L-SIGN is a receptor on liver sinusoidal endothelial cells for SARS-CoV-2 virus
Yuji Kondo, Jason L. Larabee, Liang Gao, Huiping Shi, Bojing Shao, Christopher M. Hoover, J. Michael McDaniel, Yen-Chun Ho, Robert Silasi-Mansat, Stephanie A. Archer-Hartmann, Parastoo Azadi, R. Sathish Srinivasan, Alireza R. Rezaie, Alain Borczuk, Jeffrey C. Laurence, Florea Lupu, Jasimuddin Ahamed, Rodger P. McEver, James F. Papin, Zhongxin Yu, Lijun Xia
Yuji Kondo, Jason L. Larabee, Liang Gao, Huiping Shi, Bojing Shao, Christopher M. Hoover, J. Michael McDaniel, Yen-Chun Ho, Robert Silasi-Mansat, Stephanie A. Archer-Hartmann, Parastoo Azadi, R. Sathish Srinivasan, Alireza R. Rezaie, Alain Borczuk, Jeffrey C. Laurence, Florea Lupu, Jasimuddin Ahamed, Rodger P. McEver, James F. Papin, Zhongxin Yu, Lijun Xia
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Research Article Cell biology Vascular biology

L-SIGN is a receptor on liver sinusoidal endothelial cells for SARS-CoV-2 virus

Abstract

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a pandemic. Severe disease is associated with dysfunction of multiple organs, but some infected cells do not express ACE2, the canonical entry receptor for SARS-CoV-2. Here, we report that the C-type lectin receptor L-SIGN interacted in a Ca2+-dependent manner with high-mannose–type N-glycans on the SARS-CoV-2 spike protein. We found that L-SIGN was highly expressed on human liver sinusoidal endothelial cells (LSECs) and lymph node lymphatic endothelial cells but not on blood endothelial cells. Using high-resolution confocal microscopy imaging, we detected SARS-CoV-2 viral proteins within the LSECs from liver autopsy samples from patients with COVID-19. We found that both pseudo-typed virus enveloped with SARS-CoV-2 spike protein and authentic SARS-CoV-2 virus infected L-SIGN–expressing cells relative to control cells. Moreover, blocking L-SIGN function reduced CoV-2–type infection. These results indicate that L-SIGN is a receptor for SARS-CoV-2 infection. LSECs are major sources of the clotting factors vWF and factor VIII (FVIII). LSECs from liver autopsy samples from patients with COVID-19 expressed substantially higher levels of vWF and FVIII than LSECs from uninfected liver samples. Our data demonstrate that L-SIGN is an endothelial cell receptor for SARS-CoV-2 that may contribute to COVID-19–associated coagulopathy.

Authors

Yuji Kondo, Jason L. Larabee, Liang Gao, Huiping Shi, Bojing Shao, Christopher M. Hoover, J. Michael McDaniel, Yen-Chun Ho, Robert Silasi-Mansat, Stephanie A. Archer-Hartmann, Parastoo Azadi, R. Sathish Srinivasan, Alireza R. Rezaie, Alain Borczuk, Jeffrey C. Laurence, Florea Lupu, Jasimuddin Ahamed, Rodger P. McEver, James F. Papin, Zhongxin Yu, Lijun Xia

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Figure 2

L-SIGN binds to a high-mannose–type N-glycan on the spike protein of SARS-CoV-2.

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L-SIGN binds to a high-mannose–type N-glycan on the spike protein of SAR...
(A) Protein domain structures of spike/Fc (left) (numbers represent amino acids). Spike/Fc forms dimer (right). RBD, receptor-binding domain; hFc, human IgG Fc domain. (B) Percentages of high-mannose–type versus complex/hybrid–type N-glycans in control Fc and spike/Fc proteins produced from COS-7 cells determined by MALDI-TOF MS. See also Supplemental Figure 1. (C) CBB staining of SDS-PAGE gels of COS-7 cell–derived spike/Fc under reducing conditions, with or without PNGaseF or Endo-H treatment (top). Immunoblotting of spike/Fc produced from COS-7 and HEK293T cells with or without PNGaseF or Endo-H treatment (bottom). (D) Silver staining of SDS-PAGE gel of COS-7 cell–derived purified SpikeN343Q/Fc protein or of WT spike/Fc treated with or without PNGaseF. Immunoblots and GNL lectin blots of WT and SpikeN343Q/Fc. The reduced size of SpikeN343Q/Fc protein relative to that of WT indicates the loss of an N-glycan on the RBD. (E) Representative flow cytometry plots of spikeN343Q/Fc binding to mock-, ACE2-myc3–, and L-SIGN-flag–transfected HEK293T cells (left) and quantification of the percentage of Fc protein binding (right). Dashed boxes indicate positive population. (F) Representative flow cytometry plot of spike/Fc binding to L-SIGN-flag–transfected HEK293T cells with the pretreatment of isotype IgG or anti–L-SIGN mAb (left) and quantification of the percentage of Fc protein binding (right). Isotype, isotype IgG pretreated; mAb, anti–L-SIGN mAb pretreated. (G) Representative flow cytometry plots of L-SIGN/Fc binding to spike protein–transfected HEK293T cells (left) and quantification of the percentage of Fc protein binding (right). Mannan (100 μg/ml), a mannose polymer, was used as a competitive inhibitor of L-SIGN/Fc. L-SIGNQPD/Fc is a carbohydrate recognition domain mutant of L-SIGN/Fc. EGTA (1 mM) was used as Ca2+ chelator. All experiments were repeated at least 3 times. For all analyses, n = 3. Bars indicate the mean; error bars represent mean ± SEM. Significance was calculated using a 1-way ANOVA for multiple groups: **P < 0.01.