Brown1,2 1 Department of Biology and Biochemistry, University of Bath, UK; 2 Department of Biochemistry, Cambridge University, UK; 3 Institute of Pathology, Case Western Reserve Universi
Trang 1Mapping the functional domain of the prion protein
Taian Cui1, Maki Daniels2, Boon Seng Wong3, Ruliang Li3, Man-Sun Sy3, Judyth Sassoon1
and David R Brown1,2
1 Department of Biology and Biochemistry, University of Bath, UK; 2 Department of Biochemistry, Cambridge University, UK;
3 Institute of Pathology, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA
Prion diseases such as Creutzfeldt–Jakob disease are
pos-sibly caused by the conversion of a normal cellular
glyco-protein, the prion protein (PrPc) into an abnormal isoform
(PrPSc) The process that causes this conversion is unknown,
but to understand it requires a detailed insight into the
normal activity of PrPc It has become accepted from results
of numerous studies that PrPcis a Cu-binding protein and
that its normal function requires Cu Further work has
suggested that PrPcis an antioxidant with an activity like
that of a superoxide dismutase We have shown in this
investigation that this activity is optimal for the whole
protein and that deletion of parts of the protein reduce or abolish this activity The protein therefore contains an active domain requiring certain regions such as the Cu-binding octameric repeat region and the hydrophobic core These regions show high evolutionary conservation fitting with the idea that they are important to the active domain of the protein
Keywords: copper; Creutzfeldt–Jakob disease; oxidative stress; scrapie; superoxide dismutase
Neurodegenerative diseases are a major threat to human
health One group of disease termed prion diseases [1,2]
make up a small percentage of all human neurodegenerative
diseases Prion diseases have become a major concern
because of the possibility that one particular from, variant
Creutzfeldt–Jacob disease (vCJD), might arise through
transmission of an animal disease, such as bovine
spongi-form encephalopathy [3], to humans [4] Other prion
diseases include the sheep disease scrapie [5] and inherited
forms such as Gerstmann–Stra¨ussler–Scheinker syndrome
[6] All of these disease are linked together because of the
deposition of an abnormal, protease-resistant isoform of the
prion protein in brains of individuals with these diseases
This abnormal form of the protein (PrPSc) is also suggested
to be the infectious agent in the disease on the basis of
infection studies [2]
PrPScis generated from the normal cellular isoform of the
prion protein (PrPc) which is present in the brain as a cell
surface glycoprotein [7] Each form has distinct properties
[8] Therefore understanding the basis of prion disease
revolves around understanding how the normal protein is
converted to the abnormal isoform This conversion
involves a switch in conformation from a structure rich in
a helices to one rich in b-sheet [9] Although there have been
many studies with PrPScthe study of PrPchas been limited
until recently As an evolutionarily conserved glycoprotein [10] it has been postulated that PrPc has an important function Nevertheless, knockout mice for PrPc show no gross changes in terms of development or behaviour [11] but cannot be infected with mouse-passaged scrapie [12] In contrast to this biochemical and cell biological studies have suggest that PrP-knockout mice have compromised cellular resistance to oxidative stress [13,14]
The first clue to the molecular function of PrPccame from studies that show PrPcto be a Cu-binding protein [15–20] The main Cu-binding site of the protein was shown to be within a conserved octameric repeat region, rich in histidine, located in the N terminus [10] PrPcbinds up to four atoms
of Cu at these sites with a possible fifth binding site located elsewhere in the molecule [16,18,21] Cellular expression of PrPc also facilitates Cu uptake by neurones [22] and increased extracellular Cu causes an increased turnover of PrPc[23] Binding of Cu to the protein influences its ability
to interact with other proteins such as plasminogen [24] and glycosaminoglycans [25]
Knockout of PrPccauses a decrease in cellular resistance
of neurones to oxidative stress [13,14,26] This has lead to suggestions that PrPcmight be an antioxidant Immuno-depletion of PrPc from the brain extracts leads to a reduction in superoxide dismutase (SOD) activity within the extract [27] Studies with both recombinant protein and native protein purified from the brains of mice suggest that PrPccan act as a SOD [17,28] This activity is high and requires specific binding of Cu to the octameric repeats Binding of Cu elsewhere in the protein, or Cu simply to a peptide based on the octameric repeats does not result in this activity [28] Cellular resistance to oxidative stress is influenced by the PrPcprotein and the amount of Cu bound
to it [17] Allelic differences in mouse PrPchave also been shown to influence the level of the activity of the protein, as protein with the sequence of the mouse b allele is more
Correspondence to D R Brown, Department of Biology and
Bio-chemistry, University of Bath, Calverton Down, Bath, BA2 7AY, UK.
Fax: +44 1225 826779, Tel.: +44 1225 323133,
E-mail: bssdrb@bath.ac.uk
Abbreviations: CJD, Creutzfeldt–Jacob disease; vCJD, variant
Creutzfeldt–Jacob disease; PrP c , prion protein; PrP Sc , abnormal
isoform of prion protein; rPrP, recombinant mouse prion protein;
SOD, superoxide dismutase.
(Received 28 April 2003, revised 5 June 2003, accepted 11 June 2003)
Trang 2active than that based of the sequence of the a allele [29] In
contrast, PrPSc, which binds almost no Cu has no detectable
SOD activity [30,31]
Proteins that are enzymes normally have active sites that
are essential for the enzymatic activity In this study we used
both a panel of highly specific antibodies and a series of
deletion mutants of recombinant PrP to determine which
regions of the protein are necessary for the SOD activity
We determined that the active site consists of two domains
The first includes the Cu-binding domain and the second
includes the conserved hydrophobic domain in the middle
of the protein Additionally, the C terminus of the protein is
important for this activity
Experimental methods
Production of recombinant protein
Production of recombinant mouse prion protein (rPrP) has
been described previously [28] Briefly, PCR amplified
product was cloned in the expression vector, pET-23
(Novagen) and transformed into Escherichia coli
AD494(DE3) The expressed proteins were recovered from
urea solubilized, sonicated bacterial lysate after using
immo-bilized nickel-based affinity chromatography (Invitrogen)
The eluted material was refolded by several successive rounds
of dilution in either deionized water or 1 mM CuSO4
fol-lowed by ultrafiltration and dialysis to remove unbound Cu
The final protein was typically >95% pure and was
concen-trated to 1–2 mgÆmL)1 Its identity was confirmed by
N-terminal sequencing and Western blotting using the
poly-clonal antibody to mouse PrP (DR1) Protein concentration
was determined using the Sigma BCA protein assay reagent
Mutagenesis
Deletion mutants of the rPrP were prepared using a PCR
based mutagenesis procedure involving paired
oligonucleo-tides to either insert an additional restriction site or to delete
a proportion of the gene sequence Mutagenesis was
confirmed by DNA sequencing The mouse PrP ORF was
inserted between the Nde1 site ( 5¢) and the Xho1 site ( 3¢) An
additional Xho1 site was inserted either after codon 171, or
codon 112 Removal of an Xho1 fragment by enzymatic
digestion and subsequent ligation created the deletion
mutants PrP23–112 and PrP23–171 A similar procedure
was used to produce PrP45–231, PrP90–231, PrP105–231
and PrP113–231 In this case an Nde1 site was inserted
before codon, 45, 90, 105 and 113, respectively Paired
primers were also used in mutagenesis experiments to
generate deletions of codons 35–45 (PrPD34–45), 112 to136
(PrPD112–136) and 135–150 (PrPD135–150) The
oligo-nucleotides used in these mutagenesis experiments are listed
in Table 1 Other prion protein mutations generated in a
similar way were as described previously [28,32,33] Protein
for these deletion mutants was expressed, purified and
refolded as described above for wild-type protein
SOD assays
SOD-like activity of recombinant PrP (1 lgÆmL)1) was
determined using the xanthine/xanthine oxidase/nitro-blue
tetrazolium (NBT) assay as described before [28] This assay uses superoxide production from xanthine oxidase and xanthine and detection of a coloured formazan product formed from nitro-blue tetrazolium at 560 nm The SOD-like activity was expressed as percentage inhibi-tion of formazan produced where 100% formazan product formation is the amount of nitro-blue tetrazolium reduced
by xanthine oxidase-formed radicals in control reactions without brain extracts or affinity-purified PrP All assays were performed in triplicate The proteins used were tested for their ability to reduce nitro-blue tetrazolium in the absence of xanthine oxidase None of the proteins showed any reduction of nitro-blue tetrazolium to form formazan
as measured spectrophotometrically for 5 min Also, xanthine oxidase was driven to reduce nitro-blue tetra-zolium aerobically by the addition of 50 lMxanthine to the reaction mixture A second gel-based assay was also used
to detect SOD activity Proteins (5–20 lg) were electro-phoresed on a 7% polyacrylamide gel without SDS or reducing agents After electrophoresis, the gel was soaked
in a solution of 5 mM nitro-blue tetrazolium at room temperature with rocking for 20 min The gel was then rinsed briefly with distilled water and a developing solution (30 lM riboflavin, 30 mM tetramethylethylenediamine,
40 mM potassium phosphate pH 7.8) for 15 min At this point the gel was exposed to the light until a uniform blue colour covered the gel Protein with SOD reactivity leaves the gel transparent However, if the reaction was allowed
to proceed indefinitely the contrast between these regions would be lost
Western blotting Purified proteins were electrophoresed on a 15% polyacryl-amide gel in the presence of SDS and reducing agents Proteins were blotted onto polyvinylidene fluoride (PVDF) membrane and protein detected by a specific polyclonal (DR1) or monoclonal (DM3) antibody as described previ-ously [32] This allowed verification of the size and identity
of these proteins
Table 1 Mutagenesis oligonucleotides Only forward oligonucleotides are listed The reverse oligonucleotide of the splint pair had the com-plementary sequence.
Prion protein generated Oligonucleotide PrP23–112 GCATGTGGCAGGGCTCGAGGCAGCTGGGGC
PrP23–171 GCAACCAGCTCGAGTTCGTGCACG
PrP45–231 GGGAAGCCATATGGGCAACCG
PrP90–231 GCCCCATGGCGGTGGATGGCATATGGGAGG
GGGTACCC
PrP105–231 GGAACAAGCCCAGCCATATGAAAACCAACC
TCAAGC
PrP113–231 CCAACCTCAAGCATATGGCAGGG
PrPD35–45 GGGTGGAACACCGGTGGCAACCGTTACCC
PrP112–119 CCTCAAGCATGTGGTAGTGGGGGGCC
PrPD112–136 CCAACCTCAAGCATGTGATGATCCATTTTGGC
PrPD135–150 GCGCCGTGAGCGAAAACATGTACCGC
Trang 3CD spectroscopy
CD spectra were recorded for prion proteins and peptides
using a Jasco J-810 spectropolarimeter, calibrated with
ammonium d-camphor-10-sulfonate by a method similar to
that described previously [27] Protein solutions were
prepared to contain 2 mgÆmL)1 in 10 mM sodium
phos-phate pH 7.4 These samples were measured in cuvettes of
1 mm or 0.5 mm pathlength (Hellma) The spectrum from
190 nm to 250 nm was analysed with step resolution of
0.5 nm at a temperature of 23C Five scans were averaged
and the buffer background was subtracted Spectra are
presented as molar ellipticity (h)
Results
Antibody inhibition of PrP SOD-like activity
A panel of highly specific monoclonal antibodies and
polyclonal antisera were generated against mouse PrP and
have been described previously [32,34–36] The epitopes of
these antibodies have been mapped and are listed in
Table 2 The activity of wild-type PrP is like that of a SOD
and this activity can be measured by a number of assays
The most robust and accessible method for such a study
uses spectrophotometric analysis We used an assay based
on formazan production from nitro-blue tetrazolium by
superoxide generated by xanthine oxidase and xanthine
SOD activity inhibits formazan production in the assay by
breaking down superoxide This assay was used to measure
the activity of wild-type PrP A concentration of
0.5 lgÆmL)1 PrP was found to inhibit 70% of the
formazan production in the assay This concentration was
used in further experiments in which antibodies or antisera were added in conjunction with PrP to the SOD assay The results of these experiments are shown in Fig 1 Several of the antibodies and antisera caused a concentration-dependent inhibition of the SOD-like activity of PrP The ability of the antibodies and antiserum to inhibit the SOD-like activity of PrP is sumarized in Table 2 Antibodies and antisera are listed according to the epitope to which they bind It can be clearly seen that the antibodies and antisera that inhibit PrP’s activity are clustered around two parts of the protein The first cluster is in the N terminus The second cluster is focused on the hydrophobic domain, residues 112–145 Two antibodies that bind to the C terminus also had a minor inhibitory effect on the SOD-like activity of PrP
Production of deletion mutants of PrP
On the basis of the results with antibodies, a series of PrP mutants were made to assess whether deletions of certain domains of the protein decrease the SOD-like activity of the protein The domains deleted were based on the epitopes of the antibodies that had a clear effect on SOD-like activity of PrP The mutants used in the study include the complete N- and C-terminal fragments (PrP23–112 and PrP113–231), deletions of the octameric repeat region (PrPD51–89, PrPD67–89), deletions of the hydrophobic domain (PrPD112–119, PrP112–136, PrP135–150), deletions of parts
of the N terminus (PrP90–231, PrP45–231, PrPD35–45, PrP) and deletions of the C-terminal domain (PrP23–171) These proteins are illustrated in Fig 2 A number of these proteins have been studied [28,32] The identity of the proteins was verified by Western blot with specific antibodies (Fig 3) SOD-like activity of PrP deletion mutants
The activity of the PrP mutant proteins were compared to that of the wild-type recombinant PrP using two assays that have been widely used to detect SOD activity An in-gel assay (Fig 4) and a spectrophotometric assay (Fig 5) detected high levels of activity in wild-type protein How-ever, most of the mutations tested showed either no activity
or reduced activity A summary of these findings is shown in Table 3 Some results from previously published work are included for completeness The in-gel assay showed visually that wild-type PrP has strong activity while the mutants PrPD35–45, PrP23–171, PrPD112–119 and PrP45-231 had reduced activity PrPD112–136 had no activity The spec-trophotemetric assay was performed (Fig 5) with increasing concentrations of protein from all the mutants It should be kept in mind that for mutants with large deletions the concentration of 5 lgÆmL)1 represents a higher molar concentration than wild-type protein However, these proteins except for PrP23–171 were inactive in the assay All mutants lacking the octameric repeat region were inactive Most of the mutants with small deletions showed some activity except PrPD112–136 This mutant was completely inactive Of considerable interest was the mutant PrP23–171 which despite a lack of a large amount of the C terminus did show some activity To test the stability of this activity a time-course study was carried out to compare the activity of this protein to wild-type PrP The proteins were
Table 2 Epitopes of antibodies and antisera used in the investigation of
PrP activity Numbers relate to the amino acid residue sequence of
mouse prion protein Conformational implies antibodies that bind to
the C terminus of the protein (145–231) The affinity of these
anti-bodies is sensitive to the conformation adopted by the protein.
Antibody
Ability to Inhibit Activity
Trang 4added to the assay mixture at time zero and the activity measured for 60 s After this time the activity of the protein was measured repeatedly at regular intervals for the next hour (Fig 5D) Where as wild-type PrP maintained its activity over the hour, PrP23–171 lost the majority of its activity over the same time period
CD analysis of PrP mutants
In order to determine if deletion of critical regions of PrP caused loss of activity because of structural alterations, the deletion mutants were studied using CD spectroscopy The spectra produced are shown in Fig 6 Of key interest were those with minimal deletions of protein sequence which caused significant reduction in activity of the protein The majority of the deletion mutations did not cause significant changes in the structure of PrP As suggested from previous publications the N terminus (PrP23–112) showed a spec-trum typical of a random coil (Fig 6E) All other spectra demonstrated predominantly helical content Interestingly, PrP23–171, which has deletions of two of the helical domains of the PrP protein, also possessed a high content of helical structure
Activity domains of PrP This body of research has provided two sets of results concerning regions of the prion sequence necessary for its
1 0 0 0
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Fig 1 Antibodies The activity of wild-type recombinant mouse PrP
was tested using a spectrophotometric assay based on the conversion
of nitro-blue tetrazolium to a coloured formazan product by
super-oxide generated from xanthine oxidase PrP (0.5 lgÆmL)1) was used in
the assay which inhibited the reaction by 70% (see Fig 5)
Anti-bodies and antisera were then tested for ability to block the effect of
PrP PrP activity in the presence of the antibodies was expressed as a
percentage of the activity of PrP alone Thus, decreased percentage of
control activity indicates inhibition of PrP (Top) Effect of three
antisera of equivalent titre Middle and bottom graphs show effects of
15 monoclonal antibodies Shown are the mean and SEM of at least
three experiments.
PrP23-231 PrP23-112 PrP23-171 PrP45-231 PrP90-231 PrP105-231 PrP113-231 PrP ∆35-45 PrP ∆51-89 PrP ∆51-67 PrP ∆112-119 PrP ∆112-136 PrP ∆135-151
171
45
90
105
113
35
67
119
136
135 150
231 231
231
231
231
231 231
231
231
231
112
112 51 45 23
23
23
23
23
23
Fig 2 Mutant proteins Mutants of PrP were prepared using PCR-based mutatagenesis and restriction digestion/ligation The mutations were deleted in parts of the protein that would possibly reduce the activity of PrP based on results shown in Fig 1 and Table 2 Schematic locations of the deletions as compared with the wild-type protein are shown by a space within the grey bar next to the name of the protein Numbers refer to the amino acid residues in the mouse PrP sequence.
Trang 5function Results of the use of antibodies on wild-type
protein and deletion mutants indicate the relative
importance of these domains to PrP’s SOD-like activity
Comparison of data presented in Tables 2 and 3
indicates that there are principally two domains that
are necessary for this activity The first is the Cu-binding
domain otherwise known as the octameric repeat region
The second is the hydrophobic domain in the centre of
the molecule A third domain in the N-terminal region
before the Cu-binding domain also has a strong influence
on activity Further analysis indicates that the C terminus
influences the activity to some extent but is not essential
Collectively, these results suggest that the activity of PrPc
requires N- and C-terminal domains which might interact
to form the active site These results are summarized in Fig 7
Fig 3 Western blotting Wild-type mouse PrP and nine of the deletion
mutants described were electrophoresed on a 15% polyacrylamide gel
and transferred to a membrane PrP was detected by using the antisera
DR1 which detects all of the mutants shown 1, Wild-type PrP;
2, PrP23–231; 3, PrP23–171, 4; PrPD35–45; 5, PrPD135–150;
6, PrPD112–119; 7, PrP90–231; 8, PrPD51–89; 9, PrP45–231;
10, PrPD112–136.
Fig 4 In-gel assay An in-gel assay was used to provide a visual
demonstration of the SOD-like activity of wild-type PrP and some of
the active mutants Five lg protein was electrophoresed on a native
polyacrylamide gel and stained for SOD-like activity 1, Wild type PrP
protein; 2, PrPD35–45; 3, PrPD112–136; 4, PrP23–171; 5 PrPD112–119;
6, PrP45–231.
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Fig 5 Activity of deletion mutants The nitro-blue tetrazolium/xan-thine/xanthine oxidase assay for SOD activity was used to assess the affect of deletions on the activity of recombinant PrP (A) Wild-type PrP (d), PrP23–171 (s), PrP45–231 (h), PrP90–231 (j) PrP23–112 (s) and PrP105–231 (n) (B) Wild-type PrP (d), PrP113–231 (s), PrPD35–45 (h), PrPD51–89 (j) PrPD67–89 (s) (C) Wild-type PrP (d), PrPD112–119 (s), PrPD135–150 (h), PrPD112–136 (j) Shown are the mean and and standard errors for at least three separate experi-ments (D) The activity of wild-type PrP (d) and PrP23-171 (s) were recorded over time One lg protein was added to the nitro-blue tetra-zolium/xanthine oxidase assay mixture and measured for SOD-like activity in terms of inhibition of formazan production From this zero time point the SOD-like activity was remeasured 5, 15, 30, 45 and
60 min later Results are expressed as a percentage of the time zero value at 560 nm Shown are the mean and SEM for at least three separate experiments.
Table 3 Relative activity of PrP deletion mutants Activity relative to the wild-type PrP is indicated by the number of + signs: +++++, activity equivalent to wild type; –, no activity Recombinant protein is not glycosylated but native protein has equivalent activity to recom-binant protein [17] Reactive cleavage of the disulfide bond has been shown to decrease the activity of PrP [48].
PrP deletion mutant Relative activity
Trang 6The prion protein is a Cu-binding protein Early estimates
of the affinity of Cu for PrPcsuggested that binding of Cu
was within the micromolar range [15,16] Assessment of
PrPc-mediated Cu uptake suggested that PrPcinfluences Cu
distribution within the nanomolar range [22] The
implica-tion of this is an affinity between Cu and PrPc in the
nanomolar range or lower Recent studies have also
suggested that the affinity of Cu for PrPccould be in the
femtomolar range [18] Despite these inconsistencies, the
conclusion that PrPc is a Cu-binding protein is widely
accepted The implication of this is that PrPcis somehow
involved in Cu metabolism Cu in the body is tightly linked
to redox chemistry and regulation of the balance between
the use of oxygen in respiration and possible oxidative
damage Thus, without further consideration PrPc is
implicated in regulation of cellular resistance to oxidative
stress However, there is also considerable evidence that PrPcis an antioxidant [14] This was first suggested in 1995 [13] Much of this evidence comes from studies of PrP knockout mice Changes including electrophysiological parameters [37] and altered sleeping patterns [38] in PrP knockout mice have been linked to loss of antioxidant protection [39,40] PrP knockout mice are also more susceptible to kindling agents [41] The effect of such agents
is related to the induction of oxidative stress [14] Cultured cells are also more susceptible to oxidative stress when they lack PrPc expression [13,24,42,43] PC12 cells expressing increased levels of PrPcare more resistant to oxidative stress [44] These findings were further clarified when it was shown that recombinant and native PrPccan act as superoxide dismutase [17,28] Subsequently it has been shown that this activity is dependent of the Cu binding of the protein [45] Transfection of cells to express PrPcincreases their ability to reduce intracellular levels of oxidants [43] Depletion of PrPc from cells reduces their total superoxide dismutase activity [27] Loss of PrPcexpression by cells is compensated for by specific up-regulation of other SODs including manganese SOD [46] and extracellular SOD [14] Therefore there is a strong body of evidence linking PrPcto SOD-like activity Although some scepticism about the antioxidant function of PrPcremains [47,48], there are sufficient data to consider this as a potentially important enzymatic function of this protein
Data presented in this work verifies the previous findings that PrPc is a SOD Two separate assays confirm this suggestion That the recombinant protein was effective in the in-gel assay also verifies that the protein is not a weak SOD-like protein but one with equivalent catalytic ability to that of cytoplasmic Cu/Zn SOD The specific activity of PrP has been shown previously to be about 10-fold less than that
of Cu/Zn SOD [28] However, this Cu/Zn SOD is widely recognized as a very potent catalyst given its ability to catalyse a reaction that would spontaneously occur in minutes or less, in the presence of sufficient concentration of superoxide, in the absence of the enzyme [49]
Analyses of domains in this protein necessary for the SOD-like activity used both deletion mutants and antibod-ies that recognized known epitopes In particular antibodantibod-ies DM1 and 11G5 showed the strongest inhibition of the
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Fig 6 CD of PrP mutants Wild-type and mutant PrPs were studied
by CD spectroscopy (A) Wild-type PrP (B) PrPD35–45 (C)
PrPD112–119 (D) PrP51–89 (E) PrP23–112 (F) PrPD135–150 (G)
PrPD112–136 (H) PrP23–171 Spectra are shown as molar ellipticity
(h).
Necessary Domains
N N 35
Minor Domain
Signal Sequence Octameric Repeats Hydrophobic Domain GPI Anchor Signal
Fig 7 Summary This schematic diagram, based on the results from all experiments, shows those regions of the PrP protein necessary for the SOD-like activity with Cu bound Black bars are essential for activity; grey bars show those regions that also play a role but are not essential; hatched bar indicates that the C-terminal part of PrP cor-responding to the last two helices can also influence the activity of the protein but are not essential.
Trang 7protein’s activity of suggesting that both the octameric
repeat region and the hydrophobic domain were critical for
the activity of the protein This was confirmed by deletion
mutations that showed lack of SOD-like activity when
these regions were deleted Experiments using three other
antibodies binding near the residues 35–45 also suggested
that this region was important Deletion of these residues
confirmed this finding Assays using the antibodies 7H6
and DM3 also indicated that residues 130–160 might also
play a role in the activity However deletion of residues
135–150 had no affect at all on the activity suggesting this
domain is not involved in the activity of the protein This
deletion also served as a good negative control showing
that deletion of part of PrP need not inhibit the SOD-like
activity if it is made outside the domains essential for this
activity
Inhibition of activity by two antibodies that were sensitive
to the conformation of the C terminus indicated that the
conformation of the C terminus is important to the activity
of the protein Deletion of the whole C terminus rendered
the protein inactive but surprisingly deletion of only the last
two helices (PrP23–171) did not lead to inactive protein
Further analysis indicated that this mutant was labile in its
activity and rapidly lost activity when continually exposed
to superoxide This mutant was highly soluble and
con-tained surprisingly high helical content One possibility is
that the N terminus of the protein does not contain a totally
unordered structure when associated with at least one helix
of the C terminus This would contradict findings from
NMR studies suggesting there is no structure in the N
terminus [50] We have also observed the lack of regular
secondary structure along the N terminus with CD analysis
but again this might be different when associated with other
domains of the protein Our findings concerning the
C-terminal domain support what was shown previously
Preventing the formation of the disulfide bridge in the last
two helices reduces the activity of PrP [51] Thus, although
these regions are not essential for the manifestation of the
activity they are important to maintaining that activity
Further evidence for this comes from work with the
different mouse alleles of the protein It was found that
protein generated from the mouse b allele has higher
activity from that of the a allele [29] These alleles differ
only in two amino residues one of which is residue 189 in the
second helix
That the octameric repeat region of the protein is
necessary for SOD function is clear from the fact that this
is the main Cu-binding region of the protein Although it
has been suggested that Cu binds elsewhere in the
molecule [18,21] it is not clear if this occurs in vivo and
may only occur under nonphysiological conditions or
when the N-terminal region has been cleaved off
How-ever, PrP90–231 which is equivalent to the protein studied
by Jackson et al [18] also lacked SOD activity Thus, if
Cu does bind elsewhere in the protein this is not relevant
to the protein’s antioxidant activity Deletion of only part
of the octameric repeat region renders the protein inactive
This confirms previous suggestions that binding only one
atom of Cu is not sufficient for significant SOD-like
activity of the protein [17] The importance of residues
35–45 is currently unknown However, it might be that
this region of the protein interacts with other regions of
the protein, possibly to bring the Cu-binding domain into proximity with the hydrophobic domain or the C-terminal globular domain This interpretation is supported by our earlier findings that there are interactions between the
N terminus and the C terminus of PrP Binding of a monocolonal antibody to an epitope located between residues 35 and 45 prevented the binding of another monoclonal antibody that reacts with a conformational epitope in the C terminus [52]
Analysis of the evolutionary conservation of the prion protein among mammals clearly shows that all three critical domains (residues 35–45, 51–89, 112–136) are extremely highly conserved Indeed, the region 112–126 is identical in all mammals, birds and reptiles so far sequenced [10,53,54] Therefore this report indicating that residues 112–136 constitute part of the functional domain of the protein provides a plausible explanation for the high evolutionary conservation of this region Other research has shown the importance of this region in PrP neurotoxicity [55,56] as a binding site for PrP ligands [57,58]
There are already several reports that link oxidative stress to prion disease [59–63] Also, changes in the essential metalloelements in the brains of patients with CJD and experimental mouse scrapie have also been noted [28,29] These findings suggest that changes in Cu meta-bolism and redox balance occur in prion diseases The exact nature of these changes is far from clear However, there is evidence that loss of Cu binding to PrP and consequently, loss of PrP’s antioxidant activity occur early
in the course of prion disease [31] The relevance of the findings presented here are therefore quite important in determining the changes that PrP undergoes during the course of prion disease and the possible role of the loss of its function to the disease It is known that the hydropho-bic domain spanning amino acids 112–136 form a critical site in the protein at which the protein gains b-sheet content [64] This region also spans the site at which normal metabolic cleavage occurs [65] Deletion of this region inhibits conversion of the protein to PrPSc in infected cells [66] The importance of this region to the function of the protein explains its evolutionary conserva-tion Conversion of this site to one that forms b-sheet and facilitates aggregation of the protein is known to prevent cleavage of the protein [67] and abolish antioxidant activity [31] Furthermore, interaction between this site on PrPc and PrPScor the neurotoxic peptides such as PrP106–126 also inhibits the antioxidant activity of PrPc[31] or cause the protein to change conformation [67]
In summary we have provided an insight into regions of PrPc critical to its normal antioxidant activity We have shown that regions outside the octameric repeat region are necessary for this activity These data suggest that a key domain in PrPcthat is involved in structural conversion of the protein to PrPScmay be conserved in evolution because
of its importance to this function
Acknowledgements
Thanks to Dr Laurie Irons for assistance with CD measurements This work was supported by a fellowship from the BBSRC of the UK to DRB.
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