Chicken prion tandem repeats form a stable, protease-resistant domain

Prion-linked diseases, such as mad cow disease, scrapie, and the human genetic disorder Creutzfeldt-Jakob disease, are fatal neurodegenerative diseases correlated with changes in the secondary structure of neural prion protein. We expressed recombinant chicken prion protein in Escherichia coli and purified the protein to homogeneity. Circular dichroism spectra of the 26 kDa recombinant protein closely resemble those of prion protein purified directly from healthy hamster brain. The chicken prion protein exists as a soluble, monodisperse monomer but can be forced to multimerize following lyophilization and resuspension. We analyzed the chicken prion protein domain structure by proteolysis and show that, unlike the mammalian homologues, the chicken prion protein N-terminal tandem amino acid repeats form a stable, protease-resistant domain. This domain probably represents a physiologically functional unit. As tested by both mass spectrometry and circular dichroism, the mature chicken prion protein does not bind copper, unlike synthetic peptides from the chicken prion N-terminus, suggesting that binding copper is not the physiological activity of the chicken prion. However, copper strongly destabilizes the prion protein and depresses the melting temperature by 30 degreesC, presumably by binding to the unfolded form of the prion protein. The chicken prion N-terminus may have evolved to fold without a cofactor, unlike mammalian prion proteins, whose N-termini are disordered without cofactors such as copper present. Chicken prion offers an alternative to intractable mammalian prions for structural studies of the amino-terminal domain.     

Structural aspects of Congo red as an inhibitor of protease-resistant prion protein formation

Congo red (CR) has been shown to inhibit the accumulation in scrapie-infected cells of prion protein (PrP) in the abnormal protease-resistant form (PrP-res). However, it was not clear if this effect was due to a direct interaction of CR with either PrP-res or its protease-sensitive precursor (PrP-sen) or to a less direct effect on living cells. Here we show that CR inhibits PrP-res formation in a simple cell-free reaction composed predominantly of purified PrP-res and PrP-sen. Structurally modified CR analogues were also compared in both the cell-free conversion reaction and scrapie-infected neuroblastoma cells. Methylation of the central phenyl groups at the 2,2' positions diminished the inhibitory potency by > or = 10-fold. In contrast, there was little effect of 3,3' methylation of the phenyls, deletion of one phenyl, or addition of an amido group between the phenyls. The relative activities of these compounds were well correlated in both cellular and acellular systems. Molecular modeling indicated that CR and 3,3'-methyl-CR have little rotational restriction about the biphenyl bond and can readily adopt a planar conformation, as can phenyl-CR and amido-CR. In contrast, 2,2'-methyl-CR is restricted to a nonplanar conformation of the biphenyl group. Thus, planarity and/or torsional mobility of the central phenyl rings of CR and its analogues is probably important for inhibition of PrP-res formation. On the other hand, variations in the intersulfonate distance in these molecules had little effect on PrP-res inhibition. These results indicated a high degree of structural specificity in the inhibition of PrP-res formation by CR and related compounds.     

Identification of intermediate steps in the conversion of a mutant prion protein to a scrapie-like form in cultured cells

The central causative event in infectious, familial, and sporadic forms of prion disease is thought to be a conformational change that converts the cellular isoform of the prion protein (PrPC) into the scrapie isoform (PrPSc) that is the primary constituent of infectious prion particles. To provide a model system for analyzing the mechanistic details of this critical transformation, we have previously prepared cultured Chinese hamster ovary cells that stably express mouse PrP molecules carrying mutations homologous to those seen in familial prion diseases of humans. In the present work, we have analyzed the kinetics with which a PrP molecule containing an insertional mutation associated with Creutzfeldt-Jakob disease acquires several biochemical properties characteristic of PrPSc. Within 10 min of pulse labeling, the mutant protein undergoes a molecular alteration that is detectable by a change in Triton X-114 phase partitioning and phenyl-Sepharose binding. After 30 min of labeling, a detergent-insoluble and protease-sensitive form of the protein appears. After a chase period of several hours, the protein becomes protease-resistant. Incubation of cells at 18 degrees C or treatment with brefeldin A inhibits acquisition of detergent insolubility and protease resistance but does not affect Triton X-114 partitioning and phenyl-Sepharose binding. Our results support a model in which conversion of mutant PrPs to a PrPSc-like state proceeds in a stepwise fashion via a series of identifiable biochemical intermediates, with the earliest step occurring during or very soon after synthesis of the polypeptide in the endoplasmic reticulum.     

Absence of protease-resistant prion protein in dementia characterized by neuronal loss and status spongiosus

Dementia characterized by neuronal loss and status spongiosus (DNLS) is a non-Alzheimer degenerative process which is characterized by Pick-like lobar atrophy with neuronal depletion and gliosis of the cerebral cortex, corpus striatum, medial thalamus, and substantia nigra and the absence of neuronal inclusions. To further investigate the cause and pathogenesis of DNLS, we probed cerebral homogenates from three cases of DNLS for protease-resistant prion protein to determine if DNLS could be a variant of a human prion disease. Limited proteolysis of prion proteins and guanidine thiocyanate treatment of cortical homogenates was used to enrich potential abnormal prion protein immunoreactivity. Although protease-resistant prion protein was detected in a case of sporadic Creutzfeldt-Jakob disease no abnormal prion protein was found in the cases of DNLS. We conclude that DNLS is not a human prion disease and remains an important dementia of uncertain etiology.     

A protease-resistant form of insulin-like growth factor (IGF) binding protein 4 inhibits IGF-1 actions

Smooth muscle cells (SMC) secrete a serine protease that cleaves insulin-like growth factor (IGF) binding protein (IGFBP)-4 into fragments that have low affinity for IGF-1. When IGFBP-4 is added to monolayer cultures of cell types that do not secrete this protease, IGF-1 stimulation of DNA synthesis is significantly inhibited. In contrast, if cell types that secrete this protease are used, IGFBP-4 is a much less potent inhibitor. These studies were conducted to determine whether proteolysis of IGFBP-4 accounted for its reduced capacity to inhibit IGF-1-stimulated DNA synthesis. The cleavage site in IGFBP-4 that the SMC protease uses was determined to be lysine120, histidine121. A protease-resistant mutant form of IGFBP-4 was prepared, expressed, purified, and tested for biologic activity using porcine SMC cultures. Addition of the protease-resistant mutant resulted in inhibition of DNA and cell migration responses to IGF-1. The inhibition was concentration dependent and was maximal when 500 ng/ml (20 nM) of the mutant was added with 20 ng/ml (2.8 nM) of IGF-1. When the mutant was added in the absence of IGF-1, it had no activity. The results show that cleavage of IGFBP-4 at lysine120, histidine121 results in inactivation of the ability of IGFBP-4 to bind to IGF-1. Creation of a mutant form of IGFBP-4 that was not cleaved by the protease resulted in inhibition of IGF-1-stimulated actions. The results suggest that IGFBP-4 can act as a potent inhibitor of the anabolic effects of IGF-1 and that the variables that regulate protease activity may indirectly regulate IGF-1 actions.     

Expression of pro form of prostate-specific antigen by mammalian cells and its conversion to mature, active form by human kallikrein 2

To study the expression, biosynthesis, and processing of prostate-specific antigen (PSA) in mammalian cells, recombinant PSA was expressed in Syrian hamster tumor cell line AV12-664 (AV12-PSA). Expression of PSA was monitored by the Tandem-MP PSA assay. PSA was secreted into the medium during the logarithmic phase of cell growth at >9 microg/ml and was stable. The PSA purified from spent medium of AV12-PSA cells did not exhibit any enzymatic activity and did not complex with the protease inhibitor, alpha-1-antichymotrypsin. These findings indicated that an inactive form of PSA was expressed by AV12-PSA cells. NH2-terminal sequencing confirmed the identity of the PSA purified from the spent medium of AV12-PSA cells to be pro-PSA. This demonstrates that PSA is expressed as pro-PSA by mammalian cells and suggests that pro-PSA may be present in biological fluids. Human kallikrein 2 (hK2), another member of the hK family, is also expressed predominantly in prostate epithelium. Although hK2 has been shown to exhibit trypsin-like activity, little is known about its natural substrates. Using purified proteins, we show that hK2 can convert pro-PSA to mature, enzymatically active PSA, thus establishing a physiological connection between hK2 and PSA. These findings imply that hK2 may be regulating PSA activity in vivo.     

Propagation of prion strains through specific conformers of the prion protein

Two prion strains with identical incubation periods in mice exhibited distinct incubation periods and different neuropathological profiles upon serial transmission to transgenic mice expressing chimeric Syrian hamster/mouse (MH2M) prion protein (PrP) genes [Tg(MH2M) mice] and subsequent transmission to Syrian hamsters. After transmission to Syrian hamsters, the Me7 strain was indistinguishable from the previously established Syrian hamster strain Sc237, despite having been derived from an independent ancestral source. This apparent convergence suggests that prion diversity may be limited. The Me7 mouse strain could also be transmitted directly to Syrian hamsters, but when derived in this way, its properties were distinct from those of Me7 passaged through Tg(MH2M) mice. The Me7 strain did not appear permanently altered in either case, since the original incubation period could be restored by effectively reversing the series of passages. Prion diversity enciphered in the conformation of the scrapie isoform of PrP (PrP(Sc)) (G. C. Telling et al., Science 274:2079-2082, 1996) seems to be limited by the sequence of the PrP substrates serially converted into PrP(Sc), while prions are propagated through interactions between the cellular and scrapie isoforms of PrP.     

A trend of molecular genetics on prion diseases and prion protein

Infectious amyloid filaments designated as prion rods or scrapie associated fibrils (SAF) present in brain tissues affected by transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease (CJD), Gerstmann-Str?ussler-Scheinker disease (GSS) and kuru of humans, and scrapie of sheep. A hydrophobic glycoprotein, PrPSc is a major component of SAF, and is known to be associated with the infectivity of these diseases. Both PrPSc and the normal isoform of this glycoprotein, PrPC are encoded by a single host gene, PrP gene, and the conversion of PrPC to PrPSc is a posttranslational event. Several mutations on the PrP gene are associated with variations of the phenotype and the occurrence in familial CJD and GSS.     

Circumventing tolerance to generate autologous monoclonal antibodies to the prion protein

Prion diseases are disorders of protein conformation and do not provoke an immune response. Raising antibodies to the prion protein (PrP) has been difficult due to conservation of the PrP sequence and to inhibitory activity of alpha-PrP antibodies toward lymphocytes. To circumvent these problems, we immunized mice in which the PrP gene was ablated (Prnp 0/0) and retrieved specific monoclonal antibodies (mAbs) through phage display libraries. This approach yielded alpha-PrP mAbs that recognize mouse PrP. Studies with these mAbs suggest that cellular PrP adopts an unusually open structure consistent with the conformational plasticity of this protein.     

Doxycycline control of prion protein transgene expression modulates prion disease in mice

Conversion of the cellular prion protein (PrPC) into the pathogenic isoform (PrPSc) is the fundamental event underlying transmission and pathogenesis of prion diseases. To control the expression of PrPC in transgenic (Tg) mice, we used a tetracycline controlled transactivator (tTA) driven by the PrP gene control elements and a tTA-responsive promoter linked to a PrP gene [Gossen, M. and Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89, 5547-5551]. Adult Tg mice showed no deleterious effects upon repression of PrPC expression (>90%) by oral doxycycline, but the mice developed progressive ataxia at approximately 50 days after inoculation with prions unless maintained on doxycycline. Although Tg mice on doxycycline accumulated low levels of PrPSc, they showed no neurologic dysfunction, indicating that low levels of PrPSc can be tolerated. Use of the tTA system to control PrP expression allowed production of Tg mice with high levels of PrP that otherwise cause many embryonic and neonatal deaths. Measurement of PrPSc clearance in Tg mice should be possible, facilitating the development of pharmacotherapeutics.     

Proof of the primacy of prion protein

This paper presents a case of a 5-year old girl with a congenital absence of the nose. Congenital arrhinia is a very rare malformation of the midfacial bones. The difficulties of treating a child with this abnormality are discussed.     

Effect of ultraviolet irradiation of bacteriophage f1 DNA on its conversion to replicative form by extracts of Escherichia coli

When DNA of phage chiX174 or phage f1 is used as a template after exposure to ultraviolet radiation, the conversion of single-stranded DNA to replicative form by cell-free extracts of Escherichia coli is inhibited. The extend of synthesis is proportional to the distance of a pyrimidine dimer from a specific origin of replication as calculated from the random location of dimers at various UV doses. The results therefore indicate that the initiation of DNA synthesis on these phage DNAs occurs normally at a specific site, and that chain elongation is blocked when replication reaches a photo product in the template. Reinitiation of DNA synthesis distal to the lesion does not occur.     

Astrocytic glutamate uptake and prion protein expression

Factors influencing glutamate uptake by astrocytes may indirectly influence neuronal survival. Elevated extracellular glutamate may be excitotoxic or may exacerbate neurodegeneration in various neurological diseases. By using a cell culture model, we have investigated the influence of astrocytic prion protein (PrPc) expression on glutamate uptake. Type 1 astrocytes expressing PrPc have a higher rate of Na+-dependent glutamate uptake than PrPc-deficient type 1 astrocytes. This difference is exacerbated when serum free media is used to culture the astrocytes. Further analysis suggested that a decrease in substrate affinity is responsible for the sensitivity of PrP-deficient astrocytic glutamate uptake to culture conditions. PrPc has been shown to bind copper. Greater sensitivity of cells to copper concentrations may be responsible for the decreased substrate affinity observed. PrPc-deficient cerebellar cells are more sensitive to glutamate toxicity in the presence of copper. These results show that glutamate uptake from astrocytes is dependent on PrPc expression which in turn may be related to copper metabolism.     

Mapping the prion protein using recombinant antibodies

The fundamental event in prion disease is thought to be the posttranslational conversion of the cellular prion protein (PrPC) into a pathogenic isoform (PrPSc). The occurrence of PrPC on the cell surface and PrPSc in amyloid plaques in situ or in aggregates following purification complicates the study of the molecular events that underlie the disease process. Monoclonal antibodies are highly sensitive probes of protein conformation which can be used under these conditions. Here, we report the rescue of a diverse panel of 19 PrP-specific recombinant monoclonal antibodies from phage display libraries prepared from PrP deficient (Prnp0/0) mice immunized with infectious prions either in the form of rods or PrP 27-30 dispersed into liposomes. The antibodies recognize a number of distinct linear and discontinuous epitopes that are presented to a varying degree on different PrP preparations. The epitope reactivity of the recombinant PrP(90-231) molecule was almost indistinguishable from that of PrPC on the cell surface, validating the importance of detailed structural studies on the recombinant molecule. Only one epitope region at the C terminus of PrP was well presented on both PrPC and PrPSc, while epitopes associated with most of the antibodies in the panel were present on PrPC but absent from PrPSc.     

Impaired motor coordination in mice lacking prion protein

1. Prion protein (PrPC) is a host-encoded glycoprotein constitutively expressed on the neuronal cell surface. Accumulation of its protease-resistant isoform is closely related to pathologic changes and prion propagation in the brain tissue of a series of prion diseases. However, the physiological role of PrPC remains to be elucidated. 2. After long-term observation, we noted impaired motor coordination and loss of cerebellar Purkinje cells in the aged mice homozygous for a disrupted PrP gene, a finding which strongly suggests that PrPC plays a role in the long-term survival of Purkinje cells. 3. We also describe the resistance of the PrP null mice to the prion, indicating the requirement of PrPC for both the development of prion diseases and the prion propagation.     

A prion protein fragment primes type 1 astrocytes to proliferation signals from microglia

Giliosis is a hallmark of prion disease. A neurotoxic prion peptide (PrP106-126) induces astrocyte proliferation in the presence of microglia. This peptide also directly enhances microglial proliferation in culture. We have investigated this further to understand the method by which factors released by microglia and PrP106-126 work together to enhance astrocyte proliferation. PrP106-126 in the presence of microglia specifically enhanced type 1 astrocyte proliferation but not Type 2. Astrocytes that do not express the prion protein were more sensitive to oxidative stress and the toxicity of cytosine arabinoside. In the presence of cytosine arabinoside, PrP106-126 was toxic to pure astrocyte cultures. Using conditioned medium from microglia we have shown that PrPc-expressing astrocytes proliferate in response to factors released by microglia stimulated by granulocyte/macrophage colony-stimulating factor. This response is enhanced in the presence of PrP106-126. PrPc-deficient astrocytes do not show this response. These results suggest that astrocytes are primed by PrP106-126 to respond more to factors released by proliferating microglia. Astrocytes may proliferate in this system to escape entering the cell suicide pathway.     

Strain-dependent differences in beta-sheet conformations of abnormal prion protein

Strain diversity in the transmissible spongiform encephalopathies (TSEs) has been proposed to be determined by variations in the conformation of the abnormal, protease-resistant form of prion protein (PrP-res). We have investigated whether infection of hamsters with three TSE strains resulted in the formation of PrP-res with different conformations using limited proteinase K (PK) digestion and infrared spectroscopy. PrP-res isolated from the brains of hamsters infected with the hyper (HY), drowsy (DY), and 263K TSE strains yielded similar SDS-polyacrylamide gel electrophoresis profiles prior to PK treatment. However, after limited digestion with PK, the PrP-res from the DY strain exhibited a fragmentation pattern that was distinct from that of the other two strains. Infrared spectra of HY and 263K PrP-res each had major absorption bands in the amide I region at 1626 and 1636 cm-1 both prior to and after digestion with PK. These bands were not evident in the DY PrP-res spectra, which had a unique band at 1629-1630 cm-1 and stronger band intensity at both 1616 and 1694-1695 cm-1. Because absorbances from 1616 to 1636 cm-1 of protein infrared spectra are attributed primarily to beta-sheet structures, these findings indicate that the conformations of HY and 263K PrP-res differ from DY PrP-res at least in structural regions with beta-sheet secondary structure. These results support the hypothesis that strain-specific PrP-res conformers can self-propagate by converting the normal prion protein to the abnormal conformers that induce phenotypically distinct TSE diseases.     

A prion disease with a novel 96-base pair insertional mutation in the prion protein gene

There are coding mutations in the prion protein gene in familial Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker disease, and other phenotypes that make up the inherited prion diseases. Insertional mutations consisting of two, five, six, seven, eight, and nine additional octapeptide repeat elements are seen in the inherited prion diseases and usually present as atypical dementias with considerable intrafamilial phenotypic variability. A four-octarepeat insertion was reported previously in an individual without neurodegenerative disease who died of hepatic cirrhosis. Here we report a novel four-octarepeat insertional mutation in a case with classical clinical, electroencephalographic and histopathologic features of CJD with the unusual finding of pronounced prion protein immunoreactivity of the molecular layer of the cerebellum.     

Chicken monoclonal antibodies against synthetic bovine prion protein peptide

A night course taken almost 25 years ago sparked an interest in Leonardo da Vinci that has become a passion for a London, Ont., neurosurgeon. Dr. Rolando Del Maestro now boasts one of the largest collections of da Vinci artifacts in North America.