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. 2012 Oct 26;287(44):37219-32.
doi: 10.1074/jbc.M112.395921. Epub 2012 Sep 4.

Characterization of conformation-dependent prion protein epitopes

Hae-Eun Kang  1 , Chu Chun Weng, Eri Saijo, Vicki Saylor, Jifeng Bian, Sehun Kim, Laylaa Ramos, Rachel Angers, Katie Langenfeld, Vadim Khaychuk, Carla Calvi, Jason Bartz, Nora Hunter, Glenn C Telling
Affiliations

Characterization of conformation-dependent prion protein epitopes

Hae-Eun Kang et al. J Biol Chem. .

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.

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Figures

FIGURE 1.
FIGURE 1.
Functional epitope of mAb PRC5. A, Western blot reactivity with various PrP primary structures. AHa, Armenian hamster; Sq. Mon., squirrel monkey. B, primary structures are aligned between mouse PrP residues 123 and 163. Reactive species are boxed and shaded red. The 6H4 and D18 epitopes are bold in cattle and mouse PrP, respectively. Residues 132 and 158 are boxed and shaded blue, and side chains are yellow in the tertiary structure. C, Western blots of RK13 cell extracts transfected with the following: Vector, pIRESPuro; Mo wt, mouse PrP; Elk wt, elk PrP; Elk V133, elk PrP with Val at residue 133; Elk S162, elk PrP with Ser at residue 162; Mo V132, mouse PrP with Val at residue 132; and Mo S158, mouse PrP with Sr at residue 158.
FIGURE 2.
FIGURE 2.
Functional epitope of mAb PRC7. A, Western blot reactivity with various PrP primary structures. Samples from hamsters, mink, ferret, and squirrel monkey were prion infected. Syr., Syrian; Chin., Chinese; and Arm., Armenian hamsters; Sq. Mon., squirrel monkey. B, primary structures are aligned between mouse PrP residues 152 and 198. Reactive species are boxed and shaded red. Glycans attached at residues 180 and 196 are shown as green hexagons. Residues 154 and 185 are boxed and shaded blue, and side chains are yellow in the tertiary structure. C, Western blots of RK13 cell extracts transfected with the following: Mo N154, mouse PrP with Asn at residue 154; Mo E185, mouse PrP with Glu at residue 185; Mo N154/E185, mouse PrP with Asn at residue 154 and Glu at residue 185; SHa wt, wild type Syrian hamster PrP; SHa Y155, Syrian hamster PrP with Tyr at residue 155; Bo wt, wild type cattle PrP; Bo Y166, cattle PrP with Tyr at residue 166; Bo Y166/Q197, cattle PrP with Tyr at residue 166, and Gln at residue 197.
FIGURE 3.
FIGURE 3.
Functional epitope of mAb PRC1. A, Western blot reactivity with various PrP primary structures. Deer H95 and S96, deer PrP with His and Ser at residues 95 and 96, respectively. B, primary structures are aligned between mouse PrP residues 81 and 115. Reactive species are boxed and shaded red. The HumP and POM2 epitopes are bold in cattle and mouse PrP, respectively. Amino acids controlling PRC1 reactivity are boxed and shaded blue. Arrows depict the locations of protease cleavage to generate C2 and C1. C, Western blots of RK13 cell extracts transfected with the following: Vector, pIRESPuro; Mo wt, mouse PrP; Elk wt, elk PrP; Mo 2G, mouse PrP with two Gly residues after residue 90; Elk 3G, elk PrP with three Gly residues. D, Western blots showing the reactivity of mAb PRC1 with PrP in the brains of uninfected and RML prion infected wild type mice and uninfected and CWD-infected Tg mice expressing elk PrP. Samples were treated with proteinase K (PK) or PNGase F as indicated. Positions of unglycosylated full-length (F), C1, and C2 fragments are shown.
FIGURE 4.
FIGURE 4.
Functional epitope of mAb PRC9. A, Western blot reactivity with various PrP primary structures. B, primary structures are aligned between mouse PrP residues 218 and 241. Reactive species are boxed and shaded red. The R1 epitope is bold in hamster PrP. Amino acids controlling PRC9 reactivity are boxed and shaded blue. Arrow depicts the glycosylphosphatidylinositol anchor signal peptidase cleavage site. C, Western blots of RK13 cell extracts transfected with the following: Vector, pIRESPuro; Mo wt, mouse PrP; Mo 224A and Mo 225A, mouse PrP harboring a mutation of Tyr to Ala at residue 224 and 225, respectively.
FIGURE 5.
FIGURE 5.
Effect of mAb recognition following PrP tertiary structure disruption by deletion mutagenesis. A, scheme showing the various deletion constructs and locations of mAb epitopes. Arrows or brackets indicate which deletions harbor epitope components for the various conformation-dependent mAbs. N-Linked glycan attachment sites at residues 180 and 196 are indicated by green hexagons, and the disulfide bond between cysteine residues 178 and 213 as a red line. B, Western blot reactivity of conformation-dependent and -independent mAbs with PNGase F-treated extracts of RK13 cells expressing PrP deletion constructs.
FIGURE 6.
FIGURE 6.
Effect of mAb recognition following PrP tertiary structure disruption by disulfide bond reduction. A, Western blots showing relative immunoreactivities of mAbs with brain extracts of RML-infected wild type mice in the presence (+βME) or absence (−βME) of reducing agent. Samples treated with proteinase K (PK) as indicated. B, densitometric analysis of mouse (blue) and elk (red) PrP levels in the presence (shaded bars) and absence (unshaded bars) of βME. *, p < 0.05; **, p < 0.005; ***, p < 0.001. O/R, ratio of oxidized to reduced PrP. C, Western blots demonstrating the effects of treatment with various concentrations of guanidinium hydrochloride on PrP detection in the brains of uninfected or RML-infected mice.
FIGURE 7.
FIGURE 7.
Immunoreactivity of conformation-dependent but not conformation-independent mAbs is reduced following post-transfer denaturation of PrP. Brain extracts were separated by SDS-PAGE and transferred to PVDF-FL membranes. Membranes were then either not treated or treated with 100 mm βME, 2% SDS at 55 °C for 30 min prior to immunoprobing with the indicated antibodies.
FIGURE 8.
FIGURE 8.
Effects of mutating PRC mAb epitopes on PrPSc formation. Western blots of extracts from RK13 cells expressing wild type and mutant PrP infected with CWD or RML prions. A and B, residues involved in the PRC5 epitope. C, residues involved in the PRC7 epitope. Mo N154, mouse PrP harboring mutation of Tyr to Asn at 154; Mo E185, mouse PrP harboring a mutation of Gln to Glu at 185; Mo N154/E185, mouse PrP harboring mutations of Tyr to Asn at 154 and Gln to Glu at 185. D–F, residues involved in the PRC1 epitope. Elk3G, elk PrP with three instead of two Gly residues; MoPrP2G, mouse PrP harboring two instead of three glycine residues; MoPrP2G/RML, infected with RML prions. Extracts were either treated (+) or not treated (−) with proteinase K (PK).
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