Prions, proteinase K and infectivity

Abstract

It has been described that the breakdown of β-sheets in PrP^Sc by denaturation results in loss of infectivity and PK-sensitivity, suggesting a relationship between the structure and PK-resistance. It is also known that an important fraction of total PrP^Sc is PK-sensitive and can be isolated by the method we already described. Consequently, we decided to employ the PK-sensitive fraction of PrP^Sc as a potential and useful tool for structural studies. Thus, two essential questions were addressed in our recent article. First, the difference in the infectivity between the sensitive and resistant fractions and second, whether sensitive and resistant PrP^Sc shared the same conformation or were only different size multimers with the same basic conformation. Here we discuss our latest data in light of recent infectivity studies and their possible implications on the conformation of the prion.

Keywords: :

• prion
• sensitive PrP^Sc
• resistant PrP^Sc
• infectivity
• structure

This article refers to:

Infectious Prions

About 30 y ago Prusiner coined the term “prion” derived from the words proteinaceus and infectious, indicating whereupon an infectious particle composed mainly of protein.¹ The term infectivity implies both the ability of a pathogen to pass from one host to another and the self-replication ability. In the prion literature infectivity experiments rely on the detection of the clinical signs in the inoculated animals. Most of the animals involved, excluding those receiving highly diluted doses, eventually succumb indicating that what is really being evaluated is not only infectivity but also toxicity. Thus, in most of the cases mammalian prion infectivity entails cell toxicity and host death. Sadberg and colleagues have demonstrated recently that infectivity and toxicity are two processes that can be separated and suggested the existence of two types of particles, an infectious and a toxic one.² A clear example of this uncoupling between the two processes is the so-called “subclinical infection” operationally defined by Hill and Collinge to refer to animals in which prion replication occurs but where the clinical signs of disease have not developed during a normal lifespan.³ The existence of subclinical forms of prion infection is very common in experiments involving species-barriers crossing.

However, prion infectivity is a complex issue since many different factors seem to modulate it and varies between strains and also between tissues within the same animal. For example, inocula taken from the spleen lead to a longer incubation period than the same number of LD50 units taken from the brain.⁴ Furthermore, at the molecular level the presence of non-protein components, like lipids, influences the level of infectivity.⁵

Similar to other neurodegenerative diseases such as Parkinson or Alzheimer, prion diseases are associated with the deposition of amyloid fibrils where the aggregation process confers insolubility and a degree of resistance to proteolytic digestion. However, in contrast to other amyloids, prions are practically indestructible and conventional chemical and physical decontamination methods have been proved ineffective in reducing their infectivity. This exceptional feature may explain why prions are so infectious and other amyloid-based diseases are not. Interestingly, a study of Wille and coworkers has differentially identified prion infectivity from the amyloid properties of PrP 27-30 which underlines the dependence of prion infectivity on PrP^Sc conformation.⁶

PK Sensitive PrP^Sc

In an attempt to define the nature of the scrapie agent Prusiner used a variety of hydrolytic enzymes such as proteinase K (PK) to assess its grade of infectivity after inactivation.⁷ Nowadays, prion disease diagnosis relies on the relative resistance of PrP^Sc to the non-specific protease PK in brain samples to discriminate between PrP^C and PrP^Sc, in combination with immunological detection of the main PK-resistant part of PrP^Sc (PrP27-30). Resistance to PK is not exclusive to mammalian prions but it also is present in yeast prions and, like mammalian prions, some of them are highly resistant to protease digestion (e.g., Sup35p), while others are more sensitive (e.g., Ure2p).⁸

Evidence for PK-sensitive abnormal PrP^Sc was originally reported by Safar and coworkers in infected hamsters.⁹ Sensitive PrP^Sc challenged the properties traditionally assigned to PrP^Sc such as, resistance to proteases, in particular PK, and insolubility in non-denaturing solvents. Besides, the existence of sPrP^Sc might constitute a fairly large fraction of total PrP^Sc, as much as 90% according to a study in patients with Creutzfeldt-Jakob disease (CJD).¹⁰

The elusive nature of prion infectivity and structure has been reflected in many studies. Thus, studies by Silveira and coworkers have shown that in certain PrP^Sc isolates the most infectious particles are composed of 14-28 PrP^Sc subunits while larger PrP^Sc assemblies, particularly fibrils, have very little infectivity.¹¹ Yet another research group, employing the scrapie cell assay, reported that sPrP^Sc had a small contribution to the overall infectivity of PrP^Sc.¹² On the other hand, Tzaban and co-authors have previously shown that fractions of PrP^Sc containing a small number of PrP^Sc monomers are completely destroyed by PK.¹³ As Silveira used PK to prepare their PrP^Sc isolates, which they later dispersed with detergent and/or sonication, it is impossible to determine whether they may have missed an important population of highly infectious sPrP^Sc aggregates. One important methodological difficulty present when different studies are compared arises because the properties of sPrP^Sc are indirectly inferred after a PrP^Sc sample has been treated with PK.

In this context, the method we described to isolate sPrP^Sc provides a useful tool for studying directly its infectivity and conformation.¹⁴ The method relies on the separation of a lighter fraction of PrP^Sc by means of a finely tuned centrifugation step.¹⁴ In addition, in the study we demonstrated that self-propagation of sPrP^Sc was possible by means of protein misfolding cyclic amplification assay (PMCA). Propagation in vitro does not necessarily correlate with infectivity as demonstrated by a recent study where the ratio of the infectivity titer to the amount of PK resistance PrP^Sc was much lower in the amplified material than in the brain derived sample.¹⁵

With the intention of providing a clear answer about the infectivity of the sensitive fraction, we have performed a bioassay by inoculating animals with similar amounts of four prion isolates, purified total PrP^Sc, sPrP^Sc, rPrP^Sc and infected brain homogenate at different dilutions. Our data showed that the PK-sensitive fraction of PrP^Sc is as infectious as the resistant fraction, even at higher dilutions such as 1/1000. Curiously, animals inoculated with the undiluted sPrP^Sc preparation expressed typical scrapie symptoms sooner than those inoculated with any of the other three undiluted preparations. Although these data were statistically significant, the titer of sPrP^Sc dropped dramatically in the consecutive dilutions until it reached the same level as the rest of the prion inocula.¹⁶ A higher possible rate of clearance seems not to affect the degree of infectivity of sPrP^Sc as demonstrated by the fact that it was infectious via the intracerebral route and the intraperitoneal also.

Our data are in line with the study presented by Tixador and colleagues, in which they characterized the relative levels of infectivity in different PrP^Sc fractions using infected brain homogenates as the starting material instead of purified PrP^Sc. Following fractionation of infectious crude brain homogenate in a viscous medium they found that upper fractions (slow particles) of 139H and Sc237 hamster strains contained higher infectivity than the lower ones. This infectivity was approximately 50 fold higher in the case of the139H strain.¹⁷

Taken together, the above data indicate that the aggregation state or quaternary structure of PrP^Sc is relevant for prion infectivity. Furthermore, it is very likely that additional conformational aspects, such as those derived from the tertiary structure, play an important role in determining the differential infectivity of specific prion strains. Therefore we studied whether any conformational difference between sensitive and resistant could be detected at the level of the tertiary structure. Data regarding the tertiary structure of resistant and sensitive PrP^Sc fractions, obtained by limited proteolysis and mass spectrometry, showed that both have similar PK-cleavage maps and therefore seems to share the same basic architecture.¹⁶ Confirming this result, western blot analysis with a C-terminal antibody of purified total PrP^Sc vs. sPrP^Sc showed a similar PK-cleavage pattern, although the relative intensities of some of the bands varied. Interestingly, three of the lower bands with molecular weights around 15, 10 and 6 kDa were more intense for the sensitive fraction. Such a difference is very striking, especially after considering that sPrP^Sc was treated with 5μg/ml of PK, ten times less than the amount used for the total fraction. Could these data reflect a difference in the conformation of sPrP^Sc? For instance, it might be formed by less compact oligomers and therefore be more exposed to PK attack? Interestingly enough, we have shown previously a susceptibility of sPrP^Sc to virtually complete degradation by trypsin as well as a higher immunoreactivity after guanidine hydrochloride treatment when comparing it with rPrP^Sc.¹⁴ It has been described that the prion-determining region of yeast Sup35 (NM) can form earlier oligomers that are less compact and which react with the conformational antibody A11 (which recognizes prefibrillar intermediates), whereas more mature oligomers are more compact and react to a different conformational antibody, the OC antibody (which recognizes amyloid fibrils but not monomers or soluble prefibrillar oligomers).¹⁸ Another possibility is that small oligomers are more susceptible to the presence of the detergent and/or sonication employed during the isolation protocol. It is well known that sonication and detergents have an effect on the conformation of proteins.^19,20 Thus the prion peptide 192-213 changed its secondary structure depending on the concentration and the type of detergent.²⁰

Again, the study by Tixador and coworkers highlights the importance of the tertiary structure of PrP^Sc in relation to PK sensitivity. The atypical form of field scrapie, Nor98, despite being highly sensitive to PK digestion when compared with other ovine strains showed a larger proportion of high molecular weight multimers. The authors did not interpret the results as an aggregation size difference but as a difference in its tertiary structure.¹⁷

Finally, PK-sensitivity appears to be connected with oligomer stability which in turn is directly proportional to the incubation time. Colby and coworkers demonstrated that stable recombinant amyloids produced a more stable prion strain, leading to the longest incubation time, whereas more labile amyloids generated less stable strains and shorter incubation times.²¹ Thus we have shown that the sPrP^Sc fraction is higher in the hamster prion strain drowsy than in 263K along with a longest incubation time.¹⁶

The common notion that the prion is a PK-resistant aggregate has been questioned in the last few years by several experimental studies. Therefore, it is possible that in vivo PK-resistance may not be the rule but the exception.

Abbreviations:
sPrPSc	=	sensitive prion protein Sc
rPrPSc	=	resistant prion protein Sc
PK	=	proteinase K
CJD	=	Creutzfeldt-Jakob disease
PMCA	=	protein misfolding cyclic amplification assay