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Intact glycosphingolipidomic analysis of the cell membrane during differentiation yields extensive glycan and lipid changes.

ABSTRACT: Glycosphingolipids (GSLs) are found in cellular membranes of most organisms and play important roles in cell-cell recognition, signaling, growth, and adhesion, among others. A method based on nanoflow high performance liquid chromatography-chip-quadrupole-time-of-flight mass spectrometry (nanoHPLC Chip-Q-TOF MS) was applied towards identifying and quantifying intact GSLs from a variety of samples, including cultured cell lines and animal tissue. The method provides the composition and sequence of the glycan, as well as variations in the ceramide portion of the GSL. It was used to profile the changes in the glycolipidome of Caco-2 cells as they undergo differentiation. A total of 226 unique GSLs were found among Caco-2 samples from five differentiation time-points. The method provided a comprehensive glycolipidomic profile of a cell during differentiation to yield the dynamic variation of intact GSL structures.

SUBMITTER: Wong M

PROVIDER: S-EPMC6054638 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Intact glycosphingolipidomic analysis of the cell membrane during differentiation yields extensive glycan and lipid changes.

Wong Maurice M Xu Gege G Park Dayoung D Barboza Mariana M Lebrilla Carlito B CB

Scientific reports 20180720 1

Glycosphingolipids (GSLs) are found in cellular membranes of most organisms and play important roles in cell-cell recognition, signaling, growth, and adhesion, among others. A method based on nanoflow high performance liquid chromatography-chip-quadrupole-time-of-flight mass spectrometry (nanoHPLC Chip-Q-TOF MS) was applied towards identifying and quantifying intact GSLs from a variety of samples, including cultured cell lines and animal tissue. The method provides the composition and sequence o ...[more]

PMID: 30030471

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Project description:Objective: It has been demonstrated that the transcription factors TAZ (transcriptional coactivator with PDZ-binding motif), paired box gene 8 (PAX8), and NK2 homeobox 1 (NKX2-1) are coexpressed in the nucleus of thyroid cells. Furthermore, TAZ is known to enhance the transcriptional activity of PAX8 and NKX2-1 as well as the key thyroid-specific gene, thyroglobulin (TG), suggesting a critical role for TAZ in the control of thyroid cell speciation. We previously reported that the small molecule ethacridine, identified as a TAZ activator, was able to induce thyroid-specific transcription in endodermal cells differentiated from human embryonic stem (hES) cells using activin A. Since transcription factors are epigenetically regulated in cell differentiation, we investigated the epigenetic changes in the promoter regions of these key transcription factors during in vitro differentiation of hES cells into thyrocytes. Methods: We initially profiled chromatin accessibility using the technique of Assay for Transposase Accessible Chromatin sequencing (ATAC-seq), and then examined DNA methylation and histone acetylation in the promoter regions of the three selected thyroid transcription factors and the thyroid-specific genes during hES cell differentiation. Results: ATAC-seq analysis showed enriched chromatin accessibility of TAZ, NKX2-1, and PAX8 after exposure to activin A and ethacridine. There were no methylation changes found in the NKX2-1, PAX8, and TAZ promoters by bisulfite sequencing. In contrast, acetylation of histone H4, specifically acetylation of lysine 16, was observed in each of the promoters when measured by chromatin immunoprecipitation polymerase chain reaction assays, which correlated with the activity and expression of NKX2-1 and PAX8 as well as sodium/iodide symporter, thyroid stimulating hormone receptor, and TG genes. Conclusions: These results indicate that ethacridine treatment of activin A-derived endodermal hES cells leads to enhanced chromatin accessibility, which, in turn, allows histone H4 acetylation in the regulation of active genes for speciation of thyroid follicular cells from hES cells.

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Dataset Information

Intact glycosphingolipidomic analysis of the cell membrane during differentiation yields extensive glycan and lipid changes.

Publications

Intact glycosphingolipidomic analysis of the cell membrane during differentiation yields extensive glycan and lipid changes.

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