In vitro amplification of scrapie and chronic wasting disease PrP(res) using baculovirus-expressed recombinant PrP as substrate.

Abstract

Protein misfolding cyclic amplification (PMCA) is an in vitro simulation of prion replication, which relies on the use of normal brain homogenate derived from host species as substrate for the specific amplification of abnormal prion protein, PrP(Sc). Studies showed that recombinant cellular PrP, PrP(C), expressed in Escherichia coli lacks N-glycosylation and an glycophosphatidyl inositol anchor (GPI) and therefore may not be the most suitable substrate in seeded PMCA reactions to recapitulate prion conversion in vitro. In this study, we expressed 2 PRNP genotypes of sheep, V136L141R154Q171 and A136F141R154Q171, and one genotype of white-tailed deer (Q95G96, X132,Y216) using the baculovirus expression system and evaluated their suitability as substrates in seeded-PMCA. It has been reported that host-encoded mammalian RNA molecules and divalent cations play a role in the pathogenesis of prion diseases, and RNA molecules have also been shown to improve the sensitivity of PMCA assays. Therefore, we also assessed the effect of co-factors, such as prion-specific mRNA molecules and a divalent cation, manganese, on protein conversion. Here, we report that baculovirus-expressed recombinant PrP(C) shows a glycoform and GPI-anchor profile similar to mammalian brain-derived PrP(C) and supports amplification of PrP(Sc) and PrP(CWD) derived from prion-affected animals in a single round of seeded PMCA in the absence of exogenous co-factors. Addition of species-specific in vitro transcribed PrP mRNA molecules stimulated the conversion efficiency resulting in increased PrP(Sc) or PrP(CWD) production. Addition of 2 to 20 μM of manganese chloride (MnCl2) to unseeded PMCA resulted in conversion of recombinant PrP(C) to protease-resistant PrP. Collectively, we demonstrate, for the first time, that baculovirus expressed sheep and deer PrP can serve as a substrate in protein misfolding cyclic amplification for sheep and deer prions in the absence of additional exogenous co-factors.

• Authors: Bonto Faburay, Dongseob Tark, Anumantha G Kanthasamy, Juergen A Richt
• Journal: Prion (Prion) Vol. 8 Issue 6 Pg. 393-403 ( 2014) ISSN: 1933-690X [Electronic] United States
• PMID: 25495764 (Publication Type: Journal Article, Research Support, N.I.H., Extramural)

Chemical References

• Cations
• Chlorides
• Detergents
• Manganese Compounds
• Prions
• RNA, Messenger
• Recombinant Proteins
• Polyethylene Glycols
• Manganese
• Octoxynol
• Nonidet P-40
• Type C Phospholipases
• Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
• manganese chloride

Topics

• Animals
• Baculoviridae (metabolism)
• Brain (metabolism, pathology)
• Cations
• Chlorides (chemistry)
• Cloning, Molecular
• Deer (genetics)
• Detergents (chemistry)
• Escherichia coli (metabolism)
• Genotype
• Insecta
• Manganese (chemistry)
• Manganese Compounds (chemistry)
• Microscopy, Fluorescence
• Octoxynol
• Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase (metabolism)
• Polyethylene Glycols (chemistry)
• Prion Diseases (metabolism, pathology)
• Prions (genetics, metabolism)
• Protein Folding
• RNA, Messenger (metabolism)
• Recombinant Proteins (metabolism)
• Sheep (genetics)
• Type C Phospholipases (metabolism)