Neuropathogenesis Unit, Institute for Animal Health, West Mains Road, Edinburgh EH9 3JF, UK
Correspondence
Robert A. Somerville
robert.somerville{at}bbsrc.ac.uk
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ABSTRACT |
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INTRODUCTION |
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Whatever its role in the structure of the agent, it is clear that an abnormal form of PrP, normally denoted PrPSc, is produced and deposited in infected tissues. PrPSc differs from the normal form of the protein PrPC in several respects. PrPSc tends to sediment after detergent treatment whereas PrPC is soluble, PrPSc shows greater resistance to protease digestion than PrPC although the N terminus is cleaved (Meyer et al., 1986). PrPSc is often less glycosylated at its two N-glycosylation sites than PrPC (Hill et al., 1997
; Kascsak et al., 1985
; Somerville et al., 1997
; Somerville & Ritchie, 1989
). The greatest degree of diversity in the degree of glycosylation is associated with differences in the strain of TSE agent, although other factors including tissue source (Hill et al., 1999
) and the region of brain can also have a significant effect (Somerville, 1999
).
The apparent molecular mass of the aglycosyl form of the protein also varies. The variation is due to differential cleavage of PrPSc at a series of sites between codons 80 and 100 (as numbered in the human PrP sequence), two of which, G82 and S97, tend to predominate, at least in humans (Bessen & Marsh, 1994; Hope et al., 1988
; Parchi et al., 2000
; reviewed by Schreuder & Somerville 2003)
. Several cleavage sites have been identified in the same sample (Parchi et al., 2000
). The observed banding pattern of the aglycosyl PrPSc polypeptide fragment on SDS-PAGE usually either appears as two bands or a single diffuse band, presumably corresponding to the two major protein fragments and/or an average of the migration of all the aglycosyl PrPSc in the sample (Notari et al., 2004
; Parchi et al., 2000
).
Because the diversity in glycosylation and cleavage of PrPSc are associated with different TSE strains, it has been suggested that the degree of glycosylation of PrPSc may be used as a differential diagnostic tool for TSEs (Hill et al., 1997), for example between BSE and natural scrapie in sheep (Baron et al., 2000
; Stack et al., 2002
). Time consuming traditional strain-typing techniques where incubation period and distribution of pathology are compared after passage in a panel of three or more strains of mice (Bruce et al., 1994
) would be avoided. However, before the properties of PrPSc can be exploited it is necessary to assess to what degree these properties are specified by the causal agent and by the host, including the effects of location in the brain.
In this study, we have investigated the effect of PrP genotype on the glycosylation of PrPSc and show that some TSE strains differ in the degree to which they are glycosylated after passage in a new PrP genotype, while others show little change. In some, but not all, cases PrP genotype has an effect. In a previous publication we showed that brain region can have an effect on the degree of PrPSc glycosylation (Somerville, 1999). We have also examined the effect of brain region in more TSE models. Again in some, but not all, cases brain region has an effect.
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METHODS |
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RESULTS AND DISCUSSION |
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The 301C and 301V strains were derived by serial passage in C57BL and VM mice, respectively (Bruce et al., 1994). In this case 301V shows little change in the degree of glycosylation after a single passage in SV mice. However, the degree of glycosylation of PrPSc from 301C after a single passage in VM mice is reduced compared with PrPSc from SV mice (Student's t-test of %H, P<0·005). Unfortunately, there yet has been no systematic study of the stability of other phenotypic properties of these two strains after passage in mouse PrP genotypes other than those in which they were first passaged. Perhaps other properties of 301C might diverge on serial passage in VM mice. Phenotypic changes of 87A changing to ME7 have been explained by mutation of the TSE agent's genome (Bruce & Dickinson, 1987
). Mutational change could well be responsible for the change in the properties of 22A and possibly the change in glycoform ratio of 301C.
Overall the results show that the degree to which PrPSc is glycosylated can vary with strain of agent within the same PrP genotype after passage in either SV or VM mice (Fig. 2). The strains 79A and 139A, derived from the drowsy goat source (Dickinson, 1976
), were least glycosylated, while 301C and 301V which are derived from BSE were most heavily glycosylated. There was no detectable effect of PrP genotype on the degree of glycosylation of the Prnpaa passaged 22C, 79A, 139A or the Prnpbb passaged strain 301V. However with 22A, originally passaged in Prnpbb mice, there was a small but significant difference and with 301C originally passaged in Prnpaa mice there was a larger difference.
Effect of brain region
Previously we have found a small effect of PrP genotype on ME7 for which both the overall degree of glycosylation differed and the pattern of glycosylation in the monoglycosyl band varied according to PrP genotype (Somerville, 1999). In the present experiments we examined the effect of brain region on the degree of glycosylation of PrPSc from three TSE models by using TSE strains after continuing passage in the same PrP genotype in which they were originally isolated. The effect of passage in the alternate PrP genotype was not examined. Infected brains were dissected to obtain cortex, cerebellum and medulla. Differences were observed in the degree of glycosylation of PrPSc from 301C and 301V with respect to brain region (Fig. 3
a and c) in accord with previous observations on ME7 (Somerville, 1999
). In particular, PrPSc from 301C cerebellum was significantly more highly glycosylated than PrPSc from 301C cortex (Student's t-test of %H, P<0·05) and PrPSc from 301V cerebellum was significantly more highly glycosylated than PrPSc from 301V medulla (%H, P<0·05) or cortex (%H, P<0·05). However, 22A showed no discernible difference between the three brain regions.
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Migration of the aglycosyl band
In all cases, except for 301C, the migration of the aglycosyl band was similar (Fig. 2a). With 301C the aglycosyl band migrated faster in the sample from SV mice than from VM mice; the latter showed similar migration to 301V and indeed other strains examined (Fig. 2a
and data not shown).
The aglycosyl band in the brain region samples appeared to resolve more clearly into two bands (Fig. 3b) than from whole brain samples (Fig. 2a
), possibly reflecting the major cleavage sites in the sequence and also reflecting reduced heterogeneity. Whereas the 22A samples contained similar amounts of both aglycosyl bands, the 301V samples tended to have more of the faster migrating band, except in the sample from medulla where the amounts seem similar. In contrast, the samples from 301C predominantly contained the faster migrating band with evidence of even lower Mr forms.
Strain typing using glycosylation of PrPSc
Overall the results show that the strain of TSE agent has the greatest effect on the degree of glycosylation of PrPSc. More minor effects are sometimes but not always associated with the location of PrPSc in the brain. There were also minor differences in the degree to which PrPSc was glycosylated after passage in mice of differing PrP genotypes in two cases, 22A and 301C. With the exception of 301C in SV mice, there was little variation in the aglycosyl band migration with respect to TSE strain, PrP genotype or brain region. However, 301C in SV mice had a predominance of the faster migrating aglycosyl band. These results demonstrate further that within a well-controlled and characterized experimental system, discriminatory phenotypic information about PrPSc properties can be obtained. They also further confirm that tissue type and location, at which brain are sampled, can in principle significantly affect the measurements. These data also show that passage through hosts varying in PrP genotype can have significant effects on PrPSc properties. If such changes were to occur in field cases of TSE disease they might compromise attempts to use PrPSc properties for differential diagnosis/strain typing of TSEs. For example, it has been proposed that the differences in PrPSc properties between cattle BSE and scrapie sheep PrPSc might be used in the differential diagnosis of BSE in sheep (Baron et al., 2000; Stack et al., 2002
). However, such use might be compromised by changes in the PrPSc properties of BSE on transmission to one or more PrP genotypes of sheep (Hunter et al., 2000
).
Origins of differential glycosylation
The diversity in structure of PrPSc between and within TSE models and its difference from PrPC demands an assessment of possible mechanism(s) by which structural diversity arises. As discussed previously (Somerville, 1999), PrPC is consistently highly glycosylated, but the degree of glycosylation of PrPSc varies between strains and PrPSc from some TSE models (e.g. 79A and 139A) is poorly glycosylated. Three possible models were proposed: (i) that PrP destined for the PrPSc fraction follows a separate or modified biosynthetic path from that normally followed by PrPC (biosynthetic model); (ii) that the PrP to comprise PrPSc was selected from the mature PrPC pool (selection model); or (iii) that the glycans were removed from PrPSc during its conversion from mature PrPC (degradation model). Some preference is shown for PrP with similar degrees of glycosylation to the template in in vitro conversion assays although the host cell can also be influential (Vorberg & Priola, 2002
). Hence although the in vitro system demonstrated the feasibility of a selection hypothesis, it also showed that other factors affect the degree of glycosylation, suggesting that other mechanisms for determining the degree of glycosylation of PrPSc might operate in vivo. Overall the data still favour the biosynthetic model (Somerville, 1999
).
The difference in apparent size of PrPSc fragments after digestion with proteinase K is assumed to be due to differences in the conformation of the protein, which allows the protease differential access to cleavage sites (Caughey et al., 1998). Cleavage properties are retained when PrP is converted to protease resistant forms in vitro (Bessen et al., 1995
), suggesting that a similar conversion mechanism might be followed in vivo. However, an alternative mechanism is more compatible with differences in glycosylation: that conformation of PrPSc precursors is determined during post-translational maturation of the protein. Such diversity is compatible with the different relative amounts of the aglycosyl fragments observed in different brain regions. Indeed aberrant folding in the endoplasmic reticulum could well affect subsequent glycosylation. There are sequences in the 3' untranslated region of the PrP gene that modulate protein synthesis (Goldmann et al., 1999
), suggesting a mechanism of control via 3' (or possibly 5') elements in mRNA sequence. It has recently been proposed that PrPC controls the translation of HIV proteins Env and Vpr (Leblanc et al., 2004
). It is equally feasible that PrPC also regulates the translation of other proteins including itself.
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ACKNOWLEDGEMENTS |
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Received 4 May 2004;
accepted 5 October 2004.