Abstract
Recombinant prion protein, rPrP, binds DNA. Both the KKRPK motif and the octapeptide repeat region of rPrP are essential for maximal binding. rPrP with pathogenic insertional mutations binds more DNA than wild-type rPrP. DNA promotes the aggregation of rPrP and protects its N terminus from proteinase K digestion. When rPrP is mixed with an expression plasmid and Ca(2+), the rPrP.DNA complex is taken up by mammalian cells leading to gene expression. In the presence of Ca(2+), rPrP by itself is also taken up by cells in a temperature- and pinocytosis-dependent manner. Cells do not take up rPrP(DeltaKKRPK), which lacks the KKRPK motif. Thus, rPrP is the carrier for DNA and the KKRPK motif is essential for its uptake. When mixed with DNA, a pentapeptide KKRPK, but not KKKKK, is sufficient for DNA internalization and expression. In contrast, whereas the normal cellular prion protein, PrP(C), on the cell surface can also internalize DNA, the imported DNA is not expressed. These findings may have relevance to the normal functions of PrP(C) and the pathogenic mechanisms of human prion disease.
Highlights
More than 30 different pathogenic mutations in the human PRNP gene have been identified [3]
Accumulated evidence suggests that the conversion process may require the participation of other macromolecules, such as glycosaminoglycans (6 – 8), nucleic acids [9, 10], lipids [11, 12], cellular proteins, such as chaperone proteins [13, 14], or divalent cations [15, 16]
We reported that rPrPMs with pathogenic mutations have a more exposed N terminus and bind more glycosaminoglycan (GAG) [22, 23]
Summary
More than 30 different pathogenic mutations in the human PRNP gene have been identified [3]. Binding of rPrP to DNA promotes the uptake of the rPrP1⁄7DNA complexes by mammalian cells, resulting in gene expression. For the cell surface PrPC-mediated DNA internalization assay, Cy3-labeled pEYFP-GPI plasmid
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