Fig 1.
Distinct disease duration, schematic representation, and molecular characteristics of sCJD MM1, MM2, and VV2 prions.
(a) The age (left panel) and disease duration (right panel) in 343 cases of sporadic CJD MM1, MM2, and VV2 prion disease; (b) Outline of classification of Type 1 and Type 2 human prions based on proteolytic fragmentation of PrPSc [75, 89]. Major cleavage sites by PK are indicated by arrows; GLP–glycolipid; CHO- complex N-glycosylation chains. The codes above light blue brackets represent monoclonal antibodies used in differentiation of various domains of human prions, and the numbers below these brackets indicate linear epitopes recognized by these antibodies. (c) Distinct glycosylation and Proteinase K fragmentation patterns of purified human MM1, MM2, and VV2 sCJD rPrPSc (homozygous for methionine [M] or valine [V] in codon 129) used in structural studies. To differentiate Type 1, Type 2 prions, and their C-terminal fragments, Western blots of purified rPrPSc (fraction 8; F8) of MM1, MM2, and VV2 sCJD rPrPSc were developed with mAb 12B2 (epitope residues 89–93) [90], mAb 3F4 (epitope residues 108–112) [47], and rabbit polyclonal antibody 2301 (epitope residues 220–231) [91]. (c) Distinct fragmentation patterns of purified MM1, MM2, and VV2 sCJD prions in silver stained SDS-PAGE. The symbol (#) indicates bands corresponding to PK. The molecular weights of marker proteins are in kDa.
Table 1.
Demographics, molecular characteristics, and synchrotron hydroxyl radical inactivation of different strains of sCJD prions.
Conformational stability assay (CSA) of prions is expressed in a midpoint concentration of the GdnHCl in the transition from folded to unfolded state [24, 29, 30, 39].
Fig 2.
The •OH modification rates of different antibody epitopes in recombinant human monomeric α-helical PrP (recHuPrP) and in MM1, MM2, and VV2 sCJD prions monitored with dCDI.
(a) Calibration of dCDI with non-irradiated recHuPrP and Europium-labeled mAb 6D11 (aa 93–109), 3F4 (aa 108–112), 1E5 (aa 145–149), 8H4 (aa 145–185), 5B9 (aa 157–167) and MAR1 (aa 179–214). (b) Parallel rapid drop in normalized antibody dCDI signals of synchrotron irradiated recHuPrP. Time course of epitope modification monitored in sCJD MM1 (red), MM2 (blue) and VV2 (yellow) prions with mAb (c) Eu-6D11, (d) Eu-3F4, (e)Eu-1E5, (f) Eu-8H4, (g) Eu-5B9, and (h) Eu-MAR1. (i) Half-life of different epitopes in MM1 (red), MM2 (blue) and VV2 (yellow) sCJD prions in synchrotron beam. Each dCDI datapoint is an average ± standard error obtained in triplicate experiments normalized from the time resolved fluorescence (TRF) signal of europium in counts per minute (cpm).
Fig 3.
Inactivation rate of human sCJD MM1, MM2, and VV2 sCJD prions by synchrotron •OH. The end-point seeding activity of serially diluted MM1 prions (a) before and (b) after synchrotron irradiation, end-point seeding activity of serially diluted MM2 prions (c) before and (d) after irradiation, and seeding activity of serially diluted VV2 prions (e) before and (f) after synchrotron irradiation. The sCJD prions were irradiated in synchrotron for 0 to 200 ms, serially diluted, and seeding potency of each dilution was monitored by second generation RT-QuIC [41]. The curves in a-f are thioflavin T (ThT) fluorescence averaged at each sample dilution from four wells of 96-well RT QuIC plate as described [46]s. (g) Kinetics of inactivation of MM1 (red), MM2 (blue) and VV2 (yellow) sCJD prions determined as a function of exposure time. The SD50 was calculated with Spearman-Kärber analysis of end-point dilutions at each synchrotron irradiation time point with RT QuIC as described [46].
Fig 4.
Differential •OH modification of amino acid side chains in MM1 (red), MM2 (blue) and VV2 (yellow) brain-derived sCJD prions (a) before and (b) after synchrotron irradiation. (a) Degree of modification of specific residues before synchrotron irradiation was calculated as described for synchrotron irradiated samples. (b) •OH modification half-life of specific amino acid side chains in MM1 (red), MM2 (blue) and VV2 (yellow) calculated from fitting the fraction of unmodified data using first order rate constant equation. The complete protection of residues in MM1 prions is indicated by red line; the green line indicates high modifications rates due to the solvent exposure. The values are expressed as mean ± SEM from two independent irradiation experiments and three MS/MS peptide datasets for each time point. *, p < 0.05; **, p < 0.01; and ***, p < 0.001.
Fig 5.
Relative protection factor of MM1, MM2, and VV2 sCJD prions. The protection factor for individual residues was calculated from the modification rate of •OH modification of amino acid side chains in α-helical monomeric recHuPrP divided by the modification rate of the same residues in (a) MM1, (b) MM2, and (c) VV2 prions; the different range of amino acid protection is indicated by the color scale. (d) Comparison of protection factors in three strains of sCJD prions.