Investigation of the function of SLC35F6, a lysosomal transmembrane protein

Résumé

Background: SLC35F6 is part of the SoLute Carrier family 35 (SLC35). Members of this family are known to transport nucleotide-sugars into the endoplasmic reticulum or Golgi apparatus, in exchange for exporting their corresponding nucleoside monophosphate. In these organelles, glycosyltransferases use the nucleotide-sugars as precursors to pursue the synthesis or remodeling of glycans linked to proteins or lipids. Our team identified SLC35F6 as a lysosomal membrane protein, but to our knowledge, biosynthetic glycosylation does not take place in lysosomes. Intriguingly though, we found that SLC35F6 exhibits partial homology with known nucleotide sugar transporters and metabolomic studies highlighted UDP-GlcNAc in lysosomes. Aims and methods: A first aim is to test, using fluorescence microscopy and biochemical analyses, if silencing of SLC35F6 in hepatocytes causes any detectable lysosomal abnormalities. Another aim is to investigate if SLC35F6 could transport nucleotide-sugars. We therefore tested whether processes dependent on nucleotide-sugars, including glycogen synthesis, N-glycosylation and O-GlcNacylation, are altered in hepatocytes knocked-out or knock-downed for SLC35F6. Moreover, we initiated an investigation of the expression and consequences of SLC35F6 knock-down on breast cancer cell proliferation because microarray data in the Geo Profiles database suggested possible modulation in estrogen-responsive breast cancer cells, and the report that SLC35F6 knock-down decreases the proliferation and promotes apoptosis of pancreatic cancer cells. Results: SLC35F6 deficiency did not induce any obvious lysosomal storage phenotype. A slight increase of HYAL1 activity (enzyme that degrades hyaluronic acid) was observed in Slc35f6 knock-out mice livers, without any changes for the other tested lysosomal enzymes. Regarding O-GlcNacylation and N-Glycosylation, no SLC35F6 knock-down effect were observed. Contrary to the suggested Geo profiles microarray datasets, in MCF-7 and T47D breast cancer cells, SLC35F6 expression is not controlled by estrogens. Interestingly, the investigation of a potential effect of SLC35F6 knock-down on cancer cell behavior revealed that it causes slower proliferation in MDA-MB-231 breast cancer cells, whereas little effect was observed in MCF-7 cells. Conclusion: The inactivation of SLC35F6 appears to have little effect on lysosomal morphology, lysosomal biogenesis and overall glycosylation processes in the cells. It is however possible that some changes of O-GlcNacylation or N-glycosylation would affect some proteins more than others and that our analyses were not specific enough to reveal these differences. Alternatively, the consequences in hepatocytes may be limited due to compensation by other transporters. In the future, it would be most interesting to study if SLC35F6 contributes to the transport of nucleotide sugars in MDA-MB-231, whose survival is strongly reduced after SLC35F6 knock-down. An intriguing idea is that SLC35F6 could contribute to glycocalyx synthesis at the plasma membrane of some cells after lysosomal exocytosis.

la date de réponse	17 janv. 2023
langue originale	Anglais
L'institution diplômante	Universite de Namur
Superviseur	Michel Jadot (Promoteur) & Marielle Boonen (Copromoteur)