Reversible off and on switching of prion infectivity via removing and reinstalling prion sialylation

Abstract

The innate immune system provides the first line of defense against pathogens. To recognize pathogens, this system detects a number of molecular features that discriminate pathogens from host cells, including terminal sialylation of cell surface glycans. Mammalian cell surfaces, but generally not microbial cell surfaces, have sialylated glycans. Prions or PrP(Sc) are proteinaceous pathogens that lack coding nucleic acids but do possess sialylated glycans. We proposed that sialylation of PrP(Sc) is essential for evading innate immunity and infecting a host. In this study, the sialylation status of PrP(Sc) was reduced by replicating PrP(Sc) in serial Protein Misfolding Cyclic Amplification using sialidase-treated PrP(C) substrate and then restored to original levels by replication using non-treated substrate. Upon intracerebral administration, all animals that received PrP(Sc) with original or restored sialylation levels were infected, whereas none of the animals that received PrP(Sc) with reduced sialylation were infected. Moreover, brains and spleens of animals from the latter group were completely cleared of prions. The current work established that the ability of prions to infect the host via intracerebral administration depends on PrP(Sc) sialylation status. Remarkably, PrP(Sc) infectivity could be switched off and on in a reversible manner by first removing and then restoring PrP(Sc) sialylation.

Figure 1. Experimental design and 2D analysis of sialylation status.

(A) Experimental design illustrating generation of desialylated and re-sialylated SSLOW material. To produce desialylated SSLOW, serial dsPMCAb reactions were seeded with brain-derived SSLOW and conducted using sialidase-treated NBH to a final dilution of SSLOW brain material of 10⁻²⁴-fold. To produce re-sialylated SSLOW, serial rsPMCAb was seeded with dsPMCAb products and conducted using non-treated NBH to a final dilution of original SSLOW brain material 10⁻³⁴-fold. To produce reference PMCAb-derived material, serial PMCAb reaction was seeded with brain-derived SSLOW and conducted using non-treated NBH to a final dilution of SSLOW brain material 10⁻²⁷-fold. Animals were inoculated IC with 10-fold diluted PMCAb, dsPMCAb- or rsPMCAb-derived material. (B) 2D Western blot analysis of SSLOW brain-, PMCAb-, dsPMCAb- and rsPMCAb-derived material. Black and white triangles mark diglycosylated and monoglycosylated glycoforms, respectively, whereas arrows mark the unglycosylated form. All blots were stained with 3F4 antibody. (C) Sialylation profiles of diglycosylated isoforms of SSLOW brain- (solid thin line), PMCAb- (solid bold line), dsPMCAb- (dashed line) and rsPMCAb-derived material (dotted line). Profiles were built as described in Materials and Methods using results of 2D Western blots.

Figure 2. Analysis of brains and spleens from animals inoculated with SSLOW PMCAb-, dsPMCAb- or rsPMCAb-derived materials.

Syrian hamsters were inoculated IC with 10-fold diluted PMCAb-, dsPMCAb- or rsPMCAb-derived material. (A) Brain or spleen homogenates were treated with PK and analyzed by Western blot. (B) Western blots of brain homogenates from animals inoculated with PMCAb-, dsPMCAb- or rsPMCAb-derived material or non-inoculated animals 600–670 days old. Brain homogenates were treated with increasing concentrations of PK as indicated. PK-digestion profiles for three independent animals are shown. (C) Serial PMCAb reactions were seeded with 10% brain or spleen homogenates from hamsters inoculated with dsPMCAb- or PMCAb-derived material, subjected to four serial rounds and reaction products were analyzed by Western blot. As positive controls, serial PMCAb reactions were seeded with 10⁹-fold diluted SSLOW brain material. As a negative control for cross-contamination, non-seeded serial PMCAb reactions were conducted in parallel (NS). Lane “PMCAb” refers to PMCAb reactions seeded with brain and spleen tissue from animal #8 in panel A “PMCAb-derived”. Independent serial PMCAb reactions were repeated twice for each samples and produced identical results. Black and white triangles mark diglycosylated and monoglycosylated glycoforms, respectively, whereas arrows mark the unglycosylated form. All blots were stained with 3F4 antibody.

Figure 3. Assessing secondary structure by infrared microspectroscopy.

(A) IR spectra obtained from PrP^Sc material purified from brains of three Syrian Hamster infected with SSLOW (blue), or PMCAb- (red), dsPMCAb- (green) or rsPMCAb-derived SSLOW (black). IR spectra were collected for products of three independent reactions for each format, PMCAb, dsPMCAb or rsPMCAb. Each spectrum represents a min/max normalized (tyrosine band at 1515 cm⁻¹) second derivative spectrum obtained by averaging ten individual point spectra. (B) Dendrogram analysis of conformational heterogeneity of SSLOW PrP^Sc material purified from brains of animals infected with SSLOW or PMCAb, dsPMCAb, or rsPMCAb reactions (n = 3 independent brains or reactions). Dendrogram obtained by hierarchical cluster analysis of the mean microspectra using the information content in the amide I region (1620–1690 cm⁻¹), using the D value as the interspectral distance measure, and Ward’s algorithm as the clustering method.