PrP Conformational Transitions Alter Species Preference of a PrP-specific Antibody

Abstract

The epitope of the 3F4 antibody most commonly used in human prion disease diagnosis is believed to consist of residues Met-LysHis-Met (MKHM) corresponding to human PrP-(109 -112). This assumption is based mainly on the observation that 3F4 reacts with human and hamster PrP but not with PrP from mouse, sheep, and cervids, in which Met at residue 112 is replaced by Val. Here we report that, by brain histoblotting, 3F4 did not react with PrP of uninfected transgenic mice expressing elk PrP; however, it did show distinct immunoreactivity in transgenic mice infected with chronic wasting disease. Compared with human PrP, the 3F4 reactivity with the recombinant elk PrP was 2 orders of magnitude weaker, as indicated by both Western blotting and surface plasmon resonance. To investigate the molecular basis of these species-and conformer-dependent preferences of 3F4, the epitope was probed by peptide membrane array and antigen competition experiments. Remarkably, the 3F4 antibody did not react with MKHM but reacted strongly with KTNMK (corresponding to human PrP-(106 -110)), a sequence that is also present in cervids, sheep, and cattle. 3F4 also reacted with elk PrP peptides containing KTNMKHV. We concluded that the minimal sequence for the 3F4 epitope consists of residues KTNMK, and the speciesand conformer-dependent preferences of 3F4 arise largely from the interactions between Met 112 (human PrP) or Val 115 (cervid PrP) and adjacent residues.

Characterization of Conformation-dependent Prion Protein Epitopes

“…Whereas such an approach is useful for assessing the effects of amino acid alterations on PrP Sc formation, it is not without drawbacks, because 3F4 epitope inclusion can lead to interfering effects on PrP Sc formation (55). In addition, the exact composition of the 3F4 epitope has been recently debated (56). Stable expression of PRC epitope-mutated PrP constructs in RK13 cells not only allowed us to confirm the requirement of those residues for recognition by various PRC mAbs but also to assess the effects of substitutions on conversion of PrP Sc .…”

Unaltered prion protein expression in Alzheimer disease patients

“…The epitope for mAb 6H4 has been mapped to the sequence DYEDRYYRE, corresponding to PrP residues 144-152, 25 while the epitope for 3F4 is located in the KTNMKHM, corresponding to residues 106-112. [26][27][28] Thus, while mAb 6H4 can detect the proteolytically processed sub-fragment of PrP C , referred to as C1, 3 mAb 3F4 cannot. Because di-glycosylated C1 fragments overlap with mono-and non-glycosylated forms of full-length PrP C , this could potentially affect quantification of PrP C levels and explain the discrepancy between the two mAbs.…”

Analysis of Conformational Stability of Abnormal Prion Protein Aggregates across the Spectrum of Creutzfeldt-Jakob Disease Prions

“…Given the observed rapid recovery of the original conformation of PrP Sc and the notion that PK is not significantly inhibited by GdnHCl even at a relatively high concentration (41,42), we chose to perform PK digestions in the undiluted denaturing agent (i.e., PK hydrolysis in buffer containing GdnHCl). To select the most appropriate antibody for monitoring the effect of GdnHCl on PrP Sc PK sensitivity, we compared the 3F4 antibody, which recognizes a central PrP epitope (35), with antibodies against distinct C-terminal epitopes, which are known to recognize PrP Sc truncated fragments, in addition to PrP . In contrast to 3F4, which revealed only PrP Sc 27-30, C-terminal antibodies also showed variable amounts of an 18-kDa fragment, consistent with PrP C -C1 (43), and of a 7-to 8-kDa fragment, likely generated by endogenous proteolysis of PrP C during the 1-h incubation at 37°C preceding PK digestion.…”

“…The following monoclonal mouse antibodies, immunoreactive with human PrP, were used: 3F4 at 1:30,000, which recognizes residues 106 to 110 (35); 12B2, at 1:8,000, which binds residues 89 to 93 (36); and SAF60 at 1:2,000, which reacts with residues 157 to 161 (37). In addition, the PrP Sc type 2-specific polyclonal antibody T2 (1:5,000), which binds residues 97 to 103 (7), and the rabbit antiserum 2301 (1: 3,000) to human PrP residues 220 to 231 were used.…”