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. 2022 Mar 30;14(4):e09824. doi: 10.15252/emmm.201809824

Figure EV2. VHH‐B9 binding is specific to BACE1 with an epitope identical or overlapping the one targeted by the 1A11 anti‐BACE1 monoclonal antibody.

Figure EV2

  • A
    VHH‐B9 binds to a unique exosite on BACE1. Wild‐type (WT) BACE1 ectodomain (1–460) or various mutants, including S376Q377D378/WAA (F(SQD/WAAA)), EDVATSQDD371‐379/MGAGLNYE (F(BACE2)), EVATSQD371‐378/EGS (ΔF), and GFPLNQSEVLASVG219‐232/GAG (ΔA), were purified from cultures of HEK293 cells. 200 ng purified protein was dotted onto a nitrocellulose membrane and probed with the indicated antibodies. Monoclonal antibodies 10B8, 5G7, and 1A11 were used as positive controls. 10B8 and 5G7 recognize all forms of BACE1 tested, indicating that the mutants were properly folded. 1A11 does not recognize BACE1 with mutations in loop F but recognizes Helix A mutants, as well as WT BACE1, as previously reported (Zhou et al, 2011). VHH‐B9 only weakly recognized ΔLoop F and ΔHelix A mutants, but recognized other Loop F mutants, S376Q377D378/WAA and EDVATSQDD371‐379/MGAGLNYE. Helix A is a structure adjacent to Loop F, flanking the active‐site cleft of BACE1. This suggests that VHH‐B9 binds via a conformational epitope engaging Helix A and Loop F, although direct binding to Loop F may not necessarily be needed. Binding to these structural elements, unique to BACE1, likely explains the specific inhibition seen with VHH‐B9.
  • B, C
    VHH‐B9 and monoclonal antibody 1A11 compete for binding to BACE1. Epitope binning was done by biolayer interferometry (Octet RED96; Molecular Devices). Biotinylated BACE1 was bound to streptavidin sensor tips. Afterwards, BACE1‐loaded sensors were dipped in 1A11, VHH‐B9, or kinetic buffer for 600 s to allow binding. After a 30‐s equilibration in kinetic buffer (orange vertical lines), tips were dipped in VHH‐B9 or 1A11. (B) No additional binding of VHH‐B9 was observed. (C) A low level of 1A11 binding was observed. This residual binding is most probably due to the displacement of VHH‐B9 by 1A11, suggesting that these two antibodies bind to the same or adjacent epitopes.