scholarly journals Distance of sequons to the C-terminus influences the cellular N-glycosylation of the prion protein

2003 ◽  
Vol 370 (1) ◽  
pp. 351-355 ◽  
Author(s):  
Adrian R. WALMSLEY ◽  
Nigel M. HOOPER
Keyword(s):  
Prion Protein ◽  
Signal Sequence ◽  
Prion Diseases ◽  
The Novel ◽  
Gpi Anchor ◽  
Glycosyl Phosphatidylinositol ◽  
Novel Approach ◽  
C Terminus ◽  
Human Neuronal Cells ◽  
Translational Process

Cell-specific differences in the utilization of the two N-glycosylation sequons (Asn180-Ile-Thr and Asn196-Phe-Thr) of the prion protein (PrP) have been proposed to influence the aetiology of the neurodegenerative prion diseases. As the N-glycosylation of PrP is ablated by deletion of the C-terminal glycosyl-phosphatidylinositol (GPI) anchor signal sequence, we have investigated the determinants for PrP sequon utilization in human neuronal cells using the novel approach of restoring N-glycosylation to secreted forms of PrP lacking a GPI anchor. N-glycosylation was restored to an efficiency comparable with that of GPI anchored PrP when the distance of the sequon to the C-terminus was increased so that it was sufficient to reach the active site of oligosaccharyltransferase before chain termination. Our findings indicate that sequon utilization in PrP is a co-translational process that precedes GPI anchor addition and, as such, will be greatly influenced by the dynamics of the translocon—oligosaccharyltransferase complex.

scholarly journals Characterization of four new monoclonal antibodies against the distal N-terminal region of PrPc

2014 ◽  
Author(s):  
Alessandro Didonna ◽  
Anja Colja Venturini ◽  
Katrina Hartman ◽  
Tanja Vranac ◽  
Vladka Curin Serbec ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Prion Protein ◽  
Biochemical Characterization ◽  
Prion Diseases ◽  
Physiological Role ◽  
Prion Infection ◽  
Promising Tool ◽  
C Terminus ◽  
N Terminus

Prion diseases are a group of fatal neurodegenerative disorders that affect humans and animals. They are characterized by the accumulation in the central nervous system of a pathological form of the host-encoded prion protein (PrPC). The prion protein is a membrane glycoprotein that consists of two domains: a globular, structured C-terminus and an unstructured N-terminus. The N-terminal part of the protein is involved in different functions in both health and disease. In the present work we discuss the production and biochemical characterization of a panel of four monoclonal antibodies (mAbs) against the distal N-terminus of PrPC using a well-established methodology based on the immunization of Prnp0/0 mice. Additionally, we show their ability to block prion (PrPSc) replication at nanomolar concentrations in a cell culture model of prion infection. These mAbs represent a promising tool for prion diagnostics and for studying the physiological role of the N-terminal domain of PrPC.

scholarly journals Increased Infectivity of Anchorless Mouse Scrapie Prions in Transgenic Mice Overexpressing Human Prion Protein

2015 ◽  
Vol 89 (11) ◽  
pp. 6022-6032 ◽  
Author(s):  
Brent Race ◽  
Katie Phillips ◽  
Kimberly Meade-White ◽  
James Striebel ◽  
Bruce Chesebro
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Transgenic Mice ◽  
Prion Protein ◽  
Prion Diseases ◽  
Human Species ◽  
Species Barrier ◽  
Gpi Anchor ◽  
Prion Infection ◽  
Barrier Model

ABSTRACTPrion protein (PrP) is found in all mammals, mostly as a glycoprotein anchored to the plasma membrane by a C-terminal glycosylphosphatidylinositol (GPI) linkage. Following prion infection, host protease-sensitive prion protein (PrPsen or PrPC) is converted into an abnormal, disease-associated, protease-resistant form (PrPres). Biochemical characteristics, such as the PrP amino acid sequence, and posttranslational modifications, such as glycosylation and GPI anchoring, can affect the transmissibility of prions as well as the biochemical properties of the PrPres generated. Previousin vivostudies on the effects of GPI anchoring on prion infectivity have not examined cross-species transmission. In this study, we tested the effect of lack of GPI anchoring on a species barrier model using mice expressing human PrP. In this model, anchorless 22L prions derived from tg44 mice were more infectious than 22L prions derived from C57BL/10 mice when tested in tg66 transgenic mice, which expressed wild-type anchored human PrP at 8- to 16-fold above normal. Thus, the lack of the GPI anchor on the PrPres from tg44 mice appeared to reduce the effect of the mouse-human PrP species barrier. In contrast, neither source of prions induced disease in tgRM transgenic mice, which expressed human PrP at 2- to 4-fold above normal.IMPORTANCEPrion protein (PrP) is found in all mammals, usually attached to cells by an anchor molecule called GPI. Following prion infection, PrP is converted into a disease-associated form (PrPres). While most prion diseases are species specific, this finding is not consistent, and species barriers differ in strength. The amino acid sequence of PrP varies among species, and this variability affects prion species barriers. However, other PrP modifications, including glycosylation and GPI anchoring, may also influence cross-species infectivity. We studied the effect of PrP GPI anchoring using a mouse-to-human species barrier model. Experiments showed that prions produced by mice expressing only anchorless PrP were more infectious than prions produced in mice expressing anchored PrP. Thus, the lack of the GPI anchor on prions reduced the effect of the mouse-human species barrier. Our results suggest that prion diseases that produce higher levels of anchorless PrP may pose an increased risk for cross-species infection.

scholarly journals cDNA cloning and expression in Xenopus laevis oocytes of pig renal dipeptidase, a glycosyl-phosphatidylinositol-anchored ectoenzyme

1990 ◽  
Vol 271 (3) ◽  
pp. 755-760 ◽  
Author(s):  
E Rached ◽  
N M Hooper ◽  
P James ◽  
G Semenza ◽  
A J Turner ◽  
...  
Keyword(s):  
Amino Acids ◽  
Xenopus Laevis ◽  
Signal Sequence ◽  
Polyclonal Antiserum ◽  
Cloning And Expression ◽  
Kidney Cortex ◽  
Xenopus Laevis Oocytes ◽  
Glycosyl Phosphatidylinositol ◽  
Pig Kidney ◽  
C Terminus

Clones expressing renal dipeptidase (EC 3.4.13.11) have been isolated from a pig kidney cortex cDNA library after employing the polymerase chain reaction technique to amplify a region of the dipeptidase cDNA. The complete primary sequence of the enzyme has been deduced from a full length cDNA clone. This predicts a protein of 409 amino acids, a cleavable N-terminal signal sequence of 16 residues and two N-linked glycosylation sites. At the C-terminus of the predicted sequence is a stretch of mainly hydrophobic amino acids which is presumed to direct the attachment of the glycosyl-phosphatidylinositol membrane anchor. Expression of the mRNA for pig renal dipeptidase in Xenopus laevis oocytes led to the production of a disulphide-linked dimeric protein of subunit Mr 48,600 which was recognized by a polyclonal antiserum raised to renal dipeptidase purified from pig kidney cortex. Bacterial phosphatidylinositol-specific phospholipase C released renal dipeptidase from the surface of the oocytes and converted the amphipathic detergent-solubilized form of the dipeptidase to a hydrophilic form, indicating that Xenopus laevis oocytes can process expressed proteins to their glycosyl-phosphatidylinositol anchored form.

scholarly journals Expanding spectrum of prion diseases

2020 ◽  
Vol 4 (2) ◽  
pp. 155-167
Author(s):  
Jacob I. Ayers ◽  
Nick A. Paras ◽  
Stanley B. Prusiner
Keyword(s):  
Amino Acids ◽  
Nucleic Acid ◽  
Prion Protein ◽  
Prion Diseases ◽  
Lewy Body Dementia ◽  
Cellular Prion Protein ◽  
Chromosomal Gene ◽  
Scrapie Agent ◽  
Β Sheet ◽  
Translational Process

Prions were initially discovered in studies of scrapie, a transmissible neurodegenerative disease (ND) of sheep and goats thought to be caused by slow viruses. Once scrapie was transmitted to rodents, it was discovered that the scrapie pathogen resisted inactivation by procedures that modify nucleic acids. Eventually, this novel pathogen proved to be a protein of 209 amino acids, which is encoded by a chromosomal gene. After the absence of a nucleic acid within the scrapie agent was established, the mechanism of infectivity posed a conundrum and eliminated a hypothetical virus. Subsequently, the infectious scrapie prion protein (PrPSc) enriched for β-sheet was found to be generated from the cellular prion protein (PrPC) that is predominantly α-helical. The post-translational process that features in nascent prion formation involves a templated conformational change in PrPC that results in an infectious copy of PrPSc. Thus, prions are proteins that adopt alternative conformations, which are self-propagating and found in organisms ranging from yeast to humans. Prions have been found in both Alzheimer's (AD) and Parkinson's (PD) diseases. Mutations in APP and α-synuclein genes have been shown to cause familial AD and PD. Recently, AD was found to be a double prion disorder: both Aβ and tau prions feature in this ND. Increasing evidence argues for α-synuclein prions as the cause of PD, multiple system atrophy, and Lewy body dementia.

scholarly journals α2,3 linkage of sialic acid to a GPI anchor and an unpredicted GPI attachment site in human prion protein

2020 ◽  
Vol 295 (22) ◽  
pp. 7789-7798 ◽  
Author(s):  
Atsushi Kobayashi ◽  
Tetsuya Hirata ◽  
Takashi Nishikaze ◽  
Akinori Ninomiya ◽  
Yuta Maki ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Prion Protein ◽  
Prion Diseases ◽  
Attachment Site ◽  
Cellular Prion Protein ◽  
Side Chain ◽  
Glycan Structure ◽  
Gpi Anchor ◽  
Acetylneuraminic Acid ◽  
Sialic Acid Linkage

Prion diseases are transmissible, lethal neurodegenerative disorders caused by accumulation of the aggregated scrapie form of the prion protein (PrPSc) after conversion of the cellular prion protein (PrPC). The glycosylphosphatidylinositol (GPI) anchor of PrPC is involved in prion disease pathogenesis, and especially sialic acid in a GPI side chain reportedly affects PrPC conversion. Thus, it is important to define the location and structure of the GPI anchor in human PrPC. Moreover, the sialic acid linkage type in the GPI side chain has not been determined for any GPI-anchored protein. Here we report GPI glycan structures of human PrPC isolated from human brains and from brains of a knock-in mouse model in which the mouse prion protein (Prnp) gene was replaced with the human PRNP gene. LC–electrospray ionization–MS analysis of human PrPC from both biological sources indicated that Gly229 is the ω site in PrPC to which GPI is attached. Gly229 in human PrPC does not correspond to Ser231, the previously reported ω site of Syrian hamster PrPC. We found that ∼41% and 28% of GPI anchors in human PrPCs from human and knock-in mouse brains, respectively, have N-acetylneuraminic acid in the side chain. Using a sialic acid linkage-specific alkylamidation method to discriminate α2,3 linkage from α2,6 linkage, we found that N-acetylneuraminic acid in PrPC's GPI side chain is linked to galactose through an α2,3 linkage. In summary, we report the GPI glycan structure of human PrPC, including the ω-site amino acid for GPI attachment and the sialic acid linkage type.

scholarly journals Cells Expressing Anchorless Prion Protein Are Resistant to Scrapie Infection

2009 ◽  
Vol 83 (9) ◽  
pp. 4469-4475 ◽  
Author(s):  
Kristin L. McNally ◽  
Anne E. Ward ◽  
Suzette A. Priola
Keyword(s):  
Transgenic Mice ◽  
Prion Protein ◽  
Prion Diseases ◽  
Transmitted Disease ◽  
Transmissible Spongiform Encephalopathies ◽  
Gpi Anchor ◽  
Spongiform Encephalopathies ◽  
Infected Cells

ABSTRACT The hallmark of transmissible spongiform encephalopathies (TSEs or prion diseases) is the accumulation of an abnormally folded, partially protease-resistant form (PrP-res) of the normal protease-sensitive prion protein (PrP-sen). PrP-sen is attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. In vitro, the anchor and the local membrane environment are important for the conversion of PrP-sen to PrP-res. In vivo, however, the anchor is not necessary because transgenic mice expressing anchorless PrP-sen accumulate PrP-res and replicate infectivity. To clarify the role of the GPI anchor in TSE infection, cells expressing GPI-anchored PrP-sen, anchorless PrP-sen, or both forms of PrP-sen were exposed to the mouse scrapie strain 22L. Cells expressing anchored PrP-sen produced PrP-res after exposure to 22L. Surprisingly, while cells expressing anchorless PrP-sen made anchorless PrP-res in the first 96 h postinfection, no PrP-res was detected at later passes. In contrast, when cells expressing both forms of PrP-sen were exposed to 22L, both anchored and anchorless PrP-res were detected over multiple passes. Consistent with the in vitro data, scrapie-infected cells expressing anchored PrP-sen transmitted disease to mice whereas cells expressing anchorless PrP-sen alone did not. These results demonstrate that the GPI anchor on PrP-sen is important for the persistent infection of cells in vitro. Our data suggest that cells expressing anchorless PrP-sen are not directly infected with scrapie. Thus, PrP-res formation in transgenic mice expressing anchorless PrP-sen may be occurring extracellularly.

scholarly journals NMR characterization of the pH 4 β-intermediate of the prion protein: the N-terminal half of the protein remains unstructured and retains a high degree of flexibility

2006 ◽  
Vol 401 (2) ◽  
pp. 533-540 ◽  
Author(s):  
Denis B. D. O'Sullivan ◽  
Christopher E. Jones ◽  
Salama R. Abdelraheim ◽  
Andrew R. Thompsett ◽  
Marcus W. Brazier ◽  
...  
Keyword(s):  
Prion Protein ◽  
Prion Diseases ◽  
Molten Globule ◽  
Three Dimensional ◽  
Molecular Size ◽  
Native Form ◽  
C Terminus ◽  
Nmr Characterization ◽  
Helical Protein ◽  
Β Sheet

Prion diseases are associated with the misfolding of the PrP (prion protein) from a largely α-helical isoform to a β-sheet-rich oligomer. CD has shown that lowering the pH to 4 under mildly denaturing conditions causes recombinant PrP to convert from an α-helical protein into one that contains a high proportion of β-sheet-like conformation. In the present study, we characterize this soluble pH 4 folding intermediate using NMR. 15N-HSQC (heteronuclear single-quantum correlation) studies with mPrP (mouse PrP)-(23–231) show that a total of 150 dispersed amide signals are resolved in the native form, whereas only 65 amide signals with little chemical shift dispersion are observable in the pH 4 form. Three-dimensional 15N-HSQC-TOCSY and NOESY spectra indicate that the observable residues are all assigned to amino acids in the N-terminus: residues 23–118. 15N transverse relaxation measurements indicate that these N-terminal residues are highly flexible with additional fast motions. These observations are confirmed via the use of truncated mPrP-(112–231), which shows only 16 15N-HSQC amide peaks at pH 4. The loss of signals from the C-terminus can be attributed to line broadening due to an increase in the molecular size of the oligomer or exchange broadening in a molten-globule state.

scholarly journals Inhibition of the FKBP family of peptidyl prolyl isomerases induces abortive translocation and degradation of the cellular prion protein

2016 ◽  
Vol 27 (5) ◽  
pp. 757-767 ◽  
Author(s):  
Pawel Stocki ◽  
Maxime Sawicki ◽  
Charles E. Mays ◽  
Seo Jung Hong ◽  
Daniel C. Chapman ◽  
...  
Keyword(s):  
Prion Protein ◽  
Therapeutic Strategy ◽  
Signal Sequence ◽  
Prion Diseases ◽  
Cellular Prion Protein ◽  
Potential Therapeutic Target ◽  
Subsequent Degradation ◽  
Mouse Cells ◽  
Peptidyl Prolyl Isomerases ◽  
Calcineurin Inhibition

Prion diseases are fatal neurodegenerative disorders for which there is no effective treatment. Because the cellular prion protein (PrPC) is required for propagation of the infectious scrapie form of the protein, one therapeutic strategy is to reduce PrPCexpression. Recently FK506, an inhibitor of the FKBP family of peptidyl prolyl isomerases, was shown to increase survival in animal models of prion disease, with proposed mechanisms including calcineurin inhibition, induction of autophagy, and reduced PrPCexpression. We show that FK506 treatment results in a profound reduction in PrPCexpression due to a defect in the translocation of PrPCinto the endoplasmic reticulum with subsequent degradation by the proteasome. These phenotypes could be bypassed by replacing the PrPCsignal sequence with that of prolactin or osteopontin. In mouse cells, depletion of ER luminal FKBP10 was almost as potent as FK506 in attenuating expression of PrPC. However, this occurred at a later stage, after translocation of PrPCinto the ER. Both FK506 treatment and FKBP10 depletion were effective in reducing PrPScpropagation in cell models. These findings show the involvement of FKBP proteins at different stages of PrPCbiogenesis and identify FKBP10 as a potential therapeutic target for the treatment of prion diseases.

scholarly journals PrP Knockout Cells Expressing Transmembrane PrP Resist Prion Infection

2016 ◽  
Vol 91 (2) ◽  
Author(s):  
Karen E. Marshall ◽  
Andrew Hughson ◽  
Sarah Vascellari ◽  
Suzette A. Priola ◽  
Akikazu Sakudo ◽  
...  
Keyword(s):  
Prion Protein ◽  
Signal Sequence ◽  
Transmissible Spongiform Encephalopathy ◽  
Spongiform Encephalopathy ◽  
Wild Type ◽  
Gpi Anchor ◽  
Prion Infection ◽  
Endogenous Expression ◽  
Prion Propagation ◽  
Transmembrane Sequence

ABSTRACT Glycosylphosphatidylinositol (GPI) anchoring of the prion protein (PrPC) influences PrPC misfolding into the disease-associated isoform, PrPres, as well as prion propagation and infectivity. GPI proteins are found in cholesterol- and sphingolipid-rich membrane regions called rafts. Exchanging the GPI anchor for a nonraft transmembrane sequence redirects PrPC away from rafts. Previous studies showed that nonraft transmembrane PrPC variants resist conversion to PrPres when transfected into scrapie-infected N2a neuroblastoma cells, likely due to segregation of transmembrane PrPC and GPI-anchored PrPres in distinct membrane environments. Thus, it remained unclear whether transmembrane PrPC might convert to PrPres if seeded by an exogenous source of PrPres not associated with host cell rafts and without the potential influence of endogenous expression of GPI-anchored PrPC. To further explore these questions, constructs containing either a C-terminal wild-type GPI anchor signal sequence or a nonraft transmembrane sequence containing a flexible linker were expressed in a cell line derived from PrP knockout hippocampal neurons, NpL2. NpL2 cells have physiological similarities to primary neurons, representing a novel and advantageous model for studying transmissible spongiform encephalopathy (TSE) infection. Cells were infected with inocula from multiple prion strains and in different biochemical states (i.e., membrane bound as in brain microsomes from wild-type mice or purified GPI-anchorless amyloid fibrils). Only GPI-anchored PrPC supported persistent PrPres propagation. Our data provide strong evidence that in cell culture GPI anchor-directed membrane association of PrPC is required for persistent PrPres propagation, implicating raft microdomains as a location for conversion. IMPORTANCE Mechanisms of prion propagation, and what makes them transmissible, are poorly understood. Glycosylphosphatidylinositol (GPI) membrane anchoring of the prion protein (PrPC) directs it to specific regions of cell membranes called rafts. In order to test the importance of the raft environment on prion propagation, we developed a novel model for prion infection where cells expressing either GPI-anchored PrPC or transmembrane-anchored PrPC, which partitions it to a different location, were treated with infectious, misfolded forms of the prion protein, PrPres. We show that only GPI-anchored PrPC was able to convert to PrPres and able to serially propagate. The results strongly suggest that GPI anchoring and the localization of PrPC to rafts are crucial to the ability of PrPC to propagate as a prion.

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