Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland.
Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland.
Matrix Biol. 2019 Jul;80:85-103. doi: 10.1016/j.matbio.2018.10.004. Epub 2018 Oct 27.
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/-PO 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.
透明质酸合酶（HAS1-3）从内质网（ER）到质膜（PM）的囊泡转运，以及随后返回内体和溶酶体，或者外排到细胞外囊泡，对其活性非常重要。我们通过对泛素化（K190R）、磷酸化（T110A）和 O-GlcNAc 化（S221A）位点的突变，研究了翻译后修饰如何影响 HAS2 的转运。使用 Dendra2 和 EGFP-HAS2 转染 COS1 细胞进行研究。共聚焦显微镜显示 HAS2 野生型（wt）及其 K190R 和 S221A 突变体位于 ER、高尔基体和细胞外囊泡中，而 T110A 突变体主要位于 ER 中。HA 合成减少 S221A，而 K190R 和 T110A 完全阻断。细胞表面生物素化表明 T110A 不存在于质膜中，而 S221A 接近 wt 水平，K190R 则增加到质膜中。TIRF 显微镜分析得到了类似的结果。Rab10 沉默增加了 HAS2 的 HA 分泌，可能是通过抑制酶从质膜的内吞作用，如之前报道的 HAS3。Dendra2-HAS2 构建体的绿到红光转换表明 K190R 和 S221A 的衰减比 HAS2wt 慢，而 T110A 几乎没有降解。该位点的磷酸模拟突变体 S221D 和 S221E 降解更快并阻断透明质酸合成，表明替代的 O-GlcNAc/-PO 取代可调节酶的稳定性。通过添加葡萄糖胺探测 S221 处动态 O-GlcNAc 化的作用仅增加了 HAS2wt 的半衰期。与 HAS2wt 共转染的两种无活性构建体之一的 K190R 从高尔基体的消失较慢。在这两种无活性的构建体中，K190R 与 HAS2wt 共转染抑制了 HAS2 的 HA 合成，而 T110A 则没有影响。有趣的是，HAS2 刺激的细胞外囊泡脱落依赖于 HAS2 在质膜中的存在，但与 HA 合成无关。结果表明，翻译后修饰控制 HAS2 的转运，而转运是 HAS2 活性的翻译后调节的一个组成部分。
