Further Characterization of Glycoform-Selective Prions of Variably Protease-Sensitive Prionopathy.

Weiguanliu Zhang,Mohammed Moudjou,Wen-Quan Zou,Xiangzhu Xiao,Jue Yuan,Aaron Foutz,Jan P M Langeveld,Mingxuan Ding,Tetsuyuki Kitamoto,Li Cui

doi:10.3390/pathogens10050513

Abstract

Prion is an infectious protein (PrPSc) that is derived from a cellular glycoprotein (PrPC) through a conformational transition and associated with a group of prion diseases in animals and humans. Characterization of proteinase K (PK)-resistant PrPSc by western blotting has been critical to diagnosis and understanding of prion diseases including Creutzfeldt-Jakob disease (CJD) and Gerstmann-Sträussler-Scheinker (GSS) disease in humans. However, formation as well as biochemical and biological properties of the glycoform-selective PrPSc in variably protease-sensitive prionopathy (VPSPr) remain poorly understood. Here we reveal that formation of the ladder-like PrPSc in VPSPr is a PK-dependent two-step process, which is enhanced by basic pH. Two sets of PrPSc fragments can be identified with antibodies directed against an intermediate or a C-terminal domain of the protein. Moreover, antibodies directed against specific PrP glycoforms reveal faster electrophoretic migrations of PrP fragments mono-glycosylated at residue 181 and 197 in VPSPr than those in sporadic CJD (sCJD). Finally, RT-QuIC assay indicates that PrPSc-seeding activity is lower and its lag time is longer in VPSPr than in sCJD. Our results suggest that the glycoform-selective PrPSc in VPSPr is associated with altered glycosylation, resulting in different PK-truncation and aggregation seeding activity compared to PrPSc in sCJD.

Highlights

Human prion diseases can be sporadic, inherited, or acquired by infection, includingCreutzfeldt-Jakob disease (CJD), fatal insomnia, Gerstmann-Sträussler-Scheinker (GSS)disease, kuru, and variant CJD
3F4 displayed the two additional PKresistant PrPSc (PrPres) bands including VPSPr23 and 17 kDa in the brain homogenates prepared in lysis buffer plus treated with proteinase K (PK) at 25–50 μg/mL, weakly, whereas 1E4 showed increased intensity of the three additional PrPres bands compared to the samples treated with standard lysis buffer at PK concentrations between 5 and 100 μg/mL (Figure 1, right sides of the blots)
The generation of the three bands VPSPr23, 17 and 7 was followed by gradually fading of the two PrPres VPSPr26 and 20. These findings suggest that generation of the unique ladder-like electrophoretic profile of 5 PrPres fragments in VPSPr129MM involves a two-step PK-digestion process: first, generation of mono-glycosylated (VPSPr26) and unglycosylated (VPSPr20) PrPres fragments from the full-length scrapie isoform of prion protein (PrPSc) and second, generation of VPSPr23, 17 and 7 from VPSPr26 and 20, respectively

Summary

Introduction

They are highly heterogeneous, all these diseases are associated with deposition and accumulation of a pathological and infectious scrapie prion protein termed prion or PrPSc in the central nervous system [1]. Comparing PrPC and the PK-resistant PrPSc (PrPres) by western blotting has clearly disclosed that all variably glycosylated PrPC species are able to convert into PrPres in virtually all typical human prion diseases. Detection and characterization of PrPres fragments by western blotting has significantly improved our understanding of the etiology, pathogenesis, formation of PrPSc, clinical and neuropathological alternations, diagnosis, clinical classification and identification of atypical forms of various human prion diseases [1,4,5,6,7,8,9,10,11,12]

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Conclusion