Identification and characterization of a spontaneously aggregating amyloid-forming variant of human PrP((90-231)) through phage-display screening of variants randomized between residues 101 and 112.
The N-terminal 'unstructured' region of the human prion protein [PrP((90-231))] is believed to play a role in its aggregation because mutations in this region are associated with seeding-independent deposition disorders like Gerstmann-Straussler-Scheinker disease (GSS). One way of examining the effects of such mutations is to search combinatorially derived libraries for sequence variants showing a propensity to aggregate and/or the ability to interact with prion molecules folded into a beta-sheet-based conformation (i.e., beta-PrP or PrP(Sc)). We created a library of 1.8x10(7) variants randomized between positions 101 and 112, displayed it on filamentous bacteriophage, and 'spiked' it with a approximately 25% population of phages-bearing wild-type prion (wt-PrP). Screening was performed through four rounds of biopanning and amplification against immobilized beta-PrP, and yielded three beta-PrP-binding populations: wt-PrP (26% representation) and two non-wt-PrP variants ( approximately 10% and approximately 64% representation, respectively). The remarkable enrichment of one non-wt-PrP variant (MutPrP) incorporating residues KPSKPKTNMKHM in place of KGVLTWFSPLWQ, despite its initial representation at a 5 million-fold lower level than wt-PrP, caused us to produce it and discover: (i) that it readily aggregates into thioflavin-T-binding amyloids between pH 6.0 and 9.0, (ii) that it adopts a soluble beta-sheet based monomeric structure at pH 10.0, (iii) that it is less thermally stable and more compact than wt-PrP, and (iv) that it displays significantly greater resistance to proteolysis than wt-PrP. Our results suggest that sequence variations in the 101-112 region can indeed predispose the prion for aggregation.
Verma
Archana
A
Postgraduate Institute of Medical Education and Research , Chandigarh 160012, India.
Sharma
Swati
S
Ganguly
Nirmal Kumar
NK
Majumdar
Siddharta
S
Guptasarma
Purnananda
P
Luthra-Guptasarma
Manni
M
D000595:Amino Acid Sequence; D000682:Amyloid / Q000378:metabolism* / Q000648:ultrastructure; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D011328:Prions / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D017510:Protein Folding; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D012995:Solubility; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D017550:Spectroscopy, Fourier Transform Infrared
D000682:Amyloid; D011328:Prions