1 MRC Prion Unit, Department of Neurodegenerative Disease, Institute of Neurology, University College London, Queen Square, London WC1N 3BG, UK
2 International Blood Group Reference Laboratory, Southmead Road, Bristol BS10 5NO, UK
Correspondence
John Collinge
j.collinge{at}prion.ucl.ac.uk
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ABSTRACT |
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INTRODUCTION |
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Similar NMR structures of PrPC have been reported for mouse (Riek et al., 1996), hamster (James et al., 1997
) and human (Hosszu et al., 1999
; Zahn et al., 2000
) recombinant protein residues
120231. In vitro studies have shown that PrP can adopt different conformations depending upon solvent pH and redox potential (Swietnicki et al., 1997
; Hornemann & Glockshuber, 1998
). At low pH following reduction of the disulphide bond, the folded C-terminal domain of the human prion protein can exist as a soluble monomeric
-sheet structure (Jackson et al., 1999a
). It is possible that the conversion of the
-helical form,
-PrP, to the
-sheet form,
-PrP, caused by reduction and mild acidification is relevant to the conditions that PrPC might encounter within the cell (Shyng et al., 1993
).
Distinct prion strains can be serially propagated, producing characteristic clinical features and neuropathology in defined hosts. Although the molecular basis of strains is unknown, PrPSc can be distinguished by differing physico-chemical properties of the protein (Bessen & Marsh, 1992; Collinge et al., 1996
). After PK digestion of brain homogenates from different CJD clinical phenotypes, variation in PrPSc fragment length has been observed (Collinge et al., 1996
; Parchi et al., 1996
) and glycosylation can also be used to further distinguish strains (Collinge et al., 1996
). PrP is post-translationally modified by the addition of two N-linked glycosylation sites at positions 181 and 197 in the human sequence, either or both of which can be occupied to give monoglycosylated or diglycoslyated isoforms, or remain unglycosylated. The type and combination of sugar molecules can produce a high degree of heterogeneity (Endo et al., 1989
), potentially affecting the conformation and intermolecular interactions. Crucially, both PrPSc fragment size (Collinge et al., 1996
; Telling et al., 1996
) and glycoform ratios (Collinge et al., 1996
), following PK cleavage, can be maintained on serial passage in experimental animals, arguing that these intrinsic properties of PrPSc may encode prion strain diversity. Several human PrPSc types have been identified that are associated with different phenotypes of CJD (Parchi et al., 1996
). Based both on PrPSc fragment sizes and the ratios of the three PrP glycoforms, four different PrPSc types are readily distinguishable (Collinge et al., 1996
, 1997
; Wadsworth et al., 1999
; Hill & Collinge, 2002
).
Although all the PrP glycoforms are present in PrPSc, it is not known if they can be physically or chemically separated, except when denatured, since existing mAbs do not readily immunoprecipitate PrPSc, i.e. we cannot yet determine whether a single PrPSc particle contains mixed glycoforms or only a single species. Prnp0/0 mice have been used by many groups to raise anti-PrP mAbs (Krasemann et al., 1996; Korth et al., 1997
; Zanusso et al., 1998
; Williamson et al., 1998
; Nakamura et al., 2003
), but mAbs that can readily imunoprecipitate PrPSc or recognize different glycoforms of PrP are rare. We have also used Prnp0/0 mice to raise anti-PrP mAbs, using a soluble
-helical monomer of the human PrP sequence 91231 (
-PrP91231) as antigen. We also used the novel immunogen recombinant human
-PrP91231, identical in amino acid sequence but folded into a soluble monomeric conformation and partially resistant to PK (Jackson et al., 1999b
), as antigen and produced mAbs that could efficiently immunoprecipitate PrPSc. Here, we describe these mAbs and the findings on the physical association of PrP glycoforms in normal and prion-diseased brains. We also show that mAbs raised to
-PrP91231 and reactive to residues 93105, can immunoprecipitate native PrPSc.
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METHODS |
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Preparation of brain homogenates.
Brain homogenates (10 % w/v) from uninoculated Prnp0/0, wild-type and RML-prion terminally scrapie-ill Prnp+/+ FVB/N mice were prepared, either in cold lysis buffer (PBS, 0·5 % sodium deoxycholate, 0·5 % NP40, pH 7·4) or in Dulbecco's Mg2+/Ca2+-free PBS (Gibco-BRL). Normal human and vCJD brains were homogenized to 10 % (w/v) in Mg2+/Ca2+-free PBS. The brain homogenates prepared in PBS were diluted 1 : 2 with 4 % N-laurylsarcosine, and after 10 min at 37 °C DNase (Benzonase; 50 U ml1) (Roche) and 1 mM MgCl2 were added and further incubated for 30 min at 37 °C, then diluted in PBST [PBS, 0·1 % (v/v) Tween 20] to use as antigen. To denature PrPSc, the brain homogenate, 1 % (w/v) in 4 % (w/v) SDS, was incubated at 100 °C for 14 min then diluted in PBST.
Western blotting.
The brain homogenates were electrophoresed through 16 % gels using conventional methods. Following transfer onto Immobilon-P membranes (Millipore), non-specific binding was blocked with 5 % (w/v) milk proteins in PBST and ICSM-4, ICSM-10, ICSM-18, ICSM-35 and ICSM-37 optimized at IgG 10·5 µg ml1, and ICSM-4, ICSM-18 and ICSM-35 also as biotinylated IgG 0·25 µg ml1 were used as primary antibody. Anti-mouse (Fab) IgG-horseradish peroxidase or streptavidin-horseradish peroxidase (Sigma-Aldrich) was used for detection, and visualized by Supersignal chemiluminescent ECL or ECL-Plus substrate (Pierce) and Kodak Biomax MR film.
The infected and normal brain homogenates were titrated by Western blotting for PrP detection at dilutions from 1 to 0·0001 % (w/v), and for PK digestion at 2200 µg ml1, using ICSM-18. As a negative control, mAbs BRIC-222 (IgG1) raised to CD44, anti-D (IgG1)-anti-Rh blood group related, and BRIC-126 (IgG2b) raised to CD47 were used (IBGRL).
Immunoprecipitation.
Immunoprecipitation was carried out either directly, using antibody covalently cross-linked to beads to capture PrP, or indirectly, using beads to capture antibodyantigen immune complexes. Diseased and normal brain homogenates were used, matched for their PrP signal.
For the direct method, protein-A Dynabeads or M-280 streptavidin beads (Dynal Biotech) were used. Protein-A beads have a maximum binding capacity of mouse IgG 100 µg beads ml1 (2·7x109 beads ml1). For maximum binding, we incubated 100 µg IgG per 100 µl beads, and the immobilized immunoglobulin was cross-linked to the beads using 20 mM dimethyl pimelimidate (Sigma-Aldrich). M-280 streptavidin beads (binding capacity of 50100 µg biotinylated-IgG ml1) were used with biotinylated mAbs. mAbs IgG were biotinylated by incubating 500 µl IgG (1 mg ml1) with Biotin-LC-NHS ester (2 mg ml1) (IGEN International), at a 1 : 20 molar ratio, and quenching the reaction with 20 µl 2 M glycine. Free biotin was removed using a PD-10 column (Amersham Bioscience). To allow binding of both high and low affinity mAbs, IgG-adsorbed streptavidin or cross-linked protein-A beads (2025 µl) were incubated with 100200 µl of 0·50·1 % (w/v) brain homogenates for 812 h at 4 °C, then washed briefly, 68 times in PBST (1 % Tween 20). To determine the PK-resistance of the bead-adsorbed PrP, these beads were treated with PK (20 µg ml1 for mouse and 50 µg ml1 for human PrPSc), for 1 h at 37 °C, and the reaction was stopped with 20 mM PMSF or AEBSF [4-(2-aminoethyl)-benzenesulphonyl fluoride hydrochloride] (Roche). For a direct capture of PrP2730, the brain homogenates were treated with PK before immunoprecipitation.
The indirect immunoprecipitation method was carried out by incubating anti-PrP mAbs IgG at a concentration of 23 µg ml1 with brain homogenate [200 µl 0·50·1 % (w/v)] containing a complete protease inhibitor cocktail (Roche), for 1214 h at 4 °C; to allow even the weak antibodies to bind. Protein-G Dynabeads (2025 µl) (Dynal Biotech), with a binding capacity of IgG 200 µg ml1, were then added for 2 h at room temperature, to immunoprecipitate the immune complexes. The beads were then washed four times with PBST (2 % Tween 20), four times with PBS containing 2 % Tween 20 and 2 % NP40, and once with PBS to remove the detergents. The captured PrP was visualized by Western blot.
Adsorption of PrP to beads was optimized by varying the incubation time, temperature, adsorption buffer, salt concentration, pH and detergent. Both isotype control negative mAb- and PBS-adsorbed beads were negative when using normal brain, but a small amount of non-specific binding occurred with scrapie-affected brain homogenate that could not be eradicated. Using tosyl-activated Dynabeads (Fischer et al., 2000) did not improve non-specific PrPSc binding, which occurred at longer film exposures or higher brain homogenate concentrations.
Immunocytochemistry.
RML-inoculated mice have minimal brain plaque formation; thus we used terminally scrapie-sick SJL mice, inoculated with mouse passaged-BSE prions, that produced spongiform changes associated with large discrete PrP-reactive plaques (Lloyd et al., 2004). Age matched PBS-inoculated SJL mice were used as controls.
A standard PrPSc detection protocol was used (Bell & Ironside, 1997). Briefly, formaldehyde (10 %) fixed brains were immersed in 98 % formic acid for 1 h to denature infectious prions. Treated brains were then embedded in paraffin wax and 4 µm sections were cut. These brain sections were autoclaved and treated with formic acid and 4 M guanidine thiocyanate to abolish the PrPC signal. To label PrPSc, ICSM-4, ICSM-10, ICSM-18 and ICSM-35 were used as primary antibody, followed by avidinbiotin complex, and DAB (3',3-diaminobenzedine) as the chromagen. The staining of PrP was optimized by titrating mAb IgG, using appropriate controls throughout.
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RESULTS |
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Three of the five antibodies (ICSM-4, ICSM-10 and ICSM-18) were raised to -PrP and two (ICSM-35 and ICSM-37) to
-PrP (Table 1
). As previously described (Khalili-Shirazi et al., 2005
) using a peptide ELISA, ICSM-18 recognized PrP residues 142153, ICSM-35 residues 93105 and ICSM-37 residues 97105. ICSM-4 and ICSM-10 could not be epitope-mapped by peptide ELISA. The isotype of ICSM-4, ICSM-10 and ICSM-18 was IgG1, ICSM-35 was IgG2b and ICSM-37 was IgG2a (data not shown).
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In wild-type mouse brain homogenate, a truncated PrP band 18 kDa was also recognized by ICSM-4, ICSM-10 and weakly by ICSM-18 and ICSM-37, and a truncated band at 2022 kDa was recognized in scrapie-infected mouse brain homogenate by all the mAbs (Fig. 1a
). Both the 18 kDa and 2022 kDa truncated bands were PK-sensitive and, since they were both recognized by ICSM-4, they are probably unglycosylated. A PK-sensitive 18 kDa truncated band (C1) and a PK-resistant 2022 kDa band (C2) in CJD brain have been reported previously (Chen et al., 1995
), as have minority C-terminal smaller fragments that would not be visualized by the majority of our antibodies with more N-terminal epitopes.
PrP glycoforms are associated in native PrPSc but not in PrPC
To investigate whether an individual PrPSc particle in infected brain consists of a single glycoform or a mixture, we used our glycoform-specific mAbs, ICSM-4 and ICSM-10, to capture PK-resistant PrP from infected brain homogenates. The unique properties of ICSM-4 and ICSM-10 allowed the individual PrP glycoforms, which comprise native PrPC, to be selectively captured from homogenates of normal murine brain. ICSM-4 immunoprecipitated a single band corresponding to the unglycosylated form of PrP, whereas ICSM-10 captured two principal bands corresponding to the unglycosylated and monoglycosylated isoforms (Fig. 2a). In contrast, immunoprecipitation from scrapie-infected homogenates by ICSM-4 and ICSM-10 was either negative or the three PK-resistant PrP glycoforms were only weakly captured (Fig. 2a
). PrPSc glycoforms could not be separated, regardless of the glycoform specificity of the mAb used. Thus, ICSM-4 and ICSM-10 could selectively immunoprecipitate PrPC glycoforms from normal brain homogenates, whereas PrPSc glycoforms always co-immunoprecipitated as a complex from prion-infected brain homogenates.
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The high affinity of ICSM-18 for PrPC was demonstrated not only by its strong recognition of native (Fig. 3b) and denatured PrPC (Fig. 4c
), but also by its depletion of native PrPC from a normal human brain homogenate after immunoprecipitation (Fig. 4c
).
Residues 93105 in PrPSc are resistant to proteolysis with PK, but are exposed to antibody binding
mAbs raised to -PrP, such as ICSM-35 and ICSM-37, could immunoprecipitate both native PrPC and PrPSc. Indeed, ICSM-35 had a higher affinity for native PrPSc than PrPC, while ICSM-37 had a high affinity for both native PrPC and PrPSc (Fig. 2a
). ICSM-35 and ICSM-37 immunoprecipitate all three PK-sensitive PrPC and three PK-resistant PrPSc bands from normal and prion-infected mouse (Fig. 2a
) and human brain homogenates (Fig. 3a
). As previously described (Khalili-Shirazi et al., 2005
), we showed by ELISA that these
-PrP-derived mAbs are directed to epitopes comprising residues 93105 and 97105 of PrP, which are unstructured in PrPC. The structure of PrPSc is unknown, but this N-terminal region of PrPSc is believed to be important in conferring some strain-specific properties of prions, and is thus likely to have at least a degree of conformation that may vary between strains. Structural heterogeneity within the N terminus may explain the different affinities observed for ICSM-35 and ICSM-37, for native PrPC and PrPSc.
ICSM-35 robustly immunoprecipitated native human PrPSc regardless of the method used, whether the vCJD brain homogenate was treated with PK after immunocapture (Fig. 3a) or before (Fig. 3b and c
). Although both ICSM-35 and 3F4 immunoprecipitated native PrPC from normal and vCJD human brain homogenates (Fig. 3b
), ICSM-35 and 3F4 singularly failed to immunoprecipitate PrPSc at IgG concentrations of 3 µg ml1 (Fig. 3b
) or 20 µg ml1 (Fig. 3c
).
We conclude that residues 93105 of PrP, comprising the epitope for ICSM-35, are exposed in human and mouse PrPSc. The efficiency of ICSM-35 binding to native PrPSc was evidenced by its ability to almost deplete a vCJD brain homogenate (0·25 % w/v) of its PrPSc signal (Fig. 4b). The high affinity of ICSM-35 for native PrPSc was also confirmed, when no measurable dissociation of mouse-scrapie brain-derived PrPSc was observed from the PrPSc-bound covalently coupled ICSM-35-protein-A beads, after washing the beads in excess PBST for 12 h at 4 °C (data not shown). A titration of scrapie-affected mouse brain homogenate also showed ICSM-35 capturing PrPSc from as little as 0·01 % (w/v) brain homogenate (data not shown).
The glycoform profile of PrPSc immunoprecipitated by ICSM-35 exactly matched the known glycoform profile of the infected brain. Diglycosylated dominant PrPSc bands from vCJD-affected brains and monoglycosylated dominant PrPSc from sporadic CJD-affected brain homogenates were immunoprecipitated by ICSM-35 (Fig. 5). This suggests that PrPSc aggregates are highly ordered, and the glycoform ratios specific for each strain are reflected in the prion polymer itself.
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DISCUSSION |
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Having shown that PrPSc particles in a given prion strain are composed of a characteristic spectrum of glycoforms, it is interesting to consider mechanisms that can account for this observation. With respect to the formation of the PrPSc particles, there are two mechanisms that can lead to a defined characteristic ratio of components which constitutes a prion strain. In a stoichiometric assembly process, the formation of prion particles occurs by the exact packing of the three glycoforms. For instance, a given PrP conformation and packing arrangement, which constitutes a strain, might only allow direct 2 : 4 : 2 stoichiometry of the three glycoforms, i.e. there is a discreet, whole-number molecular stoichiometry. By contrast, in a statistical or probabilistic process, a strain will be characterized by having different on and off rates for addition of each glycoform to the growing prion particle. For instance, assuming a negligible rate of dissociation, the ratio of the on-rates for each glycoform would exactly reflect the ratio in the assembled prion particle. However, unlike the case of the stoichiometric model, there would not be an exact packing arrangement that only allows precise stoichiometries; rather there would be a given probability of finding a particular glycoform at a particular location in the structure of the fibril.
Studying the appearance of PrPSc deposits in the brains of mice with terminal scrapie using our panel of mAbs showed that, regardless of the mAb's glycoform specificity, large PrP-reactive plaques were successfully recognized in the corpus callosum of diseased brain. These brain sections were treated such that native PrPC was destroyed and PrPSc was denatured; hence, the mAbs should recognize their specific PrPSc glycoforms if their epitope is available. A higher concentration of ICSM-4 than ICSM-18 or ICSM-35 was required to detect the plaques, possibly reflecting lower unglycosylated PrP expression in the brain or that the epitope of ICSM-4 is partially occluded or destroyed by tissue processing, but remains an integral component of disease related PrP. ICSM-35 was more sensitive in detecting PrPSc in the diseased brain than ICSM-18, although they both recognized all three PrPSc glycoforms. It is possible that the 93105 epitope of ICSM-35 is more frequently exposed in the plaques and PrPSc deposits in the brain than the 142153 epitope of ICSM-18.
Finally, we found that native PrPSc could strongly be immunoprecipitated from human and mouse prion-infected brain homogenates by ICSM-35 and ICSM-37, reactive to PrP residues of 93105 and 97105. Our findings with ICSM-35 and ICSM-37 are in contrast to the previous report (Peretz et al., 1997), suggesting that residues 90120 are accessible in PrPC, but cryptic in PrPSc. In that study, mAbs 3F4 (epitope aa 104113) and D13 (epitope 96104) immunoprecipitated native Syrian hamster (SHa) PrPC and denatured SHa PrP2730, but were incapable of immunoprecipitating native SHa PrP2730, and it was deduced that the 90120 region is exposed in PrPC but is largely cryptic in PrP2730 (Peretz et al., 1997
). In agreement with previous findings, we found that 3F4 could successfully immunoprecipitate human native PrPC, but failed to capture native PrP2730 from vCJD brain. ICSM-35, raised to human
- PrP91231 (epitope 93105), shows a similar pattern of PrP2730 (types 14) recognition as 3F4 in Western blots of PK-treated CJD brain homogenates. However, in contrast to 3F4, ICSM-35 and ICSM-37 (epitope 97105) efficiently immunoprecipitated all the PrP2730 glycoforms from prion-infected human and mouse brain homogenates, indicating that residues 93105 are not only exposed in native PrPC, but are at least partially also exposed in human and mouse native PrPSc. In confirmation, five further mAbs that were raised to human
-PrP91231 and could readily immunoprecipitate native PrPSc were also directed to residues 91105 (unpublished data). The only amino acid difference between SHa and human prion proteins within residues 91105 is at position 97, with human PrP containing serine (S) and hamster asparagine (N). However, since ICSM-35 and ICSM-37 both successfully capture mouse native PrP2730, which like SHa PrP has an N at amino acid position 97, this should have no bearing on immunoprecipitation of SHa and human native PrP2730. Since there are no amino acid differences between human and SHa PrP at residues 105112, it remains possible that the difference observed in PrP2730 immunoprecipitation by mAbs 3F4, D13, ICSM-35 and ICSM-37 is either due to a strain effect the conformation of this region being distinct in hamster scrapie or occluded by another ligand in hamster native PrP2730. Alternatively, the PrP molecules used to produce 3F4 and to enrich for D13 may not resemble native PrPSc in this region. In contrast, recombinant
-PrP has proven to be an extremely useful immunogen for raising mAbs with high affinity for native PrPSc. In view of our findings, we deduce that the region encompassing residues 93105 is not buried or altered in PrP2730 in prion-infected human and mouse brains, and that this region appears to be a highly antigenic epitope for mAbs raised to
-PrP91231.
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ACKNOWLEDGEMENTS |
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Received 17 June 2004;
accepted 2 June 2005.