scholarly journals Normal cellular prion protein is a ligand of selectins: binding requires LeX but is inhibited by sLeX

2007 ◽  
Vol 406 (2) ◽  
pp. 333-341 ◽  
Author(s):  
Chaoyang Li ◽  
Poki Wong ◽  
Tao Pan ◽  
Fan Xiao ◽  
Shaoman Yin ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Fusion Protein ◽  
Human Brain ◽  
Prion Protein ◽  
Fusion Proteins ◽  
Cellular Prion Protein ◽  
The Other ◽  
Dependent Manner ◽  
Peripheral Tissues ◽  
Sialyl Lex

The normal PrPC (cellular prion protein) contains sLeX [sialyl-LeX (Lewis X)] and LeX. sLeX is a ligand of selectins. To examine whether PrPC is a ligand of selectins, we generated three human PrPC–Ig fusion proteins: one with LeX, one with sLeX, and the other with neither LeX nor sLeX. Only LeX-PrPC–Ig binds E-, L- and P-selectins. Binding is Ca2+-dependent and occurs with nanomolar affinity. Removal of sialic acid on sLeX-PrPC–Ig enables the fusion protein to bind all selectins. These findings were confirmed with brain-derived PrPC. The selectins precipitated PrPC in human brain in a Ca2+-dependent manner. Treatment of brain homogenates with neuraminidase increased the amounts of PrPC precipitated. Therefore the presence of sialic acid prevents the binding of PrPC in human brain to selectins. Hence, human brain PrPC interacts with selectins in a manner that is distinct from interactions in peripheral tissues. Alternations in these interactions may have pathological consequences.

scholarly journals Activation effects of a prion protein fragment [PrP-(106-126)] on human leucocytes*

1996 ◽  
Vol 320 (2) ◽  
pp. 563-570 ◽  
Author(s):  
Luisa DIOMEDE ◽  
Silvano SOZZANI ◽  
Walter LUINI ◽  
Marina ALGERI ◽  
Luca DE GIOIA ◽  
...  
Keyword(s):  
Prion Protein ◽  
Human Lymphocytes ◽  
Cellular Prion Protein ◽  
Dependent Manner ◽  
Membrane Microviscosity ◽  
Peripheral Tissues ◽  
Cell Extracts ◽  
Dose Dependent ◽  
O2 Production

Prion-related encephalopathies are characterized by the intracerebral accumulation of an abnormal isoform of the cellular prion protein (PrPC) named scrapie prion protein (PrPSc). The pathological forms of this protein and its cellular precursor are not only expressed in the brain but also, at lower concentrations, in peripheral tissues. We recently showed that a synthetic peptide corresponding to residues 106–126 [PrP-(106–126)] of the human PrP is toxic to neurons and trophic to astrocytes in vitro. Our experiments were aimed at verifying whether PrP-(106–126) and other peptides corresponding to fragments of the amyloid protein purified from brains of patients with Gerstmann–Sträussler–Scheinker disease – namely PrP-(89–106), PrP-(106–114), PrP-(127–147) – were capable of stimulating circulating leucocytes. Native PrP expression in human lymphocytes, monocytes and neutrophils was first confirmed using PCR amplification of total RNA, after reverse transcription, and immunoblot analysis of cell extracts with anti-PrP antibodies. PrP-(106–126), but not the other peptides, increased membrane microviscosity, intracellular Ca2+ concentration and cell migration in circulating leucocytes, and O2-•production in monocytes and neutrophils. Membrane microviscosity was determined by the fluorescence polarization technique, using diphenylhexatriene as a probe, 300 s after the addition of PrP-(106–126) to the cell suspension in the concentration range 5–50 µM. The increase in intracellular Ca2+ elicited by PrP-(106–126) was dose-dependent in the range 5–500 µM. PrP-(106–126) stimulated O2-•production in monocytes and neutrophils in a dose- (10–300 µM) and time-(5–30 min) dependent manner in the presence of 10 µM dihydrocytochalasin B. Both the increase in Ca2+ concentration and the O2-•production were partially sensitive to pertussis toxin. PrP-(106–126) stimulated leucocyte migration in a dose-dependent (30–300 µM) manner and, at the highest concentration used, this migration was comparable with that elicited by 2.5 nM interleukin 8 or 10 nM fMet-Leu-Phe peptide.

Bimodal Induction of NK Cell Reactivity Against Acute Myeloid (AML) and Chronic Lymphoid Leukemia (CLL) by Fc-Engineered GITR-Fc Fusion Proteins.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2143-2143
Author(s):  
Julia Steinbacher ◽  
Benjamin J Schmiedel ◽  
Antje Werner ◽  
Tina Nuebling ◽  
Corina Buechele ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Nk Cells ◽  
Fusion Proteins ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Combined Treatment ◽  
Leukemia Cells ◽  
Target Antigen ◽  
Dependent Manner ◽  
Allogenic Stem Cell Transplantation

Abstract Abstract 2143 NK cells play an important role in anti-tumor immunity and largely contribute to the efficacy of therapeutic strategies like allogenic stem cell transplantation in AML and application of Rituximab that induces antibody-dependent cellular cytotoxicity (ADCC) in CLL. Recently, we demonstrated that the TNF family member GITR ligand (GITRL) is expressed on leukemia cells in a high proportion of AML and CLL patients and impairs direct and Rituximab-induced reactivity of NK cells which constitutively express its counterpart GITR (e.g., Buechele et al., Leukemia 2012). Here we developed a strategy to reinforce NK anti-leukemia reactivity by combining disruption of NK-inhibitory GITR-GITRL interaction with induction of ADCC against the GITRL-expressing leukemia cells using GITR-Ig fusion proteins with modified Fc moieties. Fc parts were engineered by amino acid exchange as previously described (Lazar et al., PNAS 2006; Armour et al., Eur. J. Immunol. 1999). Compared to wild type GITR-Ig (GITR-Fc-WT), our mutants (S239D/I332E and E233P/L234V/L235A/deltaG236/A327G/A330S) displayed highly enhanced (GITR-Fc-ADCC) and abrogated (GITR-Fc-KO) affinity to the Fc(gamma)RIIIa receptor (CD16) expressed on NK cells, respectively. In functional analyses of NK cells and primary leukemia cells, GITR-Fc-KO, which does not induce ADCC, already increased NK reactivity due to disruption of GITR-GITRL interaction. Treatment with GITR-Fc-WT further enhanced NK reactivity due to modest induction of ADCC, while GITR-Fc-ADCC induced highly increased NK-mediated target cell lysis, degranulation and cytokine production in a target-antigen dependent manner. With CLL cells, combined treatment with GITR-Fc-ADCC fusion protein and Rituximab caused additive effects, resulting in significantly enhanced NK cell ADCC. Notably, the effects of our fusion proteins were observed both in an allogenic setting and when employing NK cells of patients with autologous leukemia cells as targets. Our results demonstrate that Fc-engineered GITR-Fc-ADCC fusion protein may combine both neutralization of the NK-inhibitoryeffects of GITR-GITRL interaction and targeting GITRL-expressing malignant cells for NK anti-tumor reactivity and thus constitute an attractive immunotherapeutic means for the treatment of AML and CLL. Disclosures: No relevant conflicts of interest to declare.

Induction of NK Cell Reactivity Against AML Cells by Fc-Engineered RANK-Ig Fusion Proteins.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2625-2625
Author(s):  
Tina Nuebling ◽  
Benjamin J Schmiedel ◽  
Miyuki Azuma ◽  
Pascal Schneider ◽  
Ludger Grosse-Hovest ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Nk Cells ◽  
Fusion Proteins ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Residual Disease ◽  
Target Antigen ◽  
Dependent Manner ◽  
Cell Reactivity ◽  
Allogenic Stem Cell Transplantation

Abstract Abstract 2625 NK cells are cytotoxic lymphocytes that play an important role in anti-tumor immunity. A clinically important feature of NK cells is their ability to mediate antibody-dependent cellular cytotoxicity (ADCC) upon application of anti-tumor antibodies. In acute myeloid leukemia (AML) NK cells largely contribute to the therapeutic efficacy allogenic stem cell transplantation (SCT). Recently we demonstrated that AML cells functionally express the TNF family member RANK ligand (RANKL) which impairs NK cell anti-leukemia reactivity (Schmiedel et al., ASH annual meeting 2011). Here we developed a strategy to combine blocking of the NK inhibitory effects of RANKL with targeting of AML cells for NK cell ADCC. To this end we generated fusion proteins consisting of the extracellular domain of RANK and a human IgG1 Fc part that was modified by amino acid exchange. Compared to wild type RANK-Fc fusion protein (RANK-Fc-WT), our mutant RANK-Fc-ADCC (S239D/I332E) displayed highly enhanced affinity to FcγRIIIa (CD16) on NK cells. Primary AML cells expressed substantial levels of RANKL in 53 of 78 (68%) investigated patient cases, and our RANK-Ig fusion proteins bound to AML cells in a target antigen-specific manner. Treatment with both RANK-Fc-WT and RANK-Fc-ADCC clearly reduced the release of RANKL-induced immunomodulatory factors like TNF, IL-6, IL-8 and IL-10 by AML cells. When the effects of the fusion proteins on NK cell ADCC were studied we found that treatment with RANK-Fc-WT only slightly enhanced NK cell reactivity against RANKL-positive patient AML cells. However, RANK-Fc-ADCC potently induced NK cell ADCC and cytokine production in response to AML targets in a target antigen-dependent manner due to the functional properties of its engineered Fc moiety. Taken together, our Fc-engineered RANK-Fc-ADCC fusion protein may serve to modulate the cytokine milieu involved in AML pathophysiology and target RANKL-expressing leukemia cells for NK anti-tumor reactivity. Thus, RANK-Fc-ADCC constitutes an attractive immunotherapeutic means for the treatment of AML, e.g. for elimination of minimal residual disease after conventional therapy including SCT. Disclosures: No relevant conflicts of interest to declare.

Botulinum neurotoxin serotype D attacks neurons via two carbohydrate-binding sites in a ganglioside-dependent manner

2010 ◽  
Vol 431 (2) ◽  
pp. 207-216 ◽  
Author(s):  
Jasmin Strotmeier ◽  
Kwangkook Lee ◽  
Anne K. Völker ◽  
Stefan Mahrhold ◽  
Yinong Zong ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Synaptic Vesicle ◽  
Motor Neurons ◽  
Binding Sites ◽  
Botulinum Neurotoxin ◽  
Specific Binding ◽  
Binding Pocket ◽  
The Other ◽  
Carbohydrate Binding ◽  
Dependent Manner

The extraordinarily high toxicity of botulinum neurotoxins primarily results from their specific binding and uptake into neurons. At motor neurons, the seven BoNT (botulinum neurotoxin) serotypes A–G inhibit acetylcholine release leading to flaccid paralysis. Uptake of BoNT/A, B, E, F and G requires a dual interaction with gangliosides and the synaptic vesicle proteins synaptotagmin or SV2 (synaptic vesicle glycoprotein 2), whereas little is known about the cell entry mechanisms of the serotypes C and D, which display the lowest amino acid sequence identity compared with the other five serotypes. In the present study we demonstrate that the neurotoxicity of BoNT/D depends on the presence of gangliosides by employing phrenic nerve hemidiaphragm preparations derived from mice expressing the gangliosides GM3, GM2, GM1 and GD1a, or only GM3 [a description of our use of ganglioside nomenclature is given in Svennerholm (1994) Prog. Brain Res. 101, XI–XIV]. High-resolution crystal structures of the 50 kDa cell-binding domain of BoNT/D alone and in complex with sialic acid, as well as biological analyses of single-site BoNT/D mutants identified two carbohydrate-binding sites. One site is located at a position previously identified in BoNT/A, B, E, F and G, but is lacking the conserved SXWY motif. The other site, co-ordinating one molecule of sialic acid, resembles the second ganglioside-binding pocket (the sialic-acid-binding site) of TeNT (tetanus neurotoxin).

scholarly journals Strain-Dependent Prion Infection in Mice Expressing Prion Protein with Deletion of Central Residues 91–106

2020 ◽  
Vol 21 (19) ◽  
pp. 7260
Author(s):  
Keiji Uchiyama ◽  
Hironori Miyata ◽  
Yoshitaka Yamaguchi ◽  
Morikazu Imamura ◽  
Mariya Okazaki ◽  
...  
Keyword(s):  
Prion Protein ◽  
Protein Misfolding ◽  
Prion Diseases ◽  
Cellular Prion Protein ◽  
Spongiform Encephalopathy ◽  
Dependent Manner ◽  
Prion Infection ◽  
Amplification Assay ◽  
The Brain ◽  
Strain Dependent

Conformational conversion of the cellular prion protein, PrPC, into the abnormally folded isoform, PrPSc, is a key pathogenic event in prion diseases. However, the exact conversion mechanism remains largely unknown. Transgenic mice expressing PrP with a deletion of the central residues 91–106 were generated in the absence of endogenous PrPC, designated Tg(PrP∆91–106)/Prnp0/0 mice and intracerebrally inoculated with various prions. Tg(PrP∆91–106)/Prnp0/0 mice were resistant to RML, 22L and FK-1 prions, neither producing PrPSc∆91–106 or prions in the brain nor developing disease after inoculation. However, they remained marginally susceptible to bovine spongiform encephalopathy (BSE) prions, developing disease after elongated incubation times and accumulating PrPSc∆91–106 and prions in the brain after inoculation with BSE prions. Recombinant PrP∆91-104 converted into PrPSc∆91–104 after incubation with BSE-PrPSc-prions but not with RML- and 22L–PrPSc-prions, in a protein misfolding cyclic amplification assay. However, digitonin and heparin stimulated the conversion of PrP∆91–104 into PrPSc∆91–104 even after incubation with RML- and 22L-PrPSc-prions. These results suggest that residues 91–106 or 91–104 of PrPC are crucially involved in prion pathogenesis in a strain-dependent manner and may play a similar role to digitonin and heparin in the conversion of PrPC into PrPSc.

scholarly journals Sialic Acid within the Glycosylphosphatidylinositol Anchor Targets the Cellular Prion Protein to Synapses

2016 ◽  
Vol 291 (33) ◽  
pp. 17093-17101 ◽  
Author(s):  
Clive Bate ◽  
William Nolan ◽  
Harriet McHale-Owen ◽  
Alun Williams
Keyword(s):  
Sialic Acid ◽  
Prion Protein ◽  
Acyl Chain ◽  
Cellular Prion Protein ◽  
Gpi Anchor ◽  
Knock Out ◽  
Glycosylphosphatidylinositol Anchor

Although the cellular prion protein (PrPC) is concentrated at synapses, the factors that target PrPC to synapses are not understood. Here we demonstrate that exogenous PrPC was rapidly targeted to synapses in recipient neurons derived from Prnp knock-out(0/0) mice. The targeting of PrPC to synapses was dependent upon both neuronal cholesterol concentrations and the lipid and glycan composition of its glycosylphosphatidylinositol (GPI) anchor. Thus, the removal of either an acyl chain or sialic acid from the GPI anchor reduced the targeting of PrPC to synapses. Isolated GPIs (derived from PrPC) were also targeted to synapses, as was IgG conjugated to these GPIs. The removal of sialic acid from GPIs prevented the targeting of either the isolated GPIs or the IgG-GPI conjugate to synapses. Competition studies showed that pretreatment with sialylated GPIs prevented the targeting of PrPC to synapses. These results are consistent with the hypothesis that the sialylated GPI anchor attached to PrPC acts as a synapse homing signal.

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 Structural Features of Heparin and Its Interactions With Cellular Prion Protein Measured by Surface Plasmon Resonance

2020 ◽  
Vol 7 ◽  
Author(s):  
So Young Kim ◽  
Fuming Zhang ◽  
David A. Harris ◽  
Robert J. Linhardt
Keyword(s):  
Surface Plasmon Resonance ◽  
Prion Protein ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Structural Features ◽  
Cellular Prion Protein ◽  
Dependent Manner ◽  
Heparin Binding ◽  
Structural Determinants ◽  
Terminal Domain

Self-propagating form of the prion protein (PrPSc) causes many neurodegenerative diseases, such as Creutzfeldt-Jakob disease (CJD) and Gerstmann-Straussler-Scheinker syndrome (GSS). Heparin is a highly sulfated linear glycosaminoglycan (GAG) and is composed of alternating D-glucosamine and L-iduronic acid or D-glucuronic acid sugar residues. The interactions of heparin with various proteins in a domain-specific or charged-dependent manner provide key roles on many physiological and pathological processes. While GAG-PrP interactions had been previously reported, the specific glycan structures that facilitate interactions with different regions of PrP and their binding kinetics have not been systematically investigated. In this study, we performed direct binding surface plasmon resonance (SPR) assay to characterize the kinetics of heparin binding to four recombinant murine PrP constructs including full length (M23–230), a deletion mutant lacking the four histidine-containing octapeptide repeats (M23–230 Δ59–90), the isolated N-terminal domain (M23–109), and the isolated C-terminal domain (M90–230). Additionally, we found the specific structural determinants required for GAG binding to the four PrP constructs with chemically defined derivatives of heparin and other GAGs by an SPR competition assay. Our findings may be instrumental in developing designer GAGs for specific targets within the PrP to fine-tune biological and pathophysiological activities of PrP.

scholarly journals The N-terminal cleavage of cellular prion protein in the human brain

FEBS Letters ◽  
2005 ◽  
Vol 579 (28) ◽  
pp. 6333-6337 ◽  
Author(s):  
Isabelle Laffont-Proust ◽  
Baptiste A. Faucheux ◽  
Raymonde Hässig ◽  
Véronique Sazdovitch ◽  
Stéphanie Simon ◽  
...  
Keyword(s):  
Human Brain ◽  
Prion Protein ◽  
Cellular Prion Protein

scholarly journals Mutation of copper binding sites on cellular prion protein abolishes its inhibitory action on NMDA receptors in mouse hippocampal neurons

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Sun Huang ◽  
Stefanie A. Black ◽  
Junting Huang ◽  
Peter K. Stys ◽  
Gerald W. Zamponi
Keyword(s):  
Nmda Receptor ◽  
Prion Protein ◽  
Binding Sites ◽  
Hippocampal Neurons ◽  
Primary Cultures ◽  
Cellular Prion Protein ◽  
Null Mouse ◽  
Dependent Manner ◽  
Copper Binding ◽  
Copper Binding Sites

AbstractWe have previously reported that cellular prion protein (PrPC) can down-regulate NMDA receptor activity and in a copper dependent manner. Here, we employed AAV9 to introduce murine cellular prion protein into mouse hippocampal neurons in primary cultures from PrP null mice to determine the role of the six copper binding motifs located within the N-terminal domain of PrPC. The results demonstrate that viral expression of wild type PrPC lowers NMDAR activity in PrP null mouse hippocampal neurons by reducing the magnitude of non-desensitizing currents. Elimination of the last two copper binding sites alone, or in combination with the remaining four attenuates this protective effect. Thus our data suggest that copper ion interactions with specific binding sites on PrPC are critical for PrPC dependent modulation of NMDA receptor function.

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