Incomplete glycosylation during prion infection unmasks a prion protein epitope that facilitates prion detection and strain discrimination

Hae-Eun Kang,Jifeng Bian,Sarah J Kane,Sehun Kim,Vanessa Selwyn,Jenna Crowell,Jason C Bartz,Glenn C Telling

doi:10.1074/jbc.ra120.012796

Hae-Eun Kang, Jifeng Bian + Show 6 more

Open Access

https://doi.org/10.1074/jbc.ra120.012796

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Abstract

The causative factors underlying conformational conversion of cellular prion protein (PrPC) into its infectious counterpart (PrPSc) during prion infection remain undetermined, in part because of a lack of monoclonal antibodies (mAbs) that can distinguish these conformational isoforms. Here we show that the anti-PrP mAb PRC7 recognizes an epitope that is shielded from detection when glycans are attached to Asn-196. We observed that whereas PrPC is predisposed to full glycosylation and is therefore refractory to PRC7 detection, prion infection leads to diminished PrPSc glycosylation at Asn-196, resulting in an unshielded PRC7 epitope that is amenable to mAb recognition upon renaturation. Detection of PRC7-reactive PrPSc in experimental and natural infections with various mouse-adapted scrapie strains and with prions causing deer and elk chronic wasting disease and transmissible mink encephalopathy uncovered that incomplete PrPSc glycosylation is a consistent feature of prion pathogenesis. We also show that interrogating the conformational properties of the PRC7 epitope affords a direct means of distinguishing different prion strains. Because the specificity of our approach for prion detection and strain discrimination relies on the extent to which N-linked glycosylation shields or unshields PrP epitopes from antibody recognition, it dispenses with the requirement for additional standard manipulations to distinguish PrPSc from PrPC, including evaluation of protease resistance. Our findings not only highlight an innovative and facile strategy for prion detection and strain differentiation, but are also consistent with a mechanism of prion replication in which structural instability of incompletely glycosylated PrP contributes to the conformational conversion of PrPC to PrPSc.

Highlights

Scrapie of sheep and chronic wasting disease (CWD) of deer, moose, elk, and other cervids, belong to a group of interrelated, fatal, transmissible neurodegenerative diseases caused by prions
We show that monoclonal antibodies (mAbs) PRC7 preferentially reacts with PrPSc produced during prion infection, and that this selective reactivity results from hypoglycosylation and consequent exposure of the PRC7 epitope in PrPSc relative to PrPC
Our previous limited investigations of a novel conformationdependent mAb referred to as PRC7 indicated that it recognized hypoglycosylated PrP; by contrast, the conformation-dependent mAb PRC5 recognized the complete complement of full-length and proteolytically processed PrP glycoforms [24]

Summary

Introduction

Scrapie of sheep and chronic wasting disease (CWD) of deer, moose, elk, and other cervids, belong to a group of interrelated, fatal, transmissible neurodegenerative diseases caused by prions. Prion strain information is considered to be enciphered by distinct conformations of PrPSc [14, 15], our limited knowledge of PrPSc structure and inability to define strain-dependent conformational variability with any degree of precision has placed significant restrictions on our understanding of how prion proteins transmit heritable information in the absence of informational nucleic acids. In addition to their structural and biochemical differences, PrPC and PrPSc undergo diverse intracellular proteolytic processing events. Full-length, and the aforementioned amino-terminal processed forms of PrP are subject to differential glycosylation resulting in a complex distribution of upwards of 50 distinct PrPC species [20]

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