General Description of the Mammalian Prions
The mammalian prions cause scrapie and other related neurodegenerative diseases of animals and humans (Table 1). The prion diseases are also referred to as the transmissible spongiform encephalopathies (TSEs).
Prions are composed of an abnormal, pathogenic PrP isoform denoted PrPSc. The “Sc” superscript was initially derived from the term scrapie because scrapie was the prototypic prion disease. Since all of the known prion diseases (Table 1) of mammals involve aberrant metabolism of PrP similar to that observed in scrapie, we suggest using the “Sc” superscript for all abnormal, pathogenic PrP isoforms. In this context, the “Sc” superscript is used to designate the scrapie-like isoform of PrP; for those who desire a more general derivation “Sc” can equally well be derived from the term “prion sickness” (see Table 2).
A post-translational process converts the normal cellular isoform of the protein, designated PrPC, into PrPSc. Attempts to identify a chemical difference that distinguishes PrPSc from PrPC have not been successful; moreover, PrPC and PrPSc are encoded by the same single copy chromosomal gene. The conformations of the two PrP isoforms are profoundly different; PrPC has little, if any, -sheet while PrPSc has a high -sheet content.
Like viruses, prions are infectious because they stimulate a process by which more of the pathogen is produced. As prions or viruses accumulate in an infected host, they eventually cause disease. Both prions and viruses exist in different varieties or subtypes that are called strains. But many features of prion structure and replication distinguish them from viruses and all other known infectious pathogens.
Prions differ from viruses and viroids since they lack a nucleic acid genome that directs the synthesis of their progeny. Prions are composed of an abnormal isoform of a cellular protein whereas, most viral proteins are encoded by the viral genome and viroids are devoid of protein. Prions can exist in multiple molecular forms, whereas viruses exist in a single form with a distinct ultrastructural morphology. Prions are non-immunogenic, in contrast to viruses, which almost always provoke an immune response. An enlarging body of evidence argues that strains of prions are enciphered in the conformation of PrPSc; in contrast, strains of viruses and viroids have distinct nucleic acid sequences that produce pathogens with different properties.
Nomenclature of the Mammalian Prions
The terminology for prions is still evolving (Table 2). While some terms are borrowed from infectious diseases caused by viruses, others are taken from genetics and still others from the biology of protein structure as well as neuropathology. This new area of biological investigation, which has such diverse roots, creates some unique problems with terminology that are discussed below in the Nomenclature section.
Microsomal fractions from infected tissues enriched for prion infectivity contain numerous membrane vesicles (Fig.1A); detergent extraction and limited proteolysis of brain microsomes generate rod-shaped particles (Fig.1B). Most are of uniform diameter (11 nm) with mean lengths of 165 nm (range 25-550 nm). The rods are smooth, almost ribbon-like, and infrequently are twisted. The rods resemble purified amyloid, both ultrastructurally and histochemically (Fig.1B). The rods are not considered the infectious entity since large PrP 27-30 polymers are not required for infectivity (Fig.1C).
Physicochemical and Physical Properties
The Mr of PrPSc is 33-35 103. The smallest infectious prion particle is probably a dimer of PrPSc; this estimate is consistent with an ionizing radiation target size in daltons of 55 ± 9 103. Prions aggregate into particles of non-uniform size and cannot be solubilized by detergents, except under denaturing conditions where infectivity is lost. However, solubilization of PrPSc and prions can be achieved with phospholipids (Fig.1C). Prions resist inactivation by nucleases, UV-irradiation at 254 nm, treatment with psoralens, divalent cations, metal ion chelators, acid (between pH 3 and 7), hydroxylamine, formalin, boiling, and proteases. Prion infectivity is diminished by prolonged digestion with proteases, or by treatments such as urea, boiling in SDS, alkali (> pH 10), autoclaving at 132°C for more than 2 h, denaturing organic solvents (e.g., phenol), or chaotropic agents such as guanidine isocyanate.
No prion-specific nucleic acid has been detected.
PrPC is likely to be a three-helix bundle protein based on NMR structural studies of recombinant PrPs expressed in Escherichia coli. When PrPSc is formed from PrPC, the region from residue 90 to 125 seems to undergo a profound structural change where antibody epitopes once exposed become cryptic. Optical spectroscopy has shown that concurrent with the conversion of PrPC into PrPSc, there is a modest decrease in the -helical content of PrP and a large increase in -sheet. PrPSc is generally distinguished from PrPC by its different biochemical and biophysical properties. Limited proteolysis of PrPSc produces a smaller, protease-resistant molecule of about 142 amino acids, designated PrP 27-30. Under the same conditions, PrPC is completely hydrolyzed. The amino acid sequence of PrPSc that has been established by protein sequencing and mass spectrometry is identical to that deduced from the genomic DNA sequence. No proteins other than PrPSc have been consistently found in fractions enriched for prion infectivity.
PrPSc contains a glycosylinositol phospholipid (GPI) attached to amino acid residue 231 (serine) of the Syrian hamster (SHa) PrP. The lipids of the diradylglycerol moiety of the GPI anchor are not well characterized.
In addition to the GPI anchor which contains sialic acid, PrPSc has two consensus sites where it can undergo N-linked glycosylation (residues 181 and 197 of the Syrian hamster PrP). Bi-, tri- and tetra-antennary structures have been reported for the N-linked, complex type glycans of PrPSc. Some of these complex-type oligosaccharides have branched fucose residues, some have terminal sialic acid residues. Six different GPI glycans have been found, two of which are sialylated.
Genome Organization and Replication
The entire ORF of all known mammalian PrP genes is contained within a single exon. In general, PrP genes are composed of three exons, as clearly demonstrated for mouse and sheep. The PrP genes of humans and Syrian hamsters (SHa) appear to have three exons but most HuPrP and SHaPrP mRNAs are spliced from only two exons that are separated by ~10 kb. Exon-1 of the SHaPrP gene encodes a portion of the 5 untranslated leader sequence while exon-3 encodes the ORF and the 3-untranslated region. The ORF of the HuPrP gene encodes a protein of 253 amino acids while the mouse and SHa PrP genes encode proteins of 254 residues. The promoters of both the PrP genes of both animals contain 3 or 2 repeats, respectively, of G-C nonamers, but are devoid of TATA boxes. These nonamers represent a motif which may function as a canonical binding site for transcription factor Sp1.
The multiplication of prion infectivity involves the post-translational conversion of the precursor PrPC into PrPSc. Studies with mice expressing a foreign SHa, Hu, or BoPrP transgene argue that prion formation results from infecting PrPC molecules combining with homologous host-encoded PrPC molecules giving rise to new PrPSc molecules.
Additional evidence to support this proposed model for prion replication comes from studies of transgenic mice expressing chimeric or mutant PrPs where the prions produced from these transgene products have an artificial host range. In the absence of any candidate post-translational chemical modification that differentiates PrPC from PrPSc, these two isoforms remain distinguishable only by their conformations.
PrPSc is a weak antigen. The antigenicity of PrPSc is enhanced by immunization of PrP-deficient (Prnp°/°) mice which are not tolerant to PrPC. The immunoreactivity of PrPSc is significantly enhanced by denaturation. Some antibodies and recombinant Fabs bind to non-denatured PrPSc; moreover, anti-PrP antibodies have been used to neutralize prion infectivity that is dispersed into liposomes.
The prion diseases are a group of neurodegenerative disorders afflicting mammals (Table 1). The diseases are transmissible under some circumstances but, unlike other transmissible disorders, the prion diseases can also be caused by mutations in the host PrP gene. The mechanism of prion spread among sheep and goats developing natural scrapie is unknown. CWD, TME, BSE, FSE, and EUE are all thought to occur after the consumption of prion-infected materials. Similarly, kuru of the New Guinea Fore people is thought to have resulted from the consumption of brains during ritualistic cannibalism. Familial CJD, GSS, and FFI are all dominantly inherited prion diseases; five different mutations of the PrP gene have been shown to be genetically linked to development of inherited prion disease. While iotragenic CJD cases can be traced to inoculation of prions through human pituitary-derived growth hormone, cornea transplants, dura mater grafts, or cerebral electrode implants, the number of cases recorded to date is small. New variant CJD is thought to be due to the consumption of BSE tainted beef products.
Most cases of CJD are sporadic, probably the result of somatic mutation of the PrP gene or the spontaneous conversion of PrPC into PrPSc. About 10-15% of CJD cases and virtually all cases of GSS and FF1 appear to be caused by germline mutations in the PrP gene. Twenty different mutations of the PrP gene have been shown to segregate with the heritable human prion diseases (Table 3).
Strains or isolates of prions are defined by specific incubation times, distribution of vacuolar lesions, and patterns of PrPSc accumulation. Strain-specific information in prions appears to reside in the conformation of PrPSc. Prions derived from fCJD(E200K) and FF1 (Table 3) patients have PrPSc molecules with different conformations as judged by the size of the protease resistant fragments after deglycosylation. These different conformations of PrPSc were replicated in mice expressing a chimeric mouse:human PrP transgene. The origin of the inoculum determined the incubation time, the distribution of neuropathologic lesions and the pattern of PrPSc deposition in the Tg mice. Additional support for strain specific information being enciphered in the conformation of PrPSc comes from two prion strains isolated from mink dying of TME and passaged in Syrian hamsters where PrPSc from each strain exhibited different sensitivities to proteolytic digestion.
Humans carrying point mutations or inserts in their PrP genes produce mutant PrPC molecules that are believed to spontaneously convert into PrPSc. While the initial stochastic event could be inefficient, once it happens the process may become autocatalytic. The proposed mechanism is consistent with individuals harboring germline mutations who develop CNS dysfunction only in middle or old age as is seen with all of the inherited prion diseases of humans. The level of mutant PrP transgene overexpression in mice is inversely proportional to the age at which neurologic deficits appear.
The M/V polymorphism at codon 129 in HuPrP influences the incidence of sporadic and infectious CJD as well as the age of onset of inherited prion disease. Homozygosity at codon 129 is more frequent in both sporadic and infectious CJD; it also heralds an earlier age of clinical disease in people carrying pathologic mutations. Those individuals who carry the DI78N mutation develop FFI manifest by insomnia and dysautonomia if they encode M129 on the mutant allele while those who encode V129 present with fCID characterized by dementia (Table 3).
The E/K polymorphism at codon 219 in HuPrP seems to influence resistance to CJD. Individuals with wild-type (wt) PrP sequences who are heterozygous for K219 appear to be resistant to CJD. This dominant negative effect is likely to be the result of increased affinity of PrPC (K219) for protein X which is thought to function like a molecular chaperone in facilitating the conversion of PrPC into PrPSc.
The Q/R polymorphism at codon 171 in OvPrP seems to govern the resistance of sheep to natural scrapie. Sheep with wild-type (wt) PrP sequences that are heterozygous for R171 appear to be resistant to natural scrapie but scrapie can be transmitted to some of these animals by inoculation of prions. This dominant negative effect is also likely to be the result of increased affinity of PrPC (R171) for protein X. A polymorphism at codon 136 is also thought to influence the susceptibility of some breeds of sheep to scrapie.
Taxonomic Consideration of Mammalian Prions
A listing of the different animal prions is given in Table 1. Although the prions that cause TME and BSE are referred to as TME prions and BSE prions this may be unjustified, because both are thought to originate from the oral consumption of scrapie prions in sheep-derived foodstuffs and because many lines of evidence argue that the only difference among the various prions is the sequence of PrP which is dictated by the host and not the prion itself. The human prions present a similar semantic conundrum. Transmission of human prions to laboratory animals produces prions carrying PrP molecules with sequences dictated by the PrP gene of the host, not that of the inoculum.
To simplify the terminology, the generic term PrPSc is suggested in place of such terms as PrPCJD, PrPBSE, and PrPres. To distinguish PrPSc found in humans or cattle from that found in other animals, we suggest HuPrPSc or BoPrPSc instead of PrPCJD or PrPBSE, respectively (Table 1). Once human prions and thus, HuPrPSc molecules have been passaged into animals, then the prions and PrPSc are no longer of the human species unless they were formed in an animal expressing a HuPrP transgene. In the case of mutant PrPs, the mutation and any important polymorphism can be denoted in parentheses following the particular PrP isoform. For example in FFI, the pathogenic PrP isoform would be referred to as PrPSc or HuPrPSc; alternatively, if it were important to identify the mutation, then it would be written as HuPrPSc (D178N, M129) (Table 3). The term PrPres or PrP-res is derived from the protease-resistance of PrPSc but protease-resistance, insolubility, and high -sheet content should be only considered as surrogate markers of PrPSc since one or more of these may not always be present. Whether Prpres is useful in denoting PrP molecules that have been subjected to procedures that modify their resistance to proteolysis but have not been demonstrated to convey infectivity or to cause disease remains debatable.
The term PrP* has been used in two different ways. First, it has been used to identify a fraction of PrPSc molecules that are infectious. Such a designation is thought to be useful since there are ~ 105 PrPSc molecules per infectious unit. Second, PrP* has been used to designate a metastable intermediate of PrPC that is bound to protein X. It is noteworthy that neither a subset of biologically active PrPSc molecules nor a metastable intermediate of PrPC has been identified, to date.
In mice, the PrP gene denoted Prnp is now known to be identical with two genes denoted Sinc and Prn-i that were known to control the length of the incubation time in mice inoculated with prions. These findings permit a welcome simplification. A gene designated Pid-1 on mouse chromosome 17 also appears to influence experimental CJD and scrapie incubation times but information on this locus is limited.
Distinguishing among CJD, GSS, and FFI has grown increasingly difficult with the recognition that CJD, GSS, and FFI are autosomal dominant diseases caused by mutations in the PRNP gene. Initially, it was thought that a specific PrP mutation was associated with a particular clinico-neuropathologic phenotype but an increasing number of exceptions are being recognized. Multiple examples of variations in the clinico-neuropathologic phenotype within a single family where all affected members carry the same PrP mutation have been recorded. Most patients with a PrP mutation at codon 102 present with ataxia and have PrP amyloid plaques; such patients are generally given the diagnosis of GSS, but some individuals within these families present with dementia, a clinical characteristic that is usually associated with CJD. One suggestion is to label these inherited disorders as “prion disease” followed by the mutation in parenthesis while another is the use the terms FCJD and GSS followed by the mutation. In the case of FFI, describing the Dl78N mutation and M129 polymorphism seems unnecessary since this is the only known mutation-polymorphism combination that gives the FFI phenotype.
Prion: singla for proteinaceous and infectious particle.
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