Department of Biosciences, University of Salzburg, Salzburg, Austria.
Center of Molecular Biosciences & Institute of General, Inorganic and Theoretical Chemistry, University of Innsbruck, Innsbruck, Austria.
Front Immunol. 2020 Aug 18;11:1824. doi: 10.3389/fimmu.2020.01824. eCollection 2020.
Understanding, which factors determine the immunogenicity and immune polarizing properties of proteins, is an important prerequisite for designing better vaccines and immunotherapeutics. While extrinsic immune modulatory factors such as pathogen associated molecular patterns are well-understood, far less is known about the contribution of protein inherent features. Protein fold-stability represents such an intrinsic feature contributing to immunogenicity and immune polarization by influencing the amount of peptide-MHC II complexes (pMHCII). Here, we investigated how modulation of the fold-stability of the grass pollen allergen Phl p 6 affects its ability to stimulate immune responses and T cell polarization. MAESTRO software was used for prediction of stabilizing or destabilizing point mutations. Mutated proteins were expressed in , and their thermal stability and resistance to endolysosomal proteases was determined. Resulting peptides were analyzed by mass spectrometry. The structure of the most stable mutant protein was assessed by X-ray crystallography. We evaluated the capacity of the mutants to stimulate T cell proliferation , as well as antibody responses and T cell polarization in an adjuvant-free BALB/c mouse model. In comparison to wild-type protein, stabilized or destabilized mutants displayed changes in thermal stability ranging from -5 to +14°. While highly stabilized mutants were degraded very slowly, destabilization led to faster proteolytic processing . This was confirmed in BMDCs, which processed and presented the immunodominant epitope from a destabilized mutant more efficiently compared to a highly stable mutant. , stabilization resulted in a shift in immune polarization from TH2 to TH1/TH17 as indicated by higher levels of IgG2a and increased secretion of TNF-α, IFN-γ, IL-17, and IL-21. MAESTRO software was very efficient in detecting single point mutations that increase or reduce fold-stability. Thermal stability correlated well with the speed of proteolytic degradation and presentation of peptides on the surface of dendritic cells . This change in processing kinetics significantly influenced the polarization of T cell responses . Modulating the fold-stability of proteins thus has the potential to optimize and polarize immune responses, which opens the door to more efficient design of molecular vaccines.
了解哪些因素决定蛋白质的免疫原性和免疫极化特性，是设计更好疫苗和免疫疗法的重要前提。虽然已经很好地理解了病原体相关分子模式等外在免疫调节因子，但对于蛋白质固有特征的贡献知之甚少。蛋白质折叠稳定性就是这样一种内在特征，它通过影响肽-MHC II 复合物 (pMHCII) 的数量来影响免疫原性和免疫极化。在这里，我们研究了草花粉过敏原 Phl p 6 的折叠稳定性的调节如何影响其刺激免疫反应和 T 细胞极化的能力。使用 MAESTRO 软件预测稳定或不稳定的点突变。突变蛋白在 中表达，并测定其热稳定性和对内溶酶体蛋白酶的抗性。通过质谱分析所得肽。通过 X 射线晶体学评估最稳定突变体蛋白的结构。我们评估了突变体刺激 T 细胞增殖的能力，以及在无佐剂 BALB/c 小鼠模型中的抗体反应和 T 细胞极化。与野生型蛋白相比，稳定或不稳定的突变体显示出热稳定性的变化范围为-5 至+14°C。虽然高度稳定的突变体降解非常缓慢，但不稳定会导致更快的蛋白水解加工。这在 BMDC 中得到了证实，与高度稳定的突变体相比，BMDC 更有效地加工和呈递不稳定突变体的免疫显性表位。折叠稳定性的增加导致免疫极化从 TH2 向 TH1/TH17 转变，表现为 IgG2a 水平升高，TNF-α、IFN-γ、IL-17 和 IL-21 的分泌增加。MAESTRO 软件在检测增加或降低折叠稳定性的单点突变方面非常有效。热稳定性与蛋白水解降解和树突状细胞表面肽呈递的速度密切相关。这种加工动力学的变化显著影响了 T 细胞反应的极化。调节蛋白质的折叠稳定性有可能优化和极化免疫反应，为更有效地设计分子疫苗开辟了道路。
