Carbohydrate Ligands for COVID-19 Spike Proteins

Abstract

An outbreak of SARS-CoV-2 coronavirus (COVID-19) first detected in Wuhan, China, has created a public health emergency all over the world. The pandemic has caused more than 340 million confirmed cases and 5.57 million deaths as of 23 January 2022. Although carbohydrates have been found to play a role in coronavirus binding and infection, the role of cell surface glycans in SARS-CoV-2 infection and pathogenesis is still not understood. Herein, we report that the SARS-CoV-2 spike protein S1 subunit binds specifically to blood group A and B antigens, and that the spike protein S2 subunit has a binding preference for Le^a antigens. Further examination of the binding preference for different types of red blood cells (RBCs) indicated that the spike protein S1 subunit preferentially binds with blood group A RBCs, whereas the spike protein S2 subunit prefers to interact with blood group Le^a RBCs. Angiotensin converting enzyme 2 (ACE2), a known target of SARS-CoV-2 spike proteins, was identified to be a blood group A antigen-containing glycoprotein. Additionally, 6-sulfo N-acetyllactosamine was found to inhibit the binding of the spike protein S1 subunit with blood group A RBCs and reduce the interaction between the spike protein S1 subunit and ACE2.

Keywords: COVID-19; blood group; carbohydrate ligand; spike protein.

Figure 1

Binding profile of SARS-CoV-2 spike proteins with 97 biotin-PAA-sugars. (A) spike protein S1 subunit and (B) spike protein S2 subunit.

Figure 2

The binding preference of SARS-CoV-2 spike proteins to blood group A, B, and O RBCs. The histograms of spike protein S1 subunits binding with blood group A, B, and O RBCs show a 51.02%, 36.29%, and 21.07% shift in mean fluorescence intensity (MFI), respectively (red line). The histograms of spike protein S2 subunits binding with blood group A, B, and O RBCs show a 27.53%, 26.79%, and 13.26% shift in MFI, respectively (blue line).

Figure 3

The binding efficiency of the SARS-CoV-2 spike protein S1 subunit to blood group A RBCs affected by carbohydrate derivatives. (A) The histogram of spike protein S1 subunit binding with blood group A RBC cells shows a 16.44% shift in MFI without carbohydrate inhibitors. Lactose and three sulfated carbohydrate derivatives were preincubated with spike protein S1 subunit then subjected to the binding assay. The histograms of spike protein S1 subunit binding with blood group A RBC cells are shown (green line). The MFI shifts of blood group A RBC cells with carbohydrate derivatives tested are (A) lactose, 23.76%; (B) compound 1, 8.98%; (C) compound 2, 20.77%; and (D) TD139, 18.91%. (E) The relative fluorescence shows the binding efficiency of the spike protein S1 subunit to blood group A RBCs influenced by carbohydrate derivatives. (F) Structures of lactose and three sulfated carbohydrate derivatives. * indicated p < 0.05; ** indicated p < 0.01.

Figure 4

ACE2 contains blood group A antigen. (A) Western blotting analysis of ACE2 from the lung tissue of a blood group A patient. Lane 1: markers. Lane 2: the Western blotting of the lung tissue from blood group A patient immunoprecipitated with anti-ACE2 antibody and immunoblotted with anti-ACE2 antibody. Lane 3: the Western blotting of the lung tissue from blood group A patient immunoprecipitated with anti-ACE2 antibody and immunoblotted with anti-A antibody. (B) The binding inhibition assay of lactose or sulfated carbohydrate derivatives in blocking of the SARS-CoV-2 spike protein S1 subunit to ACE2. Compounds 1 and 2 showed a reduction in binding efficiency of ACE2 with the spike protein S1 subunit by 6.7% and 12.5%, respectively. TD139 exhibited no effects in spike protein S1 subunit–ACE2 interaction. * indicated p < 0.05; ** indicated p < 0.01.