Table 1.
Statistics of X-ray diffraction data collection and atomic refinement of MoPrP•Nb484 complexes and free Nb484.
Fig 1.
Crystal structure of the MoPrP(89–230) in complex with Nb484 at pH 8.0.
(A) Ribbon representation of the MoPrP•Nb484 complex shown in two orientations, the MoPrP is shown in green and Nb484 highlighted in cyan. (B) Cartoon representation of the three-stranded antiparallel β-sheet shown in red. (C) The β0-β1-α1-β2-α2-α3 topology of MoPrP(89–230).
Fig 2.
Effect of nanobodies on the prion amplification.
(A) Inhibition of prion propagation by different concentrations (1, 2, 4 and 8 μM) of Nb484 in Protein Misfolding Cyclic amplification (PMCA) for six consecutive rounds. (B) Elispot cell culture assay of prion infectivity from PMCA samples (round 6) for Nb484. (C) Inhibition of prion propagation by different concentrations (1, 2, 4 and 8 μM) of Nb862 in PMCA. (D) Elispot cell culture assay of prion infectivity from PMCA samples (round 6) for Nb862. CAD5 cells were infected with serial 10-fold dilutions of round six of PMCA products.
Fig 3.
Nb484 interacting with the hydrophobic region of PrPC.
(A) Influence of Nb484 on the interaction of PrP and POPG synthetic lipid. For rPrP+POPG+Nb484, recombinant mouse PrP was incubated with POPG before mixed with Nb484 at different molar ratios (Nb484:recPrP = 0, 1, 2, 4, or 8:1). For rPrP+Nb484+POPG, recombinant mouse PrP was incubated with Nb484 at different molar ratios (Nb484:recPrP = 0, 1, 2, 4, or 8:1) before mixed with POPG. PK-resistant PrP was detected using POM1 antibody. (B) Representative ELISA results show that both Nb484 and Nb862 bind strongly to rPrP. Nb484 has very week binding to ΔHC, but Nb862 shows strong binding signal to ΔHC. POM1 antibody was used as a primary antibody to determine the binding signal. POM1 displayed similar binding for both rPrP and ΔHC. All results are the average of 3 replicates.rPrP: MoPrP(23–230), ΔHC: MoPrPΔHC.
Fig 4.
Nb484 did not induce neurotoxicity in Tga20 mice organotypic slices comparing to POM1 antibody.
NeuN staining showing that POM1 has strong toxicity similar to the pervious results obtained by Aguzzi group [18]. Nb484 shows no neurotoxicity in Tga20 mice organotypic slices similar to the slices treated with PBS. Slices were stained with IgG1 antibodies to NeuN. The white bar is 100 μm.
Fig 5.
Superimposition of different MoPrP, HuPrP and ovine PrP structures in complex with Fab and Nb484.
(A) Front view, all prion proteins are shown in green, heavy and light chains of POM1 Fab (PDB 4H88) are shown in yellow, ICSM18 (PDB 2W9E) in pink, VRQ14 Fab (PDB 1TPX) in cyan and Nb484 in brown. (B) Top view of the superposition.