From the Plasma Proteins, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, The Netherlands (M.S., E.K., B.L.v.d.E., A.G., M.H., D.v.B., H.M., M.v.d.B., J.V., R.B.).
Cell Biology, The Netherlands Cancer Institute, Amsterdam (H.J.).
Arterioscler Thromb Vasc Biol. 2018 Jul;38(7):1549-1561. doi: 10.1161/ATVBAHA.117.310701. Epub 2018 Jun 7.
Endothelial cells store VWF (von Willebrand factor) in rod-shaped secretory organelles, called Weibel-Palade bodies (WPBs). WPB exocytosis is coordinated by a complex network of Rab GTPases, Rab effectors, and SNARE (soluble NSF attachment protein receptor) proteins. We have previously identified STXBP1 as the link between the Rab27A-Slp4-a complex on WPBs and the SNARE proteins syntaxin-2 and -3. In this study, we investigate the function of syntaxin-3 in VWF secretion.
In human umbilical vein endothelial cells and in blood outgrowth endothelial cells (BOECs) from healthy controls, endogenous syntaxin-3 immunolocalized to WPBs. A detailed analysis of BOECs isolated from a patient with variant microvillus inclusion disease, carrying a homozygous mutation in (STX3), showed a loss of syntaxin-3 protein and absence of WPB-associated syntaxin-3 immunoreactivity. Ultrastructural analysis revealed no detectable differences in morphology or prevalence of immature or mature WPBs in control versus STX3 BOECs. VWF multimer analysis showed normal patterns in plasma of the microvillus inclusion disease patient, and media from STX3 BOECs, together indicating WPB formation and maturation are unaffected by absence of syntaxin-3. However, a defect in basal as well as Ca- and cAMP-mediated VWF secretion was found in the STX3 BOECs. We also show that syntaxin-3 interacts with the WPB-associated SNARE protein VAMP8 (vesicle-associated membrane protein-8).
Our data reveal syntaxin-3 as a novel WPB-associated SNARE protein that controls WPB exocytosis.
内皮细胞将 VWF（血管性血友病因子）储存在称为 Weibel-Palade 体（WPB）的杆状分泌细胞器中。WPB 的胞吐作用由 Rab GTPase、Rab 效应物和 SNARE（可溶性 NSF 附着蛋白受体）蛋白的复杂网络协调。我们之前已经确定 STXBP1 是 WPB 上 Rab27A-Slp4-a 复合物与 SNARE 蛋白 syntaxin-2 和 -3 之间的联系。在这项研究中，我们研究了 syntaxin-3 在 VWF 分泌中的作用。
在人脐静脉内皮细胞和来自健康对照者的血液衍生的内皮细胞（BOEC）中，内源性 syntaxin-3 免疫定位到 WPB。对来自携带（STX3）纯合突变的变异微绒毛包涵体病患者的 BOEC 的详细分析显示 syntaxin-3 蛋白缺失和 WPB 相关的 syntaxin-3 免疫反应性缺失。超微结构分析显示，在对照与 STX3 BOEC 中，未成熟或成熟 WPB 的形态或发生率均无明显差异。VWF 多聚体分析显示微绒毛包涵体病患者血浆中的正常模式，以及 STX3 BOEC 中的培养基，共同表明 WPB 的形成和成熟不受 syntaxin-3 缺失的影响。然而，在 STX3 BOEC 中发现基础以及 Ca 和 cAMP 介导的 VWF 分泌缺陷。我们还表明 syntaxin-3 与 WPB 相关的 SNARE 蛋白 VAMP8（囊泡相关膜蛋白-8）相互作用。
我们的数据揭示 syntaxin-3 是一种新的 WPB 相关 SNARE 蛋白，可控制 WPB 胞吐作用。
