Parsed document review

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H1. Assay of nucleotide sugar transport activity (Golgi/ER transporter)

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H2. Introduction

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Nucleotide - sugar transporters (NSTs) are multimembrane-spanning proteins in the plane of the endoplasmic reticulum (ER) or Golgi membrane and are crucial for the synthesis of glycoconjugates ( Fig ure 1) (1 - 4). Glycosylation is performed by various types of glycosyltransferase s in the lumens of the ER and the Golgi apparatus. All glycosyltransferases require high-energy donor sugars activated by the addition of a nucleoside mono- or diphosphate (CMP, UDP or GDP), which are referred to as nucleotide sugars. Nucleotide sugars are synthesized in the cytosol (or in the nucleus in the case of CMP-sialic acid). The translocation of nucleotide sugars from the cytosol into the lumen of each compartment is mediated by NSTs. NSTs transport nucleotide sugars by coupling with the antiport of nucleoside monophosphate (NMP), which is produced because of a glycosyltransferase reaction and a subsequent luminal nucleoside diphosphatase ( NDPase ) reaction. NSTs are likely to be key components in the synthesis of glycoconjugates.

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The nucleotide sulfate, 3'-phosphoadenosine 5'-phosphosulfate (PAPS), is a universal sulfuryl donor for sulfation. In a similar fashion to nucleotide sugars, PAPS is synthesized in the cytosol by a PAPS synthetase and is translocated from the cytosol into the Golgi lumen through a PAPS transporter (PAPST) (5, 6). Thus, PAPS transporters belong to the NST family ( Fig ure 2).

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Since the original cloning of NST, which was reported in 1996, many other NSTs have been cloned and their transport activities identified ( Table s 1 , 2, 3, and 4 ). The Hugo Nomenclature committee categorizes NSTs and PAPSTs as solute carrier transporters that do not perform active transport using energy derived from adenosine triphosphate. This group of transporters comprise s the SLC35 (solute carrier 35) family of proteins ( Table s 1 - 4 ). The SLC35 family is divided into five sub-groups, A - F. In addition to these NSTs, many GDP-Man transporters have been identified in plants and fungi ( Table 4 ). Using the amino acid sequences of these transporter molecules, their phylogenetic tree was constructed ( Fig ure 2). Given that the group of GDP-Man transporters was found near the sub-groups, SLC35C and SLC35D, therefore, we added this group as a new sub-group to the SLC35 family, although it has not been identified in humans.

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There are two types of assays for NST activity: the “heterologous expression system” (2, 5 - 7) and the “ proteoliposome system” (8). The former method use s the expression of NST in a yeast expression system because the yeast microsome normally shows only low endogenous NST activity, except for GDP-Man transporter activity.

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H2. Protocol

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The yeast Saccharomyces cerevisiae is widely used for NST assays because of its low NST activities, except for GDP-mannose. Yeast expression vectors carrying cloned NST or candidate genes are used to transfect yeast, which is then grown in culture and subsequently harvested to prepare the membrane fraction. This membrane fraction is used for the assay of NST activity. The protocol comprises 1) preparation of subcellular fractionation of yeast expressing the NST of interest and 2) the NST activity assay.

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H3. Materials

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Nucleotide sugar

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Radio labeled nucleotide sugar (ex. UDP-[ 3 H] GlcNAc )

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Zymolyase (Zymolyase-100T; Seikagaku Corporation, Tokyo, Japan)

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Protease inhibitors (Complete protease inhibitor mixture tablets; Roche, Basel, Switzerland)

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Lithium acetate (Sigma-Aldrich, St. Louis, MO)

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Polyethylene Glycol 4000 ( Nacalai Tesque , Kyoto, Japan)

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Synthetic defined medium (2)

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Spheroplast buffer (1.4 M sorbitol, 50 mM of potassium phosphate , pH 7.5, 10 mM of NaN 3 , 40 mM of 2-mercaptoethanol, and Zymolyase 100T 1 - mg/g wet cells)

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Wash buffer (1.0 M sorbitol)

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Lysis buffer (0.6M sorbitol, 10 mM of triethanolamine , pH 7.2, protease inhibitors)

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Reaction mixture (20 mM of Tris-HCl , pH 7.5, 0.25 M sucrose, 5 mM of MgCl 2 , 1 mM of MnCl 2 , and 10 mM of 2-mercaptoethanol)

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Stop buffer (20 mM of Tris-HCl , pH 7.5, 0.25 M sucrose, 150 mM of KCl , and 1 mM of MgCl 2 )

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Scintillation mixture

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H3. Instruments

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Liquid scintillation counter

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HA filters (0.45 μm pore size, 24 - mm diameter)

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1225 Sampling Manifold (Merck Millipore, Burlington, MA)

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H3. Methods

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Protocol for preparation of ER or Golgi-rich membrane fraction

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Insert cDNA of NST into the yeast expression vector YEp352GAP-II (9).

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Transform yeast strain W303-1a ( MATa , ade2-1, ura3-1, his3-11, 15, trp1-1, leu2-3, 112 , and can1-100 ) using the lithium acetate procedure (10).

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Incubate the transformed yeast cells at 30 ° C in 800 m L of synthetic defined medium that lacks uracil to select for transformants; continue culture until an OD 600 of ~ 3.0 is achieved.

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Collect yeast cells by centrifugation (3,000 × g , 5 min).

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Wash with 100 m L of ice-cold 10 mM of NaN 3 .

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Suspend in 30 m L of spheroplast buffer.

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Incubate at 30 ° C for 35 min.

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Centrifuge (3,000 × g , 5 min) and collect the pellet.

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Wash 3 times with 100 m L of wash buffer to remove traces of zymolyase .

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Add 20 m L of lysis buffer.

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Homogenize using 20 - 40 strokes of the Dounce homogenizer.

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Centrifuge at 1,000 × g for 10 min and collect the supernatant.

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Centrifuge the supernatant at 10,000 × g for 15 min and collect the pellet as the P10 membrane fraction (ER-rich membrane fraction). Do not discard the supernatant.

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Centrifuge the above supernatant at 100,000 × g for 1 h , and collect the pellet as the P100 membrane fraction (Golgi-rich membrane fraction).

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Quantify the protein contents of the fractions suspended in appropriate amounts of the reaction mixture using the conventional method.

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Protocol for the assay of NST activity

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Add each unlabeled nucleotide sugar to the corresponding radiolabeled nucleotide sugar to prepare each nucleotide sugar to 1 , 000 cpm / pmol and add it up to 10 μM to the reaction mixture at s tep 2 b of Methods ; add 1,000,000 cpm of each nucleotide sugar to 100 µ L of reaction solution.

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Suspend 240 µg of proteins of P10 or P100 membrane fraction in 100 µ L of reaction mixture with radiolabeled nucleotide sugar.

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Incubate at 30 ° C for 5 min.

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Add 1 m L of ice-cold stop buffer ( Note 1 ).

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Filter through 0.45 μm of HA filters using 1225 Sampling Manifold ( Note 1 ).

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Wash with 10 m L of stop buffer ( Note 1 ).

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Air-dry and place in a vial with 4 m L of scintillation mixture.

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Count using a liquid scintillation counter.

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H3. Notes

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Perform each step quickly and in a serial order from S tep 2 d to S tep 2 f.

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H2. References

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Nishihara S. Members of the nucleotide-sugar transporter family and their functions. In: Taniguchi N., Endo T., Hart G., Seeberger P., Wong CH. (eds) Glycoscience : Biology and Medicine. 2015 pp 1253-1265. Springer, Tokyo. https://doi.org/10.1007/978-4-431-54841-6_174.

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Suda T, Kamiyama S, Suzuki M, Kikuchi N, Nakayama K, Narimatsu H, Jigami Y, Aoki T, Nishihara S. Molecular cloning and characterization of a human multisubstrate specific nucleotide-sugar transporter homologous to Drosophila fringe connection. J Biol Chem. 2004 Jun 18;279(25):26469-74. doi : 10.1074/jbc.M311353200. PMID: 15082721.

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Hadley B, Litfin T, Day CJ, Haselhorst T, Zhou Y, Tiralongo J. Nucleotide s ugar t ransporter SLC35 f amily s tructure and f unction. Comput Struct Biotechnol J. 2019 Aug 7;17:1123-1134. doi : 10.1016/j.csbj.2019.08.002. PMID: 31462968.

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Hirschberg CB. My journey in the discovery of nucleotide sugar transporters of the Golgi apparatus. J Biol Chem. 2018 Aug 17;293(33):12653-12662. doi : 10.1074/jbc.X118.004819. PMID: 30120148.

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Kamiyama S, Suda T, Ueda R, Suzuki M, Okubo R, Kikuchi N, Chiba Y, Goto S,Toyoda H, Saigo K, Watanabe M, Narimatsu H, Jigami Y, Nishihara S. Molecular cloning and identification of 3'-phosphoadenosine 5'-phosphosulfate transporter. J Biol Chem. 2003 Jul 11;278(28):25958-63. doi : 10.1074/jbc.M302439200. PMID: 12716889.

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Kamiyama S, Sasaki N, Goda E, Ui-Tei K, Saigo K, Narimatsu H, Jigami Y, Kannagi R, Irimura T, Nishihara S. Molecular cloning and characterization of a novel 3'-phosphoadenosine 5'-phosphosulfate transporter, PAPST2. J Biol Chem. 2006 Apr 21;281(16):10945-53. doi : 10.1074/jbc.M508991200. PMID:16492677.

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Roy SK, Chiba Y, Takeuchi M, Jigami Y. Characterization of Yeast Yea4p, a uridine diphosphate-N-acetylglucosamine transporter localized in the endoplasmic reticulum and required for chitin synthesis. J Biol Chem. 2000 May 5;275(18):13580-7. doi : 10.1074/jbc.275.18.13580. PMID: 10788474.

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Caffaro CE, Hirschberg CB. Nucleotide sugar transporters of the Golgi apparatus: from basic science to diseases. Acc Chem Res. 2006 Nov;39(11):805-12. doi : 10.1021/ar0400239. PMID: 17115720.

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Nakayama K, Maeda Y, Jigami Y. Interaction of GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase with GDP-mannose-4,6-dehydratase stabilizes the enzyme activity for formation of GDP-fucose from GDP-mannose. Glycobiology. 2003 Oct;13(10):673-80. doi : 10.1093/ glycob /cwg099. PMID: 12881408.

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Ito H, Fukuda Y, Murata K, Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163-8. doi : 10.1128/jb.153.1.163-168.1983. PMID: 6336730.

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H2. [figs-and-tables] Figures, Tables and Boxes Appendix

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Figure. Figure 1: Schematic diagram of nucleotide-sugar transporter (NST). Nucleotide sugar is synthesized in the cytosol or the nucleus. NST transports nucleotide sugars from the cytosol into the endoplasmic reticulum (ER)/Golgi lumens. Glycosyltransferases are then responsible for the transfer of sugars to acceptor substrates from the donor nucleotide sugars. Released nucleoside diphosphate (NDP) is hydrolyzed by nucleoside diphosphatase ( NDPase ) to form nucleoside monophosphate (NMP) and inorganic phosphate (Pi). NMP is exported in antiport with incoming nucleotide sugars. Pi exits through a phosphate transporter.

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At, Arabidopsis thaliana; Af , Aspergillus fumigatus; Bt , Bos Taurus; Ce, Caenorhabditis elegans; Ca, Candida albicans; Cgl , Candida glabrata; Cl, Canis lupus familiaris ; Cg, Cricetulus griseus; Cn, Cryptococcus neoformans; Dr, Danio rerio; Dm, Drosophila melanogaster; Eh, Entamoeba histolytica; Hs, Homo sapiens; Kl, Kluyveromyces lactis; Ld , Leishmania donovani ; Lm , Leishmania major; Mm, Mus musculus; Os , Oryza sativa Japonica; Pp, Pichia pastoris; Rn, Rattus norvegicus; Sc, Saccharomyces cerevisiae; Sp , Schizosaccharomyces pombe; Ss, Sus scrofa; Tg , Toxoplasma gondii; Tb, Trypanosoma brucei.

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Figure. Figure 2: Phylogenetic tree of the members of the solute carrier 35 (SLC35) transporter family. The phylogenetic tree was created based on amino acid sequences using the ClustalX program. Branch length indicate s evolutionary distances between members. The scale at the top represents the evolutionary distance. Human SLC35 transporters are shown in red.

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Table. Table 1: The members of nucleotide-sugar transporter (NST) family, solute carrier 35 (SLC35): Sub-group SLC35A.

Footer: * Only the NSTs, of which substrates have already been determined, are described with references. [substrates: name of transporters (reference)] Abbreviation: At, Arabidopsis thaliana; Af , Aspergillus fumigatus; Bt , Bos Taurus; Ce, Caenorhabditis elegans; Ca, Candida albicans; Cgl , Candida glabrata; Cl, Canis lupus familiaris ; Cg, Cricetulus griseus; Cn, Cryptococcus neoformans; Dr, Danio rerio; Do, Dendrobium officinale; Dm, Drosophila melanogaster; Eh, Entamoeba histolytica; Hs, Homo sapiens; Kl, Kluyveromyces lactis; Ld , Leishmania donovani ; Lm , Leishmania major; Mm, Mus musculus; Os , Oryza sativa Japonica; Pp, Pichia pastoris; Sc, Saccharomyces cerevisiae; Sp , Schizosaccharomyces pombe; Ss, Sus scrofa; Tg , Toxoplasma gondii; Tb, Trypanosoma brucei; N/A, not applicable.

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Table. Table 2: The members of nucleotide - sugar transporter (NST) family, solute carrier 35 (SLC35): Sub-group SLC35B.

Footer: * Only the NSTs, of which substrates have already been determined, are described with references. [substrates: name of transporters (reference)] Abbreviation: At, Arabidopsis thaliana; Af , Aspergillus fumigatus; Bt , Bos Taurus; Ce, Caenorhabditis elegans; Ca, Candida albicans; Cgl , Candida glabrata; Cl, Canis lupus familiaris ; Cg, Cricetulus griseus; Cn, Cryptococcus neoformans; Dr, Danio rerio; Do, Dendrobium officinale; Dm, Drosophila melanogaster; Eh, Entamoeba histolytica; Hs, Homo sapiens; Kl, Kluyveromyces lactis; Ld , Leishmania donovani ; Lm , Leishmania major; Mm, Mus musculus; Os , Oryza sativa Japonica; Pp, Pichia pastoris; Sc, Saccharomyces cerevisiae; Sp , Schizosaccharomyces pombe; Ss, Sus scrofa; Tg , Toxoplasma gondii; Tb, Trypanosoma brucei; N/A, not applicable.

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Table. Table 3: The members of nucleotide - sugar transporter (NST) family, solute carrier 35 (SLC35): Sub-groups, SLC35C , and SLC35D.

Footer: * Only the NSTs, of which substrates have already been determined, are described with references. [substrates: name of transporters (reference)] Abbreviation: At, Arabidopsis thaliana; Af , Aspergillus fumigatus; Bt , Bos Taurus; Ce, Caenorhabditis elegans; Ca, Candida albicans; Cgl , Candida glabrata; Cl, Canis lupus familiaris ; Cg, Cricetulus griseus; Cn, Cryptococcus neoformans; Dr, Danio rerio; Do, Dendrobium officinale; Dm, Drosophila melanogaster; Eh, Entamoeba histolytica; Hs, Homo sapiens; Kl, Kluyveromyces lactis; Ld , Leishmania donovani ; Lm , Leishmania major; Mm, Mus musculus; Os , Oryza sativa Japonica; Pp, Pichia pastoris; Sc, Saccharomyces cerevisiae; Sp , Schizosaccharomyces pombe; Ss, Sus scrofa; Tg , Toxoplasma gondii; Tb, Trypanosoma brucei; N/A, not applicable.

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Table. Table 4: The members of nucleotide - sugar transporter (NST) family, solute carrier 35 (SLC35): Sub-groups, GDP-Man, SLC35E , and SLC35F.

Footer: *Only the NSTs, of which substrates have already been determined, are described with references. [substrates: name of transporters (reference)] Abbreviation: At, Arabidopsis thaliana; Af , Aspergillus fumigatus; Bt , Bos Taurus; Ce, Caenorhabditis elegans; Ca, Candida albicans; Cgl , Candida glabrata; Cl, Canis lupus familiaris ; Cg, Cricetulus griseus; Cn, Cryptococcus neoformans; Dr, Danio rerio; Do, Dendrobium officinale; Dm, Drosophila melanogaster; Eh, Entamoeba histolytica; Hs, Homo sapiens; Kl, Kluyveromyces lactis; Ld , Leishmania donovani ; Lm , Leishmania major; Mm, Mus musculus; Os , Oryza sativa Japonica; Pp, Pichia pastoris; Sc, Saccharomyces cerevisiae; Sp , Schizosaccharomyces pombe; Ss, Sus scrofa; Tg , Toxoplasma gondii; Tb, Trypanosoma brucei; N/A, not applicable.