透過冷凍電子顯微鏡、質譜儀以及分子模擬探討人類冠狀病毒棘蛋白其結構與功能之關係

Abstract

人類冠狀病毒屬於網巢病毒目，其可藉由動物傳播給人類，為一種人畜共通的病原體。冠狀病毒主要通過病毒細胞膜上高度醣基化的棘蛋白與宿主細胞上的受體結合，促進病毒與宿主細胞膜的融合，達到病毒入侵的目的。醣基化修飾是一種蛋白質的轉譯後修飾，在棘蛋白的建構中扮演非常重要的角色。因此，了解醣基化棘蛋白的結構與分子動態對受體免疫辨識的影響將有助於後續的預防以及抗病毒治療的發展。在本研究中，我們主要討論屬於乙型冠狀病毒屬的三種人類冠狀病毒，包括嚴重急性呼吸道症候群冠狀病毒(SARS-CoV)、中東呼吸症候群冠狀病毒(MERS-CoV)以及人類冠狀病毒HKU1(hCoV-HKU1)。我們通過哺乳動物細胞蛋白質表達系統製備重組棘蛋白，並藉由差示掃描量熱法(DSC)以及差示掃描螢光法(DSF)分析在不同物化條件下的蛋白質穩定性，接著利用冷凍電子顯微鏡(cryo-EM)解析出受體結合區域在中東呼吸症候群冠狀病毒棘蛋白中的不同構型。為了更進一步瞭解蛋白質表面N-醣基化修飾的化學組成和結構，我們使用質譜儀鑑定在特定醣化位點上的不同醣型，並在GlycoSHIELD的幫助下建立棘蛋白的醣化模型，定量描述N-醣基化修飾對於棘蛋白表面所形成的遮蔽效應，結果表明棘蛋白表面的局部抗原性與醣基化修飾所形成的遮蔽效應間呈現負相關。綜上所述，我們的研究建立一個完整的流程探討醣基化修飾在棘蛋白表面的影響，並且具有預測潛在受體與輔助受體結合區以及抗原決定位的能力。

Human coronaviruses (hCoVs) belong to the order Nidovirales, which are zoonotic pathogens that evolve to infect humans. CoVs use highly glycosylated spike proteins to bind to host receptor(s), the first step towards host membrane fusion and viral entries. As the post-translationally modified glycans constitute a significant part of the overall structures of the spike proteins, understanding their structures and dynamics in the context of receptor and immunity recognition is essential for developing preventions and antivral treatments. Here, we focused on three hCoV spike proteins in the genus Betacoronavirus, including SARS-CoV, MERS-CoV and hCoV-HKU1. We produced the recombinant spike proteins by a mammalian protein expression system. The folding stabilities over a range of experimental conditions were evaluated by differential scanning calorimeter (DSC) and differential scanning fluorimeter (DSF). We also employed single particle analysis by cryo-electron microscopy (cryo-EM) to determine four distinct structures of the MESR-CoV spike protein that differed in the different conformations of the receptor-binding domain (RBD). To characterize the chemical compositions and structures of the surface glycans, we used mass spectrometry to determine site-specifically the glycoforms of individual N-linked glycans. We further implemented a modelling procedure by GlycoSHIELD to quantitatively describe the shielding effect of the N-glycans on the spike proteins. Our results revealed that the local antigenicity of the spike proteins negatively correlated with the extent of protein surface shielding by the N-glycans. Collectively, our study provided a robust workflow to determine the molecular structure of CoV spike proteins in the context of the shielding effect of glycosylation with the ability to predict potential (co)receptor binding sites and epitopes for antibody binding.