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Source file: g111-sugartransport.docx
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Notes: Parsed 9 headings, 88 blocks, 28 references, 133 citations, 43 issues.
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Headings: 9
Paragraphs: 58
References: 28
Citations: 133
Matched citations: 90
Open issues: 43

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H1.

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

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

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Keywords : nucleotide sugar, nucleotide sugar transporter, transport activity, Golgi apparatus , endoplasmic reticulum, the solute carrier 35 transporter family, SLC35

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H2. Introduction

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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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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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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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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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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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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

reference Order 62 word/document.xml:/w:document[1]/w:body[1]/w:p[60]

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.

reference Order 63 word/document.xml:/w:document[1]/w:body[1]/w:p[61]

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.

reference Order 64 word/document.xml:/w:document[1]/w:body[1]/w:p[62]

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.

reference Order 65 word/document.xml:/w:document[1]/w:body[1]/w:p[63]

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.

reference Order 66 word/document.xml:/w:document[1]/w:body[1]/w:p[64]

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.

reference Order 67 word/document.xml:/w:document[1]/w:body[1]/w:p[65]

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.

reference Order 68 word/document.xml:/w:document[1]/w:body[1]/w:p[66]

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.

reference Order 69 word/document.xml:/w:document[1]/w:body[1]/w:p[67]

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.

reference Order 70 word/document.xml:/w:document[1]/w:body[1]/w:p[68]

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.

reference Order 71 word/document.xml:/w:document[1]/w:body[1]/w:p[69]

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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Conflicts of Interest :

reference Order 73 word/document.xml:/w:document[1]/w:body[1]/w:p[71]

The authors declare no competing or financial interests .

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Figures, Tables and Boxes Appendix

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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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reference Order 78 word/document.xml:/w:document[1]/w:body[1]/w:p[76]

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.

reference Order 79 word/document.xml:/w:document[1]/w:body[1]/w:p[77]

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

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* Only the NSTs, of which substrates have already been determined, are described with references. [substrates: name of transporters (reference)]

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

reference Order 86 word/document.xml:/w:document[1]/w:body[1]/w:p[82]

* Only the NSTs, of which substrates have already been determined, are described with references. [substrates: name of transporters (reference)]

reference Order 87 word/document.xml:/w:document[1]/w:body[1]/w:p[83]

reference Order 88 word/document.xml:/w:document[1]/w:body[1]/w:p[84]

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

reference Order 90 word/document.xml:/w:document[1]/w:body[1]/w:p[85]

* Only the NSTs, of which substrates have already been determined, are described with references. [substrates: name of transporters (reference)]

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

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*Only the NSTs, of which substrates have already been determined, are described with references. [substrates: name of transporters (reference)]

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Extracted references

Reference list

reference Order 1 Status matched Block 68

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reference Order 4 Status matched Block 71

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.

reference Order 5 Status matched Block 72

reference Order 6 Status matched Block 73

reference Order 7 Status matched Block 74

reference Order 8 Status matched Block 75

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.

reference Order 9 Status matched Block 76

reference Order 10 Status matched Block 77

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.

reference Order 11 Status unmatched Block 78

Conflicts of Interest :

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The authors declare no competing or financial interests .

reference Order 13 Status unmatched Block 80

Figures, Tables and Boxes Appendix

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reference Order 17 Status unmatched Block 86

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

reference Order 18 Status unmatched Block 87

* Only the NSTs, of which substrates have already been determined, are described with references. [substrates: name of transporters (reference)]

reference Order 19 Status unmatched Block 88

reference Order 20 Status matched Block 89

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

reference Order 21 Status matched Block 90

* Only the NSTs, of which substrates have already been determined, are described with references. [substrates: name of transporters (reference)]

reference Order 22 Status matched Block 91

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

reference Order 24 Status matched Block 93

* Only the NSTs, of which substrates have already been determined, are described with references. [substrates: name of transporters (reference)]

reference Order 25 Status matched Block 94

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

reference Order 27 Status matched Block 96

*Only the NSTs, of which substrates have already been determined, are described with references. [substrates: name of transporters (reference)]

reference Order 28 Status matched Block 97

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unmatched_citation Status open Citation 99

Unmatched citation "20 - 40"

unmatched_citation Status open Citation 100

Unmatched citation "20 - 40"

unmatched_citation Status open Citation 101

Unmatched citation "20 - 40"

unmatched_citation Status open Citation 102

Unmatched citation "20 - 40"

unmatched_citation Status open Citation 103

Unmatched citation "20 - 40"

unmatched_citation Status open Citation 104

Unmatched citation "20 - 40"

unmatched_citation Status open Citation 105

Unmatched citation "1,000"

unmatched_citation Status open Citation 108

Unmatched citation "10,000"

unmatched_citation Status open Citation 111

Unmatched citation "100,000"

unmatched_citation Status open Citation 112

Unmatched citation "100,000"

unmatched_citation Status open Citation 114

Unmatched citation "1 , 000"

unmatched_citation Status open Citation 118

Unmatched citation "1,000,000"

unmatched_citation Status open Citation 120

Unmatched citation "100"

unmatched_citation Status open Citation 121

Unmatched citation "240"

unmatched_citation Status open Citation 122

Unmatched citation "100"

unmatched_citation Status open Citation 123

Unmatched citation "30"

unmatched_citation Status open Citation 127

Unmatched citation "0"

Detected citations

Citation match results

Order	Style	Raw text	Normalized	Match state	Reference	Confidence
1	numeric	35	35	unmatched	None	n/a
2	numeric	1	1	matched	1	1.0000
Candidate references: 1
3	numeric	1 - 4	1	matched	1	1.0000
Candidate references: 1
4	numeric	1 - 4	2	matched	2	1.0000
Candidate references: 2
5	numeric	1 - 4	3	matched	3	1.0000
Candidate references: 3
6	numeric	1 - 4	4	matched	4	1.0000
Candidate references: 4
7	numeric	3	3	matched	3	1.0000
Candidate references: 3
8	numeric	5	5	matched	5	1.0000
Candidate references: 5
9	numeric	5, 6	5	matched	5	1.0000
Candidate references: 5
10	numeric	5, 6	6	matched	6	1.0000
Candidate references: 6
11	numeric	2	2	matched	2	1.0000
Candidate references: 2
12	numeric	1 , 2, 3	1	matched	1	1.0000
Candidate references: 1
13	numeric	1 , 2, 3	2	matched	2	1.0000
Candidate references: 2
14	numeric	1 , 2, 3	3	matched	3	1.0000
Candidate references: 3
15	numeric	4	4	matched	4	1.0000
Candidate references: 4
16	numeric	35	35	unmatched	None	n/a
17	numeric	1 - 4	1	matched	1	1.0000
Candidate references: 1
18	numeric	1 - 4	2	matched	2	1.0000
Candidate references: 2
19	numeric	1 - 4	3	matched	3	1.0000
Candidate references: 3
20	numeric	1 - 4	4	matched	4	1.0000
Candidate references: 4
21	numeric	4	4	matched	4	1.0000
Candidate references: 4
22	numeric	2	2	matched	2	1.0000
Candidate references: 2
23	numeric	2, 5 - 7	2	matched	2	1.0000
Candidate references: 2
24	numeric	2, 5 - 7	5	matched	5	1.0000
Candidate references: 5
25	numeric	2, 5 - 7	6	matched	6	1.0000
Candidate references: 6
26	numeric	2, 5 - 7	7	matched	7	1.0000
Candidate references: 7
27	numeric	8	8	matched	8	1.0000
Candidate references: 8
28	numeric	1	1	matched	1	1.0000
Candidate references: 1
29	numeric	2	2	matched	2	1.0000
Candidate references: 2
30	numeric	3	3	matched	3	1.0000
Candidate references: 3
31	numeric	2	2	matched	2	1.0000
Candidate references: 2
32	numeric	1	1	matched	1	1.0000
Candidate references: 1
33	numeric	50	50	unmatched	None	n/a
34	numeric	7	7	matched	7	1.0000
Candidate references: 7
35	numeric	10	10	matched	10	1.0000
Candidate references: 10
36	numeric	3 , 40	3	matched	3	1.0000
Candidate references: 3
37	numeric	3 , 40	40	unmatched	None	n/a
38	numeric	2	2	matched	2	1.0000
Candidate references: 2
39	numeric	1	1	matched	1	1.0000
Candidate references: 1
40	numeric	1	1	matched	1	1.0000
Candidate references: 1
41	numeric	0	0	unmatched	None	n/a
42	numeric	10	10	matched	10	1.0000
Candidate references: 10
43	numeric	7	7	matched	7	1.0000
Candidate references: 7
44	numeric	20	20	matched	20	1.0000
Candidate references: 20
45	numeric	7	7	matched	7	1.0000
Candidate references: 7
46	numeric	0	0	unmatched	None	n/a
47	numeric	5	5	matched	5	1.0000
Candidate references: 5
48	numeric	2 , 1	1	matched	1	1.0000
Candidate references: 1
49	numeric	2 , 1	2	matched	2	1.0000
Candidate references: 2
50	numeric	2	2	matched	2	1.0000
Candidate references: 2
51	numeric	10	10	matched	10	1.0000
Candidate references: 10
52	numeric	2	2	matched	2	1.0000
Candidate references: 2
53	numeric	20	20	matched	20	1.0000
Candidate references: 20
54	numeric	7	7	matched	7	1.0000
Candidate references: 7
55	numeric	0	0	unmatched	None	n/a
56	numeric	150	150	unmatched	None	n/a
57	numeric	1	1	matched	1	1.0000
Candidate references: 1
58	numeric	2	2	matched	2	1.0000
Candidate references: 2
59	numeric	0	0	unmatched	None	n/a
60	numeric	24	24	matched	24	1.0000
Candidate references: 24
61	numeric	9	9	matched	9	1.0000
Candidate references: 9
62	numeric	15	15	matched	15	1.0000
Candidate references: 15
63	numeric	112	112	unmatched	None	n/a
64	numeric	10	10	matched	10	1.0000
Candidate references: 10
65	numeric	30	30	unmatched	None	n/a
66	numeric	800	800	unmatched	None	n/a
67	numeric	600	600	unmatched	None	n/a
68	numeric	3	3	matched	3	1.0000
Candidate references: 3
69	numeric	3,000	0	unmatched	None	n/a
70	numeric	3,000	3	matched	3	1.0000
Candidate references: 3
71	numeric	5	5	matched	5	1.0000
Candidate references: 5
72	numeric	100	100	unmatched	None	n/a
73	numeric	10	10	matched	10	1.0000
Candidate references: 10
74	numeric	3	3	matched	3	1.0000
Candidate references: 3
75	numeric	30	30	unmatched	None	n/a
76	numeric	30	30	unmatched	None	n/a
77	numeric	35	35	unmatched	None	n/a
78	numeric	3,000	0	unmatched	None	n/a
79	numeric	3,000	3	matched	3	1.0000
Candidate references: 3
80	numeric	5	5	matched	5	1.0000
Candidate references: 5
81	numeric	3	3	matched	3	1.0000
Candidate references: 3
82	numeric	100	100	unmatched	None	n/a
83	numeric	20	20	matched	20	1.0000
Candidate references: 20
84	numeric	20 - 40	20	matched	20	1.0000
Candidate references: 20
85	numeric	20 - 40	21	matched	21	1.0000
Candidate references: 21
86	numeric	20 - 40	22	matched	22	1.0000
Candidate references: 22
87	numeric	20 - 40	23	matched	23	1.0000
Candidate references: 23
88	numeric	20 - 40	24	matched	24	1.0000
Candidate references: 24
89	numeric	20 - 40	25	matched	25	1.0000
Candidate references: 25
90	numeric	20 - 40	26	matched	26	1.0000
Candidate references: 26
91	numeric	20 - 40	27	matched	27	1.0000
Candidate references: 27
92	numeric	20 - 40	28	matched	28	1.0000
Candidate references: 28
93	numeric	20 - 40	29	unmatched	None	n/a
94	numeric	20 - 40	30	unmatched	None	n/a
95	numeric	20 - 40	31	unmatched	None	n/a
96	numeric	20 - 40	32	unmatched	None	n/a
97	numeric	20 - 40	33	unmatched	None	n/a
98	numeric	20 - 40	34	unmatched	None	n/a
99	numeric	20 - 40	35	unmatched	None	n/a
100	numeric	20 - 40	36	unmatched	None	n/a
101	numeric	20 - 40	37	unmatched	None	n/a
102	numeric	20 - 40	38	unmatched	None	n/a
103	numeric	20 - 40	39	unmatched	None	n/a
104	numeric	20 - 40	40	unmatched	None	n/a
105	numeric	1,000	0	unmatched	None	n/a
106	numeric	1,000	1	matched	1	1.0000
Candidate references: 1
107	numeric	10	10	matched	10	1.0000
Candidate references: 10
108	numeric	10,000	0	unmatched	None	n/a
109	numeric	10,000	10	matched	10	1.0000
Candidate references: 10
110	numeric	15	15	matched	15	1.0000
Candidate references: 15
111	numeric	100,000	0	unmatched	None	n/a
112	numeric	100,000	100	unmatched	None	n/a
113	numeric	1	1	matched	1	1.0000
Candidate references: 1
114	numeric	1 , 000	0	unmatched	None	n/a
115	numeric	1 , 000	1	matched	1	1.0000
Candidate references: 1
116	numeric	10	10	matched	10	1.0000
Candidate references: 10
117	numeric	2	2	matched	2	1.0000
Candidate references: 2
118	numeric	1,000,000	0	unmatched	None	n/a
119	numeric	1,000,000	1	matched	1	1.0000
Candidate references: 1
120	numeric	100	100	unmatched	None	n/a
121	numeric	240	240	unmatched	None	n/a
122	numeric	100	100	unmatched	None	n/a
123	numeric	30	30	unmatched	None	n/a
124	numeric	5	5	matched	5	1.0000
Candidate references: 5
125	numeric	1	1	matched	1	1.0000
Candidate references: 1
126	numeric	1	1	matched	1	1.0000
Candidate references: 1
127	numeric	0	0	unmatched	None	n/a
128	numeric	1	1	matched	1	1.0000
Candidate references: 1
129	numeric	10	10	matched	10	1.0000
Candidate references: 10
130	numeric	1	1	matched	1	1.0000
Candidate references: 1
131	numeric	4	4	matched	4	1.0000
Candidate references: 4
132	numeric	2	2	matched	2	1.0000
Candidate references: 2
133	numeric	2	2	matched	2	1.0000
Candidate references: 2