Project description:In collaboration with Professor Anne Dell at the South Kensington Campus of Imperial College London, we have conducted preliminary glycomic surveys of C57BL/6 CD25+ and CD25- CD4+ T cells. Cells selected in serum-containing culture medium demonstrated unusual N-glycan profiles compared to those prepared in phosphate buffered saline (PBS). These profiles were subsequently shown to result from cellular sequestration of glycoproteins present in fetal calf serum (FCS) used to supplement the medium (1). Analysis of the N-glycan profile of murine CD4+CD25+ Tregs prepared in PBS revealed distinct differences from CD4+CD25- T cells – namely, a reduction in sialic acid capping and loss of core fucose. Preliminary studies are underway to ascertain the minimum medium required to sustain general T cell survival, whilst maintaining ‘clean’ glycomic profiles. The effect of glycoprotein withdrawal on CD25+ and CD25- CD4+ T cells in culture was investigated by selecting the cells in FCS-free medium and incubating them for six hours with either FCS-containing medium, or PBS supplemented with FCS, fetuin or fetuin-derived glycans. Early apoptosis was measured using Annexin-V (BD Biosciences, Heidelberg, Germany) and late apoptosis using 7-aminoactinomycin D (7-AAD) (BD Biosciences), in parallel with a complementary apoptosis detection method – measurement of the mitochondrial membrane potential using the BD™ Mitoscreen Kit (BD Biosciences). This apoptosis staining study demonstrated that there was a remarkably higher percentage of death in both CD25+ and CD25- CD4+ T cells cultured in PBS versus cells cultured in RPMI-1640 or medium containing FCS. Both CD25+ and CD25- CD4+ T cells were highly susceptible to apoptosis in glycoprotein-free medium, demonstrating their exquisite sensitivity to the lack of glycoprotein survival factors. Continuing studies are addressing the minimum medium required to sustain cells undergoing polyclonal activation in vitro and the impact of activation on the N-glycan profiles of Tregs versus conventional T cells. To complement this work, we used Core E resources to investigate glycosyltransferase gene expression in freshly isolated versus activated C57BL/6 CD25+ and CD25- CD4+ T cells.

Project description:Protein secretion is an essential process in cell biology. The conventional secretion pathway is well known, and involves proteins with a signal sequence being directed into the endoplasmic reticulum. Once in the lumen of the endoplasmic reticulum, proteins are trafficked through the Golgi and remain separate from the cytosol throughout their journey to the outside of the cell. More recently, proteins without a signal sequence have been found outside the cell. These proteins are secreted unconventionally, and avoid the ER-Golgi route. For both pathways, some aspects remain unclear, particularly surrounding the regulation of protein secretion. Galectin-3, an unconventionally secreted protein, was used here to investigate both conventional and unconventional protein secretion. The unconventional protein secretion of galectin-3 is poorly understood. To address this, genetic manipulation of galectin-3 was used to gain insights into the mechanism of galectin-3 secretion. It was found that galectin-3 does not require unfolding or binding to cell surface glycoproteins to be secreted. Galectin-3 was also used to assess protein secretion using a genome-wide CRISPR screen. As galectin‑3 binds to cell surface glycoproteins, galectin-3 on the cell surface was used to assess secretion of these glycoproteins. Hits were validated by a glycoprotein secretion assay. Using this pipeline, 93 hits were identified as involved in protein secretion, of which 51 were novel hits not previously associated with protein secretion. Many of the hits identified also resulted in a phenotype of altered Golgi morphology, and five of these were investigated further. Two novel hits that localised to the Golgi were identified, TMEM220 and GPR161. GPR161 likely mediates its effect on Golgi morphology by its interaction with golgin A5. This thesis presents new insights into both conventional and unconventional protein secretion. Insights into the mechanism for galaectin-3 secretion were revealed. A novel Golgi-localised regulator of secretion, GRP161, was identified, with a mechanism for action suggested. Importantly, the list of 51 novel genes identified will serve as a useful resource for other researchers investigating protein secretion.

Dataset Information

Mapping glycan-mediated galectin-3 interactions by live cell proximity labeling

Publications

Mapping glycan-mediated galectin-3 interactions by live cell proximity labeling.

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