Summary of Study ST003108

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001931. The data can be accessed directly via it's Project DOI: 10.21228/M84428 This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Perform statistical analysis  |  Show all samples  |  Show named metabolites  |  Download named metabolite data  
Download mwTab file (text)   |  Download mwTab file(JSON)   |  Download data files (Contains raw data)
Study IDST003108
Study TitleComplete absence of GLUT1 does not impair human terminal erythroid differentiation
Study SummaryThe Glucose transporter 1 (GLUT1) is one of the most abundant proteins within the erythrocyte membrane and is required for glucose and dehydroascorbic acid (Vitamin C precursor) transport. It is widely recognized as a key protein for red cell structure, function, and metabolism. Previous reports highlighted the importance of GLUT1 activity within these uniquely glycolysis-dependent cells, in particular for increasing antioxidant capacity needed to avoid irreversible damage from oxidative stress in humans. However, studies of glucose transporter roles in erythroid cells are complicated by species-specific differences between humans and mice. Here, using CRISPRmediated gene editing of immortalized erythroblasts and adult CD34+ hematopoietic progenitor cells, we generate committed human erythroid cells completely deficient in expression of GLUT1. We show that absence of GLUT1 does not impede human erythroblast proliferation, differentiation, or enucleation. This work demonstrates for the first-time generation of enucleated human reticulocytes lacking GLUT1. The GLUT1-deficient reticulocytes possess no tangible alterations to membrane composition or deformability in reticulocytes. Metabolomic analyses of GLUT1-deficient reticulocytes reveal hallmarks of reduced glucose import, downregulated metabolic processes and upregulated AMPK-signalling, alongside alterations in antioxidant metabolism, resulting in increased osmotic fragility and metabolic shifts indicative of higher oxidant stress. Despite detectable metabolic changes in GLUT1 deficient reticulocytes, the absence of developmental phenotype, detectable proteomic compensation or impaired deformability comprehensively alters our understanding of the role of GLUT1 in red blood cell structure, function and metabolism. It also provides cell biological evidence supporting clinical consensus that reduced GLUT1 expression does not cause anaemia in GLUT1 deficiency syndrome.
Institute
University of Colorado
Last NameStephenson
First NameDaniel
AddressResearch 1 South L18-1303 12801 E. 17th Ave., Aurora, Colorado, 80045, USA
Emaildaniel.stephenson@cuanschutz.edu
Phone303-724-3339
Submit Date2024-02-27
Raw Data AvailableYes
Raw Data File Type(s)mzXML, raw(Thermo)
Analysis Type DetailLC-MS
Release Date2024-03-20
Release Version1
Daniel Stephenson Daniel Stephenson
https://dx.doi.org/10.21228/M84428
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR001931
Project DOI:doi: 10.21228/M84428
Project Title:Complete absence of GLUT1 does not impair human terminal erythroid differentiation
Project Summary:The Glucose transporter 1 (GLUT1) is one of the most abundant proteins within the erythrocyte membrane and is required for glucose and dehydroascorbic acid (Vitamin C precursor) transport. It is widely recognized as a key protein for red cell structure, function, and metabolism. Previous reports highlighted the importance of GLUT1 activity within these uniquely glycolysis-dependent cells, in particular for increasing antioxidant capacity needed to avoid irreversible damage from oxidative stress in humans. However, studies of glucose transporter roles in erythroid cells are complicated by species-specific differences between humans and mice. Here, using CRISPRmediated gene editing of immortalized erythroblasts and adult CD34+ hematopoietic progenitor cells, we generate committed human erythroid cells completely deficient in expression of GLUT1. We show that absence of GLUT1 does not impede human erythroblast proliferation, differentiation, or enucleation. This work demonstrates for the first-time generation of enucleated human reticulocytes lacking GLUT1. The GLUT1-deficient reticulocytes possess no tangible alterations to membrane composition or deformability in reticulocytes. Metabolomic analyses of GLUT1-deficient reticulocytes reveal hallmarks of reduced glucose import, downregulated metabolic processes and upregulated AMPK-signalling, alongside alterations in antioxidant metabolism, resulting in increased osmotic fragility and metabolic shifts indicative of higher oxidant stress. Despite detectable metabolic changes in GLUT1 deficient reticulocytes, the absence of developmental phenotype, detectable proteomic compensation or impaired deformability comprehensively alters our understanding of the role of GLUT1 in red blood cell structure, function and metabolism. It also provides cell biological evidence supporting clinical consensus that reduced GLUT1 expression does not cause anaemia in GLUT1 deficiency syndrome.
Institute:University of Colorado
Last Name:Stephenson
First Name:Daniel
Address:Research 1 South L18-1303 12801 E. 17th Ave., Aurora, Colorado, 80045, USA
Email:daniel.stephenson@cuanschutz.edu
Phone:303-724-3339

Subject:

Subject ID:SU003223
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606

Factors:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Sample source Assay
SA333676DS1-095-007L-cultured reticulocytes 01CF19
SA333677DS1-095-014-cultured reticulocytes 01CF19
SA333678DS1-095-013-cultured reticulocytes 01CF19
SA333679DS1-095-007L+cultured reticulocytes 01CF19
SA333680DS1-095-015-cultured reticulocytes 01CF19
SA333681DS1-095-008L-cultured reticulocytes 01CF19
SA333682DS1-095-010L+cultured reticulocytes 01CF19
SA333683DS1-095-013L+cultured reticulocytes 01CF19
SA333684DS1-095-014L+cultured reticulocytes 01CF19
SA333685DS1-095-015L+cultured reticulocytes 01CF19
SA333686DS1-095-012L+cultured reticulocytes 01CF19
SA333687DS1-095-011L+cultured reticulocytes 01CF19
SA333688DS1-095-009L+cultured reticulocytes 01CF19
SA333689DS1-095-012-cultured reticulocytes 01CF19
SA333690DS1-095-008L+cultured reticulocytes 01CF19
SA333691DS1-095-009-cultured reticulocytes 01CF19
SA333692DS1-095-014L-cultured reticulocytes 01CF19
SA333693DS1-095-013L-cultured reticulocytes 01CF19
SA333694DS1-095-011-cultured reticulocytes 01CF19
SA333695DS1-095-009+cultured reticulocytes 01CF19
SA333696DS1-095-015+cultured reticulocytes 01CF19
SA333697DS1-095-014+cultured reticulocytes 01CF19
SA333698DS1-095-011+cultured reticulocytes 01CF19
SA333699DS1-095-012+cultured reticulocytes 01CF19
SA333700DS1-095-013+cultured reticulocytes 01CF19
SA333701DS1-095-008+cultured reticulocytes 01CF19
SA333702DS1-095-007+cultured reticulocytes 01CF19
SA333703DS1-095-008-cultured reticulocytes 01CF19
SA333704DS1-095-015L-cultured reticulocytes 01CF19
SA333705DS1-095-010-cultured reticulocytes 01CF19
SA333706DS1-095-007-cultured reticulocytes 01CF19
SA333707DS1-095-009L-cultured reticulocytes 01CF19
SA333708DS1-095-012L-cultured reticulocytes 01CF19
SA333709DS1-095-011L-cultured reticulocytes 01CF19
SA333710DS1-095-010L-cultured reticulocytes 01CF19
SA333711DS1-095-010+cultured reticulocytes 01CF19
SA333712DS1-095-003L-cultured reticulocytes 04CF29
SA333713DS1-095-002L-cultured reticulocytes 04CF29
SA333714DS1-095-006L-cultured reticulocytes 04CF29
SA333715DS1-095-005L-cultured reticulocytes 04CF29
SA333716DS1-095-004L-cultured reticulocytes 04CF29
SA333717DS1-095-001L+cultured reticulocytes 04CF29
SA333718DS1-095-001-cultured reticulocytes 04CF29
SA333719DS1-095-002-cultured reticulocytes 04CF29
SA333720DS1-095-003-cultured reticulocytes 04CF29
SA333721DS1-095-006+cultured reticulocytes 04CF29
SA333722DS1-095-005+cultured reticulocytes 04CF29
SA333723DS1-095-002+cultured reticulocytes 04CF29
SA333724DS1-095-003+cultured reticulocytes 04CF29
SA333725DS1-095-004+cultured reticulocytes 04CF29
SA333726DS1-095-004-cultured reticulocytes 04CF29
SA333727DS1-095-005-cultured reticulocytes 04CF29
SA333728DS1-095-004L+cultured reticulocytes 04CF29
SA333729DS1-095-005L+cultured reticulocytes 04CF29
SA333730DS1-095-006L+cultured reticulocytes 04CF29
SA333731DS1-095-003L+cultured reticulocytes 04CF29
SA333732DS1-095-002L+cultured reticulocytes 04CF29
SA333733DS1-095-006-cultured reticulocytes 04CF29
SA333734DS1-095-001+cultured reticulocytes 04CF29
SA333735DS1-095-001L-cultured reticulocytes 04CF29
SA333664DS1-095-017+Red blood cells 01CF19
SA333665DS1-095-018+Red blood cells 01CF19
SA333666DS1-095-016-Red blood cells 01CF19
SA333667DS1-095-017-Red blood cells 01CF19
SA333668DS1-095-018-Red blood cells 01CF19
SA333669DS1-095-016L+Red blood cells 01CF19
SA333670DS1-095-017L+Red blood cells 01CF19
SA333671DS1-095-017L-Red blood cells 01CF19
SA333672DS1-095-018L-Red blood cells 01CF19
SA333673DS1-095-016L-Red blood cells 01CF19
SA333674DS1-095-018L+Red blood cells 01CF19
SA333675DS1-095-016+Red blood cells 01CF19
Showing results 1 to 72 of 72

Collection:

Collection ID:CO003216
Collection Summary:For flow cytometry, cells were fixed (1% paraformaldehyde, 0.0075% glutaraldehyde) to prevent antibody agglutination, except for assays using GLUT1.RBD. Samples were labelled with primary antibodies in PBSAG (PBS with 1% (w/v) Glucose and 0.5%(w/v) |Bovine Serum Albumin (BSA)) supplemented with extra 1%(w/v) BSA for 30min, in the dark. METAFORA’s GLUT1.RBD was incubated at 37°C and all remaining antibodies incubated at 4°C. Cells were washed 2x PBSAG and, if required, incubated with appropriate secondary antibody under the same conditions as described for primary. Cells were washed 2x with PBSAG and analysed on a Miltenyi MACSQuant 10 flow cytometer. Data was analysed using FlowJo v10.7 (FlowJo). Reticulocytes were identified by gating on the Hoechst-negative population. 4 Cells were sorted using a BD Influx Cell Sorter (BD Biosciences). BEL-A CRISPR edited populations were single cell sorted based on viability (DRAQ7 negativity). Primary GLUT1 KO cultures were sorted on days 6 or 7 of differentiation using DRAQ7 and the eGFP-fused GLUT1.RBD to purify the negative population with a gate based on non-targeting guide control cells.
Sample Type:Cultured cells

Treatment:

Treatment ID:TR003232
Treatment Summary:Filtered reticulocytes (1-2 x105/well) were incubated in decreasing NaCl concentrations (0.9–0%) for 10min at 37°C. Lysis was stopped by adding 4x volume of PBSAG. Live cells, considered as having a normal FSC/SSC profile as defined by the 0.9% NaCl control, were counted by flow cytometry using the MACSQuant10

Sample Preparation:

Sampleprep ID:SP003229
Sampleprep Summary:Extraction of metabolites and lipids from red cell pellets was as follows: 36 µL of cold MeOH:MeCN:H2O (5:3:2, v:v:v) was added to all pellets (pellet size pre-determined to be 10 million prior to extraction). Samples were vortexed at 4°C for 30 minutes. Insoluble material was pelleted by centrifugation (18000g, 10 min) and supernatants were isolated for analysis by UHPLC-MS for metabolomics and lipidomics.
Sampleprep Protocol Filename:Catarina_Freire_Lipidomics_and_Metabolomics_Methods.pdf

Combined analysis:

Analysis ID AN005087 AN005088 AN005089 AN005090
Analysis type MS MS MS MS
Chromatography type Reversed phase Reversed phase Reversed phase Reversed phase
Chromatography system Thermo Vanquish Thermo Vanquish Thermo Vanquish Thermo Vanquish
Column Phenomenex Kinetex C18 (150 x 2.1mm,1.7um) Phenomenex Kinetex C18 (150 x 2.1mm,1.7um) Phenomenex Kinetex C18 (30 x 2.1mm, 1.7um) Phenomenex Kinetex C18 (30 x 2.1mm, 1.7um)
MS Type ESI ESI ESI ESI
MS instrument type Orbitrap Orbitrap Orbitrap Orbitrap
MS instrument name Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap
Ion Mode POSITIVE NEGATIVE POSITIVE NEGATIVE
Units area area area area

Chromatography:

Chromatography ID:CH003843
Chromatography Summary:Metabolomics Positive
Methods Filename:Catarina_Freire_Lipidomics_and_Metabolomics_Methods.pdf
Instrument Name:Thermo Vanquish
Column Name:Phenomenex Kinetex C18 (150 x 2.1mm,1.7um)
Column Temperature:45
Flow Gradient:0 min - 0.45 ml/min - 5% B, 0.5 min - 0.45ml/min - 5% B, 1.1 min - 0.45ml/min - 95% B, 2.75 min - 0.45ml/min - 95% B, 3 min - 0.45ml/min - 5% B, 5min - 0.45ml/min - 5%B
Flow Rate:0.45 ml/min
Solvent A:0.1% Formic Acid in Water
Solvent B:0.1% Formic Acid in ACN
Chromatography Type:Reversed phase
  
Chromatography ID:CH003844
Chromatography Summary:Metabolomics Negative
Methods Filename:Catarina_Freire_Lipidomics_and_Metabolomics_Methods.pdf
Instrument Name:Thermo Vanquish
Column Name:Phenomenex Kinetex C18 (150 x 2.1mm,1.7um)
Column Temperature:45
Flow Gradient:0 min - 0.45 ml/min - 0% B, 0.5 min - 0.45ml/min - 0% B, 1.1 min - 0.45ml/min - 100% B, 2.75 min - 0.45ml/min - 100% B, 3 min - 0.45ml/min - 0% B, 5min - 0.45ml/min - 0%B
Flow Rate:0.45 ml/min
Solvent A:5% ACN 95% Water 1mM Ammonium Acetate
Solvent B:95% ACN 5% Water 1mM Ammonium Acetate
Chromatography Type:Reversed phase
  
Chromatography ID:CH003845
Chromatography Summary:Lipidomics Positive
Methods Filename:Catarina_Freire_Lipidomics_and_Metabolomics_Methods.pdf
Instrument Name:Thermo Vanquish
Column Name:Phenomenex Kinetex C18 (30 x 2.1mm, 1.7um)
Column Temperature:50
Flow Gradient:0 min - 0.3ml/min - 30%B, 3 min - 0.3ml/min - 100%B, 4.2min - 0.3ml/min - 100%B, 4.3min - 0.4ml/min - 30%B, 4.9min - 0/4ml/min - 30%B, 5 min - 0.3ml/min 30%B
Flow Rate:0.3-0.4ml/min
Solvent A:75:25 H2O:ACN 5mM NH4OAc
Solvent B:90:10 iPrOH:ACN 5mM NH4OAc
Chromatography Type:Reversed phase
  
Chromatography ID:CH003846
Chromatography Summary:Lipidomics Negative
Methods Filename:Catarina_Freire_Lipidomics_and_Metabolomics_Methods.pdf
Instrument Name:Thermo Vanquish
Column Name:Phenomenex Kinetex C18 (30 x 2.1mm, 1.7um)
Column Temperature:50
Flow Gradient:0 min - 0.3ml/min - 10%B, 3 min - 0.3ml/min - 95%B, 4.2min - 0.3ml/min - 95%B, 4.3min - 0.45ml/min - 10%B, 4.9min - 0.4ml/min - 10%B, 5 min - 0.3ml/min 10%B
Flow Rate:0.3-0.45ml/min
Solvent A:75:25 H2O:ACN 5mM NH4OAc
Solvent B:90:10 iPrOH:ACN 5mM NH4OAc
Chromatography Type:Reversed phase

MS:

MS ID:MS004824
Analysis ID:AN005087
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:El-Maven used for data analysis. Orbitrap scanning in full MS mode from 65-975 m/z at 60,000 resolution, with 50 Arb sheath gas, 10 Arb auxiliary gas, and 3.4 kV spray voltage.
Ion Mode:POSITIVE
  
MS ID:MS004825
Analysis ID:AN005088
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:El-Maven used for data analysis. Orbitrap scanning in full MS mode from 65-975 m/z at 60,000 resolution, with 50 Arb sheath gas, 10 Arb auxiliary gas, and 3 kV spray voltage.
Ion Mode:NEGATIVE
  
MS ID:MS004826
Analysis ID:AN005089
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Lipidsearch used for data analysis. Orbitrap scanning using data dependent MS2 (top 10) from 125-1500 m/z at 17,500 resolution, with 45 Arb sheath gas, 25 Arb auxiliary gas, and 4 kV spray voltage.
Ion Mode:POSITIVE
  
MS ID:MS004827
Analysis ID:AN005090
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Lipidsearch used for data analysis. Orbitrap scanning using data dependent MS2 (top 10) from 125-1500 m/z at 17,500 resolution, with 45 Arb sheath gas, 25 Arb auxiliary gas, and 4 kV spray voltage.
Ion Mode:NEGATIVE
  logo