Cellular prion protein localizes to the nucleus of endocrine and neuronal cells and interacts with structural chromatin components.

Several physiological processes have been purported for cellular prion protein (PrP(C)). However, the physiological function of PrP(C) is still unclear and the cellular localization of PrP(C) remains a subject of debate. PrP(C) is expressed in a wide range of tissues including islets of Langerhans. We previously demonstrated that the function of PrP(C) is associated with blood glucose regulation. Little is known of the function of PrP(C) in islet cells and specifically in ß-cells. To get first insight into the putative role of PrP(C) in ß-cells, we used far-Western immunoblotting and MS to identify candidate PrP(C)-interacting proteins. We also used Western blot, immunofluorescence (IF) and protein overlay IF to characterize the sub-cellular localization of PrP(C). Here we demonstrate in vivo that PrP(C) is abundant in the nuclear lamina of endocrine and neuronal cells and interacts with histone H1(0), histone H3 and lamin B1. The interaction of PrP(C) with histone H3 suggests that it is involved in transcriptional regulation in the nucleus. This study reveals new avenues for the elucidation of the physiological function of PrP(C) in endocrine and neuronal cells as well as the molecular mechanisms leading to prion diseases.

Conditional expression of full-length humanized anti-prion protein antibodies in Chinese hamster ovary cells.

Because of their high antigen specificity and metabolic stability, genetically engineered human monoclonal antibodies are on the way to becoming one of the most promising medical diagnostics and therapeutics. In order to establish an in vitro system capable of producing such biosimilar antibodies, we used human constant chain sequences to design the novel human antibody expressing vector cassette pMAB-ABX. A bidirectional tetracycline (tet)-controllable promotor was used for harmonized expression of immunoglobulin type G (IgG) heavy and light chains. As an example we used anti-prion protein (anti-PrP) IgGs. Therefore, the variable heavy (V(H)) and light chain (V(L)) sequences of anti-PrP antibodies, previously generated in our laboratory by DNA immunization of prion protein knock-out mice, were isolated from murine hybridoma cell lines and inserted into pMAB-ABX vector. After transfection of Chinese hamster ovary (CHO) cells, a number of stable antibody producing cell clones were selected. One cell line (pMAB-ABX-13F10/3B5) stably expressing the recombinant humanized antibody (rechuAb) 13F10/3B5 was selected for detailed characterization by Western blot, immunofluorescence, and flow cytometric analyses. The full-length recombinant humanized IgG antibody showed a high level of expression in the cytoplasm. In conclusion, the new cell system described here is a suitable tool to produce functional intact full-length humanized IgG antibodies.

Increase in CD230 (cellular prion protein) fluorescence on blood lymphocytes in bovine spongiform encephalopathy-infected nonhuman primates.

BACKGROUND: The cellular prion protein (PrP(c)) plays a central role in prion diseases such as variant Creutzfeldt-Jakob disease. This disease can be transmitted by blood transfusion. However, the exact kinetics of blood infectivity and the blood fraction carrying infectivity have not yet been identified. STUDY DESIGN AND METHODS: Simian PrP(c) epitopes were mapped by flow cytometry using monoclonal antibodies (MoAbs). A whole blood/no wash protocol was established, validated, and applied to investigate peripheral blood cell-associated PrP(c) expression profiles in bovine spongiform encephalopathy (BSE)-infected cynomolgus monkeys and age-/sex-matched controls. In addition, physiologic expression patterns on blood cells and in lymphoid tissues were determined. RESULTS: In BSE-infected macaques, blood lymphocyte-associated PrP(c) fluorescence gradually increased years before the onset of clinical signs (p(F test) < 0.0001). The increase in fluorescence intensity was detectable with MoAb 12F10, whereas we failed to detect an increase with 3F4. In parallel, plasma concentrations of soluble CD230 also increased. Centrifugation of lymphocytes almost completely eliminated differences between infected and noninfected animals, most likely caused by a partial loss in cell-associated CD230 into the plasma supernatant. CONCLUSION: Blood lymphocytes from asymptomatically infected as well as diseased macaques were characterized by increased CD230 fluorescence, and phosphatidylinositol-phospholipase C-resistant PrP molecules contributed at least partially to this increase. Conformational changes within PrP(c) molecules may be the underlying mechanism for the increased PrP(c) fluorescence. This cell-associated phenomenon contributed at least partially to an increase in soluble plasma-derived PrP(c) levels. It is not yet known whether these changes reflect infectivity.

Inducible expression of chimpanzee prion protein (PrP) in murine PrP knock-out cells.

In transmissible spongiform encephalopathy (TSE) pathogenesis the cellular prion protein (PrP(C)) is converted into its pathogenic PrP(Sc) isoform. Prion protein gene (Prnp) deficient mice (PrP(0/0)) are resistant to PrP(Sc) infection, but following reconstitution of Prnp they regain their susceptibility to infection. Therefore, it is challenging to simulate this natural situation in a cell culture model. We have previously reported the inducible stable expression of a human PrP(C) in murine 3T3 cells. In this study, we used murine PrP(0/0) cells stably expressing exemplarily the chimpanzee Prnp under the control of inducible tetracycline (Tet) system. The Prnp was integrated using a lentiviral vector. Its expression in the engineered PrP(0/0)Chimp1/Tet-Off cell line was analyzed by Western blot (Wb) and fluorescence activated cell sorting (FACS) analyses. PrP(C) was partially purified by using immobilized metal affinity chromatography (IMAC). Compared to all the other cell systems which possess an endogenous PrP(C) expression, here described cell line contains only an overexpressing species specific PrP(C) expression which is tightly regulated and can be turned-off at any time without showing any endogenous host PrP(C) expression. Consequently, a contamination of the isolated PrP(C) is impossible. This cell line potentially offers a new tool for simulation of mice bioassays widely used in TSE infection studies.

Time-course studies of 14-3-3 protein isoforms in cerebrospinal fluid and brain of primates after oral or intracerebral infection with bovine spongiform encephalopathy agent.

Experimental transmission of bovine spongiform encephalopathy (BSE) to cynomolgus monkeys (Macaca fascicularis) is an animal model for variant Creutzfeldt-Jakob disease (vCJD). The presence of 14-3-3 proteins in cerebrospinal fluid (CSF) samples indicates neuronal destruction and is therefore used as a clinical biomarker. However, time-course studies using 14-3-3 proteins have not been performed until now in simian vCJD. The main goals of this study were to determine isoform patterns, to examine kinetics and to correlate the clinical course with the occurrence of this biomarker in simian vCJD. In monkeys dosed intracerebrally with BSE, the earliest clinical sign of illness was a drop in body weight that was detected months before the onset of mild neurological signs. Macaques dosed orally or intracerebrally with BSE developed neurological signs 4.3 (3.7-4.6) and 4.8 (2.9-6.0) years post-infection, respectively. 14-3-3beta- and -gamma-positive CSF samples were found around the time of onset of mild neurological signs, but not earlier. In contrast, 14-3-3epsilon and -eta isoforms were not detectable. 14-3-3 levels increased with time and were positively correlated with the degree of neurological symptoms. Post-mortem examination of brain samples revealed a positive correlation between PrP res and 14-3-3epsilon levels. Interestingly, florid plaques characteristic of human vCJD could not be detected in diseased monkeys. It was concluded that analysis of 14-3-3 proteins in CSF is a reliable tool to characterize the time course of brain degeneration in simian vCJD. However, there are differences in the clinical course between orally and intracerebrally infected animals that may influence the detection of other biomarkers.

Identification of prion protein binding proteins by combined use of far-Western immunoblotting, two dimensional gel electrophoresis and mass spectrometry.

The cellular prion protein (PrP(C)), a highly conserved glycoprotein predominantly expressed by neuronal cells, can convert into an abnormal isoform (PrP(Sc)) and provoke a transmissible spongiform encephalopathy. In spite of many studies, the physiological function of PrP(C) remains unknown. Recent findings suggest that PrP(C) is a multifunctional protein participating in several cellular processes. Using recombinant human PrP as a probe, we performed far-Western immunoblotting (protein overlay assay) to detect cellular PrP(C) interactors. Brain extracts of wild-type and PrP knockout mice were screened by far-Western immunoblotting for PrP-specific interactions. Subsequently, putative ligands were isolated by 2-DE and identified by MALDI-TOF MS, enabling identification of heterogeneous nuclear ribonucleoprotein A2/B1 and aldolase C as novel interaction partners of PrP(C). These data provide the first evidence of a molecule indicating a mechanism for the predicted involvement of PrP(C) in nucleic acid metabolisms. In summary, we have shown the successful combination of 2-DE with far-Western immunoblotting and MALDI-TOF MS for identification of new cellular binding partners of a known protein. Especially the application of this technique to investigate other neurodegenerative diseases is promising.

Separation of native prion protein (PrP) glycoforms by copper-binding using immobilized metal affinity chromatography (IMAC).

The conformational conversion of the normal cellular prion protein (PrPC) into the pathology-associated PrPSc isoform is a key event in TSEs (transmissible spongiform encephalopathies). The host PrPC molecule contains two N-linked glycosylation sites and binds copper under physiological conditions. In contrast with PrPC, PrPSc is insoluble in non-ionic detergents and does not bind to Cu2+ ions. Hence, we utilized copper binding to separate and characterize both PrP isoforms. Infected and uninfected murine brain and bovine stem brain specimens were treated with the mild non-ionic detergent n-octyl-beta-D-glucopyranoside (octylglucoside) to maintain the native PrP conformations during isolation. The solubilized homogenates were loaded on to Cu2+-saturated IMAC (immobilized metal affinity chromatography) columns and eluted using the chelating agent EDTA. Fractions were separated by SDS/PAGE and analysed by immunoblotting using anti-PrP monoclonal antibodies for glycosylation profiling. Whereas native PrPC and denatured PrPSc were retained by a Cu2+-loaded resin, native PrPSc and PrPres [PK (proteinase K)-resistant PrP] passed through the column. We demonstrate here that the IMAC technique is appropriate to isolate and partially purify PrPC from healthy brains in its native-like and biologically relevant glycosylated copper-binding forms. The IMAC technique is also well suited for the separation of native PrPC from aggregated PrPSc in infected brains. Our results indicate that in contrast with PrPSc in uninfected as well as infected brains, PrPC is predominantly present in the glycosylated forms.

Tetracycline-regulated highly inducible expression of the human prion protein in murine 3T3 cells.

To provide an in vitro system that allows inducible or conditional overexpression of human prion protein (PrP), we have established a tetracycline (Tc)-regulated system in murine 3T3 L1 fibroblast cells. A replacement-type gene targeting vector cassette was constructed to express the human fatal familial insomnia (FFI) prion protein gene (PRNP) under control of a Tc-responsive element. Following stable integration of the vector into 3T3 Tet-Off cells, we have isolated and characterised six 3T3 L1 pTet-Off FFI clones. These clones were analysed by PCR and their expression level was determined by Western blot using species specific monoclonal antibodies (anti-mouse and human 3B5, 4F2, 12F10, 11C6, 8G8, and 14D3; anti-mouse l3). Addition of the antibiotic Tc to the culture medium turned off expression of human PrP. This supression was repeatedly reversible. However, no significant transcriptional leakiness of repressed PminCMV promoter was observed. In the absence of Tc, expression of human PrP was induced 10- to 20-fold as estimated from densitometric analyses. PrP was analysed by Proteinase K (PK) digestions and found to be PK sensitive. Subcellular fractionation revealed that PrP was located mainly in the cytoplasmic membrane fraction. Furthermore, we partially purified PrP by PrP-specific copper-binding. After immobilised metal affinity chromatography, majority of PrP showed a molecular weight consistent with non-glycosylated PrP. These clones offer a new tool to facilitate the investigation of PrP interaction with potential cellular ligands and PrP ex vivo propagation.