1 Introduction
1.7 PrP Sc specific antibodies
An important tool for detection of PrPSc has yet been missing, a pathogen-specific antibody (Ab). Since PrPC and PrPSc share the same peptide sequence, it is difficult to generate an Ab which can distinguish between PrPC and PrPSc. Up to now this can be circumvented by hydrated autoclaving of brain tissues in citrate buffer followed by a treatment with concentrated formic acid before expose to prion protein specific Abs [145]. Moreover, in eukaryotic cells PrPSc could be treated with 6 M GdnHCl [134], 3 M GdnSCN [131] or 96 % formic acid [138] prior to staining . These procedures not only denature PrPSc to inactivate the infectious agent but uncover an Ab specific epitope that was buried before within the molecule [148].
The only difference known so far between PrPC and PrPSc lies in its biochemical and biophysical properties. PrPSc does not share the same secondary structure with PrPC. Thus, the host immune system´s ability to recognise PrPSc as foreign or pathological may be restricted to conformational epitopes. Additionally, the conformation and aggregation state
Introduction 18
of the PrPSc glycoprotein severely limits exposure of the polypeptide surface to Abs. These structural features and the high conservation of PrP peptide sequence in mammals have limited attempts to generate PrPSc specific Abs. Thus, years of intense efforts led to several reports of PrPSc specific Abs: 15B3 [149], Abs with tyrosine-tyrosine-arginine (YYR) motifs [150], V5B2 [151] and motif grafted Abs IgG 19-33 [152], IgG 98-112 and IgG 136-158 [153].
15B3, derived from Prnp-/- mice immunised with full-length recombinant bovine PrP, immunoprecipitates PrPSc from brain homogenates without PK digestion and denaturation it specifically recognises PrPSc in human, bovine and murine brain homogenates. Recently, it was observed that 15B3 precipitated both PrPSc and aggregated insoluble forms of PrP not necessarily being infectious [154]. These insoluble forms were produced partially by point mutations within the prion protein (P101L, D177N, E199K), by PG14, a prion protein with 9 octarepeats, by cytoplasmatic PrP, upon Cu2+ treatment and by purified recombinant PrP aggregates. In all cases 15B3 could precipitate the insoluble aggregate in brain homogenates and in cell culture. Ab 15B3 was reported to stain purified aggregates of recombinant PrP.
Another publication also revealed that 15B3 immunoprecipitated mutant PrP P101L [155].
15B3 is an immunglobuline of class M which are often designated to recognise the specific conformation of a molecule. This also holds true for 15B3. The 15B3 epitope consists of three distinct peptide sequences: 142-148, 162-170 and 214-226 (human sequence) which are distributed all over the prion protein sequence.
Figure 5: Mapping of the 15B3 epitope onto the NMR structure of PrPC. In yellow, epitope 1 with aa 142-248;
in violet, epitope 2 with aa 162-170 and in cyan, epitope 3 with aa 214-226 [149].
Induction of β-sheet structures in recombinant PrP is associated with increasing solvent accessibility of tyrosines which were hidden before within the protein. Conserved YYX motifs were conserved in human, sheep, mouse, hamster and bovine PrP. They could be found at amino acid residues 149-151, 162-164 and 225-227 (human sequence). Therefore, Paramithiotis et al. conclude that the increased solvent exposure of tyrosyl side chains in
sheet rich recombinant PrP might involve at least one bi-tyrosine motif. If recombinant β-sheet rich models share some structural features of PrPSc, antibody access to one or several YYR motifs may provide a PrPSc-selective conformational epitope. YYR Abs were derived from mice immunised with CYYRRYYRYY peptide or from rabbit immunised with CYYR peptide.
Positive IgM Abs (from mice, later transformed into chimeric IgG) and IgG (from rabbit) were tested in immunoprecipitation (IP), plate capture immunoassay and flow cytometry and recognise the pathological isoform of the prion protein PrPSc but not PrPC. But also recognision of misfolded PrP with YYR Abs could be observed like with 15B3 and motif grafted Abs [154].The epitopes of 15B3 and YYR partially overlap. YYR motifs could be found in the 15B3 epitope. The similarity of the epitopes may be the reason why both Abs are IgMs. Tyr repeats have been reported to define a dominant B-cell epitope [156], suggesting that the high IgM frequency within this region may be the result of a specific mouse immune response to YYR antigens since immunised rabbits with YYR repeats only reveal IgGs [150].
V5B2, raised against a human PrP sequence at 214-226, is IgG antibody and recognises PrPSc selectively without any pretreatment. Serbec et al. used V5B2 in dot blot, IP, ELISA and IHC of human brain tissues [151]. The mAb selectively detected PrPSc in sCJD and vCJD brain samples. For IHC, cryo sections of cerebellum were used immediately for staining, but also formaldehyde fixed, formic acid treated and paraffin embedded brain sections were used.
Cryo sections displayed staining of PrPSc plaques in sCJD cases, uninfected brain sections were not stained (Figure 6). But vCJD and sCJD brain samples fixed with formaldehyde and treated with formic acid, probably to reduce the infectivity of the samples and not the antigenecity, showed a more pronounced staining of PrPSc plaques. A 13-residue synthetic peptide (214-226 in human PrP sequence) was used for immunisation of mice. V5B2 favoured binding to oligomeric forms or to fibril like aggregates of the peptid in solution and to PrPSc aggregates itself [157]. Perhaps it acts like all other designated PrPSc specific Abs and recognises soluble PrP aggregates which do not have to be infectious.
Introduction 20
Figure 6: Staining of PrPSc in human brain with V5B2. (a) sCJD case with PrPSc plaques in the cerebellum, stained with V5B2. (b) Control brain stained with V5B2 shows no signs of staining [151].
For prion propagation a direct and specific interaction between PrPC and PrPSc is proposed.
PrPSc replications could be inhibited in cell culture and in vivo by certain PrP peptides, PrP specifc Abs or their Fab fragments [153, 158-162]. The inhibitory effect may be due to binding to distinct regions of PrPC that likely interact (in)directly with PrPSc and thus prevent assembly of the prion replicative complex. In another work the inhibitory PrP sequences/epitopes were grafted into a recipient Ab scaffold [152, 153]. Only three of them, namely IgG 19-33, IgG 89-112 and IgG 136-158 efficiently recognise mouse, human and hamster PrPSc, but not PrPC, in IP. As these Abs are designated to detect PrPSc exclusively they were also used for IHC in prion infected brain tissues [163] These finding suggests that residues 19-33, 89-112 and 136-158 within the prion protein are key components of the PrPC-PrPSc complex. They bind selectively PrPSc indicating that these three epitopes are hidden within the PrPC molecule. Upon conversion they are presented at the surface of PrPSc and could therefore react with motif grafted Abs. But is could recently be shown that these motif grafted Abs detect not only PrPSc but also aggregated insoluble PrP in brain tissue, in cell culture and in a purified form [154]. Since 15B3, Abs mit YYR motif and motif grafted Abs do not only recognise PrPSc but also insoluble PrP aggregates, the use of these Abs as PrPSc specific is limited. On the other hand the use of them is extended to other inherited prion diseases which do not necessarily develop infectivity and protease resistance of PrP. In both applications lack of PrPC staining is of great advantage.