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-Lys-His-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 species- and conformer-dependent preferences of 3F4 arise largely from the interactions between Met(112) (human PrP) or Val(115) (cervid PrP) and adjacent residues.

FIGURE 1.

Histoblotting of PrP in brain tissue from normal and CWD-infected cervidized Tg mice treated without (A–F) or with PK (G–L). A, B, G, and H, probed with 3F4. C, D, I, and J, probed with 8H4. E, F, K, and L, probed with 6H4. Unlike 8H4 and 6H4 antibodies that detected both PrP^C and PrP^Sc, 3F4 detected PrP in CWD-infected brain samples (B and H) but not PrP in non-infected normal controls (A and G). The reactivity of 3F4 with untreated or PK-treated PrP^Sc in the brain histoblots was highly reproducible.

FIGURE 2.

Elk PrP from normal and CWD-infected brain tissue probed by Western blotting with 3F4. A, PrP in 10 μl of brain homogenate from normal and CWD-infected elk was probed with 3F4 (upper panel) and 6H4 (lower panel). Although 6H4 detected PrP in brain samples from both normal and CWD-infected elk, no elk PrP was detected in these samples with 3F4. B, PrP^Sc enriched by g5p from 100 μl of homogenate each of two infected elk brains was subjected to Western blotting with 3F4. PrP was detectable in both PK-treated and untreated samples by 3F4. Two CWD cases including case 1 and case 2 were examined. Brain homogenate from a sCJD case was used as a control.

FIGURE 3.

Detection of the recombinant full-length human, elk, and mouse PrP by Western blotting. A, Western blotting of a series of concentrations of recombinant human, elk, and mouse PrP (rePrP) from 1 to 64 ng probed with 3F4. Compared with human PrP, elk PrP revealed much lower 3F4 immunoreactivity. There was no detectable 3F4 immunoreactivity with mouse rePrP. The concentration of protein was determined with a spectrophotometer (Ultrospec 3000, Amersham Biosciences). B, intensity of rePrP as a function of concentration of the protein examined by using densitometric analysis of rePrP on Western blots done by three independent experiments. The affinity of 3F4 for the elk PrP was much lower than that for human PrP (∼98% less).

FIGURE 4.

Comparative analysis of the interactions between antibody 3F4 and antigen PrP by surface plasmon resonance spectroscopy. Sensorgrams of the kinetic measurements of the interaction of antibody 3F4 and human, elk, and mouse PrP (A–C). Antibody 3F4 was immobilized on the surface of the CM5 sensor chip as described under ”Experimental Procedures.“ Each PrP was injected at concentrations of 0.5, 1, 2, 5, 10, 25, and 50 nm (from bottom to top).

FIGURE 5.

Blocking of 3F4 binding to human PrP by synthetic elk PrP peptides. A, ten 10-mer elk PrP peptides between residues 101 and 120, a 20-mer human prion peptide (Con. huPrP101–119), and a random 19-mer peptide were synthesized and examined. B, the blocking of 3F4 binding to PK-treated human PrP^Sc by synthetic peptides was detected with Western blotting. Peptides in lanes 6–11 partially blocked the binding of 3F4 to PrP^Sc (blocking by ∼60 to ∼80% detected by densitometric analysis). Peptide in lane 1 is the human PrP peptide used as a positive control that completed blocked 3F4 binding. Peptides in lanes 2–5 revealed no detectable inhibition of 3F4 binding.

FIGURE 6.

Epitope mapping by peptide membrane array. The peptides with different lengths containing the entire or partial human sequence MKHM or elk sequence MKHV were synthesized on the amino-PEG cellulose membranes. The membranes were probed with 3F4 followed by HRP-conjugated sheep anti-mouse IgG secondary antibody. The membranes incubated without 3F4 but with the secondary antibody alone were used as negative controls (data not shown). A, effect of adding residues at the C terminus of the 4-mer human sequence MKHM (Hu(M)) or elk sequence MKHV (Elk(V)) on the 3F4 binding. No 3F4 immunoreactivity was detectable in all peptides examined. B, effect of adding residues at the N terminus of the two 4-mer peptides (Hu(M), human sequence MKHM; Elk(V), elk sequence MKHV) on the 3F4 binding. C, effect of adding residues at the C terminus of 5-mer KTNMK either with Met (Hu(M), human sequence) or Val (Elk(V), elk sequence) on 3F4 binding. D, effect of replacing Met at residue 112 of the human sequence with 19 individual amino acids on 3F4 binding. The sequences of the peptides examined are shown under each array. In B–D, the histogram next to the peptide sequences represent 3F4 immunoreactivity of each spot using the arbitrary unit detected by densitometric analysis (each bar represents the average of three measurements). In B–D, the individual blue bar represents each human sequence with Met¹¹², whereas the red bar represents each elk sequence with Val¹¹⁵. The number in the front of each peptide sequence matches the number of the spot shown in the array. The sequences with the light green background are shared by each individual set of peptides examined.

FIGURE 6.

Epitope mapping by peptide membrane array. The peptides with different lengths containing the entire or partial human sequence MKHM or elk sequence MKHV were synthesized on the amino-PEG cellulose membranes. The membranes were probed with 3F4 followed by HRP-conjugated sheep anti-mouse IgG secondary antibody. The membranes incubated without 3F4 but with the secondary antibody alone were used as negative controls (data not shown). A, effect of adding residues at the C terminus of the 4-mer human sequence MKHM (Hu(M)) or elk sequence MKHV (Elk(V)) on the 3F4 binding. No 3F4 immunoreactivity was detectable in all peptides examined. B, effect of adding residues at the N terminus of the two 4-mer peptides (Hu(M), human sequence MKHM; Elk(V), elk sequence MKHV) on the 3F4 binding. C, effect of adding residues at the C terminus of 5-mer KTNMK either with Met (Hu(M), human sequence) or Val (Elk(V), elk sequence) on 3F4 binding. D, effect of replacing Met at residue 112 of the human sequence with 19 individual amino acids on 3F4 binding. The sequences of the peptides examined are shown under each array. In B–D, the histogram next to the peptide sequences represent 3F4 immunoreactivity of each spot using the arbitrary unit detected by densitometric analysis (each bar represents the average of three measurements). In B–D, the individual blue bar represents each human sequence with Met¹¹², whereas the red bar represents each elk sequence with Val¹¹⁵. The number in the front of each peptide sequence matches the number of the spot shown in the array. The sequences with the light green background are shared by each individual set of peptides examined.