Prion Glycoprotein:  Structure, Dynamics, and Roles for the Sugars

Rudd, Pauline M.; Wormald, Mark R.; Wing, David R.; Prusiner, Stanley B.; Dwek, Raymond A.

doi:10.1021/bi002625f

Cited by 125 publications

(121 citation statements)

References 53 publications

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“…The less dense packing of a trimeric assembly would give the sugars additional room in the lattice. The intrinsic flexibility of the sugar side chains of PrP may allow them to rotate in and out of the lattice plane, depending on steric constraints (36). This notion is supported by the observation that Nanogold-labeled prion rods show a relatively dense covering of gold labels on the surface of the rods (data not shown).…”

Section: Methodsmentioning

confidence: 82%

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Structural studies of the scrapie prion protein by electron crystallography

Wille

Michelitsch

Guénebaut

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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387

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Because the insolubility of the scrapie prion protein (PrP Sc ) has frustrated structural studies by x-ray crystallography or NMR spectroscopy, we used electron crystallography to characterize the structure of two infectious variants of the prion protein. Isomorphous two-dimensional crystals of the N-terminally truncated PrP Sc (PrP 27-30) and a miniprion (PrP Sc 106) were identified by negative stain electron microscopy. Image processing allowed the extraction of limited structural information to 7 Å resolution. By comparing projection maps of PrP 27-30 and PrP Sc 106, we visualized the 36-residue internal deletion of the miniprion and localized the N-linked sugars. The dimensions of the monomer and the locations of the deleted segment and sugars were used as constraints in the construction of models for PrP Sc . Only models featuring parallel ␤-helices as the key element could satisfy the constraints. These low-resolution projection maps and models have implications for understanding prion propagation and the pathogenesis of neurodegeneration.electron microscopy ͉ image processing ͉ Nanogold labeling ͉ parallel ␤-helix ͉ amyloid structure C reutzfeldt-Jakob disease (CJD), bovine spongiform encephalopathy (BSE), scrapie, and other spongiform encephalopathies are caused by an aberrantly folded isoform (PrP Sc ) of the prion protein (PrP) (1). Replication of prions includes a profound change in the conformation of the cellular isoform of PrP (PrP C ) to form the highly insoluble PrP Sc . The insolubility of PrP Sc has thwarted attempts to investigate its structure by either x-ray crystallography or NMR spectroscopy. Our knowledge about the structure of PrP Sc is therefore rather limited (2).After treatment with proteinase K (PK), PrP Sc loses the N-terminal residues 23 to Ϸ89 (forming PrP 27-30), but retains infectivity. During purification, PrP 27-30 polymerizes into rod-shaped filaments with the tinctorial properties of amyloid (3, 4). X-ray fibril diffraction illustrated the amyloid nature of PrP 27-30; characteristic 4.7 Å reflections indicative of cross-␤ structure were observed (5). Optical spectroscopy revealed that PrP Sc and PrP 27-30 are substantially enriched in ␤-sheet structure (6-9). This finding is in sharp contrast to the predominantly ␣-helical fold of the three-helix-bundle structure of PrP C as determined by NMR spectroscopy and x-ray crystallography on refolded recombinant PrP (10 -18). Owing to the lack of high-resolution structural information for PrP Sc , predictive methods have been used to develop molecular models to codify the existing spectroscopic, immunological, and biochemical data (19).In attempts to simplify the structural analysis of PrP Sc , we systematically deleted parts of the prion protein. One of these constructs containing only 106 residues, PrP106 (⌬23-88, ⌬141-176), supported the propagation of prions (20,21). Transgenic mice expressing only PrP106 develop a histologically accurate neurodegenerative prion disease after inoculation with prions, and the resulting prions can...

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Section: Methodsmentioning

confidence: 82%

“…By comparison, PrP 27-30 has a longer peptide chain and is a mix of un-, mono-, and diglycosylated forms (36). As these crystals display a complex mixture of negative and positive staining, it is important to consider the staining behavior of the different constructs.…”

Section: Methodsmentioning

confidence: 99%

Structural studies of the scrapie prion protein by electron crystallography

Wille

Michelitsch

Guénebaut

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

387

384

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“…For example, fluctuations in metal ion concentrations (19,20), glycosylphosphatidylinositol anchor stability (21), extracellular molecules such as glycosaminoglycans (22, 23), pH (24), N-linked glycosylation (18,25), and the redox environment have all been proposed to be implicated (18,26,27). Some of these factors (e.g., variations in pH, posttranslational modifications, or redox environment in the secretory pathway) (13, 17) could also be associated with protein localization or processing within the cell.…”

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confidence: 99%

The interplay of glycosylation and disulfide formation influences fibrillization in a prion protein fragment

Bosques

Imperiali

2003

Proc. Natl. Acad. Sci. U.S.A.

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It is now accepted that the structural transition from cellular prion protein (PrP C ) to proteinase K-resistant prion protein scrapie (PrP Sc ) is the major event leading to transmissible spongiform encephalopathies. Although the mechanism of this transition remains elusive, glycosylation has been proposed to impede the PrP C to PrP Sc conversion. To address the role of glycosylation, we have prepared glycosylated and unglycosylated peptides derived from the 175-195 fragment of the human prion protein. Comparison of the structure, aggregation kinetics, fibril formation capabilities, and redox susceptibility of Cys-179 has shown that the N-linked glycan (at Asn-181) significantly reduces the rate of fibrillization by promoting intermolecular disulfide formation via Cys-179. Furthermore, the aggressive fibrillization of a C179S mutant of this fragment highlights the significant role of disulfide stability in retarding the rate of fibril formation. The implications of these studies are discussed in the context of fibril formation in the intact prion protein.S pongiform encephalopathies are a group of fatal neurodegenerative diseases that can be manifested sporadically, genetically inherited, or in some cases transmitted (1-3). In contrast to many diseases where the nature of the infectious particle is virus or bacterium, currently, the only agent associated with these conditions is the structural isoform of the cellular prion protein (PrP C ) known as the scrapie conformation (PrP Sc ) (1, 4). PrP C is an N-linked glycoprotein that is normally attached to the cell membrane by a glycosylphosphatidylinositol anchor (5). PrP C also contains an intramolecular disulfide bond (Cys179OCys-214) that provides structural stability to the C terminus of the protein (6, 7). Secondary structure analyses have shown that PrP C is a predominantly helical protein, whereas PrP Sc is mainly ␤-sheet, suggesting that the conversion into PrP Sc involves a major structural change (8). Although the structure of the unglycosylated PrP C monomer has been solved by NMR spectroscopy (7, 9), elucidation of the PrP Sc structure has been hampered by its highly aggregated state.As with many membrane-associated proteins, initiation of the biosynthesis of PrP begins with translocation into the endoplasmic reticulum (ER) where the amino-terminal signal sequence is cleaved (1). The ER houses the machinery for asparagine-linked glycosylation as well as for disulfide formation (10, 11). Proteins in the secretory pathway that are misfold in the ER (12, 13) are subject to retrograde transport into the cytosol, where they are degraded by proteasomes (14, 15). In particular, protein isoforms (or mutants) that are less stable during their maturation are most likely to undergo this retrograde transport (14). Lindquist and coworkers have recently shown that inhibition of the proteasome causes PrP to accumulate in the cytosol, where it can adopt a PrP Sc -like conformation (16,17). Furthermore, expression of an unglycosylated, cytosolic form of PrP has extreme...

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“…However, as the exact structure of these GPIs was not determined, it remains possible that subtle differences between GPI-PrP c and GPI-PrP Sc exist. It is worth noting that the GPI anchor attached to Thy-1 is modified differently from that of PrP c (Rudd et al, 2001) and that GPI anchors isolated from Thy-1 failed to stimulate PGE 2 production or to activate apoptotic pathways. Thus, although it is not known whether PGE 2 production and the initiation of apoptotic pathways in neurons is a unique property of GPIs attached to PrP c , this activity is not shared by all GPIs.…”

Section: Discussionmentioning

confidence: 99%