Effects of mutations in the post-translational modification sites on the trafficking of hyaluronan synthase 2 (HAS2)
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CitationMelero-Fernandez de Mera, RM. Arasu, UT. Kärnä, R. Oikari, S. Rilla, K. Vigetti, D. Passi, A. Heldin, P. Tammi, MI. Deen, AJ. (2019). Effects of mutations in the post-translational modification sites on the trafficking of hyaluronan synthase 2 (HAS2). Matrix biology, 80, 85-103. 10.1016/j.matbio.2018.10.004.
Vesicular trafficking of hyaluronan synthases (HAS1-3) from endoplasmic reticulum (ER) through Golgi to plasma membrane (PM), and either back to endosomes and lysosomes, or out into extracellular vesicles, is important for their activities. We studied how post-translational modifications affect the trafficking of HAS2 by mutagenesis of the sites of ubiquitination (K190R), phosphorylation (T110A) and O-GlcNAcylation (S221A), using Dendra2- and EGFP-HAS2 transfected into COS1 cells. Confocal microscopy showed HAS2 wild type (wt) and its K190R and S221A mutants in ER, Golgi and extracellular vesicles, while the T110A mutant remained mostly in the ER. HA synthesis was reduced by S221A, while completely blocked by K190R and T110A. Cell-surface biotinylation indicated that T110A was absent from PM, while S221A was close to the level of wt, and K190R was increased in PM. TIRF microscopy analysis gave similar results. Rab10 silencing increased HA secretion by HAS2, likely by inhibiting endocytosis of the enzyme from PM, as reported before for HAS3. Green-to-red photo-conversion of Dendra2-HAS2 constructs suggested slower decay of K190R and S221A than HAS2 wt, while T110A was barely degraded at all. S221D and S221E, the phosphomimetic mutants of this site, decayed faster and blocked hyaluronan synthesis, suggesting alternative O-GlcNAc/-PO4 substitution to regulate the stability of the enzyme. Probing the role of dynamic O-GlcNAcylation at S221 by adding glucosamine increased the half-life of only HAS2 wt. The Dendra2·HAS2 disappearance from Golgi was slower for K190R. Of the two inactive constructs, K190R co-transfected with HAS2 wt suppressed, whereas T110A had no effect on HA synthesis. Interestingly, the HAS2-stimulated shedding of extracellular vesicles was dependent on HAS residence in PM but independent of HA synthesis. The results indicate that post-translational modifications control the trafficking of HAS2, and that trafficking is an integral part of the post-translational regulation of HAS2 activity.