嚴重急性呼吸道症候群冠狀病毒棘蛋白質參與ACE2脫離之分子機制

Abstract

嚴重急性呼吸道症候群冠狀病毒 (Severe acute respiratory syndrome coronavirus, SARS-CoV) 是導致嚴重急性呼吸道症候群 (SARS) 的病原體，在2002年至2003年間於全世界造成了至少8000例的感染、接近10%的死亡率。SARS-CoV主要傳播途徑為飛沫傳染與密切的接觸，初期症狀類似感冒所常見的發高燒、頭痛和肌肉痠痛等，進一步會引起患者肺部浸潤、纖維化等非典型肺炎症狀。血管收縮素轉化酵素2 (angiotensin-converting enzyme 2, ACE2)，被證實為SARS-CoV感染宿主時重要的細胞表面受器。先前的研究發現，SARS-CoV和SARS-CoV棘蛋白質 (spike) 施予都會誘發a disintegrin and metalloproteinase 17 (ADAM17) 對ACE2作用，切除其extracellular domain，造成ACE2脫離 (shedding)。由本實驗室先前之研究已知，SARS-CoV類病毒顆粒和SARS-CoV棘蛋白質均會透過ACE2進一步引發下游的訊息傳遞，活化extracellular signal regulated kinase (ERK) 與activator protein 1 (AP-1) 使chemokine C-C motif ligand 2 (CCL2) 的表現量增加。ADAM17的活化與CCL2的表現量增加在引發宿主的發炎及免疫反應上，扮演著相當重要的角色。為了探討病毒感染時參與在ACE2 shedding過程中的訊息傳遞路徑，實驗利用會表現SARS-CoV spike蛋白質的重組桿狀病毒感染昆蟲細胞Sf9，表現並純化全長的spike蛋白質作為研究材料。對能夠被SARS-CoV感染的非洲綠猴腎臟細胞株Vero E6施予此經純化的spike蛋白質，並利用西方墨點法以及偵測ACE2的活性來分析ACE2 shedding的程度。實驗結果發現，利用此一系統表現與純化的SARS-CoV spike確實能夠導致Vero E6細胞株ACE2的shedding。透過施予各種kinase的抑制劑，進一步實驗發現ERK以及protein kinase C (PKC) 可能參與在spike蛋白質所導致的ACE2 shedding過程當中。本篇研究模擬病毒感染時的作用，顯示spike蛋白質與ACE2交互作用後可能會透過ERK以及PKC活化ADAM17進而增加ACE2 shedding。這些結果對SARS-CoV感染時所引起的發炎及免疫反應等致病性提供了可能的分子機制。

Severe acute respiratory syndrome (SARS) is an emerging infectious disease caused by SARS-coronavirus (SARS-CoV). Epidemic of SARS in 2002–2003 results in over 8000 infected cases with approximately 10% mortality. SARS-CoV spreads primarily through droplets (respiratory secretions) and close person-to-person contact. SARS symptoms usually start off like those of the common cold including high fever, headache and muscle aches. As the illness progresses, people may develop cellular infiltration, fibrosis, cytokine secretion and accumulation of leukocytes in lungs. Angiotensin converting enzyme 2 (ACE2) was identified to be the human cell surface receptor to which SARS-CoV binds through the viral spike protein. In addition, both SARS-CoV and the viral spike protein can induce the proteolytic activity of a disintegrin and metalloproteinase 17 (ADAM17) to target ACE2 and produced a catalytically active soluble form of ACE2 ectodomain shedding from the plasma membrane. Furthermore, previous studies in our laboratory have demonstrated that the interaction between SARS-CoV spike protein and the ACE2 receptor can mediate the phosphorylation of extracellular signal regulated kinase (ERK) and activator protein 1 (AP-1), resulting in an upregulation of chemokine C-C motif ligand 2 (CCL2) expression. Both CCL2 upregulation and ADAM17 activation play important roles in inflammatory regulation and immune responses. To understand the signaling pathways that are involved in ACE2 shedding, SARS-CoV spike protein was expressed in insect sf9 cells following an infection with the recombinant baculovirus expressing the full-length SARS-CoV spike protein. The recombinant spike protein was purified and incubated with Vero E6 cells. Degree of ACE2 shedding was then analyzed by Western blot analysis and ACE2 activity assay. Results showed that SARS-CoV spike protein purified from the sf9 expression system did mimic natural infection of SARS-CoV to induce shedding of the ACE2 ectodomain. In addition, ERK and protein kinase C (PKC) inhibitors, but not JNK inhibitor reduced ACE2 shedding. These results indicate that ERK and PKC signaling pathways may be involved in the ADAM17-mediated cleavage of ACE2 ectodomain. Taken together, the interaction between spike and ACE2 induces ACE2 shedding through PKC- and ERK-mediated activation of ADAM17. This study provides a possible mechanism in the association between ADAM17 activation and SARS-CoV-induced inflammation and immune responses.