Sialylation Controls Prion Fate in Vivo

Abstract

Prions or PrP^Sc are proteinaceous infectious agents that consist of misfolded, self-replicating states of a sialoglycoprotein called the prion protein or PrP^C The current work tests a new hypothesis that sialylation determines the fate of prions in an organism. To begin, we produced control PrP^Sc from PrP^C using protein misfolding cyclic amplification with beads (PMCAb), and also generated PrP^Sc with reduced sialylation levels using the same method but with partially desialylated PrP^C as a substrate (dsPMCAb). Syrian hamsters were inoculated intraperitoneally with brain-derived PrP^Sc or PrP^Sc produced in PMCAb or dsPMCAb and then monitored for disease. Animals inoculated with brain- or PMCAb-derived PrP^Sc developed prion disease, whereas administration of dsPMCAb-derived PrP^Sc with reduced sialylation did not cause prion disease. Animals inoculated with dsPMCAb-derived material were not subclinical carriers of scrapie, as no PrP^Sc was detected in brains or spleen of these animals by either Western blotting or after amplification by serial PMCAb. In subsequent experiments, trafficking of brain-, PMCAb-, and dsPMCAb-derived PrP^Sc to secondary lymphoid organs was monitored in wild type mice. PrP^Sc sialylation was found to be critical for effective trafficking of PrP^Sc to secondary lymphoid organs. By 6 hours after inoculation, brain- and PMCAb-derived PrP^Sc were found in spleen and lymph nodes, whereas dsPMCAb-derived PrP^Sc was found predominantly in liver. This study demonstrates that the outcome of prion transmission to a wild type host is determined by the sialylation status of the inoculated PrP^Sc Furthermore, this work suggests that the sialylation status of PrP^Sc plays an important role in prion lymphotropism.

Keywords: Fourier transform IR (FTIR); N-linked glycans; N-linked glycosylation; cyclic amplification; prion; prion disease; protein misfolding; secondary lymphoid organs; sialic acid; sialylation; spleen.

FIGURE 1.

2D Western analysis of sialylation status of brain-, PMCAb-, and dsPMCAb-derived PrP^Sc. A, 2D Western blotting analysis of 263K brain-, PMCAb-, and dsPMCAb-derived material. 2D Western analysis of 263K brain-derived material denatured and then treated with sialidase is provided as a reference. Black and white triangles mark diglycosylated and monoglycosylated glycoforms, respectively, whereas arrows mark the unglycosylated form. All blots were stained with 3F4 antibody. HyperSia, hyper-sialylated; HypoSia, hypo-sialylated; pI, isoelectric point. B, sialylation profiles of diglycosylated isoforms of 263K brain- (solid thick line), PMCAb- (solid thin line), or dsPMCAb-derived material (dashed line). Profiles were built as described under “Experimental Procedures” using results of 2D Western blots.

FIGURE 2.

Western blotting and sPMCAb analysis of brains and spleens from animals inoculated with 263K brain-, PMCAb-, or dsPMCAb-derived material. Syrian hamsters were inoculated i.p. with 10⁴-diluted 263K brain material, or 10-fold diluted PMCAb- or dsPMCAb-derived material (n = 6). The scrapie brain material was diluted 10⁴-fold to match the amount of PK-resistant material to that in the 10-fold diluted PMCAb- and dsPMCAb-derived samples. Animals were euthanized at the terminal stage of the disease, or at 352 days after inoculation if no symptoms were evident. A, 10% brain or spleen homogenates were treated with PK and analyzed by Western blotting. B, sPMCAb reactions were seeded with 10% brain or spleen homogenates from hamsters inoculated with dsPMCAb-derived material and then subjected to four serial rounds, and reaction products were analyzed by Western blotting. As positive controls, sPMCAb reactions were seeded with 10⁹-fold diluted 263K brain material. As negative controls for cross-contamination, non-seeded (NS) sPMCAb reactions were conducted in parallel. 3F4 antibody was used for staining in all Western blots.

FIGURE 3.

Assessing secondary structure by infrared microspectroscopy. A, IR spectra obtained from PrP^Sc materials purified from brains of three Syrian Hamster infected with 263K (blue), three independent PMCAb reactions (red), or three independent dsPMCAb reactions (green), each seeded with brain-derived 263K. Each spectrum represents a minimum/maximum normalized (tyrosine band at 1515 cm⁻¹) second derivative spectrum obtained by averaging 10 individual point spectra. AU, arbitrary units. B, analysis of the conformational heterogeneity of 263K PrP^Sc material purified from brains of animals infected with 263K or PMCAb or dsPMCAb reactions (n = 3 independent brains or reactions). Dendrogram was obtained by hierarchical cluster analysis of the mean IR microspectra using the information content in the amide I region (1610–1670 cm⁻¹), D-values as the inter-spectral distance measure, and Ward's algorithm as the clustering method. 2nd deriv, second derivative; vec norm, vector normalized.

FIGURE 4.

Analysis of PrP^Sc trafficking to SLOs, liver and kidney upon intraperitoneal inoculation. A, 263K brain-, PMCAb-, or dsPMCAb-derived materials were administered via intraperitoneal injection to C57Bl mice, and then the amounts of scrapie material in spleen, lymph nodes, liver, kidney, or brain were analyzed 2, 6, and 18 h after inoculation by Western blotting (n = 3 per animal group per each time point). 0.05% 263K scrapie brain homogenate (ScBH) treated with PK served as a reference in all Western blots. Western blots were stained with Ab3531 antibody. B and C, dynamics of PrP^Sc accumulation in spleen (B) and lymph nodes (C) for brain- (black circles), PMCAb- (white circles), or dsPMCAb-derived material (triangles) are presented on the top plots, while statistical analysis of PrP^Sc amounts in spleen (B) or lymph nodes (C) at 18 h after inoculation are shown on the bottom plots. For each animal group/tissue, the signal intensities were normalized per the intensities of 0.05% scrapie brain homogenate on the same Western blots. Statistical analysis was performed as described under “Experimental Procedures.” Data are presented as means ± S.D., * indicates significant differences (p < 0.05), whereas # indicates lack of significant differences (p > 0.05) (n = 3).