Abstract
The coexistence of multiple strains or subtypes of the disease-related isoform of prion protein (PrP) in natural isolates, together with the observed conformational heterogeneity of PrP amyloid fibrils generated in vitro, indicates the importance of probing the conformation of single particles within heterogeneous samples. Using an array of PrP-specific antibodies, we report the development of a novel immunoconformational assay. Uniquely, application of this new technology allows the conformation of multimeric PrP within a single fibril or particle to be probed without pretreatment of the sample with proteinase K. Using amyloid fibrils prepared from full-length recombinant PrP, we demonstrated the utility of this assay to define (i) PrP regions that are surface-exposed or buried, (ii) the susceptibility of defined PrP regions to GdnHCl-induced denaturation, and (iii) the conformational heterogeneity of PrP fibrils as measured for either the entire fibrillar population or for individual fibrils. Specifically, PrP regions 159-174 and 224-230 were shown to be buried and were the most resistant to denaturation. The 132-156 segment of PrP was found to be cryptic under native conditions and solvent-exposed under partially denaturing conditions, whereas the region 95-105 was solvent-accessible regardless of the solvent conditions. Remarkably, a subfraction of fibrils showed immunoreactivity to PrPSc-specific antibodies designated as IgGs 89-112 and 136-158. The immunoreactivity of the conformational epitopes was reduced upon exposure to partially denaturing conditions. Unexpectedly, PrPSc -specific antibodies revealed conformational polymorphisms even within individual fibrils. Our studies provide valuable new insight into fibrillar substructure and offer a new tool for probing the conformation of single PrP fibrils.
Highlights
Misfolding and aggregation of the prion protein (PrP)4 has been linked to several fatal neurodegenerative diseases, including Creutzfeldt-Jakob dis
Using amyloid fibrils prepared from fulllength recombinant PrP, we demonstrated the utility of this assay to define (i) PrP regions that are surface-exposed or buried, (ii) the susceptibility of defined PrP regions to GdnHCl-induced denaturation, and (iii) the conformational heterogeneity of PrP fibrils as measured for either the entire fibrillar population or for individual fibrils
Design of the Dual Color Immunoconformational Assay—Our assay consisted of double staining using different pairings of PrP-specific antibodies, where the reference Ab was specific to the epitope that is solvent-accessible, but the second Ab was specific to the epitope for which we wanted to assess the conformation (Fig. 2a)
Summary
Misfolding and aggregation of the prion protein (PrP) has been linked to several fatal neurodegenerative diseases, including Creutzfeldt-Jakob dis-. Prion maladies manifest themselves in sporadic, familial, or infectious forms [2] These diseases, including sporadic CJD, display substantial variations in clinical symptoms, in neuropathological profile, and in age at onset of disease [3, 4]. This broad pathological and clinical heterogeneity is believed to be related, at least in part, to conformational variations in the disease-related isoforms of PrP (PrPSc). Similar to the high heterogeneity of PrPSc subtypes generated in sporadic CJD, spontaneous polymerization of PrP in vitro under single growth conditions produces a range of amyloid fibrillar types [20]. Solid state NMR was used to determine the threedimensional structure of fibrils produced from several amyloidogenic
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