The location of the chimeric proteins in stably transfected Chinese hamster ovary cells was determined by immunofluorescence, immunoelectron microscopy, and by the extent of processing of their N-linked glycans. A trans-dominant Golgi retention signal was identified within the C-terminal region of E2. When the transmembrane (TM) and cytoplasmic (CT) domains of VSV G were replaced with those of RV E2, the hybrid protein (G-E2TMCT+) was retained in the Golgi. Transport of G-E2TMCT+ to the Golgi was rapid (t1/2 = 10-20 min). The G-E2TMCT+ protein was determined to be distal to or within the medial Golgi based on acquisition of endo H resistance but proximal to the trans-Golgi network since it lacked sialic acid. Deletion analysis revealed that only the TM domain of E2 was required for Golgi targeting. Although the cytoplasmic domain of E2 was not necessary for Golgi retention, it was required for efficient transport of VSV G-RV chimeras out of the endoplasmic reticulum. When assayed in sucrose velocity sedimentations gradients, the Golgi-retained G-E2TMCT+ protein behaved as a dimer. Unlike virtually all other Golgi targeting signals, the E2 TM domain does not contain any polar amino acids. The TM and CT domains of E1 were not required for targeting of E2 and E1 to the Golgi indicating that a heterodimer of two integral membrane proteins can be retained in the Golgi by a single retention signal.
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Ahuja, Shivani, James Cahill, Kimberly Hartfield, and Matthew R. Whorton. "Inhibition of CMP-sialic acid transport by endogenous 5-methyl CMP." PLOS ONE 16, no. 6 (June 3, 2021): e0249905. http://dx.doi.org/10.1371/journal.pone.0249905.
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Nucleotide-sugar transporters (NSTs) transport nucleotide-sugar conjugates into the Golgi lumen where they are then used in the synthesis of glycans. We previously reported crystal structures of a mammalian NST, the CMP-sialic acid transporter (CST) (Ahuja and Whorton 2019). These structures elucidated many aspects of substrate recognition, selectivity, and transport; however, one fundamental unaddressed question is how the transport activity of NSTs might be physiologically regulated as a means to produce the vast diversity of observed glycan structures. Here, we describe the discovery that an endogenous methylated form of cytidine monophosphate (m5CMP) binds and inhibits CST. The presence of m5CMP in cells results from the degradation of RNA that has had its cytosine bases post-transcriptionally methylated through epigenetic processes. Therefore, this work not only demonstrates that m5CMP represents a novel physiological regulator of CST, but it also establishes a link between epigenetic control of gene expression and regulation of glycosylation.
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Moreira, Lílian de Oliveira, Arnaldo Feitosa Braga Andrade, Márcio Damasceno Vale, Sônia Maria Silva Souza, Raphael Hirata, Lídia Maria Oliveira Buarque Asad, Nasser Ribeiro Asad, Luiz Henrique Monteiro-Leal, José Osvaldo Previato, and Ana Luiza Mattos-Guaraldi. "Effects of Iron Limitation on Adherence and Cell Surface Carbohydrates of Corynebacterium diphtheriae Strains." Applied and Environmental Microbiology 69, no. 10 (October 2003): 5907–13. http://dx.doi.org/10.1128/aem.69.10.5907-5913.2003.
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ABSTRACT Iron limitation may cause bacterial pathogens to grow more slowly; however, it may also stimulate these microorganisms to produce greater tissue damage, given that many virulence factors are controlled by the iron supply in the environment. The present study investigated the influence of low iron availability on the expression of proteins and surface sugar residues of two toxigenic strains of Corynebacterium diphtheriae subsp. mitis and evaluated their adherence to human group B erythrocytes and HEp-2 cells. A comparison was made between bacteria grown in (i) Trypticase soy broth (TSB), (ii) TSB treated with dipyridyl to deplete free iron, and (iii) TSB enriched with FeCl3. The effects of iron concentration on adhesive properties were different for strains 241 and CDC-E8392, of the sucrose-fermenting and non-sucrose-fermenting biotypes, respectively. Iron-limited conditions enhanced interaction of strain 241 with erythrocytes and HEp-2 cells. Inhibition assays suggested the involvement of nonfimbrial protein combination 67-72p on hemagglutination of diphtheria bacilli grown under iron-limited conditions. Conversely, iron limitation inhibited adherence to glass and expression of electron-dense material on the bacterial surface. Lectin binding assays demonstrated a reduction in the number of sialic acid residues and an increase in d-mannose and d-galactose residues on the surfaces of both strains. Thus, iron exerts a regulatory role on adhesive properties of diphtheria bacilli, and low iron availability modulates the expression of C. diphtheriae surface carbohydrate moieties. The significant changes in the degree of lectin binding specific for d-mannose, d-galactose and sialic acid residues may have an effect on binding of host cells. The expression of dissimilar microbial virulence determinants may be coordinately controlled by common regulatory systems. For C. diphtheriae, the present results imply regulation of adherence and slime production as part of a global response to iron-limited environmental conditions that includes derepression of genes for the synthesis of cytotoxin and siderophores and for transport of the Fe(III)-siderophore complexes.
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Takashima, Shou, Junichi Seino, Takeshi Nakano, Kazuhito Fujiyama, Masafumi Tsujimoto, Nobuhiro Ishida, and Yasuhiro Hashimoto. "Analysis of CMP-sialic acid transporter-like proteins in plants." Phytochemistry 70, no. 17-18 (December 2009): 1973–81. http://dx.doi.org/10.1016/j.phytochem.2009.09.017.
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Johnston, P. A., A. Stieber, and N. K. Gonatas. "A hypothesis on the traffic of MG160, a medial Golgi sialoglycoprotein, from the trans-Golgi network to the Golgi cisternae." Journal of Cell Science 107, no. 3 (March 1, 1994): 529–37. http://dx.doi.org/10.1242/jcs.107.3.529.
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We have reported that MG160, an intrinsic membrane sialoglycoprotein of the Golgi apparatus (GA), resides in the medial cisternae of the organelle (Gonatas et al. (1989) J. Biol. Chem. 264, 646–653). In order to resolve the question whether MG160 acquires sialic acid residues in the trans cisternae or trans-Golgi network (TGN) prior to its retrograde transport, we have examined the effects of brefeldin A (BFA) on the post-translational processing of MG160, and the distribution of internalized wheat germ agglutinin covalently linked with HRP (WGA-HRP), which labels the TGN (Gonatas et al. (1977) J. Cell Biol. 73, 1–13). In BFA-treated PC12 cells, MG160 acquires resistance to endo H, but fails to be sialylated. This effect occurs in parallel with the redistribution of MG160 into an ER compartment dispersed throughout the cytoplasm including the nuclear envelope, and the collapse of the WGA-HRP-labelled TGN into vesicles and tubules surrounding the centriole. These results suggest that MG160 is not sialylated in BFA-treated cells because it is sequestered from the sialyltransferase enzyme(s), presumably located in the TGN, and provide evidence supporting the hypothesis for a retrograde transport pathway that recycles resident GA proteins, including MG160, between the Golgi cisternae and the TGN. To examine further the above hypothesis we studied cells treated with BFA and then allowed to recover from the effect of the drug for various lengths of time. After 15 minutes of recovery, cisternae of the Golgi apparatus, typically found in the pericentriolar region, are labeled by both MG160 and WGA-HRP.(ABSTRACT TRUNCATED AT 250 WORDS)
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Ma, Cheng, Hong-Yuan Tsai, Qi Zhang, Lakmini Senavirathna, Lian Li, Lih-Shen Chin, Ru Chen, and Sheng Pan. "An Integrated Proteomic and Glycoproteomic Investigation Reveals Alterations in the N-Glycoproteomic Network Induced by 2-Deoxy-D-Glucose in Colorectal Cancer Cells." International Journal of Molecular Sciences 23, no. 15 (July 26, 2022): 8251. http://dx.doi.org/10.3390/ijms23158251.
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As a well-known glycolysis inhibitor for anticancer treatment, 2-Deoxy-D-glucose (2DG) inhibits the growth and survival of cancer cells by interfering with the ATP produced by the metabolism of D-glucose. In addition, 2DG inhibits protein glycosylation in vivo by competing with D-mannose, leading to endoplasmic reticulum (ER) stress and unfolded protein responses in cancer cells. However, the molecular details underlying the impact of 2DG on protein glycosylation remain largely elusive. With an integrated approach to glycoproteomics and proteomics, we characterized the 2DG-induced alterations in N-glycosylation, as well as the cascading impacts on the whole proteome using the HT29 colorectal cancer cell line as a model system. More than 1700 site-specific glycoforms, represented by unique intact glycopeptides (IGPs), were identified. The treatment of 2DG had a broad effect on the N-glycoproteome, especially the high-mannose types. The glycosite occupancy of the high-mannose N-glycans decreased the most compared with the sialic acid and fucose-containing N-glycans. Many of the proteins with down-regulated high-mannose were implicated in functional networks related to response to topologically incorrect protein, integrin-mediated signaling, lysosomal transport, protein hydroxylation, vacuole, and protein N-glycosylation. The treatment of 2DG also functionally disrupted the global cellular proteome, evidenced by significant up-regulation of the proteins implicated in protein folding, endoplasmic reticulum, mitochondrial function, cellular respiration, oxidative phosphorylation, and translational termination. Taken together, these findings reveal the complex changes in protein glycosylation and expression underlying the various effects of 2DG on cancer cells, and may provide insightful clues to inform therapeutic development targeting protein glycosylation.
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Abdel-Motal, Ussama, Shixia Wang, Shan Lu, Kim Wigglesworth, and Uri Galili. "Increased Immunogenicity of Human Immunodeficiency Virus gp120 Engineered To Express Galα1-3Galβ1-4GlcNAc-R Epitopes." Journal of Virology 80, no. 14 (July 15, 2006): 6943–51. http://dx.doi.org/10.1128/jvi.00310-06.
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ABSTRACT The glycan shield comprised of multiple carbohydrate chains on the human immunodeficiency virus (HIV) envelope glycoprotein gp120 helps the virus to evade neutralizing antibodies. The present study describes a novel method for increasing immunogenicity of gp120 vaccine by enzymatic replacement of sialic acid on these carbohydrate chains with Galα1-3Galβ1-4GlcNAc-R (α-gal) epitopes. These epitopes are ligands for the natural anti-Gal antibody constituting ∼1% of immunoglobulin G in humans. We hypothesize that vaccination with gp120 expressing α-gal epitopes (gp120αgal) results in in vivo formation of immune complexes with anti-Gal, which targets vaccines for effective uptake by antigen-presenting cells (APC), due to interaction between the Fc portion of the antibody and Fcγ receptors on APC. This in turn results in effective transport of the vaccine to lymph nodes and effective processing and presentation of gp120 immunogenic peptides by APC for eliciting a strong anti-gp120 immune response. This hypothesis was tested in α-1,3-galactosyltransferase knockout mice, which produce anti-Gal. Mice immunized with gp120αgal produced anti-gp120 antibodies in titers that were >100-fold higher than those measured in mice immunized with comparable amounts of gp120 and effectively neutralized HIV. T-cell response, measured by ELISPOT, was much higher in mice immunized with gp120αgal than in mice immunized with gp120. It is suggested that gp120αgal can serve as a platform for anti-Gal-mediated targeting of additional vaccinating HIV proteins fused to gp120αgal, thereby creating effective prophylactic vaccines.
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Mohamed, M., A. Ashikov, M. Guillard, J. H. Robben, S. Schmidt, B. van den Heuvel, A. P. M. de Brouwer, et al. "Intellectual disability and bleeding diathesis due to deficient CMP-sialic acid transport." Neurology 81, no. 7 (July 19, 2013): 681–87. http://dx.doi.org/10.1212/wnl.0b013e3182a08f53.
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Chiaramonte, Molly, Jennifer L. Koviach, Chad Moore, Vidhya V. Iyer, Carston R. Wagner, Randall L. Halcomb, Wayne Miller, Paul Melançon, and Robert D. Kuchta. "Inhibition of CMP-Sialic Acid Transport into Golgi Vesicles by Nucleoside Monophosphates†." Biochemistry 40, no. 47 (November 2001): 14260–67. http://dx.doi.org/10.1021/bi011262w.
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Powell, L. D., S. W. Whiteheart, and G. W. Hart. "Cell surface sialic acid influences tumor cell recognition in the mixed lymphocyte reaction." Journal of Immunology 139, no. 1 (July 1, 1987): 262–70. http://dx.doi.org/10.4049/jimmunol.139.1.262.
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Abstract The Ia+ B cell lymphoma, AKTB-1b, fails to stimulate thymic lymphocytes in a one-way mixed lymphocyte reaction unless pretreated with sialidase or inhibitors of N-linked oligosaccharide processing. A comparison of different sialidases and sialyltransferases suggests that the removal of only a subset of total surface sialic acid, rather than net desialylation of the cell surface, is required. Three sialidases were compared, including Vibrio cholerae (VC) and Clostridium perfringens (CP), which will cleave alpha 2-3, alpha 2-6, and alpha 2-8, sialic acid linkages, and Newcastle Disease virus (NDV), which will remove only alpha 2-3 and alpha 2-8 linked sialic acid. When treated with equivalent units of sialidase, CP-, VC-, and NDV-treated cells were 24-fold, sixfold, and threefold better stimulators than untreated cells. In contrast, VC released 1.3-fold and 2.5-fold more sialic acid per cell than did CP or NDV, respectively. Furthermore, VC was superior in reducing the levels of binding of the sialic acid-specific lectin, Limulus polyphemus agglutinin, in exposing Gal beta 1-3GalNAc and Gal beta 1-4GlcNAc residues, and in desialylating gangliosides. Two-dimensional gel analysis indicated that VC and CP were both equal and superior to NDV in the desialylation of iodinatable cell-surface proteins, including H-2Kk, I-A beta k, and a highly sialylated 65,000 dalton protein of unknown identity. Maximal resialylation of CP-treated cells with exogenously added CMP-NANA and either the alpha 2-3(Gal beta 1-3GalNAc) or alpha 2-6(Gal beta 1-4GlcNAc) sialyltransferase did not reduce the stimulatory capacity of these cells. However, resialylation of VC-treated cells with just CMP-NANA alone resulted in 49% reversal of their stimulatory capacity, and no additional reversal could be achieved with either of the sialyltransferases. Although the alpha 2-6(Gal beta 1-4GlcNAc) sialyltransferase was capable of adding back approximately 10% of the sialic acid removed, the endogenous activity added back approximately 0.1% of the total sialic acid removed. SDS-PAGE gels of the sialylated cells indicated that the exogenously added sialyltransferase labeled many different proteins, whereas the endogenous activity labeled far fewer proteins, predominantly in 46,000 and 25,000 m.w. range. Both the desialylation and resialylation data suggest that the sialidase-dependent stimulation is due to the desialylation of specific membrane structures. Together with previous studies, these data suggest that the sialic acids involved are probably alpha 2-6 linked to N-linked glycosyl moieties.
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Liao, Si-Ming, Bo Lu, Xue-Hui Liu, Zhi-Long Lu, Shi-Jie Liang, Dong Chen, Frederic A. Troy, Ri-Bo Huang, and Guo-Ping Zhou. "Molecular Interactions of the Polysialytransferase Domain (PSTD) in ST8Sia IV with CMP-Sialic Acid and Polysialic Acid Required for Polysialylation of the Neural Cell Adhesion Molecule Proteins: An NMR Study." International Journal of Molecular Sciences 21, no. 5 (February 26, 2020): 1590. http://dx.doi.org/10.3390/ijms21051590.
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Polysialic acid (polySia) is an unusual glycan that posttranslational modifies neural cell adhesion molecule (NCAM) proteins in mammalian cells. The up-regulated expression of polySia-NCAM is associated with tumor progression in many metastatic human cancers and in neurocognitive processes. Two members of the ST8Sia family of α2,8-polysialyltransferases (polySTs), ST8Sia II (STX) and ST8Sia IV (PST) both catalyze synthesis of polySia when activated cytidine monophosphate(CMP)-Sialic acid (CMP-Sia) is translocate into the lumen of the Golgi apparatus. Two key polybasic domains in the polySTs, the polybasic region (PBR) and the polysialyltransferase domain (PSTD) areessential forpolysialylation of the NCAM proteins. However, the precise molecular details to describe the interactions required for polysialylation remain unknown. In this study, we hypothesize that PSTD interacts with both CMP-Sia and polySia to catalyze polysialylation of the NCAM proteins. To test this hypothesis, we synthesized a 35-amino acid-PSTD peptide derived from the ST8Sia IV gene sequence and used it to study its interaction with CMP-Sia, and polySia. Our results showed for the PSTD-CMP-Sia interaction, the largest chemical-shift perturbations (CSP) were in amino acid residues V251 to A254 in the short H1 helix, located near the N-terminus of PSTD. However, larger CSP values for the PSTD-polySia interaction were observed in amino acid residues R259 to T270 in the long H2 helix. These differences suggest that CMP-Sia preferentially binds to the domain between the short H1 helix and the longer H2 helix. In contrast, polySia was principally bound to the long H2 helix of PSTD. For the PSTD-polySia interaction, a significant decrease in peak intensity was observed in the 20 amino acid residues located between the N-and C-termini of the long H2 helix in PSTD, suggesting a slower motion in these residues when polySia bound to PSTD. Specific features of the interactions between PSTD-CMP-Sia, and PSTD-polySia were further confirmed by comparing their 800 MHz-derived HSQC spectra with that of PSTD-Sia, PSTD-TriSia (DP 3) and PSTD-polySia. Based on the interactions between PSTD-CMP-Sia, PSTD-polySia, PBR-NCAM and PSTD-PBR, these findingsprovide a greater understanding of the molecular mechanisms underlying polySia-NCAM polysialylation, and thus provides a new perspective for translational pharmacological applications and development by targeting the two polysialyltransferases.
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Chammas, R., J. M. McCaffery, A. Klein, Y. Ito, L. Saucan, G. Palade, M. G. Farquhar, and A. Varki. "Uptake and incorporation of an epitope-tagged sialic acid donor into intact rat liver Golgi compartments. Functional localization of sialyltransferase overlaps with beta-galactosyltransferase but not with sialic acid O-acetyltransferase." Molecular Biology of the Cell 7, no. 11 (November 1996): 1691–707. http://dx.doi.org/10.1091/mbc.7.11.1691.
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The transfer of sialic acids (Sia) from CMP-sialic acid (CMP-Sia) to N-linked sugar chains is thought to occur as a final step in their biosynthesis in the trans portion of the Golgi apparatus. In some cell types such Sia residues can have O-acetyl groups added to them. We demonstrate here that rat hepatocytes express 9-O-acetylated Sias mainly at the plasma membranes of both apical (bile canalicular) and basolateral (sinusoidal) domains. Golgi fractions also contain 9-O-acetylated Sias on similar N-linked glycoproteins, indicating that O-acetylation may take place in the Golgi. We show here that CMP-Sia-FITC (with a fluorescein group attached to the Sia) is taken up by isolated intact Golgi compartments. In these preparations, Sia-FITC is transferred to endogenous glycoprotein acceptors and can be immunochemically detected in situ. Addition of unlabeled UDP-Gal enhances Sia-FITC incorporation, indicating a substantial overlap of beta-galactosyltransferase and sialyltransferase machineries. Moreover, the same glycoproteins that incorporate Sia-FITC also accept [3H]galactose from the donor UDP-[3H]Gal. In contrast, we demonstrate with three different approaches (double-labeling, immunoelectron microscopy, and addition of a diffusible exogenous acceptor) that sialyltransferase and O-acetyltransferase machineries are much more separated from one another. Thus, 9-O-acetylation occurs after the last point of Sia addition in the trans-Golgi network. Indeed, we show that 9-O-acetylated sialoglycoproteins are preferentially segregated into a subset of vesicular carriers that concentrate membrane-bound, but not secretory, proteins.
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Harvey, B. E., and P. Thomas. "Inhibition of CMP-Sialic Acid Transport in Human Liver and Colorectal Cancer Cell Lines by a Sialic Acid Nucleoside Conjugate (KI-8110)." Biochemical and Biophysical Research Communications 190, no. 2 (January 1993): 571–75. http://dx.doi.org/10.1006/bbrc.1993.1086.
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Milla, M. E., and C. B. Hirschberg. "Reconstitution of Golgi vesicle CMP-sialic acid and adenosine 3'-phosphate 5'-phosphosulfate transport into proteoliposomes." Proceedings of the National Academy of Sciences 86, no. 6 (March 1, 1989): 1786–90. http://dx.doi.org/10.1073/pnas.86.6.1786.
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Igarashi, Michihiro, Yoshiaki Komiya, and Masanori Kurokawa. "Fluorographic analyses of the axonal transport of CMP-sialic acid and gangliosides in frog sciatic nerve." Neuroscience Research Supplements 3 (January 1986): S102. http://dx.doi.org/10.1016/0921-8696(86)90216-1.
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Wattenberg, B. W. "Glycolipid and glycoprotein transport through the Golgi complex are similar biochemically and kinetically. Reconstitution of glycolipid transport in a cell free system." Journal of Cell Biology 111, no. 2 (August 1, 1990): 421–28. http://dx.doi.org/10.1083/jcb.111.2.421.
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Glycolipid transport between compartments of the Golgi apparatus has been reconstituted in a cell free system. Transport of lactosylceramide (galactose beta 1-4-glucose-ceramide) was followed from a donor to an acceptor Golgi population. The major glycolipid in CHO cells is GM3 (sialic acid alpha 2-3 galactose beta 1-4-glucose-ceramide). Donor membranes were derived from a Chinese hamster ovary (CHO) cell mutant (Lec2) deficient in the Golgi CMP-sialic acid transporter, and therefore contained lactosylceramide as the predominant glycolipid. Acceptor Golgi apparatus was prepared from another mutant, Lec8, which is defective in UDP-Gal transport. Thus, glucosylceramide is the major glycolipid in Lec8 cells. Transport was measured by the incorporation of labeled sialic acid into lactosylceramide (present originally in the donor) by transport to acceptor membranes, forming GM3. This incorporation was dependent on ATP, cytosolic components, intact membranes, and elevated temperature. Donor membranes were prepared from Lec2 cells infected with vesicular stomatitus virus (VSV). These membranes therefore contain the VSV membrane glycoprotein, G protein. Donor membranes derived from VSV-infected cells could then be used to monitor both glycolipid and glycoprotein transport. Transport of these two types of molecules between Golgi compartments was compared biochemically and kinetically. Glycolipid transport required the N-ethylmaleimide sensitive factor previously shown to act in glycoprotein transport (Glick, B. S., and J. E. Rothman. 1987. Nature [Lond.]. 326:309-312; Rothman, J. E. 1987. J. Biol. Chem. 262:12502-12510). GTP gamma S inhibited glycolipid and glycoprotein transport similarly. The kinetics of transport of glycolipid and glycoprotein were also compared. The kinetics of transport to the end of the pathway were similar, as were the kinetics of movement into a defined transport intermediate. It is concluded that glycolipid and glycoprotein transport through the Golgi occur by similar if not identical mechanisms.
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Hurtado-Ziola, Nancy, Justin L. Sonnenburg, and Ajit Varki. "Differential Expression and Function of the CD33-Related Siglecs between Humans and Great Apes." Blood 104, no. 11 (November 16, 2004): 1466. http://dx.doi.org/10.1182/blood.v104.11.1466.1466.
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Abstract The Siglecs (Sialic acid-binding Immunoglobulin Superfamily Lectins) are a recently discovered family of mammalian glycan-binding proteins that have been shown to recognize the terminal sialic acids of glycoproteins and glycolipids. The CD33-Related Siglecs (CD33rSiglecs, namely Siglec-3, -5 through -11 and -XII in humans) are a subgroup of these molecules, which are thought to be primarily expressed on cells of the innate immune system. All CD33rSiglecs are type-1 transmembrane proteins with an N-terminal sialic acid-recognizing V-set domain followed by a variable number of C-2 set domains, a transmembrane region and a cytosolic C-terminal domain that usually has two tyrosine-based signaling motifs, one of which conforms to a canonical negative regulatory ITIM motif. Although the true function of the CD33rSiglecs has yet to be discovered, available data are most consistent with an inhibitory signaling role in the innate immune response, mediated by recognition of host sialic acids as “self”. CD33rSiglecs also interact with sialic acids on the same cell surface, typically resulting in “masking” of their sialic acid-binding sites. Our recent studies have shown that humans and non-human primates have a similar clustered localization of CD33rSiglec genes, and that true orthologs can generally be identified within each cluster (Angata et al., PNAS, in press). However, humans no longer express CMP-sialic acid hydroxylase (CMAH) the enzyme required to generate one of the potential CD33rSiglec sialic acid ligands called N-glycolylneuraminic acid (Neu5Gc), from its precursor N-acetylneuraminic acid (Neu5Ac). This genetic change occurred after our last common ancestor with the great apes, and dramatically altered the “Sialome” (the sialic acid makeup of a specific species) of humans when compared to that of the great apes. While great ape blood cells express about equal amounts of Neu5Ac and Neu5Gc, human blood cells express almost exclusively Neu5Ac. We also recently discovered that preferential recognition of Neu5Gc is the ancestral condition of most or all of the great ape (chimpanzee and gorilla) CD33rSiglecs (Sonnenburg JL, Altheide TK, Varki A. Glycobiology.14:339–46, 2004). We therefore reasoned that the sudden and major change in the sialome of our hominid ancestors could have had a significant impact on the evolution, binding specificities and expression patterns of CD33rSiglecs. Indeed, we have found that all human CD33rSiglecs can recognize both Neu5Ac and Neu5Gc. This presumably represents an evolutionarily-selected “relaxation” in binding specificity that was necessary to “remask” the Siglecs that had lost their Neu5Gc ligands. Also, there are differences in CD33rSiglec expression on monocytes and neutrophils between humans and great apes (chimp, bonobo, gorilla and orangutan). Furthermore, while great ape cells often show multiple populations with different signal intensities, humans express a single bright peak for each Siglec in flow cytometry. Surprisingly, while humans showed almost no CD33rSiglec expression on lymphocytes, the great apes show a moderate to high expression of some Siglecs on these cells. Total leukocyte expression of some CD33rSiglecs also shows differences between humans and great apes. Overall, CD33rSiglecs appear to be rapidly evolving in primates, with an apparent further acceleration of changes in humans. Additional studies are needed to define the mechanistic details, as well as the implications for human health and disease.
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Jackson, Ronald J., Diana F. Hall, and Peter J. Kerr. "Myxoma Virus Encodes an α2,3-Sialyltransferase That Enhances Virulence." Journal of Virology 73, no. 3 (1999): 2376–84. http://dx.doi.org/10.1128/jvi.73.3.2376-2384.1999.
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A 4.7-kb region of DNA sequence contained at the right end of the myxoma virus EcoRI-G2 fragment located 24 kb from the right end of the 163-kb genome has been determined. This region of the myxoma virus genome encodes homologs of the vaccinia virus genes A51R, A52R, A55R, A56R, and B1R; the myxoma virus gene equivalents have been given the prefix M. The MA55 gene encodes a protein belonging to the kelch family of actin-binding proteins, while the MA56 gene encodes a member of the immunoglobulin superfamily related to a variety of cellular receptors and adhesion molecules. A novel myxoma virus early gene, MST3N, is a member of the eukaryotic sialyltransferase gene family located between genes MA51 and MA52. Detergent lysates prepared from myxoma virus-infected cell cultures contained a virally encoded sialyltransferase activity that catalyzed the transfer of sialic acid (Sia) from CMP-Sia to an asialofetuin glycoprotein acceptor. Analysis of the in vitro-sialylated glycoprotein acceptor by digestion withN-glycosidase F and by lectin binding suggested that the MST3N gene encodes an enzyme with Galβ1,3(4)GlcNAc α2,3-sialyltransferase specificity for the N-linked oligosaccharide of glycoprotein. Lectin binding assays demonstrated that α2,3-sialyltransferase activity is expressed by several known leporipoxviruses that naturally infect Sylvilagus rabbits. The sialyltransferase is nonessential for myxoma virus replication in cell culture; however, disruption of the MST3N gene caused attenuation in vivo. The possible implications of the myxoma virus-expressed sialyltransferase in terms of the host’s defenses against infection are discussed.
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Ahuja, Shivani, and Matthew R. Whorton. "Structural basis for mammalian nucleotide sugar transport." eLife 8 (April 15, 2019). http://dx.doi.org/10.7554/elife.45221.
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Nucleotide-sugar transporters (NSTs) are critical components of the cellular glycosylation machinery. They transport nucleotide-sugar conjugates into the Golgi lumen, where they are used for the glycosylation of proteins and lipids, and they then subsequently transport the nucleotide monophosphate byproduct back to the cytoplasm. Dysregulation of human NSTs causes several debilitating diseases, and NSTs are virulence factors for many pathogens. Here we present the first crystal structures of a mammalian NST, the mouse CMP-sialic acid transporter (mCST), in complex with its physiological substrates CMP and CMP-sialic acid. Detailed visualization of extensive protein-substrate interactions explains the mechanisms governing substrate selectivity. Further structural analysis of mCST’s unique lumen-facing partially-occluded conformation, coupled with the characterization of substrate-induced quenching of mCST’s intrinsic tryptophan fluorescence, reveals the concerted conformational transitions that occur during substrate transport. These results provide a framework for understanding the effects of disease-causing mutations and the mechanisms of this diverse family of transporters.
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Bohlender, Lennard L., Juliana Parsons, Sebastian N. W. Hoernstein, Christine Rempfer, Natalia Ruiz-Molina, Timo Lorenz, Fernando Rodríguez Jahnke, et al. "Stable Protein Sialylation in Physcomitrella." Frontiers in Plant Science 11 (December 18, 2020). http://dx.doi.org/10.3389/fpls.2020.610032.
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Recombinantly produced proteins are indispensable tools for medical applications. Since the majority of them are glycoproteins, their N-glycosylation profiles are major determinants for their activity, structural properties and safety. For therapeutical applications, a glycosylation pattern adapted to product and treatment requirements is advantageous. Physcomitrium patens (Physcomitrella, moss) is able to perform highly homogeneous complex-type N-glycosylation. Additionally, it has been glyco-engineered to eliminate plant-specific sugar residues by knock-out of the β1,2-xylosyltransferase and α1,3-fucosyltransferase genes (Δxt/ft). Furthermore, Physcomitrella meets wide-ranging biopharmaceutical requirements such as GMP compliance, product safety, scalability and outstanding possibilities for precise genome engineering. However, all plants, in contrast to mammals, lack the capability to perform N-glycan sialylation. Since sialic acids are a common terminal modification on human N-glycans, the property to perform N-glycan sialylation is highly desired within the plant-based biopharmaceutical sector. In this study, we present the successful achievement of protein N-glycan sialylation in stably transformed Physcomitrella. The sialylation ability was achieved in a Δxt/ft moss line by stable expression of seven mammalian coding sequences combined with targeted organelle-specific localization of the encoded enzymes responsible for the generation of β1,4-galactosylated acceptor N-glycans as well as the synthesis, activation, transport and transfer of sialic acid. Production of free (Neu5Ac) and activated (CMP-Neu5Ac) sialic acid was proven. The glycosidic anchor for the attachment of terminal sialic acid was generated by the introduction of a chimeric human β1,4-galactosyltransferase gene under the simultaneous knock-out of the gene encoding the endogenous β1,3-galactosyltransferase. Functional complex-type N-glycan sialylation was confirmed via mass spectrometric analysis of a stably co-expressed recombinant human protein.
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Reinke, Stefan O., Gerhard Lehmer, Stephan Hinderlich, and Werner Reutter. "Regulation and pathophysiological implications of UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE) as the key enzyme of sialic acid biosynthesis." Biological Chemistry 390, no. 7 (July 1, 2009). http://dx.doi.org/10.1515/bc.2009.073.
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AbstractThe key enzyme for the biosynthesis ofN-acetylneuraminic acid, from which all other sialic acids are formed, is the bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE). GNE is a highly conserved protein found throughout the animal kingdom. Its highest expression is seen in the liver and placenta. GNE is regulated by a variety of biochemical means, including tetramerization promoted by the substrate UDP-GlcNAc, phosphorylation by protein kinase C and feedback inhibition by CMP-Neu5Ac, which is defect in the human disease sialuria. GNE knock-out in mice leads to embryonic lethality, emphasizing the crucial role of this key enzyme for sialic acid biosynthesis. The metabolic capacity to synthesize sialic acid and CMP-sialic acid upon ManNAc loads is amazingly high. An additional characteristic of GNE is its interaction with proteins involved in the regulation of development, which might play a crucial role in the hereditary inclusion body myopathy. Due to the importance of increased concentrations of tumor-surface sialic acid, first attempts to find inhibitors of GNE have been successful.
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Barnard, Karen N., Brynn K. Alford-Lawrence, David W. Buchholz, Brian R. Wasik, Justin R. LaClair, Hai Yu, Rebekah Honce, et al. "Modified Sialic Acids on Mucus and Erythrocytes Inhibit Influenza A Virus Hemagglutinin and Neuraminidase Functions." Journal of Virology 94, no. 9 (February 12, 2020). http://dx.doi.org/10.1128/jvi.01567-19.
Full textAbstract:
ABSTRACT Sialic acids (Sia) are the primary receptors for influenza viruses and are widely displayed on cell surfaces and in secreted mucus. Sia may be present in variant forms that include O-acetyl modifications at C-4, C-7, C-8, and C-9 positions and N-acetyl or N-glycolyl at C-5. They can also vary in their linkages, including α2-3 or α2-6 linkages. Here, we analyze the distribution of modified Sia in cells and tissues of wild-type mice or in mice lacking CMP-N-acetylneuraminic acid hydroxylase (CMAH) enzyme, which synthesizes N-glycolyl (Neu5Gc) modifications. We also examined the variation of Sia forms on erythrocytes and in saliva from different animals. To determine the effect of Sia modifications on influenza A virus (IAV) infection, we tested for effects on hemagglutinin (HA) binding and neuraminidase (NA) cleavage. We confirmed that 9-O-acetyl, 7,9-O-acetyl, 4-O-acetyl, and Neu5Gc modifications are widely but variably expressed in mouse tissues, with the highest levels detected in the respiratory and gastrointestinal (GI) tracts. Secreted mucins in saliva and surface proteins of erythrocytes showed a high degree of variability in display of modified Sia between different species. IAV HAs from different virus strains showed consistently reduced binding to both Neu5Gc- and O-acetyl-modified Sia; however, while IAV NAs were inhibited by Neu5Gc and O-acetyl modifications, there was significant variability between NA types. The modifications of Sia in mucus may therefore have potent effects on the functions of IAV and may affect both pathogens and the normal flora of different mucosal sites. IMPORTANCE Sialic acids (Sia) are involved in numerous different cellular functions and are receptors for many pathogens. Sia come in chemically modified forms, but we lack a clear understanding of how they alter interactions with microbes. Here, we examine the expression of modified Sia in mouse tissues, on secreted mucus in saliva, and on erythrocytes, including those from IAV host species and animals used in IAV research. These Sia forms varied considerably among different animals, and their inhibitory effects on IAV NA and HA activities and on bacterial sialidases (neuraminidases) suggest a host-variable protective role in secreted mucus.