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British Medical Bulletin 66:21-33 (2003)
© 2003 The British Council
Biochemistry and structure of PrPC and PrPSc
Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany
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Abstract |
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 Top Abstract IntroductionChemical properties of PrP...Chemical properties of PrPScStructure of PrPC and...Conformational transitions of...ConclusionsAcknowledgementsReferences |
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In a brief historical description, it is shown that the prion model was developed from the biochemical and biophysical properties of the scrapie infectious agent. The biochemical properties of the prion protein which is the major, if not only, component of the prion are outlined in detail. PrP is a host-encoded protein which exists as PrPC (cellular) in the non-infected host, and as PrPSc (scrapie) as the major component of the scrapie infectious agent. An overview of the purification techniques is given. Although chemically identical, the biophysical features of PrPSc are drastically different in respect to solubility, structure, and stability; furthermore, specific lipids and a polyglucose scaffold were found in prions, whereas for nucleic acids their absence could be proven. The structure of recombinant PrP in solution is known from spectroscopic studies and with high resolution from NMR analysis. Structural models of PrPSc were derived recently from electron microscopic analysis of two-dimensional crystals. Conformational transitions of PrP in vitro were studied with different techniques in order to mimic the natural PrPC to PrPSc conversion. Spontaneous transitions can be induced by solvent changes, but at present infectivity cannot be induced in vitro.
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Introduction |
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TopAbstractIntroductionChemical properties of PrP...Chemical properties of PrPScStructure of PrPC and...Conformational transitions of...ConclusionsAcknowledgementsReferences |
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The early history of the prion model is the history of the biochemical and biophysical properties of the scrapie infectious agent. In searching for a virus, no viral features were found; however, highly enigmatic properties of the infectious agent were demonstrated, such as absence of particles in the electron microscope, no immune response during the infection, and high resistance of the agent against chemical and physical treatment. As early as 1966 from inactivation studies using ionising and UV-irradiation, Alper et al1 concluded that the target size of the scrapie infectious agent (50–150 kDa) is too small for a virus but more characteristic of a protein. Many experimental results were left unexplained until Prusiner took up the inactivation studies and performed systematic analysis using not only chemical and physical, but also enzymatic procedures. He summarized the results under two groups: (i) procedures which modify or destroy nucleic acids do not inactivate scrapie infectivity; and (ii) procedures which modify or destroy proteins inactivate the infectivity. From that, he came to the conclusion that the scrapie infectious agent could not be a virus but a novel proteinaceous type of an agent, which he termed ‘prion’ (proteinaceous infectious particle)2.
To confirm a protein-like agent, the protein (one or more molecular species) had to be purified and characterized. A hydrophobic and, in mild detergents, insoluble protein of molecular weight 33–35 kDa could be highly enriched. In a collaborative research project involving the laboratories of Prusiner, Hood and Weissmann using the Syrian hamster as the experimental animal, the sequence of a peptide fragment was found; from this, the DNA-sequence was determined, first in a cDNA-library and later in a genomic library. The major component of the prion was shown to be a host-encoded protein, later called prion protein3,4. In one sense, the finding raised doubts about the prion model, because no foreign information, even a foreign protein, was found; alternatively, it supported the possibility, which indeed was mentioned earlier as a purely theoretical possibility5, that rather than a self-replicating protein, an infection-induced synthesis of a host protein might be the basis for prion amplification.
The strong, but not complete, resistance of prions to degradation by proteinase K (and other proteases) had supported the hypothesis of prions. When, however, the prion protein was identified as encoded in the host genome, the protein was found also in the non-infected host6. Its resistance against proteinase K digestion was barely measurable, so that a clear biochemical distinction between two isoforms of the prion protein could be drawn: the cellular prion protein PrPC in the non-infected organism, and the scrapie isoform PrPSc as the major component of the purified infectious agent. It should be emphasized that proteinase K resistance of PrP was used as a biochemical marker for infectivity, and often ‘PrPSc’ and ‘PrPres’ (for resistance) were used synonymously. Later, however, it was shown, that infectivity and proteinase K resistance do not correlate in all cases; therefore, in this chapter, PrPSc is used for the property of infectivity and PrPres for proteinase K resistance, respectively.
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Chemical properties of PrP and recombinant PrP |
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TopAbstractIntroductionChemical properties of PrP...Chemical properties of PrPScStructure of PrPC and...Conformational transitions of...ConclusionsAcknowledgementsReferences |
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A few biochemical features of PrP were the basis of the prion hypothesis in the early days. In the mean time, PrP is one of the most intensively studied proteins, its chemical properties are well known whereas the biological function of PrPC is still under discussion as outlined elsewhere in this volume.
Plate I summarizes the amino acid sequence, processing, and post-translational modifications of PrP from the hamster. The final, processed form of PrP contains amino acids 23–231 from the original translation product of 253 amino acids. Peptide 1–22 is cleaved as signal peptide during trafficking, and peptide 232–253 is replaced by the glycosyl-phosphatidylinositol-anchor (for details see elsewhere in this volume). The cellular form is attached via the anchor to the outer membrane. Asparagine residues 181 and 197 carry highly branched glycosyl groups with sialic acid substitutions. PrP is always isolated as a mixture of three forms – unglycosylated, with one glycosyl-, and with two glycosyl-groups. A disulphide bridge is formed between Cys179 and Cys214. As indicated in Plate I, PrP contains 2 hexarepeats and 5 octarepeats in its N-terminus (see Prusiner7 and Weissmann8).
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The amino acid sequence is the same whether derived from the genomic DNA sequence or directly by peptide sequencing. PrPC and PrPSc are identical with respect to all chemical features described in Plate I. Note, however, that the glycosyl groups are heterogeneous, and only typical glycosyl groups are depicted in Plate I. Since it is nearly impossible to compare quantitatively distributions of different glycosyl groups, the chemical identity of PrPC and PrPSc is not completely safe in this respect.
Purification of PrP was attempted first in efforts to purify the infectious agent from the brain of infected animals. This became possible only after the hamster was introduced as experimental animal where there is a relatively short incubation time of 3–5 months instead of one or more years in the case of mouse or sheep. Furthermore, in the hamster the infectious dose (ID50) could be determined not only by end-point titration using about 50 animals for one value, but also by the incubation time assay9 in 4–6 hamsters. The purification procedure consists of a homogenization of the tissue and a series of precipitations and differential centrifugations. In the standard procedure, the solvent contains detergents, and a protein digestion step with proteinase K is included. Thus, the proteinase K resistance of PrPSc was essential for the purification, and PrP 27–30 was obtained as the purified product10. The criteria for optimization are the total yield of infectivity and the specific infectivity (i.e. ID50/g of material).
Besides the need for PrPSc purification, for many studies purified PrPC is also required. Although the amount of PrPC in brain is very high in contrast to other tissues, it represents less than 0.1% of the total central nervous system proteins. For example, from 100 hamster brains only a few micrograms of purified PrPC were obtained in an optimal procedure.
Obviously, expression of recombinant PrP in Escherichia coli was required. First attempts to isolate PrP from E. coli yielded either low expression levels or low purification efficiency because of the instability and insolubility of the protein. Furthermore, the disulphide bond in PrP, which is essential for correct folding of PrP, is not formed under the reducing conditions in the cytoplasm of E. coli. Attempts to fuse PrP with proteins that target the product in the periplasm and thereby spontaneously form the disulphide bond in vivo were not effective in producing larger quantities of PrP. Therefore, recombinant PrP was purified and afterwards reconstituted by denaturing under harsh conditions (8 M urea or 5 M guanidinium hydrochloride), the disulphide bond was formed under oxidizing conditions, and than PrP was renatured by dialysing out the denaturant11. This strategy is now used for the purification of recombinant PrP in high amounts.
Eukaryotic expression systems are of particular interest because they yield post-translationally modified PrP, i.e. a PrP which is very similar or identical with PrPC. Many eukaryotic cell systems including those of yeast and mammals were tested for expression of PrP. However, in most of these cases, difficulties occurred (unstable transfection, low levels of expression, insolubility of the protein, or difficulties in purifying PrP) and prevented those procedures becoming standard.
Finally, a transgenic mammalian cell system of Chinese hamster ovary (CHO) cells was established that generated PrPC at high levels of expression (
14-fold higher than in hamster brain). By this method, purified PrPC in amounts of 10–100 µg can be prepared12. Although this yield is still rather low in comparison to the yield of recombinant PrP purified from E. coli cells, purified PrPC is now available in amounts sufficient for some biophysical studies.
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Chemical properties of PrPSc |
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TopAbstractIntroductionChemical properties of PrP...Chemical properties of PrPScStructure of PrPC and...Conformational transitions of...ConclusionsAcknowledgementsReferences |
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Purified prions, either in the form of ‘full-length’ PrPSc or as PrP 27–30, are insoluble, even in mild detergents. In electron micrographs, fibrillar structures also called prion rods are visible (Fig. 1). After staining with Congo Red, they show the typical fluorescence birefringence of amyloids13. Similar structures were detected in thin sections of the brain of infected animals, which were called originally scrapie associated fibrils (SAF)14. It should be noted that, in the brains of CJD or Kuru victims, PrP deposits can be detected as diffuse deposits, amyloidic fibres, condensed plaques, or florid plaques.
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The high tendency to aggregate correlates with a PrPSc-specific resistance against digestion with proteinase K. In Plate II, the Western blot of an SDS-gel electrophoresis of PrPC and PrPSc, without and with proteinase K digestion, respectively, is depicted. The characteristic three bands of PrP (i.e. without, with one and with two glycosyl groups) are visible; they disappear completely after proteinase K digestion of PrPC which results in small peptides. In the case of PrPSc, however, the bands remain nearly undiminished in intensity although shifted to lower molecular weight. These are the N-terminally truncated forms of PrPSc, called PrP 27–30. From the right panel of Plate II, it can be seen that the ‘full-length’-PrP is cleaved around amino acid 90. The cleavage product (PrP 27–30) is fully infectious and, indeed, it is also the product of the purification procedure described above10. Furthermore, it should be noted that all presently available routine tests for BSE and scrapie are based on the proteinase K resistance of PrP 27–30.
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The prion model might well be explained on the basis of conformational changes of the prion protein which are induced directly or indirectly by the invading PrPSc. The phenomenon of prion strains, however, is hard to explain in a similar manner. Although different physicochemical properties were found with different prion strains15, these could not be attributed to different conformations of single PrP molecules but only to the highly aggregated and insoluble PrP. Even if the principal replication features of prions did not depend on nucleic acids, it was argued that at least the strain specificity might be determined by nucleic acid molecules16. Many attempts had been undertaken to find nucleic acids, all without success, which did not prove, however, the absence of nucleic acids. One series of studies was arranged in a way that nucleic acids either would have been found or would be excluded as essential components to prion infectivity17,18. Using highly purified infectious material, the number of nucleic acid molecules irrespective of their chemical nature and structure but depending on their size were determined quantitatively and related to the number of infectious units. For all nucleic acids larger than about 80 (in later work 50) nucleotides, less than one molecule of nucleic acid was determined per infectious unit. Consequently, infectious units exist without a nucleic acid – and the virus hypothesis was disproven finally with quantitative biophysical methods.
The lipophilic nature of highly purified prions suggested that not only a glycolipid anchor is linked to PrP but, in addition, lipids might be associated non-covalently with PrP. A chemical analysis revealed specific lipids which amounted to around 1% of purified prions19. These are sphingomyelin, 
-hydroxy-cerebroside and cholesterol depending on the method of purification. Both lipids are known to be components of the outside of the cell membrane in caveolae-like sites, where PrPC also accumulates. It could not be shown that the lipids are essential for infectivity, but their presence in prions might indicate the origin of prions, namely the site of PrPC accumulation on the outside of the cell membrane.
Early experiments, in which prion rods were digested extensively with proteinase K, had shown that the rod-like structure was maintained in electron micrographs even if PrP was digested by more than 99%20. This result had pointed to an additional structural component. It was identified much later as polymeric sugar consisting of -1,4-linked and 1,4,6-branched polyglucose21. Thus, this sugar component is clearly different from the glycosyl groups which are attached covalently to PrP. A schematic presentation is given in Figure 2. Since the polysaccharide amounted to up to 10% (w/w) of highly purified prions, it might be regarded as a structural scaffold contributing to the high chemical and physical stability of prions.
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Structure of PrPC and PrPSc |
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TopAbstractIntroductionChemical properties of PrP...Chemical properties of PrPScStructure of PrPC and...Conformational transitions of...ConclusionsAcknowledgementsReferences |
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It has been indicated above that PrPC and PrPSc are different in respect to solubility, fibril formation, proteinase K resistance and other features. These differences could be either a consequence of ligands bound to one isoform but not to the other or a consequence of a different secondary and tertiary structure of PrPC and PrPSc, respectively. First indications for clear differences in the secondary structure came from spectroscopic measurements applying circular dichroism and infrared spectroscopy22,23. From those measurements,
-helix and ß-sheet contents could be determined. The numbers given in Table 1 are not very accurate, since the analysis on PrPSc had to be carried out on insoluble samples; however, they show clearly that PrPC is dominated by -helices and has only little ß-sheet content, whereas PrPSc, i.e. ‘full-length’, or PrP 27–30 are characterized by similar amounts of -helices and ß-sheets.
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Natural PrPC could not be used to apply NMR spectroscopy or X-ray analysis for determination of the exact three-dimensional structure. The amount of material available was too small, the samples were not sufficiently pure, and the concentration was too low. These three hurdles were only overcome with recombinant PrP. In addition, recombinant PrP was synthesized in a form labelled with 15N or 13C. Even then, it took several years before the groups of Wüthrich24 and a little later of James25 analysed the structure of the C-terminal part of PrP, i.e. amino acids 121–231. As shown in Plate III for PrP of mouse and of man26, the structure consists of three
-helices (amino acids 144–154, 175–193 and 200–219) and a small antiparallel ß-sheet (amino acids 128–131 and 161–164). When the structure of ‘full-length’ PrP (i.e. amino acids 23–231) was analysed it was evident that the C-terminal part (i.e. amino acids 126–231) contained the complete globular part of the structure, whereas the N-terminus (i.e. amino acids 23–125) was more or less flexible27. Close to the small ß-sheet, the disulphide bridge connects helix 2 and helix 3. The region between the ß-sheet and helix 2 (amino acids 166–171) could be determined only with less accuracy possibly because of some structural flexibility. This region, however, is of particular functional interest. Different lines of evidence such as antibody binding, transgenic animals with mutations in that region, binding of the hypothetical factor X, etc. argue that the species barrier might be localized in that region. Furthermore, the minor differences in the structure of mouse and hamster PrP on the one hand and of bovine28 and human PrP on the other are restricted to that part of the molecule.
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It should be noted that the structure described above is the best description of the PrPC structure presently available, but that it was obtained from recombinant PrP. The glycosyl-groups of PrPC probably do not alter the structure significantly, but the fact that PrPC is attached to the membrane by the glycolipid anchor might have more influence, particularly if one considers the structure of the N-terminus which was found unstructured when free in solution (see above). Furthermore, it is known that the N-terminus and, in particular, the octarepeat of the N-terminus bind 4–6 copper ions in a co-operative manner which definitely would induce more structure than presently known from the NMR analysis.
The NMR-structure of PrP is a monomeric structure. Several reports in the literature indicate that PrP in its -helical structure can form dimers under physiological or close to physiological conditions29,30. Dimers in solution show the intact intramolecular disulphide bridge. Consequently, dimerization is not induced by oxidizing the disulphide bridge and reforming it in an intermolecular structure. The latter situation was, however, found in a recent crystal structure of a dimer of PrP (120–231)31; dimers were formed by domain swapping and intermolecular disulphide bridging. Whether this structure is a consequence of the long-time crystallization, or might indicate a physiological state, cannot be stated at present.
As mentioned above, PrPSc or PrP 27–30 are not accessible to structural analysis by NMR or X-ray analysis because of their insolubility. Attempts were made32 to use the structure described above as a starting model, change -helices into ß-sheets and, in this way, develop a model for PrPSc. These models assume that helix 2 and helix 3 are unchanged in accordance with antibody binding data, but they are incomplete in the sense that they are models for isolated molecules whereas PrPSc as well as PrP 27–30 were found only in aggregated forms. Thus, one has to assume that the PrPSc structure is stabilized by intermolecular interactions.
A new approach was followed by Wille from Prusiner's laboratory who was able to prepare two-dimensional crystalline-like arrays of PrPSc- or PrPSc-like molecules33. Those samples were studied by electron microscopy and, because of the crystalline-like arrangement, images could be reconstructed from the repetitive unit with fairly high resolution. A hexagonal symmetry was visible, but it could not be decided whether one unit is built from 3 or 6 molecules. The electron density map could be fitted best if, instead of ß-sheets, a ß-helix was assumed. The structure of the ß-helix type is known from other fibrillar proteins; spectroscopically, ß-sheets and ß-helices cannot be differentiated, so that the new model would not contradict earlier spectroscopic studies. The model is depicted in Plate 1V. In summary, the ß-helical N-terminus is located in the inner part of the hexagonal unit, with the helices 2 and 3 at the outer side and the glycosyl-groups pointing into the space between the hexagonal units. Most probably, the model shown in Plate IV is not the final description of the structure of PrPSc, but it is the best model currently available and takes into account both electron microscopic and spectroscopic data as well as the intermolecular stabilization of the PrPSc structure.
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Conformational transitions of PrP in vitro |
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TopAbstractIntroductionChemical properties of PrP...Chemical properties of PrPScStructure of PrPC and...Conformational transitions of...ConclusionsAcknowledgementsReferences |
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Structural and other chemical and physical properties of PrPC and PrPSc in the purified state have been described above. PrPC was characterized by
-helical structure, solubility as a monomeric or dimeric molecule, and proteinase K sensitivity. Since no functional test for PrPC is available, it is more accurate to speak about a PrPC-like conformation when the such properties are found. Similarly, for PrPSc, a ß-sheet-rich structure, insoluble aggregates, and proteinase K resistance are typical, but these features are not sufficient for PrPSc, because PrPSc stands for infectivity and, presently, the re-establishment of infectivity has not been achieved. Thus, the properties of PrPSc as mentioned above are those of a PrPSc-like molecule and do not infer infectivity. Conformational transitions might be either denaturation processes of PrPC or PrPSc, transitions between PrPC and PrPSc induced by varying solvent conditions, or induction of the PrPC to PrPSc transition by an existing seed of PrPSc (as described in detail elsewhere in this volume).
The denaturation of the globular domain of recombinant PrP (121–231) by addition of up to 8 M urea was analyzed quantitatively by recording the ellipticity at 222 nm34. One co-operative transition was obtained at pH 7.0 with a free energy of -28.6 kJ/mol for structure formation. At pH 4.0, an intermediate state could be identified with lowered -helicity and increased ß-sheet content; at present, it cannot be decided whether the intermediate would also be an intermediate on the pathway to PrPSc-like conformations. From the reversibility of the denaturation process, it was concluded that the PrPC-like conformation is the state of lowest free energy in buffer without detergent. This conclusion might be restricted, however, to the fragment PrP (121–231).
Similar experiments, but also taking into account the mechanism of re-folding by kinetic analysis, were carried out on recombinant PrP (89–231) from mouse which is the recombinant equivalent of PrP 27–3035. In this fragment, ß-sheet-rich oligomers and even fibrils were formed at pH 3.6. However, after switching from the fully denaturing conditions of 10 M urea to native conditions without urea, first the monomeric -helical conformation and from that (in a very slow process of hours or even days) ß-sheet oligomers and finally fibrils were formed. The presence of urea during the incubation speeded up the formation of the ß-sheet-rich conformation, and the presence of 5 M urea directly induced formation of ß-sheet-rich oligomers without running through the -helical state. Consequently, at acidic pH, folding of PrP to the -helical state is under kinetic control and the thermodynamically stable state is the ß-sheet rich state.
More close to the natural conditions of the PrPC to PrPSc transition were experiments in which the PrPC-like, -helical state was established first as it is in the non-infected organism36. Then the transition to the PrPSc-like conformation was induced by slightly denaturing conditions, (e.g. 1 M guanidinium hydrochloride). Also, these experiments were carried out at acidic pH (4.0), and the conversion process could be induced by a wide variety of conditions combining mild denaturants and different salts. In accordance with renaturation experiments (see above), it was found that ß-sheet formation is always connected with aggregation and that the most stable state, at least at acidic pH, is the ß-sheet-rich aggregated state. An exception was shown under conditions reducing the disulphide bridge; it was reported that acidic and reducing conditions could induce a ß-sheet-rich and monomeric state37. It is not known whether this finding is relevant to PrPC to PrPSc conversion in nature because the intramolecular disulphide bridge is present in both states and in all other conversion experiments the disulphide bridge was not opened transiently38.
The earliest studies on the in vitro conversion were carried out with natural PrP and at neutral pH39. Infectious PrP 27–30 was converted to an -helical, oligomeric and non-infectious form by addition of 0.3% sodium-dodecylsulphate (SDS). Further addition of 30% acetonitrile or mere dilution of SDS to 0.01%40, re-established a ß-sheet-rich, aggregated and partially proteinase K-resistant conformation. Although natural PrP was used which was infectious before the conversion, infectivity could not be re-established. These experiments were closest to natural conditions if the low concentrations of SDS were regarded as a membrane-like environment. It was also shown that the conversion occurs in steps, first fast formation of ß-sheet structure concomitant with forming small oligomers, then larger oligomers in minutes to an hour, and finally large insoluble aggregates in hours to days. Applying the same conversion system to recombinant PrP (90–231), systematic studies of the influence of varying SDS concentrations were carried out and several intermediate states described (Fig. 3)30. In 0.06-0.1% SDS, an -helical dimer is present as a thermodynamically stable state which is converted in a co-operative manner in 0.04–0.06% SDS to a ß-sheet-rich oligomeric state. As recently determined (Nagel-Steger et al, unpublished), the oligomeric state with 12–16 molecules is of particular interest for further biophysical studies since it is stable in solution. In low SDS concentrations (< 0.02% SDS), large insoluble aggregates (see above) are formed which also remain stable after the SDS has been washed out completely. In buffer without detergent at pH 7, the PrPC-like as well as the PrPSc-like conformations can be established, but the stable state is the PrPSc-like conformation, and the conversion can be induced by different detergents, even in very low concentrations (see also Xiong et al41).
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Conclusions |
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TopAbstractIntroductionChemical properties of PrP...Chemical properties of PrPScStructure of PrPC and...Conformational transitions of...ConclusionsAcknowledgementsReferences |
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What is the conclusion from all the in vitro conversion studies? Evidently, the thermodynamically stable state is the PrPSc-like state, and this would be true for lysosomal acidic pH or the cell-surface neutral pH. A high activation barrier renders the transition very slow (i.e. hours, days or even weeks). Formation of the ß-sheet-rich structure is always correlated with oligomerization at least if the naturally occurring disulphide bridge is intact. Whether ß-sheet oligomers are intermediates on the pathway to PrPSc or a dead-end product is at present an artificial discussion because the PrPSc-like state as available is not infectious and a final decision on the right pathway cannot be made. If the PrPC-conformation is not the thermodynamically stable state, but only metastable, the question remains why a transition to PrPSc does not occur much more frequently in nature as a spontaneous transition. It is, however, not a discrepancy if one takes into account that PrPC is anchored in the lipid membrane, and all studies described were carried out in aqueous buffer. Consequently, as an additional transition of PrPC, one has to include the distribution between the aqueous and the lipid phase which would definitely stabilize PrPC in nature to prevent a spontaneous transition.
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Acknowledgements |
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TopAbstractIntroductionChemical properties of PrP...Chemical properties of PrPScStructure of PrPC and...Conformational transitions of...ConclusionsAcknowledgementsReferences |
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The author thanks his co-workers E Birkmann, C Dumpitak, K Elfrink, H Gruber, and Drs K Jansen and J Schell for their help in preparing the manuscript.
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Footnotes |
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Correspondence to: Prof. Dr Detlev Riesner, Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Universitäts Strasse 1, 40225 Düsseldorf, Germany
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