Characterization of conformation-dependent prion protein epitopes.

Jifeng Bian,Rachel Angers,Katie Langenfeld,Carla Calvi,Nora Hunter,Hae-Eun Kang,Vicki Saylor,Chu Chun Weng,Vadim Khaychuk,Laylaa Ramos,Sehun Kim,Jason Bartz,Eri Saijo,Glenn C. Telling

doi:10.1074/jbc.m112.395921

Abstract

Whereas prion replication involves structural rearrangement of cellular prion protein (PrP(C)), the existence of conformational epitopes remains speculative and controversial, and PrP transformation is monitored by immunoblot detection of PrP(27-30), a protease-resistant counterpart of the pathogenic scrapie form (PrP(Sc)) of PrP. We now describe the involvement of specific amino acids in conformational determinants of novel monoclonal antibodies (mAbs) raised against randomly chimeric PrP. Epitope recognition of two mAbs depended on polymorphisms controlling disease susceptibility. Detection by one, referred to as PRC5, required alanine and asparagine at discontinuous mouse PrP residues 132 and 158, which acquire proximity when residues 126-218 form a structured globular domain. The discontinuous epitope of glycosylation-dependent mAb PRC7 also mapped within this domain at residues 154 and 185. In accordance with their conformational dependence, tertiary structure perturbations compromised recognition by PRC5, PRC7, as well as previously characterized mAbs whose epitopes also reside in the globular domain, whereas conformation-independent epitopes proximal or distal to this region were refractory to such destabilizing treatments. Our studies also address the paradox of how conformational epitopes remain functional following denaturing treatments and indicate that cellular PrP and PrP(27-30) both renature to a common structure that reconstitutes the globular domain.

Highlights

Despite structural reorganization during disease, conformational prion protein epitopes remain undefined
In accordance with their conformational dependence, tertiary structure perturbations compromised recognition by PRC5, PRC7, as well as previously characterized monoclonal antibodies (mAbs) whose epitopes reside in the globular domain, whereas conformation-independent epitopes proximal or distal to this region were refractory to such destabilizing treatments
Involvement of Residues Required for mAb Binding on protease-resistant counterpart of the pathogenic scrapie form (PrPSc) Formation—Having confirmed the roles of specific amino acid residues in the epitopes of different PRC mAbs, we investigated the effects of mutations at these positions on the ability of either elk or mouse PrPC to be converted into PrPSc

Summary

Background

Despite structural reorganization during disease, conformational prion protein epitopes remain undefined. The involvement of specific amino acid residues in such hypothesized conformational epitopes has not been described This inference raises a conundrum [13], because all such antibodies bind PrP in Western blots, where PrP is defined by investigators as denatured (12, 13, 15, 18 –22). We used a directed molecular evolution approach [23] to create shuffled genes expressing novel PrP epitopes, and by determining the reactivities of the resulting mAbs against a large panel of PrP primary structures and polymorphic variants thereof, we mapped and confirmed by mutational analysis the involvement of specific residues in epitope binding and PrPC to PrPSc conversion

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION