Gow James, Yang Yujie, Govindraj Mohan, Guo Changjiang
Department of Pharmacology & Toxicology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, New Jersey, USA.
Appl In Vitro Toxicol. 2020 Sep 1;6(3):90-98. doi: 10.1089/aivt.2020.0009. Epub 2020 Sep 17.
Recognition of fungal surface β-glucan by pattern recognition receptor Dectin-1 is a critical process for fungal clearance in the lung. In humans, persistent fungal infection is observed in individuals with particular Dectin-1 polymorphism. We have identified that nitric oxide (NO) modifies critical cysteines in pattern recognition molecules to disassemble and alter protein function. There is a hydrophobic -nitrosylation motif present in surfactant protein-D (SP-D) that is also present in Dectin-1. We hypothesized that Dectin-1 can be modified by nitrosative stress potentially leading to impairment of fungal clearance. Recombinant Dectin-1 was incubated with l-nitrosocysteine (L-SNOC) and -nitrosylated Dectin-1 was detected by Biotin-switch assay. Cells of a murine macrophage line (Raw 264.7) were incubated with -nitroso-glutathione (GSNO) and Dectin-1 shedding from the cell surface was determined by Western blot. Dectin-1 quaternary structure was determined by native gel electrophoresis. Dectin-1 function was assayed by NF-κB activity and IL-6 mRNA real-time polymerase chain reaction (PCR). Phagocytic activity was measured by fluorescence labeled zymosan beads. Dectin-1 was -nitrosylated by l-nitrosocysteine (L-SNOC) as determined by Biotin-switch assay, resulting in structural disruption. We used Western blotting and flow cytometry to demonstrate that incubation of a murine macrophage cell line (Raw 264.7 cells) with GSNO reduced the surface Dectin-1 expression as a result of shedding to the media. The shedding of Dectin-1 is due to formation of -nitrosothiol (SNO)-Dectin-1 and disruption of the Dectin-1 oligomeric complex. GSNO also induces Dectin-1 shedding from the cell surface. The functional significance of GSNO treatment of macrophages is shown by reduced β-glucan-mediated signaling in terms of NF-κB function and IL-6 expression. Finally, it was demonstrated that GSNO treatment reduces the capability of macrophages to phagocytose zymosan. These data provide mechanistic data to support the role of Dectin-1 nitrosylation as a mediator of reduced fungal clearance in the face of increased NO exposure.
模式识别受体Dectin-1对真菌表面β-葡聚糖的识别是肺部清除真菌的关键过程。在人类中，具有特定Dectin-1多态性的个体中会观察到持续性真菌感染。我们已经确定，一氧化氮（NO）会修饰模式识别分子中的关键半胱氨酸，从而分解并改变蛋白质功能。表面活性蛋白-D（SP-D）中存在一个疏水的亚硝基化基序，Dectin-1中也存在该基序。我们推测Dectin-1可能会被亚硝化应激修饰，从而可能导致真菌清除受损。将重组Dectin-1与L-亚硝基半胱氨酸（L-SNOC）一起孵育，并通过生物素转换法检测亚硝基化的Dectin-1。将小鼠巨噬细胞系（Raw 264.7）的细胞与亚硝基谷胱甘肽（GSNO）一起孵育，并通过蛋白质印迹法测定细胞表面Dectin-1的脱落情况。通过非变性凝胶电泳确定Dectin-1的四级结构。通过NF-κB活性和IL-6 mRNA实时聚合酶链反应（PCR）测定Dectin-1的功能。通过荧光标记的酵母聚糖珠测量吞噬活性。通过生物素转换法确定，Dectin-1被L-亚硝基半胱氨酸（L-SNOC）亚硝基化，导致结构破坏。我们使用蛋白质印迹法和流式细胞术证明，用GSNO孵育小鼠巨噬细胞系（Raw 264.7细胞）会导致Dectin-1脱落至培养基中，从而降低其表面表达。Dectin-1的脱落是由于亚硝基硫醇（SNO）-Dectin-1的形成以及Dectin-1寡聚复合物的破坏。GSNO还会诱导Dectin-1从细胞表面脱落。就NF-κB功能和IL-6表达而言，GSNO处理巨噬细胞的功能意义表现为β-葡聚糖介导的信号传导减少。最后，证明GSNO处理会降低巨噬细胞吞噬酵母聚糖的能力。这些数据提供了机制数据，以支持Dectin-1亚硝基化在面对NO暴露增加时作为真菌清除减少的介质的作用。
