Formation of Soluble Oligomers and Amyloid Fibrils with Physical Properties of the Scrapie Isoform of the Prion Protein from the C-terminal Domain of Recombinant Murine Prion Protein mPrP-(121-231).

Prion diseases are fatal neurodegenerative disorders associated with conformational conversion of the cellular prion protein, PrPC into a misfolded, protease-resistant form, PrPSc. Here we show, for the first time, the oligomerization and fibrillization of the C-terminal domain of murine PrP, mPrP-(121–231), which lacks the entire unstructured N-terminal domain of the protein. In particular, the construct we used lacks amino acid residues 106–120 from the so-called amyloidogenic core of PrP (residues 106–126). Amyloid formation was accompanied by acquisition of resistance to proteinase K digestion. Aggregation of mPrP-(121–231) was investigated using a combination of biophysical and biochemical techniques at pH 4.0, 5.5, and 7.0 and at 37 and 65 °C. Under partially denaturing conditions (65 °C), aggregates of different morphologies ranging from soluble oligomers to mature amyloid fibrils of mPrP-(121–231) were formed. Transmission electron microscopy analysis showed that roughly spherical aggregates were readily formed when the protein was incubated at pH 5.5 and 65 °C for 1 h, whereas prolonged incubation led to the formation of mature amyloid fibrils. Samples incubated at 65 °C at pH 4.0 or 7.0 presented an initial mixture of oligomers and protofibrils or fibrils. Electrophoretic analysis of samples incubated at 65 °C revealed formation of sodium dodecyl sulfate-resistant oligomers (dimers, trimers, and tetramers) and higher molecular weight aggregates of mPrP-(121–231). These results demonstrate that formation of an amyloid form with physical properties of PrPsc can be achieved in the absence of the flexible N-terminal domain and, in particular, of residues 106–120 of PrP and does not require other cellular factors or a PrPsc template. [ABSTRACT FROM AUTHOR]