Design of anti- and pro-aggregation variants to assess the effects of methionine oxidation in human prion protein

Abstract

Prion disease is characterized by the alpha-->beta structural conversion of the cellular prion protein (PrP(C)) into the misfolded and aggregated "scrapie" (PrP(Sc)) isoform. It has been speculated that methionine (Met) oxidation in PrP(C) may have a special role in this process, but has not been detailed and assigned individually to the 9 Met residues of full-length, recombinant human PrP(C) [rhPrP(C)(23-231)]. To better understand this oxidative event in PrP aggregation, the extent of periodate-induced Met oxidation was monitored by electrospray ionization-MS and correlated with aggregation propensity. Also, the Met residues were replaced with isosteric and chemically stable, nonoxidizable analogs, i.e., with the more hydrophobic norleucine (Nle) and the highly hydrophilic methoxinine (Mox). The Nle-rhPrP(C) variant is an alpha-helix rich protein (like Met-rhPrP(C)) resistant to oxidation that lacks the in vitro aggregation properties of the parent protein. Conversely, the Mox-rhPrP(C) variant is a beta-sheet rich protein that features strong proaggregation behavior. In contrast to the parent Met-rhPrP(C), the Nle/Mox-containing variants are not sensitive to periodate-induced in vitro aggregation. The experimental results fully support a direct correlation of the alpha-->beta secondary structure conversion in rhPrP(C) with the conformational preferences of Met/Nle/Mox residues. Accordingly, sporadic prion and other neurodegenerative diseases, as well as various aging processes, might also be caused by oxidative stress leading to Met oxidation.

Fig. 1.

Methionine oxidation, PrP^c structure, and chemical analogs. (A) Met oxidation to its (S)- and (R)-sulfoxide forms [Met(O)], which, in vivo, are catalytically reduced to Met by Met-sulfoxide reductase (MsrA and MsrB) (28); further oxidation of Met(O) leads irreversibly to the Met-sulfone [(Met(O₂)]. (B) 3D structure of rhPrP^C (125–231) (PDB accession no. 1QM0); and (C) surface representation of this folded part of PrP with Met residues highlighted in yellow. Except for Met-205/206, which are buried inside the PrP^C structure, all other Met residues (including Met-109 and Met-112, which are part of the unstructured N-terminal domain) are solvent-exposed; therefore, in principle, susceptible to oxidation. (D) Met-analogs Nle and Mox used for replacement studies.

Fig. 2.

Periodate-dependent aggregation of Met-rhPrP^C determined by cross-correlation amplitude [G(0)]. Each data point represents the mean of 4 parallel samples.

Fig. 3.

The extent of Met oxidation was evaluated from the integrated peak areas: (bar 1) without periodate, (bar 2) with 5 eq of periodate, (bar 3) with 25 eq of periodate (soluble fraction), and (bar 4) with 25 eq of periodate (pellet fraction).

Fig. 4.

Mass spectra of Met-rhPrP^C (black), Nle-rhPrP^C (blue), and Mox-rhPrP^C (red). Accompanying peaks are most probably unspecifically bound Na⁺-adducts from buffer.

Fig. 5.

Aggregation properties. (A) In vitro aggregation of Met-rhPrP^C compared with Nle-rhPrP^C and Mox-rhPrP^C in the absence of oxidants. (B) Aggregation tendency as a function of periodate concentration. The intrinsic aggregation tendency of all 3 samples in the absence of periodate is arbitrarily set to 100%. Data points represent the mean of 6 different measurements. Larger variations in Mox-rhPrP^C (red triangles) sample are due to its extreme aggregation-propensity. See Materials and Methods for details (blue circles, Nle-rhPrP^C; black squares, Met-rhPrP^C).

Fig. 6.

Secondary structure analysis and thermal denaturation. (A) CD spectra of Met-rhPrP^C and its Nle- and Mox-variants at 37 °C and 0.2 mg/mL in 10 mM Mes at pH 6.0. (B) Thermal denaturation monitored by the changes of dichroic intensities at 222 nm in function of temperature.