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

Warner, R.G.; Hundt, C.; Weiß, Stefan; Turnbull, Jeremy E.

doi:10.1074/jbc.m110406200

Cited by 190 publications

(171 citation statements)

References 59 publications

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“…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.…”

Section: The Mechanism Of Copper-supported Autocleavage Of Hs Inmentioning

confidence: 54%

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

Mani

Cheng

Havsmark

et al. 2003

Journal of Biological Chemistry

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Copper are generally bound to proteins, e.g. the prion and the amyloid ␤ proteins. We have previously shown that copper ions are required to nitrosylate thiol groups in the core protein of glypican-1, a heparan sulfate-substituted proteoglycan. When S-nitrosylated glypican-1 is then exposed to an appropriate reducing agent, such as ascorbate, nitric oxide is released and autocatalyzes deaminative cleavage of the glypican-1 heparan sulfate side chains at sites where the glucosamines are N-unsubstituted. These processes take place in a stepwise manner, whereas glypican-1 recycles via a caveolin-1-associated pathway where copper ions could be provided by the prion protein. Here we show, by using both biochemical and microscopic techniques, that (a) the glypican-1 core protein binds copper(II) ions, reduces them to copper(I) when the thiols are nitrosylated and reoxidizes copper(I) to copper(II) when ascorbate releases nitric oxide; (b) maximally S-nitrosylated glypican-1 can cleave its own heparan sulfate chains at all available sites in a nitroxyl ion-dependent reaction; (c) free zinc(II) ions, which are redox inert, also support autocleavage of glypican-1 heparan sulfate, probably via transnitrosation, whereas they inhibit copper(II)-supported degradation; and (d) copper(II)-loaded but not zinc(II)-loaded prion protein or amyloid ␤ peptide support heparan sulfate degradation. As glypican-1 in prion null cells is poorly S-nitrosylated and as ectopic expression of cellular prion protein restores S-nitrosylation of glypican-1 in these cells, we propose that one function of the cellular prion protein is to deliver copper(II) for the S-nitrosylation of recycling glypican-1.

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Section: The Mechanism Of Copper-supported Autocleavage Of Hs Inmentioning

confidence: 54%

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

Mani

Cheng

Havsmark

et al. 2003

Journal of Biological Chemistry

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“…Although a more distal site has also been proposed (14), there seems to be a connection between metal-binding status and the conformation of PrP (15)(16)(17). 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).…”

mentioning

confidence: 99%

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

Rhie

Kirby

Sayer

et al. 2003

Journal of Biological Chemistry

173

148

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We have isolated artificial ligands or aptamers for infectious prions in order to investigate conformational aspects of prion pathogenesis. The aptamers are 2 -fluoro-modified RNA produced by in vitro selection from a large, randomized library. One of these ligands (aptamer SAF-93) had more than 10-fold higher affinity for PrP Sc than for recombinant PrP C and inhibited the accumulation of PrP res in near physiological cell-free conversion assay. To understand the molecular basis of these properties and to distinguish specific from nonspecific aptamer-PrP interactions, we studied deletion mutants of bovine PrP in denatured, ␣-helix-rich and ␤-sheet-rich forms. We provide evidence that, like scrapie-associated fibrils (SAF), the ␤-oligomer of PrP bound to SAF-93 with at least 10-fold higher affinity than did the ␣-form. This differential affinity could be explained by the existence of two binding sites within the PrP molecule. Site 1 lies within residues 23-110 in the unstructured N terminus and is a nonspecific RNA binding site found in all forms of PrP. The region between residue 90 and 110 forms a hinge region that is occluded in the ␣-rich form of PrP but becomes exposed in the denatured form of PrP. Site 2 lies in the region C-terminal of residue 110. This site is ␤-sheet conformation-specific and is not recognized by control RNAs. Taken together, these data provide for the first time a specific ligand for a disease conformation-associated site in a region of PrP critical for conformational conversion. This aptamer could provide tools for the further analysis of the processes of PrP misfolding during prion disease and leads for the development of diagnostic and therapeutic approaches to TSEs.

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“…The underlying mechanisms have not been determined. Since heparan sulphates can bind numerous molecules, including PrP C [29,49,149], one possibility is that the heparan sulphate moieties of proteoglycans could help bring together partners critically involved in the conversion process, such as PrP C , PrP Sc , and other possible cofactors. Inhibition of the expression (either by antisense RNA and siRNA) or function (by antibodies) of the 37 kDa/67 kDa laminin receptor (LRP/LR) prevents PrP res accumulation in infected N2a and GT1 cells [78].…”

Section: Cell Models Propagating Naturally-occurring Prion Isolatesmentioning

confidence: 99%

“…Monoclonal antibody 6H4 (recognising residues [144][145][146][147][148][149][150][151][152] and Fab fragments of D18 and D13 mAbs (recognising residues 132-156 and 95-103, respectively) potently inhibit prion multiplication in N2a cells [41,102]. SAF34 and SAF61 mAbs, which recognise the octarepeat region and residues 114-152 respectively, impaired prion propagation in infected neuroblastoma cells with an increase of the turn-over of PrP C as a proposed mechanism for prion inhibition [104].…”

Section: Passive Immunisation With Anti-prp C Antibodiesmentioning

confidence: 99%

Cell models of prion infection

Vilette

2007

Vet. Res.

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-Due to recent renewal of interest and concerns in prion diseases, a number of cell systems permissive to prion multiplication have been generated in the last years. These include established cell lines, neuronal stem cells and primary neuronal cultures. While most of these models are permissive to experimental, mouse-adapted strains of prions, the propagation of natural field isolates from sheep scrapie and chronic wasting disease has been recently achieved. These models have improved our knowledge on the molecular and cellular events controlling the conversion of the PrP C protein into abnormal isoforms and on the cell-to-cell spreading of prions. Infected cultured cells will also facilitate investigations on the molecular basis of strain identity and on the mechanisms that lead to neurodegeneration. The ongoing development of new cell models with improved characteristics will certainly be useful for a number of unanswered critical issues in the prion field.

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

Cited by 190 publications

References 59 publications

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

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

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

Cell models of prion infection

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