Munohistochemical analyses of scrapie-infected GPI2 tg mouse brain. (a) Haematoxylin-eosin staining of the hippocampal formation. (b) IHC staining (antibody 6H4) of the hippocampal formation. C. Kaplan-Meier survival curves of wild-type mice (C57BL/6) inoculated with 2 of brain homogenate from scrapie-infected GPI2 PrPSc (green line) and a negative control inoculated with PBS (red line). doi:10.1371/journal.pone.0050111.gPrPSc became more susceptible to proteolytic digestion in a guanidine-concentration dependent manner. At concentrations above 1 M, the 10.2 and, to a lesser extent, 12 and 8 kDa bands (N152-S232/M153-S232, G141-S232, and Y162-S232) predominate. Above 3 M guanidine, which renders the unfolding irreversible [18], almost no PK-resistant material remains (Figure 5). These results mirror those of the PK time course (vide supra), i.e. all of the bands are derived from the progressive N-terminal digestion of a progenitor peptide. In their original report, Kocisko et al. identified in SHaPrPSc partially unfolded with guanidine, a highly stable PKresistant core starts before position 143 and continues to the Cterminus [18]. Sajnani et al. also detected a resistant SHaPrPSc core starting at position 139/142 [13].DiscussionWe present a complete survey of susceptibility to limited proteolysis of a PrPSc strain (Figure S6). The map of PKsusceptible spots: 116?18, 133?34, 141, 152?53, 162, 169, and 179, strongly suggests regions corresponding to loops and turns, while nicks at 81, 85, and 89 signal the frontier Title Loaded From File between thestructured C-terminal and unstructured N-terminal domains of PrPSc. Given the high proportion of b-sheet secondary sctructure derived from FTIR analyses, it is logical to conclude that PKresistant stretches flanking these spots most likely are strands of bsheet. Our results are in excellent agreement with our previous studies of wild-type PrPSc [13]. Our experiments with two different SHaPrPSc strains showed the sequence stretches 23?6 (263K), 23?01 (Dy), 117?19, 131?42, and the region around 154 ( = mouse M153) to be sensitive to PK. In the present study, besides confirming these regions as being PK-sensitive, we identified three additional PK cleavage sites in the C-terminal region of GPIPrPSc (Y162, S169 and V179). We did not find evidence of any PK-resistant Title Loaded From File peptide with an Nterminus beginning beyond V179. This is not a consequence of technical limitations, since the Tricine-based SDS-PAGE allows identification of peptides as small as 3.5 kDa (Figure 3). Instead, either this region is completely resistant to PK, or no stable PKresistant cores remain if PK cleaves beyond that point. Our results also agree with several studies describing aminoterminally truncated PK-resistant peptides in human CJD PrPSc.Structural Organization of Mammalian PrionsFigure 2. MALDI-TOF spectrum of PK-treated purified GPI2 PrPSc. Doubly-charged ions from peptides with m/z 16371 and 17148 are indicated (*). Low resolution in the .16 kDa region precluded identifying unmarked peaks. A scheme of GPI- PrP sequence with PK cleavage points (color coded) and secondary structure of PrPC is included at the top: (octarepeats ( ), b-sheets (c), and a-helices (I)); epitopes of the mAbs used are also indicated. doi:10.1371/journal.pone.0050111.gTable 1. PK-resistant fragments in GPI2 PrPSc.WESTERN BLOT Band 1 2 3 4 5 6 7 kDa 17 14.6 13 12 10.2 8 6.MALDI-TOF Peak (Da) 17148 16726 16371 13606 13463 12173 12041 11171 9687 9573 8358 7436 6274 Theoretic.Munohistochemical analyses of scrapie-infected GPI2 tg mouse brain. (a) Haematoxylin-eosin staining of the hippocampal formation. (b) IHC staining (antibody 6H4) of the hippocampal formation. C. Kaplan-Meier survival curves of wild-type mice (C57BL/6) inoculated with 2 of brain homogenate from scrapie-infected GPI2 PrPSc (green line) and a negative control inoculated with PBS (red line). doi:10.1371/journal.pone.0050111.gPrPSc became more susceptible to proteolytic digestion in a guanidine-concentration dependent manner. At concentrations above 1 M, the 10.2 and, to a lesser extent, 12 and 8 kDa bands (N152-S232/M153-S232, G141-S232, and Y162-S232) predominate. Above 3 M guanidine, which renders the unfolding irreversible [18], almost no PK-resistant material remains (Figure 5). These results mirror those of the PK time course (vide supra), i.e. all of the bands are derived from the progressive N-terminal digestion of a progenitor peptide. In their original report, Kocisko et al. identified in SHaPrPSc partially unfolded with guanidine, a highly stable PKresistant core starts before position 143 and continues to the Cterminus [18]. Sajnani et al. also detected a resistant SHaPrPSc core starting at position 139/142 [13].DiscussionWe present a complete survey of susceptibility to limited proteolysis of a PrPSc strain (Figure S6). The map of PKsusceptible spots: 116?18, 133?34, 141, 152?53, 162, 169, and 179, strongly suggests regions corresponding to loops and turns, while nicks at 81, 85, and 89 signal the frontier between thestructured C-terminal and unstructured N-terminal domains of PrPSc. Given the high proportion of b-sheet secondary sctructure derived from FTIR analyses, it is logical to conclude that PKresistant stretches flanking these spots most likely are strands of bsheet. Our results are in excellent agreement with our previous studies of wild-type PrPSc [13]. Our experiments with two different SHaPrPSc strains showed the sequence stretches 23?6 (263K), 23?01 (Dy), 117?19, 131?42, and the region around 154 ( = mouse M153) to be sensitive to PK. In the present study, besides confirming these regions as being PK-sensitive, we identified three additional PK cleavage sites in the C-terminal region of GPIPrPSc (Y162, S169 and V179). We did not find evidence of any PK-resistant peptide with an Nterminus beginning beyond V179. This is not a consequence of technical limitations, since the Tricine-based SDS-PAGE allows identification of peptides as small as 3.5 kDa (Figure 3). Instead, either this region is completely resistant to PK, or no stable PKresistant cores remain if PK cleaves beyond that point. Our results also agree with several studies describing aminoterminally truncated PK-resistant peptides in human CJD PrPSc.Structural Organization of Mammalian PrionsFigure 2. MALDI-TOF spectrum of PK-treated purified GPI2 PrPSc. Doubly-charged ions from peptides with m/z 16371 and 17148 are indicated (*). Low resolution in the .16 kDa region precluded identifying unmarked peaks. A scheme of GPI- PrP sequence with PK cleavage points (color coded) and secondary structure of PrPC is included at the top: (octarepeats ( ), b-sheets (c), and a-helices (I)); epitopes of the mAbs used are also indicated. doi:10.1371/journal.pone.0050111.gTable 1. PK-resistant fragments in GPI2 PrPSc.WESTERN BLOT Band 1 2 3 4 5 6 7 kDa 17 14.6 13 12 10.2 8 6.MALDI-TOF Peak (Da) 17148 16726 16371 13606 13463 12173 12041 11171 9687 9573 8358 7436 6274 Theoretic.
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