Syntaxin 5 determines Weibel-Palade body size and Von Willebrand factor secretion by controlling Golgi architecture

Abstract Von Willebrand factor (VWF) is a multimeric hemostatic protein primarily synthesized in endothelial cells (ECs). VWF is stored in endothelial storage organelles, the Weibel-Palade bodies (WPBs), whose biogenesis strongly depends on VWF anterograde trafficking and Golgi architecture. Elongated WPB morphology is correlated to longer VWF strings with better adhesive properties. We previously identified the SNARE SEC22B, which is involved in anterograde ER-to-Golgi transport, as a novel regulator of WPB elongation. To elucidate novel determinants of WPB morphology we explored endothelial SEC22B interaction partners in a mass spectrometry-based approach, identifying the Golgi SNARE Syntaxin 5 (STX5). We established STX5 knockdown in ECs using shRNA-dependent silencing and analyzed WPB and Golgi morphology, using confocal and electron microscopy. STX5-depleted ECs exhibited extensive Golgi fragmentation and decreased WPB length, which was associated with reduced intracellular VWF levels, and impaired stimulated VWF secretion. However, the secretion incompetent organelles in shSTX5 cells maintained WPB markers such as Angiopoietin 2, P-selectin, Rab27A, and CD63. Taken together, our study has identified SNARE protein STX5 as a novel regulator of WPB biogenesis

摘要 冯·维勒布兰德因子（Von Willebrand factor, VWF）是一种多聚体止血蛋白，主要由内皮细胞（endothelial cells, ECs）合成。VWF储存在内皮储存细胞器韦贝尔-帕拉德小体（Weibel-Palade bodies, WPBs）中，其生物发生过程高度依赖VWF的顺向运输与高尔基体结构。WPB的细长形态与更长的VWF多聚体链及更佳的黏附特性相关。本团队此前已鉴定出参与内质网-高尔基体顺向运输的SNARE蛋白SEC22B，其为调控WPB伸长的新型调节因子。为阐明调控WPB形态的新型决定因素，本研究通过基于质谱的蛋白质组学方法筛选内皮细胞中SEC22B的相互作用蛋白，鉴定出高尔基体SNARE蛋白突触融合蛋白5（Syntaxin 5, STX5）。本研究通过短发夹RNA（short hairpin RNA, shRNA）介导的沉默技术在内皮细胞中构建了STX5敲低模型，并利用共聚焦显微镜与电子显微镜分析WPB与高尔基体的形态。STX5敲低的内皮细胞呈现出广泛的高尔基体碎片化现象，且WPB长度缩短，这与细胞内VWF水平降低以及刺激诱导的VWF分泌受损相关。不过，在STX5敲低细胞中，分泌功能受损的细胞器仍保留有WPB的标志性蛋白，例如血管生成素2（Angiopoietin 2）、P-选择素（P-selectin）、Rab27A以及CD63。综上，本研究鉴定出SNARE蛋白STX5是调控WPB生物发生的新型调节因子。