Structural Organization of Mammalian Prions as Probed by Limited Proteolysis

Ester Vázquez-Fernández,Adriana Ramos,Christopher J Silva,Irina Dynin,Lothar Stitz,Miguel A Pastrana,Benjamin Petsch,Jana Alonso,Jesús R Requena,Enric Vidal

doi:10.1371/journal.pone.0050111

Abstract

Elucidation of the structure of PrPSc continues to be one major challenge in prion research. The mechanism of propagation of these infectious agents will not be understood until their structure is solved. Given that high resolution techniques such as NMR or X-ray crystallography cannot be used, a number of lower resolution analytical approaches have been attempted. Thus, limited proteolysis has been successfully used to pinpoint flexible regions within prion multimers (PrPSc). However, the presence of covalently attached sugar antennae and glycosylphosphatidylinositol (GPI) moieties makes mass spectrometry-based analysis impractical. In order to surmount these difficulties we analyzed PrPSc from transgenic mice expressing prion protein (PrP) lacking the GPI membrane anchor. Such animals produce prions that are devoid of the GPI anchor and sugar antennae, and, thereby, permit the detection and location of flexible, proteinase K (PK) susceptible regions by Western blot and mass spectrometry-based analysis. GPI-less PrPSc samples were digested with PK. PK-resistant peptides were identified, and found to correspond to molecules cleaved at positions 81, 85, 89, 116, 118, 133, 134, 141, 152, 153, 162, 169 and 179. The first 10 peptides (to position 153), match very well with PK cleavage sites we previously identified in wild type PrPSc. These results reinforce the hypothesis that the structure of PrPSc consists of a series of highly PK-resistant β-sheet strands connected by short flexible PK-sensitive loops and turns. A sizeable C-terminal stretch of PrPSc is highly resistant to PK and therefore perhaps also contains β-sheet secondary structure.

Highlights

Prions are the etiological agents responsible for a diverse set of transmissible fatal neurodegerative diseases of humans and animals, characterized by an abnormal accumulation of prion protein (PrP) [1,2], primarily in the brain
In our previous study [13], we demonstrated the usefulness of combining limited proteolysis and mass spectrometry (MS) to obtain structural information about two strains of hamster PrPSc
The presence of PrPSc was confirmed by digesting a portion of some of these brains, after suitable homogenization, with proteinase K (PK) and analyzing the result by Western blot (Figure 1A and S1)

Summary

Introduction

Prions are the etiological agents responsible for a diverse set of transmissible fatal neurodegerative diseases of humans and animals, characterized by an abnormal accumulation of prion protein (PrP) [1,2], primarily in the brain. Prions replicate by converting the normal non-infectious cellular prion protein (PrPC) into a prion (PrPSc), via a poorly characterized post-translational conformational transformation. PrP contains approximately 209 amino acids (numbered 23–231 after cleavage of a 22– mer signal peptide) and has four covalent post-translational modifications: two asparagine N-linked glycans at residues N180 and N196, a disulfide bridge between residues C178–C213 and a glycosylphosphatidylinositol (GPI) anchor attached to the Cterminus of the protein (residue S231) [2,3]. Mouse PrPC is a monomer, while PrPSc is a heterogeneous multimer [2,3]. There have been no demonstrated covalent differences between mouse PrPSc and PrPC. The only difference between PrPSc and PrPC is conformational; they are isoforms [2]

Methods

Results

Conclusion