Characterization and polyanion-binding properties of purified recombinant prion protein

Abstract

Certain polysulphated polyanions have been shown to have prophylactic effects on the progression of transmissible spongiform encephalopathy disease, presumably because they bind to prion protein (PrP). Until now, the difficulty of obtaining large quantities of native PrP has precluded detailed studies of these interactions. We have over-expressed murine recombinant PrP (recPrP), lacking its glycophosphoinositol membrane anchor, in modified mammalian cells. Milligram quantities of secreted, soluble and partially glycosylated protein were purified under non-denaturing conditions and the identities of mature-length aglycosyl recPrP and two cleavage fragments were determined by electrospray MS. Binding was assessed by surface plasmon resonance techniques using both direct and competitive ligand-binding approaches. recPrP binding to immobilized polyanions was enhanced by divalent metal ions. Polyanion binding was strong and showed complex association and dissociation kinetics that were consistent with ligand-directed recPrP aggregation. The differences in the binding strengths of recPrP to pentosan polysulphate and to other sulphated polyanions were found to parallel their in vivo anti-scrapie and in vitro anti-scrapie-specific PrP formation potencies. When recPrP was immobilized by capture on metal-ion chelates it was found, contrary to expectation, that the addition of polyanions promoted the dissociation of the protein.

Prion, Amyloid β-derived Cu(II) Ions, or Free Zn(II) Ions Support S-Nitroso-dependent Autocleavage of Glypican-1 Heparan Sulfate

“…7A, filled symbols). Contact between copper-loaded PrP C and the HS chains of Gpc-1 should be necessary for transfer of copper ions, as it is known that interaction with HS weakens Cu(II) binding to PrP C (12)(13)(14)(15). In keeping with this proposal, the addition of an excess HS chains inhibited contact between PrP C and Gpc-1 and precluded cleavage of HS (Fig.…”

Characterization of 2′-Fluoro-RNA Aptamers That Bind Preferentially to Disease-associated Conformations of Prion Protein and Inhibit Conversion

“…In the absence of other signals we concluded with 99% confidence that the fragment 110 -230 was what caused the second band on the gel. Similar proteolysis digestion has been reported for recombinant murine prion protein (20,43). When the blot derived from this gel was probed with [ 32 P]SAF-93 (Fig.…”

“…Sulfated glycans interact with PrP (18) of which heparin and heparan sulfate have been shown to have three broad binding sites: residues 23-52, 53-93, and 110 -123 (19). The binding of heparin and copper comap to residues 53-93 and, interestingly, heparin binding seems to be enhanced by copper ions (19,20). Large and small nucleic acids (21)(22)(23)(24) also interact with PrP.…”

Identification of the Heparan Sulfate Binding Sites in the Cellular Prion Protein

“…Prion protein is known to bind copper (II) ions in vitro, and a linkage between divalent cation and polyanion binding activities has been demonstrated previously (15). This prompted us to undertake a second series of biosensor experiments in which GST::haPrP c was mixed with 10 M copper (II) before injection onto heparin ( Fig.…”

“…The basis for the anti-prion activity of these compounds is uncertain; one suggestion is that they compete with endogenous HSPGs for prion, another that they stabilize conformations of PrP sc , which are not favored as templates for PrP c ⅐PrP sc conversion (10). Sulfated GAGs and lipopolyamines (a group of molecules that reverse many of the actions of heparin) modulate the expression of PrP Sc in cultured cells (11)(12)(13), and several investigators have described direct interactions between cellular or recombinant prion and GAGs (14,15). Of particular significance is the detection of a glucose polysaccharide in prion rods purified from scrapieinfected hamster brains (16).…”