通过共聚焦分析研究严重急性呼吸综合征冠状病毒2（SARS-CoV-2）刺突蛋白与人原代肝细胞及永生化类肝细胞上脱唾液酸糖蛋白受体的结合情况。

Binding of the SARS-CoV-2 Spike Protein to the Asialoglycoprotein Receptor on Human Primary Hepatocytes and Immortalized Hepatocyte-Like Cells by Confocal Analysis.

作者信息

Collins Daniel P, Steer Clifford J

机构信息

CMDG, LLC, Saint Paul, MN, USA.

Department of Medicine, University of Minnesota Medical School, Minneapolis, MN, USA.

出版信息

Hepat Med. 2021 Apr 14;13:37-44. doi: 10.2147/HMER.S301979. eCollection 2021.

BACKGROUND

The SARS-CoV-2 virus may have direct or indirect effects on other human organs beyond the respiratory system and including the liver, via binding of the spike protein. This study investigated the potential direct interactions with the liver by comparing the binding of SARS-CoV-2 spike proteins to human AT2-like cells, primary human hepatocytes and immortalized hepatocyte-like hybrid cells. Receptors with binding specificity for SARS-CoV-2 spike protein on AT2 cells and hepatocytes were identified.

METHODS

The specific binding of biotinylated spike and spike 1 proteins to undifferentiated human E12 MLPC (E12), E12 differentiated alveolar type 2 (AT2) cells, primary human hepatocytes (PHH) and E12 human hepatocyte-like hybrid cells (HLC) was studied by confocal microscopy. We investigated the expression of ACE-2, binding of biotinylated spike protein, biotinylated spike 1 and inhibition of binding by unlabeled spike protein, two neutralizing antibodies and an antibody directed against the hepatocyte asialoglycoprotein receptor 1 (ASGr1).

RESULTS

E12 MLPC did not express ACE-2 and did not bind either of spike or spike 1 proteins. AT2-like cells expressed ACE-2 and bound both spike and spike 1. Both PHH and HLC did not express ACE-2 and did not bind spike 1 protein. However, both PHH and HLC actively bound the spike protein. Biotinylated spike protein binding was inhibited by unlabeled spike but not spike 1 protein on PHH and HLC. Two commercial neutralizing antibodies blocked the binding of the spike to PHH and HLC but only one blocked binding to AT2. An antibody to the hepatocyte ASGr1 blocked the binding of the spike protein to PHH and HLC.

CONCLUSION

The absence of ACE-2 receptors and inhibition of spike binding by an antibody to the ASGr1 on both PHH and HLC suggested that the spike protein interacts with the ASGr1. The differential antibody blocking of spike binding to AT2, PHH and HLC indicated that neutralizing activity of SARS-CoV-2 binding might involve additional mechanisms beyond RBD binding to ACE-2.

背景

严重急性呼吸综合征冠状病毒2（SARS-CoV-2）病毒可能通过刺突蛋白的结合，对呼吸系统以外的其他人体器官产生直接或间接影响，包括肝脏。本研究通过比较SARS-CoV-2刺突蛋白与人类AT2样细胞、原代人肝细胞和永生化肝细胞样杂交细胞的结合情况，调查其与肝脏潜在的直接相互作用。确定了AT2细胞和肝细胞上对SARS-CoV-2刺突蛋白具有结合特异性的受体。

方法

通过共聚焦显微镜研究生物素化刺突蛋白和刺突1蛋白与未分化的人E12中胚层肺祖细胞（E12）、E12分化的肺泡Ⅱ型（AT2）细胞、原代人肝细胞（PHH）和E12人肝细胞样杂交细胞（HLC）的特异性结合。我们研究了血管紧张素转换酶2（ACE-2）的表达、生物素化刺突蛋白、生物素化刺突1的结合以及未标记的刺突蛋白、两种中和抗体和一种针对肝细胞去唾液酸糖蛋白受体1（ASGr1）的抗体对结合的抑制作用。

结果

E12中胚层肺祖细胞不表达ACE-2，也不结合刺突蛋白或刺突1蛋白。AT2样细胞表达ACE-2，并结合刺突蛋白和刺突1蛋白。原代人肝细胞和肝细胞样杂交细胞均不表达ACE-2，也不结合刺突1蛋白。然而，原代人肝细胞和肝细胞样杂交细胞均能有效结合刺突蛋白。在原代人肝细胞和肝细胞样杂交细胞上，未标记的刺突蛋白可抑制生物素化刺突蛋白的结合，但未标记的刺突1蛋白则不能。两种商用中和抗体可阻断刺突蛋白与原代人肝细胞和肝细胞样杂交细胞的结合，但只有一种可阻断其与AT2细胞的结合。一种针对肝细胞ASGr1的抗体可阻断刺突蛋白与原代人肝细胞和肝细胞样杂交细胞的结合。