Full-length PrPC Research Articles

The Humanization and Maturation of an Anti-PrPc Antibody.

The cellular prion protein (PrPc) is a cell surface glycoprotein that is highly expressed in a variety of cancer tissues in addition to the nervous system, and its elevated expression is correlated to poor prognosis in many cancer patients. Our team previously found that patients with colorectal cancer (CRC) with high-level PrPc expression had significantly poorer survival than those with no or low-level PrPc expression. Mouse antibodies for PrPc inhibited tumor initiation and liver metastasis of PrPc-positive human CRC cells in mouse model experiments. PrPc is a candidate target for CRC therapy. In this study, we newly cloned a mouse anti-PrPc antibody (Clone 6) and humanized it, then affinity-matured this antibody using a CHO cell display with a peptide antigen and full-length PrPc, respectively. We obtained two humanized antibody clones with affinities toward a full-length PrPc of about 10- and 100-fold of that of the original antibody. The two humanized antibodies bound to the PrPc displayed significantly better on the cell surface than Clone 6. Used for Western blotting and immunohistochemistry, the humanized antibody with the highest affinity is superior to the two most frequently used commercial antibodies (8H4 and 3F4). The two new antibodies have the potential to be developed as useful reagents for PrPc detection and even therapeutic antibodies targeting PrPc-positive cancers.

β-Cleavage of the prion protein in the human eye: Implications for the spread of infectious prions and human ocular disorders

Recently, we reported β-cleavage of the prion protein (PrPC) in human ocular tissues. Here, we explored whether this is unique to the human eye, and its functional implications. A comparison of the cleavage pattern of PrPC in human ocular tissues with common nocturnal and diurnal animals revealed mainly β-cleavage in humans, and mostly full-length PrPC in animal retinas. Soluble FL PrPC and N-terminal fragment (N2) released from β-cleavage was observed in the aqueous and vitreous humor (AH & VH). Expression of human PrPC in ARPE-19 cells, a human retinal pigmented epithelial cell line, also showed β-cleaved PrPC. Surprisingly, β-cleavage was not altered by a variety of insults, including oxidative stress, suggesting a unique role of this cleavage in the human eye. It is likely that β-cleaved C- or N-terminal fragments of PrPC protect from various insults unique to the human eye. On the contrary, β-cleaved C-terminus of PrPC is susceptible to conversion to the pathological PrP-scrapie form, and includes the binding sites for β1-integrin and amyloid-β, molecules implicated in several ocular disorders. Considering the species and tissue-specific cleavage of PrPC, our data suggest re-evaluation of prion infectivity and other ocular disorders of the human eye conducted in mouse models.

Molecular Dynamics Simulations of the Full-Length Prion Protein

Many serious medical conditions are caused by the accumulation of amyloid aggregates in tissues and organs. One of the most well-known amyloidogenic proteins is the prion protein (PrP), which may undergo conformational change between the normal cellular isoform PrPC and the aggregation-prone isoform PrPSc. Elucidation of this conformational transition is necessary for understanding the onset and propagation of prion diseases. However, the flexibility of PrP hinders its research by the experimental methods of protein structure determination. Here, we implement de novo protein modelling and molecular dynamics simulations to predict the interdomain interactions of the full-length PrPC. Our theoretical findings can serve as the basis for mutational analysis and for further studies of the amyloidogenic behavior of the prion protein.

Application of high-throughput, capillary-based Western analysis to modulated cleavage of the cellular prion protein

The cellular prion protein (PrPC) is a glycoprotein that is processed through several proteolytic pathways. Modulators of PrPC proteolysis are of interest because full-length PrPC and its cleavage fragments differ in their propensity to misfold, a process that plays a key role in the pathogenesis of prion diseases. PrPC may also act as a receptor for neurotoxic, oligomeric species of other proteins that are linked to neurodegeneration. Importantly, the PrPC C-terminal fragment C1 does not contain the reported binding sites for these oligomers. Western blotting would be a simple end point detection method for cell-based screening of compound libraries for effects on PrPC proteolysis or overall expression level. However, traditional Western blotting methods provide unreliable quantification and have only low throughput. Consequently, we explored capillary-based Western technology as a potential alternative; we believe that this study is the first to report analysis of PrPC using such an approach. We successfully optimized the detection and quantification of the deglycosylated forms of full-length PrPC and its C-terminal cleavage fragments C1 and C2, including simultaneous quantification of β-tubulin levels to control for loading error. We also developed and tested a method for performing all cell culture, lysis, and deglycosylation steps in 96-well microplates prior to capillary Western analysis. These advances represent steps along the way to the development of an automated, high-throughput screening pipeline to identify modulators of PrPC expression levels or proteolysis.

Prion protein is essential for the RE1 silencing transcription factor (REST)-dependent developmental switch in synaptic NMDA receptors

It is important that the correct amounts of GluN2 subunits are maintained, as they determine NMDAR functional properties, which are crucial to neuronal communication, synaptogenesis and cognitive function. The transcriptional repressor RE1 silencing transcription factor (REST) is critical for the postnatal developmental switch in NMDARs. However, the mechanisms triggering REST and the link between NMDARs and REST are unclear. Here we show a new physiological essential role for cellular prion protein (PrPC) in REST-dependent homeostasis and the developmental switch of NMDARs. REST and REST-associated proteins were overactivated in the hippocampi of Prnp knockout mice (Prnp0/0) compared with wild-type Prnp (Prnp+/+) mice. This coincided with the disruption of the normal developmental switch from GluN2B-to-GluN2A in vivo. PrPC co-located with REST under physiological environments and mediated the translocation of REST in conditioners of NMDARs in vitro in Prnp+/+ hippocampal neurons. Regardless of whether REST was knocked down or overexpressed, deletion of PrPC not only disrupted REST-mediated distribution of mitochondria, but also prevented REST-regulated expression of GluN2B and GluN2A in Prnp0/0. Importantly, these effects were rescued after overexpression of full-length PrPC through restoration of NMDAR2 subunits and their distributions in dendritic processes in Prnp0/0. Consistently, knockdown of PrPC in Prnp+/+ had a similar effect on Prnp0/0. Furthermore, PrPC colocalized with both GluN2B and GluN2A in Prnp+/+. For the first time, we demonstrate that PrPC is essential for REST-regulated NMDARs. Confirming the regulation of NMDAR-modulating mechanisms could provide novel therapeutic targets against dysfunctions of glutamatergic transmission in the nervous system.

Loss of Cellular Sialidases Does Not Affect the Sialylation Status of the Prion Protein but Increases the Amounts of Its Proteolytic Fragment C1.

The central molecular event underlying prion diseases involves conformational change of the cellular form of the prion protein (PrPC), which is a sialoglycoprotein, into the disease-associated, transmissible form denoted PrPSc. Recent studies revealed a correlation between the sialylation status of PrPSc and incubation time to disease and introduced a new hypothesis that progression of prion diseases could be controlled or reversed by altering the sialylation level of PrPC. Of the four known mammalian sialidases, the enzymes that cleave off sialic acid residues, only NEU1, NEU3 and NEU4 are expressed in the brain. To test whether cellular sialidases control the steady-state sialylation level of PrPC and to identify the putative sialidase responsible for desialylating PrPC, we analyzed brain-derived PrPC from knockout mice deficient in Neu1, Neu3, Neu4, or from Neu3/Neu4 double knockouts. Surprisingly, no differences in the sialylation of PrPC or its proteolytic product C1 were noticed in any of the knockout mice tested as compared to the age-matched controls. However, significantly higher amounts of the C1 fragment relative to full-length PrPC were detected in the brains of Neu1 knockout mice as compared to WT mice or to the other knockout mice. Additional experiments revealed that in neuroblastoma cell line the sialylation pattern of C1 could be changed by an inhibitor of sialylatransferases. In summary, this study suggests that targeting cellular sialidases is apparently not the correct strategy for altering the sialylation levels of PrPC, whereas modulating the activity of sialylatransferases might offer a more promising approach. Our findings also suggest that catabolism of PrPC involves its α-cleavage followed by desialylation of the resulting C1 fragments by NEU1 and consequent fast degradation of the desialylated products.

Prion protein functions as a ferrireductase partner for ZIP14 and DMT1

Excess circulating iron is stored in the liver, and requires reduction of non-Tf-bound iron (NTBI) and transferrin (Tf) iron at the plasma membrane and endosomes, respectively, by ferrireductase (FR) proteins for transport across biological membranes through divalent metal transporters. Here, we report that prion protein (PrPC), a ubiquitously expressed glycoprotein most abundant on neuronal cells, functions as a FR partner for divalent-metal transporter-1 (DMT1) and ZIP14. Thus, absence of PrPC in PrP-knock-out (PrP–/–) mice resulted in markedly reduced liver iron stores, a deficiency that was not corrected by chronic or acute administration of iron by the oral or intraperitoneal routes. Likewise, preferential radiolabeling of circulating NTBI with 59Fe revealed significantly reduced uptake and storage of NTBI by the liver of PrP–/– mice relative to matched PrP+/+ controls. However, uptake, storage, and utilization of ferritin-bound iron that does not require reduction for uptake were increased in PrP–/– mice, indicating a compensatory response to the iron deficiency. Expression of exogenous PrPC in HepG2 cells increased uptake and storage of ferric iron (Fe3+), not ferrous iron (Fe2+), from the medium, supporting the function of PrPC as a plasma membrane FR. Coexpression of PrPC with ZIP14 and DMT1 in HepG2 cells increased uptake of Fe3+ significantly, and surprisingly, increased the ratio of N-terminally truncated PrPC forms lacking the FR domain relative to full-length PrPC. Together, these observations indicate that PrPC promotes, and possibly regulates, the uptake of NTBI through DMT1 and Zip14 via its FR activity. Implications of these observations for neuronal iron homeostasis under physiological and pathological conditions are discussed.

Copper(II) sequentially loads onto the N-terminal amino group of the cellular prion protein before the individual octarepeats.

The cellular prion protein (PrPC) binds to Cu2+ ions in vivo, and a misfolded form of PrPC is responsible for a range of transmissible spongiform encephalopathies. Recently, disruption of Cu2+ homeostasis in mice has been shown to impart resistance to scrapie infection. Using full-length PrPC and model peptide fragments, we monitor the sequential loading of Cu2+ ions onto PrPC using visible circular dichroism. We show the N-terminal amino group of PrPC is not the principal binding site for Cu2+; however, surprisingly, it has an affinity for Cu2+ tighter than that of the individual octarepeat binding sites present within PrPC. We re-evaluate what is understood about the sequential loading of Cu2+ onto the full-length protein and show for the first time that Cu2+ loads onto the N-terminal amino group before the single octarepeat binding sites.

Lactoferrin from bovine colostrum regulates prolyl hydroxylase 2 activity and prevents prion protein-mediated neuronal cell damage via cellular prion protein

Prion disorders are associated with the conversion of normal cellular prion protein (PrPc) to the abnormal scrapie isoform of prion protein (PrPsc). Recent studies have shown that expression of normal PrPc is regulated by hypoxia-inducible factor-1 alpha (HIF-1α), and that lactoferrin increases full-length PrPc on the cell surface. Lactoferrin is an 80-kDa iron-binding glycoprotein with various biological activities, including iron-chelating ability. HIF-1α and the associated ubiquitin–proteasome pathway are regulated by HIF prolyl-hydroxylases 2 (PHD2). We hypothesized that lactoferrin regulates PHD2 expression and enzymatic activity, and the PHD2 regulation promotes HIF-1α stability and prevention of neuronal cell death mediated by prion protein (PrP) residues (106–126). Lactoferrin prevented PrP (106–126)-induced neurotoxicity by the induction of PrPc expression via promoting HIF-1α stability in neuronal cells. Our results demonstrated that lactoferrin prevented PrP (106–126)-induced neurotoxicity via the up-regulation of HIF-1α stability determined by PHD2 expression and enzymatic activity. These findings suggest that possible therapies such as PHD2 inhibition, or promotion of lactoferrin secretion, may have clinical benefits in neurodegenerative diseases, including prion disease.

Anchorless 23–230 PrPC Interactomics for Elucidation of PrPC Protective Role

Accumulation of conformationally altered cellular proteins (i.e., prion protein) is the common feature of prions and other neurodegenerative diseases. Previous studies demonstrated that the lack of terminal sequence of cellular prion protein (PrPC), necessary for the addition of glycosylphosphatidylinositol lipid anchor, leads to a protease-resistant conformation that resembles scrapie-associated isoform of prion protein. Moreover, mice overexpressing the truncated form of PrPC showed late-onset, amyloid deposition, and the presence of a short protease-resistant PrP fragment in the brain similar to those found in Gerstmann-Sträussler-Scheinker disease patients. Therefore, the physiopathological function of truncated_/anchorless 23-230 PrPC (Δ23-230 PrPC) has come into focus of attention. The present study aims at revealing the physiopathological function of the anchorless PrPC form by identifying its interacting proteins. The truncated_/anchorless Δ23-230 PrPC along with its interacting proteins was affinity purified using STrEP-Tactin chromatography, in-gel digested, and identified by quadrupole time-of-flight tandem mass spectrometry analysis in prion protein-deficient murine hippocampus (HpL3-4) neuronal cell line. Twenty-three proteins appeared to interact with anchorless Δ23-230 PrPC in HpL3-4 cells. Out of the 23 proteins, one novel protein, pyruvate kinase isozymes M1/M2 (PKM2), exhibited a potential interaction with the anchorless Δ23-230 form of PrPC. Both reverse co-immunoprecipitation and confocal laser-scanning microscopic analysis confirmed an interaction of PKM2 with the anchorless Δ23-230 form of PrPC. Furthermore, we provide the first evidence for co-localization of PKM2 and PrPC as well as PrPC-dependent PKM2 expression regulation. In addition, given the involvement of PrPC in the regulation of apoptosis, we exposed HpL3-4 cells to staurosporine (STS)-mediated apoptotic stress. In response to STS-mediated apoptotic stress, HpL3-4 cells transiently expressing 23-230-truncated PrPC were markedly less viable, were more prone to apoptosis and exhibited significantly higher PKM2 expressional regulation as compared with HpL3-4 cells transiently expressing full-length PrPC (1-253 PrPC). The enhanced STS-induced apoptosis was shown by increased caspase-3 cleavage. Together, our data suggest that the misbalance or over expression of anchorless Δ23-230 form of PrPC in association with the expressional regulation of interacting proteins could render cells more prone to cellular insults-stress response, formation of aggregates and may ultimately be linked to the cell death.

The PrPC C1 fragment derived from the ovine A136R154R171PRNP allele is highly abundant in sheep brain and inhibits fibrillisation of full-length PrPC protein in vitro

Expression of the cellular prion protein (PrPC) is crucial for the development of prion diseases. Resistance to prion diseases can result from reduced availability of the prion protein or from amino acid changes in the prion protein sequence. We propose here that increased production of a natural PrP α-cleavage fragment, C1, is also associated with resistance to disease. We show, in brain tissue, that ARR homozygous sheep, associated with resistance to disease, produced PrPC comprised of 25% more C1 fragment than PrPC from the disease-susceptible ARQ homozygous and highly susceptible VRQ homozygous animals. Only the C1 fragment derived from the ARR allele inhibits in-vitro fibrillisation of other allelic PrPC variants. We propose that the increased α-cleavage of ovine ARR PrPC contributes to a dominant negative effect of this polymorphism on disease susceptibility. Furthermore, the significant reduction in PrPC β-cleavage product C2 in sheep of the ARR/ARR genotype compared to ARQ/ARQ and VRQ/VRQ genotypes, may add to the complexity of genetic determinants of prion disease susceptibility.

Release of Full-Length PrP(C) from Cultured Neurons Following Neurotoxic Challenges.

The susceptibility of the normal cellular prion protein isoform, cellular prion protein (PrPC), to proteolytic digestion has been well documented. In addition, a link between PrPC and the cytosolic protease, calpain, has been reported although the specifics of the interaction remain unclear. We performed in vitro and in cell-based studies to examine this relationship. We observed that human recombinant PrP (HrPrP) was readily cleaved by calpain-1 and -2, and we have identified and defined the targeted cleavage sites. In contrast, HrPrP was resistant to caspase-3 digestion. Unexpectedly, when brain lysates from PrPC-expressing mice were treated with calpain, no appreciable loss of the intact PrPC, nor the appearance of PrPC breakdown products (BDPs) were observed, even though alpha II-spectrin was converted to its signature calpain-induced BDPs. In addition, when rat cerebrocortical neuronal cultures (RtCNC) were subjected to the two neurotoxins at subacute levels, maitotoxin (MTX) and N-methyl-d-aspartate (NMDA), PrPC-BDPs were also not detectable. However, a novel finding from these cell-based studies is that apparently full-length, mature PrPC is released into culture media from RtCNC challenged with subacute doses of MTX and NMDA. Calpain inhibitor SNJ-1945 and caspase inhibitor IDN-6556 did not attenuate the release of PrPC. Similarly, the lysosomal protease inhibitor, NH4Cl, and the proteasome inhibitor, lactacystin, did not significantly alter the integrity of PrPC or its release from the RtCNC. In conclusion, rat neuronal PrPC is not a significant target for proteolytic modifications during MTX and NMDA neurotoxic challenges. However, the robust neurotoxin-mediated release of full-length PrPC into the cell culture media suggests an unidentified neuroprotective mechanism for PrPC.

Y145Stop is sufficient to induce de novo generation prions using protein misfolding cyclic amplification

A point mutation in Prnp that converts tyrosine (Y) at position 145 into a stop codon leading to a truncated prion molecule as found in an inherited transmissible spongiform encephalopathy (TSE), Gertsmann-Sträussler-Scheincker syndrome, suggests that the N-terminus of the molecule (spanning amino acids 23–144) likely plays a critical role in prion misfolding as well as in protein-protein interactions. We hypothesized that Y145Stop molecule represents an unstable part of the prion protein that is prone to spontaneous misfolding. Utilizing protein misfolding cyclic amplification (PMCA) we show that the recombinant polypeptide corresponding to the Y145Stop of sheep and deer PRNP can be in vitro converted to PK-resistant PrPSc in presence or absence of preexisting prions. In contrast, recombinant protein full-length PrPC did not show a propensity for spontaneous conformational conversion to protease resistant isoforms. Further, we show that seeded or spontaneously misfolded Y145Stop molecules can efficiently convert purified mammalian PrPC into protease resistant isoforms. These results establish that the N-terminus of PrPC molecule corresponding to residues 23–144 plays a role in seeding and misfolding of mammalian prions.

Spontaneous generation of anchorless prions in transgenic mice

Some prion protein mutations create anchorless molecules that cause Gerstmann-Sträussler-Scheinker (GSS) disease. To model GSS, we generated transgenic mice expressing cellular prion protein (PrP(C)) lacking the glycosylphosphatidyl inositol (GPI) anchor, denoted PrP(ΔGPI). Mice overexpressing PrP(ΔGPI) developed a late-onset, spontaneous neurologic dysfunction characterized by widespread amyloid deposition in the brain and the presence of a short protease-resistant PrP fragment similar to those found in GSS patients. In Tg(PrP,ΔGPI) mice, disease onset could be accelerated either by inoculation with brain homogenate prepared from spontaneously ill animals or by coexpression of membrane-anchored, full-length PrP(C). In contrast, coexpression of N-terminally truncated PrP(Δ23-88) did not affect disease progression. Remarkably, disease from ill Tg(PrP,ΔGPI) mice transmitted to mice expressing wild-type PrP(C), indicating the spontaneous generation of prions.

The Cellular Prion Protein Prevents Copper‐Induced Inhibition of P2X4 Receptors

Although the physiological function of the cellular prion protein (PrPC) remains unknown, several evidences support the notion of its role in copper homeostasis. PrPC binds Cu2+ through a domain composed by four to five repeats of eight amino acids. Previously, we have shown that the perfusion of this domain prevents and reverses the inhibition by Cu2+ of the adenosine triphosphate (ATP)-evoked currents in the P2X4 receptor subtype, highlighting a modulatory role for PrPC in synaptic transmission through regulation of Cu2+ levels. Here, we study the effect of full-length PrPC in Cu2+ inhibition of P2X4 receptor when both are coexpressed. PrPC expression does not significantly change the ATP concentration-response curve in oocytes expressing P2X4 receptors. However, the presence of PrPC reduces the inhibition by Cu2+ of the ATP-elicited currents in these oocytes, confirming our previous observations with the Cu2+ binding domain. Thus, our observations suggest a role for PrPC in modulating synaptic activity through binding of extracellular Cu2+.

Recombinant prion protein induces rapid polarization and development of synapses in embryonic rat hippocampal neurons in vitro

While a beta-sheet-rich form of the prion protein (PrPSc) causes neurodegeneration, the biological activity of its precursor, the cellular prion protein (PrPC), has been elusive. We have studied the effect of purified recombinant prion protein (recPrP) on rat fetal hippocampal neurons in culture. Overnight exposure to Syrian hamster or mouse recPrP, folded into an alpha-helical-rich conformation similar to that of PrPC, resulted in a 1.9-fold increase in neurons with a differentiated axon, a 13.5-fold increase in neurons with differentiated dendrites, a fivefold increase in axon length, and the formation of extensive neuronal circuitry. Formation of synaptic-like contacts was increased by a factor of 4.6 after exposure to recPrP for 7 days. Neither the N-terminal nor C-terminal domains of recPrP nor the PrP paralogue doppel (Dpl) enhanced the polarization of neurons. Inhibitors of protein kinase C (PKC) and of Src kinases, including p59Fyn, blocked the effect of recPrP on axon elongation, while inhibitors of phosphatidylinositol 3-kinase showed a partial inhibition, suggesting that signaling cascades involving these kinases are candidates for transduction of recPrP-mediated signals. The results predict that full-length PrPC functions as a growth factor involved in development of neuronal polarity.

Truncated Prion Protein and Doppel Are Myelinotoxic in the Absence of Oligodendrocytic PrPC

The cellular prion protein PrP(C) confers susceptibility to transmissible spongiform encephalopathies, yet its normal function is unknown. Although PrP(C)-deficient mice develop and live normally, expression of amino proximally truncated PrP(C) (DeltaPrP) or of its structural homolog Doppel (Dpl) causes cerebellar degeneration that is prevented by coexpression of full-length PrP(C). We now report that mice expressing DeltaPrP or Dpl suffer from widespread leukoencephalopathy. Oligodendrocyte-specific expression of full-length PrP(C) under control of the myelin basic protein (MBP) promoter repressed leukoencephalopathy and vastly extended survival but did not prevent cerebellar granule cell (CGC) degeneration. Conversely, neuron-specific PrP(C) expression under control of the neuron-specific enolase (NSE) promoter antagonized CGC degeneration but not leukoencephalopathy. PrP(C) was found in purified myelin and in cultured oligodendrocytes of both wild-type and MBP-PrP transgenic mice but not in NSE-PrP mice. These results identify white-matter damage as an extraneuronal PrP-associated pathology and suggest a previously unrecognized role of PrP(C) in myelin maintenance.

Proteolytic processing and glycosylation influence formation of porcine prion protein complexes

High level of heterogeneity seems to be a ubiquitous feature of mammalian PrPs (prion proteins) and may be relevant to the pathogenesis of prion diseases. In the present study, we describe the heterogeneity of PrP(C) (cellular form of PrP) from porcine brain. It was disclosed and characterized by a combination of one-dimensional PAGE and two-dimensional PAGE analyses with enzymic deglycosylation and copper-affinity experiments. We found that the identified two main populations of porcine PrP(C) consist of diglycosylated forms and correspond to the full-length (molecular mass 32-36 kDa) and proteolytically modified protein (molecular mass 25-30 kDa), known as C1. The two populations were fully separated during Cu2+-loaded immobilized metal affinity chromatography, indicating different affinity for copper ions. The more basic forms, migrating as species of higher molecular mass, exhibited stronger affinity for copper ions, whereas those with more acidic pI and of lower molecular mass were low-affinity Cu2+-binding molecules and thus could represent N-terminally truncated PrP(C). Size-exclusion chromatography revealed that most of the PrP(C) molecules in porcine brain extracts exist in the form of high-molecular-mass complexes (probably with other proteins). The heterogeneity of porcine PrP(C), resulting from proteolytic modification and glycosylation, influences its ability to assemble into these complexes. N-truncated molecules dominate over full-length PrP(C) in fractions of molecular mass over the range 65-130 kDa, whereas the full-length species are the major forms of PrP(C) present in the monomeric fraction and in complexes above 130 kDa. Two-dimensional PAGE analysis indicated that the complexed PrP(C) differs in the composition of pI forms from the monomers.

Overexpression of nonconvertible PrPc delta114-121 in scrapie-infected mouse neuroblastoma cells leads to trans-dominant inhibition of wild-type PrP(Sc) accumulation.

One hallmark of prion diseases is the accumulation of the abnormal isoform PrP(Sc) of a normal cellular glycoprotein, PrPc, which is characterized by a high content of beta-sheet structures and by its partial resistance to proteinase K. It was hypothesized that the PrP region comprising amino acid residues 109 to 122 [PrP(109-122)], which spontaneously forms amyloid when it is synthesized as a peptide but which does not display significant secondary structure in the context of the full-length PrPc molecule, should play a role in promoting the conversion into PrP(Sc). By using persistently scrapie-infected mouse neuroblastoma (Sc+-MNB) cells as a model system for prion replication, we set out to design dominant-negative mutants of PrPc that are capable of blocking the conversion of endogenous, wild-type PrPc into PrP(Sc). We constructed a deletion mutant (PrPc delta114-121) lacking eight codons that span most of the highly amyloidogenic part, AGAAAAGA, of PrP(109-122). Transient transfections of mammalian expression vectors encoding either wild-type PrPc or PrPc delta114-121 into uninfected mouse neuroblastoma cells (Neuro2a) led to overexpression of the respective PrPc versions, which proved to be correctly localized on the extracellular face of the plasma membrane. Transfection of Sc+-MNB cells revealed that PrPc delta114-121 was not a substrate for conversion into a proteinase K-resistant isoform. Furthermore, its presence led to a significant reduction in the steady-state levels of PrP(Sc) derived from endogenous PrPc. Thus, we showed that the presence of amino acids 114 to 121 of mouse PrPc plays an important role in the conversion process of PrPc into PrP(Sc) and that a deletion mutant lacking these codons indeed behaves as a dominant-negative mutant with respect to PrP(Sc) accumulation. This mechanism could form a basis for a new gene therapy and/or a prevention concept for prion diseases.

Truncated Forms of the Human Prion Protein in Normal Brain and in Prion Diseases

The cellular form of the prion protein (PrPc) is a glycoprotein anchored to the cell membrane by a glycosylphosphatidylinositol moiety. An aberrant form of PrPc that is partially resistant to proteases, PrPres, is a hallmark of prion diseases, which in humans include Cruetzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome, and fatal familial insomnia. We have characterized the major forms of PrP in normal and pathological human brains. A COOH-terminal fragment of PrPc, designated C1, is abundant in normal and CJD brains as well as in human neuroblastoma cells. Sequence analysis revealed that C1 contains alternative NH2 termini starting at His-111 or Met-112. Like PrPc, C1 is glycosylated, anchored to the cell membrane, and is heat-stable. Consistent with the lack of the NH2-terminal region of PrPc, C1 is more acidic than PrPc and does not bind heparin. An additional fragment longer than C1, designated C2, is present in substantial amounts in CJD brains. Like PrPres, C2 is resistant to proteases and is detergent-insoluble. Our data indicate that C1 is a major product of normal PrPc metabolism, generated by a cleavage that disrupts the neurotoxic and amyloidogenic region of PrP comprising residues 106-126. This region remains intact in C2, suggesting a role for C2 in prion diseases.