Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, Hungary.
ELKH-SE Immune-Proteogenomics Extracellular Vesicle Research Group, Budapest, Hungary.
J Extracell Vesicles. 2021 Sep;10(11):e12140. doi: 10.1002/jev2.12140.
In this study we tested whether a protein corona is formed around extracellular vesicles (EVs) in blood plasma. We isolated medium-sized nascent EVs of THP1 cells as well as of Optiprep-purified platelets, and incubated them in EV-depleted blood plasma from healthy subjects and from patients with rheumatoid arthritis. EVs were subjected to differential centrifugation, size exclusion chromatography, or density gradient ultracentrifugation followed by mass spectrometry. Plasma protein-coated EVs had a higher density compared to the nascent ones and carried numerous newly associated proteins. Interactions between plasma proteins and EVs were confirmed by confocal microscopy, capillary Western immunoassay, immune electron microscopy and flow cytometry. We identified nine shared EV corona proteins (ApoA1, ApoB, ApoC3, ApoE, complement factors 3 and 4B, fibrinogen α-chain, immunoglobulin heavy constant γ2 and γ4 chains), which appear to be common corona proteins among EVs, viruses and artificial nanoparticles in blood plasma. An unexpected finding of this study was the high overlap of the composition of the protein corona with blood plasma protein aggregates. This is explained by our finding that besides a diffuse, patchy protein corona, large protein aggregates also associate with the surface of EVs. However, while EVs with an external plasma protein cargo induced an increased expression of TNF-α, IL-6, CD83, CD86 and HLA-DR of human monocyte-derived dendritic cells, EV-free protein aggregates had no effect. In conclusion, our data may shed new light on the origin of the commonly reported plasma protein 'contamination' of EV preparations and may add a new perspective to EV research.
在这项研究中，我们测试了细胞外囊泡（EVs）在血浆中是否形成了蛋白质冠。我们分离了 THP1 细胞和 Optiprep 纯化的血小板中等大小的新生 EVs，并将其在来自健康受试者和类风湿关节炎患者的 EV 耗尽的血浆中孵育。EVs 经过差速离心、分子筛层析或密度梯度超速离心，然后进行质谱分析。与新生 EVs 相比，带有血浆蛋白的 EVs 具有更高的密度，并且携带许多新结合的蛋白质。通过共聚焦显微镜、毛细管 Western 免疫测定、免疫电子显微镜和流式细胞术证实了血浆蛋白与 EVs 之间的相互作用。我们鉴定了 9 种共同的 EV 冠蛋白（ApoA1、ApoB、ApoC3、ApoE、补体因子 3 和 4B、纤维蛋白原α链、免疫球蛋白重链γ2 和γ4 链），它们似乎是血浆中 EVs、病毒和人工纳米颗粒的共同冠蛋白。本研究的一个意外发现是蛋白质冠的组成与血浆蛋白质聚集体高度重叠。这可以解释为我们发现，除了弥散的、斑片状的蛋白质冠外，大的蛋白质聚集体也与 EVs 的表面结合。然而，虽然带有外部血浆蛋白货物的 EV 会诱导人单核细胞来源的树突状细胞中 TNF-α、IL-6、CD83、CD86 和 HLA-DR 的表达增加，但 EV 游离的蛋白质聚集体没有影响。总之，我们的数据可能为普遍报道的 EV 制剂中血浆蛋白质“污染”的来源提供新的见解，并为 EV 研究增添新的视角。
