N<i>-</i>linked glycosylation of N48 is required for equilibrative nucleoside transporter 1 (ENT1) function

Bicket, Alex; Coe, Imogen R.

doi:10.1042/bsr20160063

Cited by 12 publications

(11 citation statements)

References 55 publications

(62 reference statements)

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“…Indeed, one of the templates used for the 3D modelling, namely ENT1, forms dimeric structures. 26,31 Moreover, Figure S1A shows, in some cases the presence of a faint band at a molecular mass about double that of the protein monomer, still in favor of the presence of formation of a dimeric structure that is maintained also after treatment of the samples with the SDS for gel running. However, additional investigations are in progress using biophysical methods to ascertain this possibility.…”

Section: Discussionmentioning

confidence: 97%

Impact of natural mutations on the riboflavin transporter 2 and their relevance to human riboflavin transporter deficiency 2

et al. 2021

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Riboflavin transporter deficiency 2 (RTD2) is a rare neurological disorder caused by mutations in the Solute carrier family 52 member 2 (Slc52a2) gene encoding human riboflavin transporter 2 (RFVT2). This transporter is ubiquitously expressed and mediates tissue distribution of riboflavin, a water-soluble vitamin that, after conversion into FMN and FAD, plays pivotal roles in carbohydrate, protein, and lipid metabolism. The 3D structure of RFVT2 has been constructed by homology modeling using three different templates that are equilibrative nucleoside transporter 1 (ENT1), Fucose: proton symporter, and glucose transporter type 5 (GLUT5). The structure has been validated by several approaches. All known point mutations of RFVT2, associated with RTD2, have been localized in the protein 3D model. Six of these mutations have been introduced in the recombinant protein for functional characterization. The mutants W31S, S52F, S128L, L312P, C325G, and M423V have been expressed in E. coli, purified, and reconstituted into proteoliposomes for transport assay.All the mutants showed impairment of function. The K m for riboflavin of the mutants increased from about 3 to 9 times with respect to that of WT, whereas V max was only marginally affected. This agrees with the improved outcome of most RTD2 patients after administration of high doses of riboflavin.

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Section: Discussionmentioning

confidence: 97%

Impact of natural mutations on the riboflavin transporter 2 and their relevance to human riboflavin transporter deficiency 2

et al. 2021

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“…In this regard, several studies have either suggested or demonstrated that human ENT (hENT) function can be ABBREVIATIONS: CID, collision-induced dissociation; CKII, casein kinase 2; Cl-Ado, chloroadenosine; ENT, equilibrative nucleoside transporter; ETD, electron transfer dissociation; HA, hemagglutinin; HEK-293, human embryonic kidney 293; hENT1, human equilibrative nucleoside transporter 1; hENT2, human equilibrative nucleoside transporter 2; His, histidine; Hx, hypoxanthine; LC, liquid chromatography; MS, mass spectrometry; MS/MS, tandem mass spectrometry; MYTH, membrane yeast 2 hybrid; NanoBiT, NanoLuc Binary Technology; PDD, phorbol 12,13-didecanoate; PP1, protein phosphatase 1; PPI, protein-protein interaction; TTM, tautomycin; Ubq, ubiquitin regulated by phosphorylation, glycosylation, ethanol, and calmodulin binding (8)(9)(10)(11) and even proposed oligomerization of ENT1 (9). However, there are still many unsolved gaps in the bigger picture of ENT regulation.…”

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confidence: 99%

Oligomerization of equilibrative nucleoside transporters: a novel regulatory and functional mechanism involving PKC and PP1

et al. 2018

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Equilibrative nucleoside transporters (ENTs) translocate nucleosides and nucleobases across plasma membranes, as well as a variety of anti‐cancer, ‐viral, and ‐parasite nucleoside analogs. They are also key members of the purinome complex and regulate the protective and anti‐inflammatory effects of adenosine. Despite their important role, little is known about the mechanisms involved in their regulation. We conducted membrane yeast 2‐hybrid and coimmunoprecipitation studies and identified, for the first time to our knowledge, the existence of protein‐protein interactions between human ENT1 and ENT2 (hENT1 and hENT2) proteins in human cells and the formation of hetero‐ and homo‐oligomers at the plasma membrane and the submembrane region. The use of NanoLuc Binary Technology allowed us to analyze changes in the oligomeric status of hENT1 and hENT2 and how they rapidly modify the uptake profile for nucleosides and nucleobases and allow cells to respond promptly to external signals or changes in the extracellular environment. These changes in hENTs oligomerization are triggered by PKC activation and subsequent action of protein phosphatase 1.—Grañe‐Boladeras, N., Williams, D., Tarmakova, Z., Stevanovic, K., Villani, L. A., Mehrabi, P., Siu, K. W. M., Pastor‐Anglada, M., Coe, I. R. Oligomerization of equilibrative nucleoside transporters: a novel regulatory and functional mechanism involving PKC and PP1. FASEB J. 33, 3841–3850 (2019). http://www.fasebj.org

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“…It means the large-scale flexible movement of the extracellular loop may be directly involved in the functional structural contraction of hENT1. Previous studies have suggested that the extracellular loop contains N-linked glycosylation sites and is involved in membrane trafficking and protein folding ( Sundaram et al., 2001 ; Bicket and Coe, 2016 ). Here, simulation data suggests that the extracellular loop may play an important role in the functional structural contraction of hENT1 and will be further clarified in the following analyses.…”

Section: Resultsmentioning

confidence: 99%

Insight into the nucleoside transport and inhibition of human ENT1

Han

Zhou

et al. 2022

Current Research in Structural Biology

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N-linked glycosylation of N48 is required for equilibrative nucleoside transporter 1 (ENT1) function

Abstract: Our study confirmed that Asn48 of hENT1 is the only N-glycosylated residue when expressed in HEK293 cells, and loss of the N-glycan resulted in less hENT1 at the plasma membrane, as well as a loss of function and protein–protein self-interaction.

Cited by 12 publications

References 55 publications

Impact of natural mutations on the riboflavin transporter 2 and their relevance to human riboflavin transporter deficiency 2

Impact of natural mutations on the riboflavin transporter 2 and their relevance to human riboflavin transporter deficiency 2

Oligomerization of equilibrative nucleoside transporters: a novel regulatory and functional mechanism involving PKC and PP1

Insight into the nucleoside transport and inhibition of human ENT1

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