Vitamin D 2 interacts with Human PrP(c) (90-231) and breaks PrP(c) oligomerization in vitro

Abstract

PrP(sc), the pathogenic isoform of PrP(c), can convert PrP(c) into PrP(sc) through direct interactions. PrP(c) oligomerization is a required processing step before PrP(sc) formation, and soluble oligomers appear to be the toxic species in amyloid-related disorders. In the current study, direct interactions between vitamin D 2 and human recombinant PrP(c) (90-231) were observed by Biacore assay, and 3F4 antibody, specific for amino acid fragment 109-112 of PrP(c), inhibited this interaction. An ELISA study using3F4 antibody showed that PrP(c) (101-130), corresponding sequence to human PrP, was affected by vitamin D 2, supporting the results of Biacore studies and suggesting that the PrP(c) sequence around the 3F4 epitope was responsible for the interaction with vitamin D 2. Furthermore, the effects of vitamin D 2 on disruption of PrP(c) (90-231) oligomerization were elucidated by dot blot analysis and differential protease k susceptibilities. While many chemical compounds have been proposed as potential therapeutic agents for the treatment of scrapie, most of these are toxic. However, given the safety and blood brain barrier permeability of vitamin D 2, we propose that vitamin D 2 may be a suitable agent to target PrP(c) in the brain and therefore is a potential therapeutic candidate for prion disease.

Keywords: PrPc; PrPsc; oligomerization; prion disease; vitamin D2.

Figure 1. Affinity of V-D to PrP, as measured using the Biacore system. (A) The interaction between PrP^c (90–231) and V-D₂ showed high binding. (B) The interaction between PrP^c (90–231) and V-D₃ showed no binding affinity. (C) The interaction between PrP^c (90–231) and V-D₂, after saturating with the3F4 mAb. (D) The interaction between PrP^c (101–130) and V-D₂, after saturating with the 3F4 mAb. Experiments were performed at least 3 times and similar results were obtained. The figures express the representative results of the experiments.

Figure 2. Reactivity of mAbs against PrP^c with V-D₂ by ELISA. (A) The 3F4 epitope on PrP^c was affected by V-D₂ in a dose-dependent manner. The blue line indicates signals for Hu-rPrP^c (90–231), and the red line indicates signals for PrP^c (101–130). (B) The SAF70 epitope on PrP^c was not affected by V-D₂. The blue line indicates signals for Hu-rPrP^c (90–231), and the green line indicates signals for PrP^c (141–170). Each experiment was performed 3 times by triplicates, and the values express means ± SD *p < 0.006,**p < 0.003 vs. PrP^c(101–130) without V-D₂, †p < 0.005, ††p < 0.01, †††p < 0.05 vs. Hu-rPrP^c(90–231) without V-D₂.

Figure 3. Sensitivity of PrP^c to protease K following incubation with V-D₂. V-D₂ increased the sensitivity of Hu-rPrP^c (90–231) (A) and PrP^c (101–130) (B) to PK in a dose-dependent manner. The average pixel density of each spot was measured by NIH image analysis after subtracting the mean background pixel density from that of the test spots. Experiments were performed 3 times by duplicates, and the representative results were expressed. Values are means ± SD of three experiments. *p < 0.05, **p < 0.02, ***p < 0.01 vs. Hu-rPrP^c (90–231) or PrP^c (101–130) with PK in the absence of V-D₂.