Journal: Journal of Advanced Research

Article Title: Key β1-4 galactosylated glycan receptors of SARS-CoV-2 and its inhibitor from the galactosylated glycoproteins of bovine milk

doi: 10.1016/j.jare.2024.12.010

Figure Lengend Snippet: Glycan profiles of the SARS-CoV-2 S1 and the ACE2 receptor. (A) Schematic diagram illustrating the process of preparing antibody-overlay lectin microarrays. (B, C) Scanned images were obtained for the analysis of glycopatterns from the SARS-CoV-2-S1 (B) and ACE2 (C). HEK293-expressing recombinant proteins of S1 and ACE2 were incubated with lectin microarrays. Subsequently, the microarrays were incubated with biotin-labeled primary antibodies and Cy3-labeled streptavidin. The representative lectins that recognized β1-4 galactosylated glycans (ECA and MAL-I), agalactosylated glycans (GSL-II and STL), bisected and bi-antennary N-glycans (PHA-E), oligo-mannose type N-glycans (ConA and HHL), fucosylation (PSA and LCA) and α-2,3 linked sialic acid (MAL-II) were marked with white frames. (D, E) Analysis of glycopatterns on S1 (D) and ACE2 (E). The lectins were classified according to their glycan binding preferences. The NFIs of each lectin were obtained from three biological replicates. The proportion of main types of glycans was calculated by diverging the sum of the NFIs of the lectins that recognized this type of glycan by the total NFIs of all lectins. Blue square: GlcNAc; yellow circle: galactose; yellow square: GalNAc; green circle: mannose; red triangle: fucose; purple diamond: sialic acid. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The primary antibodies used were as follows: a mouse monoclonal antibody against ACE2 (Proteintech, China), a rabbit polyclonal antibody against the SARS-CoV-2 S protein (ABclonal, China), and a mouse monoclonal antibody against GAPDH (Abways, China).

Techniques: Glycoproteomics, Expressing, Recombinant, Incubation, Labeling, Binding Assay

Journal: Journal of Advanced Research

Article Title: Key β1-4 galactosylated glycan receptors of SARS-CoV-2 and its inhibitor from the galactosylated glycoproteins of bovine milk

doi: 10.1016/j.jare.2024.12.010

Figure Lengend Snippet: Role of N-glycans in the interaction between S1 and ACE2. (A) Schematic diagram illustrating the process of manufacturing the SRAS-CoV-2-related recombinant protein microarrays. (B, C) The N-glycans on S1 of SARS-CoV-2/1 and ACE2 were removed by PNGase F glycosidase. The roles of N-glycans in the interaction between the SARS-CoV-2-S1 /ACE2 (B) and the SARS-CoV-1-S1/ACE2 (C) were evaluated using protein microarrays. Statistical analysis of the relative fluorescence intensities was conducted by comparing the PNGase F-treated S1 and ACE2 to the intact glycosylated protein using one-way ANOVA with Dunnett multiple comparisons. The data were obtained from three biological replicates and presented as the mean ± SD (error bars), and the p values were indicated. (D) MD simulation of the interaction between the trimeric S protein and ACE2. The distances between the N-glycosites and the center of the binding interface (represented by the green globule) within 50 Å were marked with red spheres. Other N-glycosites were marked with yellow spheres. (E) The interactions of glycans at specific sites and GRDs (marked with a red frame) may be involved in the binding of the S protein to ACE2. (F) MD simulated the interactions of glycans at specific sites and GRDs. The distances between the terminal glycans on these sites and the three GRDs on the ACE and S1 subunit were monitored during a 100 ns MD simulation. The distances of N546-GRD1, N322-GRD2, and N53-GRD2 fluctuated between 1 and 15 Å, while the distances of N343-GRD3 and N165-GRD3 fluctuated between 20 and 35 Å. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The primary antibodies used were as follows: a mouse monoclonal antibody against ACE2 (Proteintech, China), a rabbit polyclonal antibody against the SARS-CoV-2 S protein (ABclonal, China), and a mouse monoclonal antibody against GAPDH (Abways, China).

Techniques: Recombinant, Fluorescence, Binding Assay

Journal: Journal of Advanced Research

Article Title: Key β1-4 galactosylated glycan receptors of SARS-CoV-2 and its inhibitor from the galactosylated glycoproteins of bovine milk

doi: 10.1016/j.jare.2024.12.010

Figure Lengend Snippet: β1-4 galactosylated N-glycans of ACE2 mediated the binding of S1 of SARS-CoV-2 and its variants. (A) Molecular docking analysis of S1 and ACE2 with various saccharides. The potential binding capacities of S1 of SARS-CoV-2 (Wuhan-Hu-1 strain, wild type) and its variants (Delta and Omicron), as well as ACE2, to various saccharides were predicted by molecular docking analysis. The saccharides were listed in columns, S1 and ACE2 were listed in rows. The different binding abilities were represented by the values of binding free energy, which were indicated by the color of each square: red: high affinity, blue: low affinity, Xyl: xylose, Glc: glucose; Man: mannose; GlcNAc: N-acetylglucosamine, GalNAc: N-acetylgalactosamine; SA: sialic acid. (B) Validation of β1-4 galactosylation level in intact and de-β1-4galactosylated ACE2. After β1-4 galactosidase treatment, the level of β1-4 galactosylation on ACE2 was detected by lectin blotting of MAL-I. The protein level of ACE2 served as the control. (C) Scanning images of protein microarrays incubated with 1 μg of intact or de-β1-4galactosylated ACE2. (D) Effect of β1-4 galactosylation of ACE2 on the binding of S1 to ACE2. The relative fluorescence intensities were statistically analyzed by comparing the de-β1-4galactosylated ACE2 to intact ACE2 using an unpaired t test with Welch's correction. The data were obtained from three biological replicates and presented as the mean ± SD (error bars), and the p values were indicated. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The primary antibodies used were as follows: a mouse monoclonal antibody against ACE2 (Proteintech, China), a rabbit polyclonal antibody against the SARS-CoV-2 S protein (ABclonal, China), and a mouse monoclonal antibody against GAPDH (Abways, China).

Techniques: Binding Assay, Biomarker Discovery, Control, Incubation, Fluorescence

Journal: Journal of Advanced Research

Article Title: Key β1-4 galactosylated glycan receptors of SARS-CoV-2 and its inhibitor from the galactosylated glycoproteins of bovine milk

doi: 10.1016/j.jare.2024.12.010

Figure Lengend Snippet: Evaluation of the ability of free saccharides to block S1 and ACE2 binding. (A, B) Scanning images of protein microarrays. ACE2 was mixed with GalNAc (A) or Galβ1-3GalNAc (B), and the inhibitory effect of saccharides was evaluated using protein microarrays. (C, D) Effect of GalNAc (C) and Galβ-1,3GalNAc (D) on the interaction between S1 of SARS-CoV-2/1 and ACE2. The binding signals were extracted, and the relative fluorescence intensities were compared with those of the controls using one-way ANOVA with Dunnett multiple comparisons. The data were obtained from three biological replicates and presented as the mean ± SD (error bars), and the p values were indicated.

Article Snippet: The primary antibodies used were as follows: a mouse monoclonal antibody against ACE2 (Proteintech, China), a rabbit polyclonal antibody against the SARS-CoV-2 S protein (ABclonal, China), and a mouse monoclonal antibody against GAPDH (Abways, China).

Techniques: Blocking Assay, Binding Assay, Fluorescence

Journal: Journal of Advanced Research

Article Title: Key β1-4 galactosylated glycan receptors of SARS-CoV-2 and its inhibitor from the galactosylated glycoproteins of bovine milk

doi: 10.1016/j.jare.2024.12.010

Figure Lengend Snippet: Evaluation of isolated glycoproteins for the inhibition of S1 and ACE2 binding. (A) The scanned image was obtained from the lectin microarray analysis of glycoproteins isolated from bovine milk. The representative lectins that recognized β1-4 galactosylated glycans (ECA and MAL-I), agalactosylated glycans (GSL-II), bisected N-glycans (PHA-E), high-mannose glycans (ConA), fucosylation (AAL, PSA, and LCA), α2-3 linked sialic acid (MAL-II), and α2-6 linked sialic acid (SNA) were marked with white frames. (B) Analysis of glycopatterns on isolated glycoproteins. The lectins were classified according to their glycan binding preferences. The NFIs of each lectin were obtained from three biological replicates. The proportion of galactosylated glycans was calculated by diverging the sum of the NFIs of the lectins that recognized Gal/GalNAc by the total NFIs. (C, D) Evaluation of the effect of intact and de-sialylated isolated glycoproteins on the interaction between S1 of SARS-CoV-2/1 and ACE2. The intact isolated glycoproteins (C) or de-sialylated isolated glycoproteins (D) were mixed with ACE2 and incubated with protein microarrays. The relative binding intensities of each group were compared with those of the control group, and any significant differences between groups were determined using one-way ANOVA with Dunnett multiple comparisons. The data were obtained from three biological replicates and presented as the mean ± SD (error bars), and the p values were indicated. (E) Inhibition curves for intact isolated glycoproteins (upper) and de-sialylated isolated glycoproteins (lower). Four-parameter inhibition curves were generated, and the particular IC50 values for intact isolated glycoproteins and de-sialylated isolated glycoproteins were indicated in this graph. The data were obtained from three biological replicates and presented as the mean ± SD (error bars).

Article Snippet: The primary antibodies used were as follows: a mouse monoclonal antibody against ACE2 (Proteintech, China), a rabbit polyclonal antibody against the SARS-CoV-2 S protein (ABclonal, China), and a mouse monoclonal antibody against GAPDH (Abways, China).

Techniques: Isolation, Inhibition, Binding Assay, Microarray, Glycoproteomics, Incubation, Control, Generated