The binding of a monoclonal antibody to the apical region of SCARB2 blocks EV71 infection

Figures

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  Figure 1. The monoclonal antibody JL2 binds human SCARB2. (A) Cells overexpressing human SCARB2 and SCARB2 knockout cells were lysed and analyzed by Western blot using the anti-human SCARB2 polyclonal antibody (pAb) (top panel) and JL2 (middle panel). The lowest panel: β-actin was used as a loading control. (B) Surface staining of 293-hSCARB2 cells. Open area represents staining with JL2 and shaded area represents isotype control staining. (C) The binding of 293-hSCARB2 cells with different concentrations of JL2 (square) and the IgG2a isotype control (circle)

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  Figure 2. JL2 blocks EV71 infection. (A) GFP-positive cell counts among 293-hSCARB2 cells pretreated with JL2 (square) or the isotype control (circle) before EV71-GFP infection. The data are shown as the mean ± SEM of triplicates. (B) The replication of wild type EV71 in 293-hSCARB2 cells in the presence of JL2 (2 μg/mL) or isotype control. The data are presented as the mean ± SEM of triplicates. *P < 0.05, **P < 0.01, and ***P < 0.001 by Student’s t test

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  Figure 3. Binding site mapping of JL2 and human SCARB2. (A) An alignment of the mouse and human SCARB2 with the sequences of the 5 α-helices in the apical region highlighted. (B) The structure of the 5 α-helices in the apical region of human SCARB2 at neutral pH. (C) The sketch map of the SCARB2 chimeras. The numbers represent the amino acid sequences of human SCARB2 in the chimeras. (D) 293-SCARB2-KO cells transfected with SCARB2 chimeras were stained by JL2 (open bars) or the isotype control (filled bars). The data shown are representative of 3 independent experiments

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  Figure 4. The complex structure of JL2 Fab bound to SCARB2 (at pH 7.0). (A) The overall complex structure. The JL2 Fab binds to the head of SCARB2 (Domain III) at a perpendicular angle with helices α2, α5, and α14 involved in the interaction. Domain I, II, and III of SCARB2 are colored in orange, violet and yellow, respectively. Glycans are shown as sticks. (B and C) Cartoon representation of the interacting residues in SCARB2 (B) and Fab (C). The residues in SCARB2 involved in the interactions with JL2 Fab are shown as sticks (B). The residues in JL2 Fab involved in the interactions with SCARB2 are highlighted in bright colors and labeled on the surface (C)

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  Figure 5. The analysis of the detailed interactions between the JL2 Fab and SCARB2. (A) Five polypeptide elements, three from the heavy chain (purple) and two from the light chain (marine), surround the domain III of SCARB2. Ten residues, S28, T30, G31, Y32, Y54, Y55, R100, S102, Y105, and Y107, in the heavy chain and four residues, D31, Y32, Y50, and I55, in the light chain interact with SCARB2 (with distance less than 4.5 Å). The residues in JL2 Fab involved in the interactions with SCARB2 are shown as sticks. (B) Similar to (A), a semi-transparent surface rendering of SCARB2 presents the detailed interactions. All residues involved in the interactions are shown as sticks