Characterization of Recombinant, Membrane-attached Full-length Prion Protein

Heike Eberl,Peter Tittmann,Rudi Glockshuber

doi:10.1074/jbc.m400952200

Abstract

An abnormal isoform, PrP(Sc), of the normal cellular prion protein (PrP(C)) is the major component of the causative agent of prion diseases. Both isoforms were found to possess the same covalent structures, including a C-terminal glycosylphosphatidylinositol anchor, but different secondary and tertiary structures. In this study, a variant of full-length PrP with an unpaired cysteine at the C terminus was recombinantly produced in Escherichia coli, covalently coupled to a thiol-reactive phospholipid, and incorporated into liposomes to serve as a model for studying possible changes in structure and stability of recombinant PrP upon membrane attachment. Covalent coupling of PrP to liposomes did not result in significant structural changes observable by far-UV circular dichroism. Moreover, limited proteolysis experiments failed to detect changes in the stability of liposome-bound PrP relative to soluble PrP. These data suggest that the requirement of raft localization for the PrP(C) to PrP(Sc) conversion, observed previously in cell culture models, is not because of a direct influence of raft lipids on the structure and stability of membranebound PrP(C) but caused by other factors, e.g. increased local PrP concentrations or high effective concentrations of membrane-associated conversion factors. The availability of recombinant PrP covalently attached to liposomes provides the basis for systematic in vitro conversion assays with recombinant PrP on the surface of membranes. In addition, our results indicate that the three-dimensional structure of mammalian PrP(C) in membranes is identical to that of recombinant PrP in solution.

Highlights

An abnormal isoform, PrPSc, of the normal cellular prion protein (PrPC) is the major component of the causative agent of prion diseases
A variant of full-length PrP with an unpaired cysteine at the C terminus was recombinantly produced in Escherichia coli, covalently coupled to a thiol-reactive phospholipid, and incorporated into liposomes to serve as a model for studying possible changes in structure and stability of recombinant PrP upon membrane attachment
This paper is available on line at http://www.jbc.org was termed PrP-SS-PDHPE and allowed, for the first time, the comparison of soluble, recombinant PrP with recombinant PrP covalently incorporated into liposomes

Summary

EXPERIMENTAL PROCEDURES

Expression Plasmid Construction—First, a C-terminal six-amino acid linker consisting of five glycine residues and one cysteine residue was fused to the genetic sequence of the murine full-length prion protein. Samples testing the effect of PC/Chol liposomes on the stability of PrP-(23–231) and covalently coupled PrP-SS-PDHPE and the respective controls contained 7.2 ␮M protein and lipid concentrations of 2.5 mM in the case of coupled PrP-SS-PDHPE and 2.2 mM in the case of the uncoupled PrP-(23–231) in 20 ␮l of buffer (for trypsin digestion, 50 mM NH4HCO3/ HCl, pH 7.8; for proteinase K digestion, 100 mM MOPS/NaOH, pH 7.4, 150 mM NaCl). Electron Microscopy Analysis—Samples of liposomes in the presence and absence of PrP-(23–231) and with coupled PrP-SS-PDHPE were adsorbed to glow-discharged carbon-coated copper grids These were washed twice with deionized water, negatively stained with 2% (w/v) uranyl acetate, and air-dried after removal of excess liquid. Light Scattering Analysis—Samples of liposomes (10 ␮l, lipid concentration, 1 mM) were measured for 15 min at 25 °C in 0.15-cm cuvettes using a Protein Solutions DynaPro Molecular Sizing Instrument containing a Temperature Controlled Micro Sampler unit, in order to investigate liposomal integrity under various conditions (0 –9 M urea; 0 –5 M guanidinium chloride; after heating to 90 °C)

RESULTS

Cleavage sites as identified by Edman sequencing

DISCUSSION