Primary Human Colon Epithelial Cells (pHCoEpiCs) Do Express the Shiga Toxin (Stx) Receptor Glycosphingolipids Gb3Cer and Gb4Cer and Are Largely Refractory but Not Resistant towards Stx
Abstract
:1. Introduction
2. Results
2.1. Detection of Stx-Binding GSLs in pHCoEpiCs by Means of Immunochemical Analysis
2.2. Mass Spectrometric Determination of Stx Receptors Gb3Cer and Gb4Cer of pHCoEpiCs
2.3. Detection of Gb3Cer, Gb4Cer, and Cholesterol in Detergent-Resistant Membranes (DRMs) and nonDRMs Prepared from pHCoEpiCs
2.4. Gb3Cer and Gb4Cer Lipoforms Derived from Detergent-Resistant Membranes (DRMs) of pHCoEpiCs Grown under Low-Serum and Serum-Free Conditions
2.5. Identification of DRM and nonDRM Phospholipids
2.6. Stx-Mediated Cellular Damage of pHCoEpiCs
3. Discussion
4. Materials and Methods
4.1. Breeding of Primary Human Colon Epithelial Cells (pHCoEpiCs)
4.2. Treatment of pHCoEpiCs with Stx and Cytotoxicity Assay
4.3. Preparation of DRM and nonDRM Fractions
4.4. Lipid Extraction and Isolation of Glycosphingolipids from Total Cells
4.5. Analysis of Phospholipids and Glycosphingolipids in DRM and nonDRM Fractions
4.6. Affinity-Purified Stx Subtypes, Antibodies and Reference Lipid Mixtures
4.7. Thin-Layer Chromatography, Lipid Staining and Overlay Immunodetection
4.8. Electrospray Ionization Mass Spectrometry of Glycosphingolipids and Phospholipids
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Compound 2 | Ceramide | Formula | m/zexp 3 | m/zcalc 3 |
---|---|---|---|---|
Gb3Cer | d18:1, C16:0 | C52H97NO18Na | 1046.67 | 1046.6603 |
Gb3Cer | d18:1, C24:1 | C60H111NO18Na | 1156.77 | 1156.7699 |
Gb3Cer * | d18:1, C24:0 | C60H113NO18Na | 1158.78 | 1158.7855 |
Gb4Cer | d18:1, C16:0 | C60H110N2O23Na | 1249.74 | 1249.7397 |
Gb4Cer | d18:1, C22:1 | C66H120N2O23Na | 1331.82 | 1331.8180 |
Gb4Cer | d18:1, C22:0 | C66H122N2O23Na | 1333.84 | 1333.8336 |
Gb4Cer | d18:1, C24:2 | C68H122N2O23Na | 1357.83 | 1357.8336 |
Gb4Cer | d18:1, C24:1 | C68H124N2O23Na | 1359.85 | 1359.8493 |
Gb4Cer * | d18:1, C24:0 | C68H126N2O23Na | 1361.87 | 1361.8649 |
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Detzner, J.; Püttmann, C.; Pohlentz, G.; Humpf, H.-U.; Mellmann, A.; Karch, H.; Müthing, J. Primary Human Colon Epithelial Cells (pHCoEpiCs) Do Express the Shiga Toxin (Stx) Receptor Glycosphingolipids Gb3Cer and Gb4Cer and Are Largely Refractory but Not Resistant towards Stx. Int. J. Mol. Sci. 2021, 22, 10002. https://doi.org/10.3390/ijms221810002
Detzner J, Püttmann C, Pohlentz G, Humpf H-U, Mellmann A, Karch H, Müthing J. Primary Human Colon Epithelial Cells (pHCoEpiCs) Do Express the Shiga Toxin (Stx) Receptor Glycosphingolipids Gb3Cer and Gb4Cer and Are Largely Refractory but Not Resistant towards Stx. International Journal of Molecular Sciences. 2021; 22(18):10002. https://doi.org/10.3390/ijms221810002
Chicago/Turabian StyleDetzner, Johanna, Charlotte Püttmann, Gottfried Pohlentz, Hans-Ulrich Humpf, Alexander Mellmann, Helge Karch, and Johannes Müthing. 2021. "Primary Human Colon Epithelial Cells (pHCoEpiCs) Do Express the Shiga Toxin (Stx) Receptor Glycosphingolipids Gb3Cer and Gb4Cer and Are Largely Refractory but Not Resistant towards Stx" International Journal of Molecular Sciences 22, no. 18: 10002. https://doi.org/10.3390/ijms221810002