RESUMO
Purification of prions from scrapie-infected hamster brain yielded a protein that was not found in a similar fraction from uninfected brain. The protein migrated with an apparent molecular size of 27,000 to 30,000 daltons in sodium dodecyl sulfate polyacrylamide gels. The resistance of this protein to digestion by proteinase K distinguished it from proteins of similar molecular weight found in normal hamster brain. Initial results suggest that the amount of this protein correlates with the titer of the agent.
Assuntos
Encéfalo/patologia , Proteínas do Tecido Nervoso/isolamento & purificação , Scrapie/patologia , Animais , Química Encefálica , Centrifugação com Gradiente de Concentração , Cricetinae , Eletroforese em Gel de Poliacrilamida , Endopeptidase K , Endopeptidases/metabolismo , Peso Molecular , Príons/crescimento & desenvolvimento , Ovinos , Ativação ViralRESUMO
The "modified host protein" model of scrapie proposes that the transmissible agent is composed of the degradation-resistant protein, Sp33-37, and that clinical and pathologic signs result from neurotoxic accumulations of this protein. Sp33-37 is an abnormal, amyloidogenic isoform of the normally occurring cellular protein Cp33-37. This study investigated the tissue distribution of Cp33-37 in hamster. In brain, Cp33-37 was most concentrated in the hippocampal formation. Immunohistochemical studies localized Cp33-37 to neurons and surrounding neuropil in hippocampus; septal, caudate, and thalamic nuclei; dorsal root ganglia cells; and large-diameter dorsal root axons. In non-neuronal hamster tissues, Cp33-37 was detected in circulating leukocytes, heart, skeletal muscle, lung, intestinal tract, spleen, testis, ovary, and some other organs. The presence of Cp33-37 in extracerebral tissues indicates that its function is not unique to brain. These results indicate that the molecular substrate for the production of Sp33-37, the scrapie agent, and scrapie amyloid is present in a variety of cerebral and extracerebral sites.
Assuntos
Príons/metabolismo , Animais , Encéfalo/metabolismo , Cricetinae , Mucosa Gástrica/metabolismo , Imuno-Histoquímica , Pulmão/citologia , Pulmão/metabolismo , Pulmão/ultraestrutura , Microscopia Imunoeletrônica , Proteínas PrPSc , Medula Espinal/citologia , Medula Espinal/metabolismo , Estômago/citologia , Distribuição TecidualRESUMO
Prion protein (PrP) is the only known constituent of the agents (called prions) that cause fatal neurodegenerative diseases in animals and humans. PrP derives from a host protein encoded by a single copy gene having three known exons in mice, cattle and sheep but only two exons in hamsters and humans. We have identified and sequenced the missing exon from the hamster PrP gene. The new hamster PrP exon is 83% identical to mouse exon 2 and 76% identical to exon 2 from cattle and sheep. PrP mRNAs containing the new exon 2 (mRNA[1+2+3]) were expressed in the colliculi, frontal cortex and hippocampus of normal hamsters at approximately 30% to approximately 50% of the levels of the mRNA without exon 2 (mRNA[1+3]). Expression of PrP mRNA[1+2+3] was increased in the colliculi beginning 49 days after inoculation with scrapie prions and reached a level 2.5 times normal by day 77. Increased expression of PrP mRNA[1+2+3] in the colliculi correlated with expression of glial fibrillary acidic protein (GFAP) mRNA. Expression of GFAP and PrP mRNAs was not significantly increased in the hippocampus or the frontal cortex during the disease. Our study shows that exon 2 plays a role in regulating the cellular expression of hamster PrP and suggests that mRNA[1+2+3] may be preferentially expressed in hamster astrocytes.
Assuntos
Cricetinae/genética , Éxons , Expressão Gênica , Príons/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Scrapie/genética , Animais , Sequência de Bases , Encéfalo/metabolismo , Bovinos , Feminino , Camundongos , Dados de Sequência Molecular , Scrapie/metabolismo , Ovinos , Fatores de Tempo , Distribuição TecidualRESUMO
Antibodies specific for GABA, glutamate and taurine were used to study the distribution of these amino acid neurotransmitters during the progression of scrapie in hamsters. Immunohistochemical distribution of glutamate and taurine were unaffected in scrapie hamsters compared with controls, but the distribution of GABA was altered by 21 days after inoculation. We found both a greater number of neurons showing GABA-like immunoreactivity and more intense staining in those neurons in scrapie-inoculated hamster brains, particularly in the hippocampus, inferior colliculus, frontal cortex and cerebellum. The overall concentrations of aspartate, GABA, glutamate and taurine, measured in seven different brain regions by PITC-amino acid analysis, were not significantly different between normal and scrapie-affected hamsters. The subtle alteration in GABA metabolism detected in this scrapie model suggests that PrPSc interacts directly with a component of the GABA system.
Assuntos
Encéfalo/metabolismo , Príons , Scrapie/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Encéfalo/patologia , Cricetinae , Feminino , Imuno-Histoquímica , Neurotransmissores/metabolismo , Scrapie/patologia , Fatores de TempoRESUMO
Infectious bovine rhinotracheitis (IBR) virus was purified by rate zonal and isopycnic centrifugation in potassium tartrate gradients. Viral nucleocapsids were isolated from purified virions by treatment with the nonionic detergent Triton X-100 followed by high speed centrifugation. This treatment was shown to produce a suspension of 74% completely de-enveloped nucleocapsids, 24% incompletely de-enveloped nucleocapsids, and 2% whole virions. The viral nucleocapsids contained DNA and banded at a density of 1.25 g/cm3. Analysis of the viral polypeptides by gradient SDS-polyacrylamide gel electrophoresis revealed that 33 virion proteins, ranging in molecular weight from 13,000 to 275,000 dalton, were present in the complete virus particle. Detergent treatment of the virus quantitatively removed two of the major proteins (vp8, 90,000 dalton, and vp13, 73,000 dalton) and partially removed eleven other proteins. Fifteen viral polypeptides appeared to remain firmly associated with the viral nucleocapsids.
Assuntos
Herpesvirus Bovino 1/análise , Proteínas Virais/análise , Animais , Capsídeo/análise , Bovinos , Células Cultivadas , Centrifugação , Eletroforese em Gel de Poliacrilamida/métodos , Herpesvirus Bovino 1/ultraestrutura , Microscopia Eletrônica , Timidina/metabolismoRESUMO
Critical parameters affecting sensitivity and specificity of an enzyme-linked immunosorbent assay (ELISA) for avian infectious bronchitis virus (IBV) were evaluated and optimized. The use of purified IBV as antigen at 50 ng protein/well and high-ionic-strength serum dilution buffer has resulted in a test with minimal nonspecific binding of chicken immunoglobulins and very high sensitivity. Optimum conditions for serum dilution, conjugate dilution, and substrate incubation were determined for minimizing background and nonspecific reactions. The use of this test in a controlled challenge study with chickens vaccinated with live IBV demonstrated its effectiveness in monitoring circulating antibody levels to infectious bronchitis. The IBV ELISA, which is rapid, inexpensive, highly sensitive, and capable of handling very large numbers of samples, should provide the poultry industry with a reliable means for IBV flock monitoring.
Assuntos
Anticorpos Antivirais/análise , Galinhas/imunologia , Coronaviridae/imunologia , Ensaio de Imunoadsorção Enzimática , Técnicas Imunoenzimáticas , Vírus da Bronquite Infecciosa/imunologia , Animais , Anticorpos Antivirais/biossíntese , Antígenos Virais/normas , Vacinação/veterináriaAssuntos
Príons/isolamento & purificação , Scrapie/microbiologia , Animais , Bioensaio , Síndrome de Creutzfeldt-Jakob/microbiologia , Cricetinae , Humanos , Camundongos , Modelos Biológicos , Peso Molecular , Proteínas do Tecido Nervoso/isolamento & purificação , Proteína PrP 27-30 , Proteínas PrPSc , Príons/química , Príons/genética , Príons/patogenicidade , Ovinos/microbiologiaRESUMO
Prion titres were measured in the lungs and brains of Syrian hamsters after intraperitoneal inoculation with sucrose gradient-purified 263K prions (approximately 10(8) LD50). Prions were detected in the lung of one hamster on day 7, but were not detected in the lungs of any other hamster until day 71. Prions were detected in the lungs of all hamsters sampled thereafter but titres remained low through day 127. Prions were first detected in the brain on day 35 and brain titres increased exponentially until day 127 with a doubling time of about 4.5 days. On day 133, titres averaged 10(8.0) LD50/g in brain and 10(5.0) LD50/g in lung. Two out of the five remaining hamsters were clinically normal but prion titres were not significantly different from those in the clinically affected hamsters. Thus, significant prion titres may be found outside the CNS in clinically normal hamsters.
Assuntos
Encéfalo/metabolismo , Pulmão/metabolismo , Príons/farmacocinética , Scrapie/metabolismo , Animais , Cricetinae , Feminino , MesocricetusRESUMO
The scrapie agent is still not completely characterized biochemically and ultrastructurally, but its requirement for a functional protein has been established. Purification of the scrapie agent by methods using digestion with proteinase K yields a glycoprotein with an apparent mass of 27-30 kDa (PrP 27-30). In contrast, a 33-37 kDa glycoprotein, called Sp33-37, is the major protein component isolated from scrapie-affected brain when protease digestion is not used. Sp33-37 is the product of a normal host gene and is a larger form of PrP 27-30. We propose a model in which Sp33-37, a modified host protein, is the critical component of the scrapie agent; a non-host nucleic acid is not part of the agent. We postulate that Sp33-37, perhaps in concert with other unidentified host components, is capable of inducing the disease and directing the production of more of itself by acting on the normal protein directly or by affecting one of the steps in protein processing. Agent replication requires that: 1) a constant supply of the substrate protein Cp33-37 is available, 2) aggregates of Sp33-37 are resistant to degradation and accumulate in cells or cell membranes, and 3) membrane damage and cell death facilitate spread to adjacent cells. The model predicts that disease can be transmitted by the scrapie agent or initiated by a spontaneous metabolic error resulting in accumulation of the abnormal protein.
Assuntos
Encéfalo/metabolismo , Glicoproteínas/metabolismo , Modelos Biológicos , Scrapie/etiologia , Animais , Fenômenos Químicos , Química , Genótipo , Humanos , Peso Molecular , Príons/classificação , Príons/genética , Príons/metabolismo , Proteínas Virais/metabolismo , Replicação ViralRESUMO
Scrapie is the best understood of the transmissible spongiform encephalopathies. These neurologic disorders include the human diseases kuru and Creutzfeldt-Jakob disease and are caused by pathogens with unique biological and molecular properties. One major protein, protease-resistant protein (PrP)-27-30, is present in fractions isolated from scrapie-infected hamster brain that contain highly purified scrapie agent. PrP-27-30 appears to be the major protein component of the hamster scrapie agent. An antiserum generated to electrophoretically purified hamster scrapie PrP-27-30 identified higher molecular weight proteins in immunoblots of homogenates of uninfected hamster and mouse brains. Antibodies to hamster and mouse scrapie agent proteins were obtained by immunoaffinity purification of this antiserum. These antibodies to hamster and mouse PrPs recognized a 54-kDa protein present in uninfected brain homogenates. Antibodies immunoaffinity purified from this antiserum using whole immunoblots of normal brain antigens also identified the 54-kDa protein and PrPs. Our findings demonstrate that scrapie agent proteins share epitopes with normal proteins and suggest that the 54-kDa protein is the normal protein precursor of the scrapie agent PrPs.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Príons , Precursores de Proteínas/metabolismo , Proteínas Virais/metabolismo , Animais , Encéfalo/imunologia , Encéfalo/microbiologia , Cricetinae , Reações Cruzadas , Epitopos , Camundongos , Peso Molecular , Proteínas do Tecido Nervoso/imunologia , Proteína PrP 27-30 , Príons/imunologia , Precursores de Proteínas/imunologia , Proteínas Virais/imunologiaRESUMO
The major scrapie prion protein, designated PrP 27-30, exhibited both charge and size heterogeneity after purification from infected hamster brains. Eight or more discrete charge isomers of PrP 27-30 with isoelectric points ranging from approximately pH 4.6 to 7.9 were found by using non-equilibrium pH gradient electrophoresis in the first dimension followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. The charge isomers were detected by silver staining as well as by radioiodination. The procedures used to disaggregate PrP 27-30 before electrophoresis in the first dimension do not appear to be responsible for the charge heterogeneity. However, heating PrP 27-30 to 100 degrees C for 15 min in 0.1 N NaOH or 0.1 N HCl resulted in modification of the protein and alteration of its electrophoretic pattern. A PrP 27-30 fragment (molecular weight, 17,100 to 21,900) obtained by cyanogen bromide cleavage also exhibited charge and size heterogeneity. Periodic acid-Schiff staining of PrP 27-30 electrophoresed into sodium dodecyl sulfate-polyacrylamide gels demonstrated that carbohydrate residues are attached to the protein. Digestion of PrP 27-30 with neuraminidase and endo-beta-N-acetylglucosaminidase H resulted in significant changes in the isoelectric pH of PrP 27-30 isomers, whereas digestion with alkaline phosphatase had no effect. Our results demonstrate that PrP 27-30 is a sialoglycoprotein; this is consistent with several properties of this protein and of the scrapie prion.
Assuntos
Príons/análise , Sialoglicoproteínas/análise , Proteínas Virais/análise , Fosfatase Alcalina , Centrifugação com Gradiente de Concentração , Fenômenos Químicos , Química , Brometo de Cianogênio , Eletroforese em Gel de Poliacrilamida , Glicosídeo Hidrolases , Concentração de Íons de Hidrogênio , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase , Peso Molecular , Neuraminidase , Reação do Ácido Periódico de Schiff , Sialoglicoproteínas/isolamento & purificação , Proteínas Virais/isolamento & purificaçãoRESUMO
Fractions purified from scrapie-infected hamster brain contain a unique protein, designated PrP. It was labeled with N-succinimidyl 3-(4-hydroxy-5-[125I]-iodophenyl) propionate, which did not alter the titer of the scrapie prion. The concentration of PrP was found to be directly proportional to the titer of the infectious prion. Both PrP and prion infectivity were resistant for 2 hr at 37 degrees C to hydrolysis by proteinase K under nondenaturing conditions. Prolonging the digestion resulted in a concomitant decrease in both PrP and the scrapie prion. When the amino-acid-specific proteases trypsin or SV-8 protease were used instead of proteinase K, no change in either PrP or the prion was detected. The parallel changes between PrP and the prion provide evidence that PrP is a structural component of the infectious prion. Our findings also suggest that the prion contains only one major protein, namely PrP.
Assuntos
Príons/análise , Proteínas Virais/análise , Endopeptidase K , Endopeptidases/metabolismo , Peptídeo Hidrolases/metabolismo , Tripsina/metabolismo , Proteínas Virais/metabolismo , Proteínas Estruturais ViraisRESUMO
A major protein was identified that purifies with the scrapie agent extracted from infected hamster brains. The protein, designated PrP 27-30, was differentiated from other proteins in purified fractions containing the scrapie agent by its microheterogeneity (Mr 27000-30000) and its unusual resistance to protease digestion. PrP 27-30 was found in all fractions enriched for scrapie prions by discontinuous sucrose gradient sedimentation or sodium dodecyl sarcosinate-agarose gel electrophoresis. It is unlikely that PrP 27-30 is a pathologic product because it was found in fractions isolated from the brains of hamsters sacrificed prior to the appearance of histopathology. If PrP 27-30 is present in normal brain, its concentration must be 100-fold lower than that found in equivalent fractions from scrapie-infected hamsters. Three protease-resistant proteins similar to PrP 27-30 were found in fractions obtained by discontinuous sucrose gradient sedimentation of scrapie-infected mouse brain. These proteins were not evident in corresponding fractions prepared from normal mouse brain. One-dimensional peptide maps comparing PrP 27-30 and normal hamster brain proteins of similar molecular weight demonstrated that PrP 27-30 has a primary structure which is distinct from these normal proteins. Heating substantially purified scrapie fractions to 100 degrees C in sodium dodecyl sulfate inactivated the prion and rendered PrP 27-30 susceptible to protease digestion. Though the scrapie agent appears to be hydrophobic, PrP 27-30 remained in the aqueous phase after extraction with organic solvents, indicating that it is probably not a proteolipid. PrP 27-30 is the first structural component of the scrapie prion to be identified.
Assuntos
Química Encefálica , Proteínas do Tecido Nervoso/análise , Scrapie/metabolismo , Serina Endopeptidases , Proteínas Virais/análise , Animais , Centrifugação com Gradiente de Concentração , Cricetinae , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , Endopeptidases , Temperatura Alta , Camundongos , Peso Molecular , Fragmentos de Peptídeos , Peptídeo Hidrolases/farmacologia , Príons , Desnaturação Proteica , Ovinos , Fatores de TempoRESUMO
The scrapie agent protein (Sp33-37) is a degradation-resistant protein that aggregates into fibrils and amyloid plaques. This protein is derived from a normal cellular protein (Cp33-37). Understanding the mechanism responsible for the conversion of Cp33-37 to Sp33-37 may explain scrapie agent replication. Cp33-37 was extracted from normal hamster brain and purified 2700-fold by an immunoaffinity method. Both Cp33-37 purified from normal hamster brain and Sp33-37 purified from scrapie-affected hamster brain had apparent masses of 33-37 kilodaltons and displayed microheterogeneity characteristic of glycoproteins. Cp33-37 was completely digested by proteinase K under conditions that resulted in conversion of Sp33-37 to the protease-resistant fragment PrP27-30. Cp33-37 did not cause scrapie when inoculated intracerebrally into hamsters. Fractions containing purified Sp33-37 had average titers of greater than 10(11) LD50 of the scrapie agent/mg of protein; these titers were not diminished by proteinase K. These results indicate that altered sensitivity to proteolysis in vitro reflects an intrinsic difference between Sp33-37 and Cp33-37.
Assuntos
Química Encefálica , Proteínas do Tecido Nervoso/isolamento & purificação , Precursores de Proteínas/isolamento & purificação , Scrapie/etiologia , Animais , Fracionamento Celular , Centrifugação com Gradiente de Concentração , Cromatografia de Afinidade , Cricetinae , Feminino , Proteínas do Tecido Nervoso/análise , Proteína PrP 27-30 , Precursores de Proteínas/análiseRESUMO
The grey tremor mouse is an autosomal recessive mutant characterized by a phenotype of unusual pigmentation, neurological abnormalities and early death. These mice have a spongiform encephalopathy similar to scrapie and Creutzfeldt-Jakob disease. Although the disease is clearly heritable, the grey tremor mouse spongiform pathology has also been transmitted by inoculation of genetically normal mice with diseased brain homogenates. The possibility that a scrapie-like agent is involved has been proposed. We examined brain homogenates from grey tremor mice, scrapie-affected mice and normal mice for the presence of the mouse scrapie agent protein (MoSp33-37) and its normal cellular homologue. All untreated homogenates contained one or both isoforms of this protein as detected on immunoblots. Grey tremor mouse brain homogenates, when protease-treated, showed no evidence of MoSp33-37. A purification method for MoSp33-37 concentrated it in samples from scrapie-affected mice, but this protein was not detected in grey tremor or normal mice. These results suggest that it is unlikely that the scrapie agent is involved in grey tremor disease.
Assuntos
Encefalopatias/microbiologia , Camundongos Mutantes Neurológicos/microbiologia , Príons/análise , Proteínas Virais/análise , Animais , Encefalopatias/genética , Camundongos , Camundongos Endogâmicos C57BL/microbiologia , Príons/patogenicidade , Scrapie/microbiologiaRESUMO
Purification of the scrapie agent by methods using digestion with proteinase K yields a protein product, PrP-27-30, with an apparent mass of 27-30 kDa (D. C. Bolton et al. (1982) Science 218, 1309-1311; S. B. Prusiner et al. (1982) Biochemistry 21, 6942-6950). In contrast, a 33-37 kDa glycoprotein, HaSp33-37, was the major protein component isolated from scrapie-affected hamster brain by a procedure that did not use protease digestion. The purified fractions containing HaSp33-37 had greater than 10(11) LD50 units of the scrapie agent per milligram of protein. Proteinase K digestion of HaSp33-37 gave a product indistinguishable from PrP-27-30 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The amino acid sequence of the first 22 residues of HaSp33-37 was determined. The sequence coincided with that predicted for the N-terminus of the precursor to PrP-27-30 (K. Basler et al. (1986) Cell 46, 417-428; N. K. Robakis et al. (1986) Proc. Natl. Acad. Sci. USA 83, 6377-6381) after processing by signal protease. HaSp33-37 was digested with N alpha-tosyl-L-phenylalanine chloromethyl ketone-trypsin to produce a 29-32 kDa protein fragment; following digestion this fraction retained complete biological activity. The amino terminal sequence of the 29-32 kDa protein corresponded to a position intermediate between the amino termini of HaSp33-37 and PrP-27-30. We conclude that HaSp33-37 is the intact form of the scrapie agent protein and that PrP-27-30 is produced by proteinase K degradation when this enzyme is introduced during isolation of the scrapie agent.
Assuntos
Encéfalo/microbiologia , Príons/patogenicidade , Proteínas Virais/isolamento & purificação , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Cricetinae , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Peso Molecular , Proteínas PrPScRESUMO
Studies were conducted to determine whether accumulation of the scrapie agent protein Sp33-37 in brain correlated with the appearance of the scrapie agent or with pathology. The concentrations of the scrapie agent and Sp33-37 were measured in purified fraction P5 isolated from hamster brains at weekly intervals after inoculation. The scrapie agent concentration in fraction P5 was approximately 10(-1) LD50/g brain 1 day post-inoculation and increased to 10(9.4) LD50/g at day 77. Sp33-37 was first detected in P5 at day 21, when the agent titre was 10(3.9) LD50/g. Sp33-37 concentration increased in concert with the scrapie agent concentration, although the apparent rate of increase was somewhat lower for the protein than for the agent. The histopathological evidence of disease, consisting of mild vacuolation and gliosis, was first seen at 35 days, but was not conspicuous until 49 to 56 days post-inoculation. Vacuolation and gliosis increased until termination of the experiment at day 77. Amyloid plaques were first detected at 56 days and were widespread at day 77. Clinical disease was first seen in these animals at day 66, with an average onset at day 71. Control animals inoculated with buffer alone showed some mild gliosis, but were otherwise normal. The fact that Sp33-37 purified with the scrapie agent isolated from brain 14 days prior to detectable (light microscopic) pathology supports the theory that Sp33-37 is the major structural component of the scrapie agent and not solely a product of the pathology.
Assuntos
Encéfalo/microbiologia , Príons/isolamento & purificação , Scrapie/patologia , Animais , Western Blotting , Encéfalo/metabolismo , Cricetinae , Eletroforese em Gel de Poliacrilamida , Feminino , Proteínas PrPSc , Scrapie/metabolismoRESUMO
The scrapie agent protein (Sp33-37 or PrPSc) is the disease-associated isoform of a normal cellular membrane protein (Cp33-37 or PrPC) of unknown function. We report that normal human lymphocytes and lymphoid cell lines, but not erythrocytes or granulocytes, express PrPC mRNA and protein. PrPC is detectable on the surface of lymphocytes; the surface immunoreactivity is sensitive to phosphatidylinositol-specific phospholipase C, indicating glycosyl-phosphatidylinositol membrane anchorage. Lymphocyte PrPC surface abundance is increased by cell activation, and polyclonal antibodies to PrPC suppress mitogen-induced activation. We conclude that PrPC is a lymphocyte surface molecule that may participate in cell activation.
Assuntos
Ativação Linfocitária , Linfócitos/imunologia , Príons/genética , Proteínas Virais/genética , Animais , Anticorpos Monoclonais , Linhagem Celular , Eritrócitos/microbiologia , Citometria de Fluxo , Imunofluorescência , Glicolipídeos/metabolismo , Glicosilfosfatidilinositóis , Granulócitos/microbiologia , Humanos , Soros Imunes , Cinética , Linfócitos/microbiologia , Fosfatidilinositóis/metabolismo , Proteínas PrPSc , Príons/isolamento & purificação , RNA Mensageiro/genética , Proteínas Virais/análise , Proteínas Virais/metabolismoRESUMO
Scrapie is a degenerative, neurological disorder caused by a slow infectious agent or prion. Extensively purified preparations of prions were denatured by boiling in sodium dodecyl sulfate and the major protein component (PrP 27-30) was isolated by preparative HPLC size exclusion chromatography after proteinase K digestion. The purified PrP 27-30 molecules were not infectious. Ultraviolet absorption spectra of purified PrP 27-30 demonstrated the absence of covalently linked polynucleotides. Amino acid composition studies showed that PrP 27-30 contains at least 17 naturally occurring amino acids. A single N-terminal amino acid sequence for PrP 27-30 was obtained; the sequence is N-Gly-Gln-Gly-Gly-Gly-Thr-His-Asn-Gln-Trp-Asn-Lys-Pro-Ser-Lys and it does not share homology with any known proteins. The same amino acid sequence was found when an extensively purified preparation of prions aggregated into rods and containing approximately 10(9.5) ID50 U/ml was sequenced directly. Knowledge of the amino acid sequence should permit determination of the genetic origin and replication mechanism of prions.
Assuntos
Príons/análise , Proteínas Virais/isolamento & purificação , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Encéfalo/microbiologia , Cricetinae , Eletroforese em Gel de Poliacrilamida , Peso Molecular , Espectrofotometria UltravioletaRESUMO
Scrapie is a transmissible neurodegenerative disease of sheep and goats. An abnormal host protein, Sp33-37, is the major protein component of the scrapie agent and the only known disease- or agent-specific macromolecule. Two monoclonal antibodies (MAbs), 4H8 (immunoglobulin G2b [IgG2b]) and 6B11 (IgG1), produced by immunizing mice with the intact hamster 263K scrapie agent protein, Sp33-37Ha, were found to have species specificity similar to that reported previously for MAb 3F4 (IgG2a), which was produced by using PrP-27-30 as the immunogen (R. J. Kascsak, R. Rubenstein, P. A. Merz, M. Tonna-DeMasi, R. Fersko, R. I. Carp, H. M. Wisniewski, and H. Diringer, J. Virol. 61:3688-3693, 1987). These antibodies all bound to Sp33-37 derived from hamster but not from mouse cells. Competitive binding assays demonstrated that all three MAbs bound to the same or overlapping sites on Sp33-37Ha. The molecular location of the epitope for these antibodies was determined to within 10 residues by using an antigen competition enzyme-linked immunosorbent assay in which synthetic peptides spanning Sp33-37Ha residues 79 to 93 or 84 to 93 specifically inhibited binding of these antibodies to plates coated with purified Sp33-37Ha. A synthetic peptide with the mouse-specific sequence (83 to 92) that differed from the hamster sequence by substitution at two positions (MetHa-87----LeuMo-86 and MetHa-90----ValMo-89) did not inhibit antibody binding to Sp33-37Ha. MAb 3F4 binding to hamster Sp33-37 was eliminated by chemical modification of Sp33-37Ha with diethylpyrocarbonate or succinic anhydride and by cleavage with CNBr or trypsin. The effect of diethylpyrocarbonate on MAb 3F4 binding was not reversed by hydroxylamine treatment. MAb 3F4 binding was not affected by prolonged exposure of Sp33-37Ha to 70% formic acid or by boiling in sodium dodecyl sulfate. We conclude that the epitope for these MAbs is a linear determinant that includes Met-87, Lys-88, and Met-90 and that Met-90 is probably the major species-specific determinant.