Leu138 in bovine prion peptide fibrils is involved in seeding discrimination related to codon 129 M ⁄ V polymorphism in the prion peptide seeding experiment Tai-Yan Liao, Lily Y.-L. Lee and Rita P.-Y. Chen
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan
Keywords amyloid; bovine spongiform encephalopathy; codon 129; species barrier; vCJD
Correspondence R. P.-Y. Chen, No. 128, Sec 2, Academia Rdoa, Nankang, Taipei 115, Taiwan Fax: +886 2 2788 9759 Tel: +886 2 2785 5696 E-mail: pyc@gate.sinica.edu.tw
(Received 2 June 2011, revised 19 August 2011, accepted 13 September 2011)
doi:10.1111/j.1742-4658.2011.08353.x
The risk of acquiring variant Creutzfeldt–Jakob disease is closely related to polymorphism at codon 129 of the human prion gene, because almost all variant Creutzfeldt–Jakob disease patients are Met ⁄ Met homozygotes. Although animal transmission experiments corroborated this seeding dis- crimination, the origin of the differential seeding efficiency of the bovine prion seed for human codon 129 polymorphism remained elusive. Here, we used a short prion protein (PrP) peptide as a model system to test whether seeding discrimination can be found in this simple system. We used a previ- ously developed ‘seed-titration method’ and time-resolved CD spectroscopy to compare sequence-dependent seeding efficiency regarding codon 129 poly- morphism. Our results showed that the Met fi Val substitution on the human PrP (huPrP) peptide decreased seeding efficiency by 10 times when fibrils formed from bovine PrP (bPrP) peptide were used as the seed. To explore whether the different seeding barrier is due to the chemical and struc- tural properties of Met and Val or whether another residue is involved in this peptide model, we constructed three bPrP mutants, V112M, L138I and N143S, in each of which one residue was replaced by the corresponding human residue. Our data showed that Leu138 in the bPrP seed might be the key residue causing the different seeding efficiencies related to 129M ⁄ V poly- morphism and the interference effect of huPrP129V in the huPrP129M ⁄ V mixture. We propose a ‘surface competition hypothesis’ to explain the big seeding barrier caused by 129V in the PrP peptide seeding experiment.
Structured digital abstract l huPrP aggregates with bPrP by circular dichroism (View Interaction 1, 2) l bPrP aggregates with bPrP by circular dichroism (View interaction) l bPrP aggregates with bPrP by electron microscopy (View interaction) l bPrP aggregates with bPrP by fluorescence technology (View interaction) l huPrP aggregates with huPrP by electron microscopy (View interaction) l huPrP aggregates with huPrP by fluorescence technology (View interaction) l huPrP aggregates with huPrP by circular dichroism (View interaction)
FEBS Journal 278 (2011) 4351–4361 ª 2011 The Authors Journal compilation ª 2011 FEBS
4351
Abbreviations bPrP, peptide corresponding to bovine PrP protein sequence 111–147; huPrP129M, peptide corresponding to human PrP protein sequence 108–144 with a methionine at position 129; huPrP129M ⁄ V, a 1 : 1 mixture of huPrP129M and huPrP129V; huPrP129V, peptide corresponding to human PrP protein sequence 108–144 with a valine at position 129; L138I, mutant bPrP peptide with a Leu-138 fi Ile mutation; N143S, mutant bPrP peptide with an Asn-143 fi Ser mutation; PrP, prion protein; ThT, thioflavin T; V112M, mutant bPrP peptide with a Val-112 fi Met mutation; vCJD, variant Creutzfeldt–Jakob disease.
T.-Y. Liao et al. Seeding discrimination of codon-129 M ⁄ V
Introduction
event in prion formation. This process is well explained by a nucleation-dependent polymerization model [12,22–24]. For spontaneous amyloidogenesis, the con- centration of seed gradually increases during the lag time until a critical point is reached which allows rapid elongation to occur. Interaction between the incoming monomer and the seed stabilizes the cross-b structure and thus facilitates subsequent amyloid elongation. In the elongation phase, the rate of elongation depends on the elongation rate constant (ke), the molar concen- tration of the seed (the number of free ends) and the concentration of the monomer which is able to associ- ate with the seed. If seed is added exogenously during the lag time, this period is shortened to different extents or even eliminated completely, depending on the amount of seed added [25,26].
residue 129.
(M ⁄ M) at
Prion disease is a transmissible neurodegenerative dis- order. The interspecies transmission barrier associated with the ‘pathogen’, namely proteinaceous particles named prions, is a very interesting phenomenon in prion diseases [1,2]. The principal component of the prion is the aberrant ‘scrapie’ form (PrPSc) produced by structural conversion of a constantly expressed cell- surface protein, the prion protein (PrP). It has been known for some time that the transmission barrier for prion diseases, often called the ‘species barrier’, is related to differences in the PrP primary sequence between the inoculum and the host, especially at sequence 90–145 [3–9]. The species barrier might be due to sequence divergence or to different amyloid structures [10]. Many prion biologists have examined how sequence affects the prion transmission barrier. For example, residue 139 has been found to be a key residue affecting the transmission efficiency between hamster and mouse [9,11,12]. In addition, variant Cre- utzfeldt–Jakob disease (vCJD) transmission seems to be strongly dependent on amino acid sequence varia- tion (polymorphism) at It has been reported that hPrP-sen with 129M has higher PrPSc conversion than hPrP-sen with 129V in a cell-free con- version assay [13]. Bishop et al. [14] used a transgenic mouse model to predict susceptibility to bovine spongi- form encephalopathy and vCJD. Although they failed to observe transmission of bovine spongiform encepha- lopathy, possibly because of the low transmission effi- ciency and long incubation time, they did observe a gradation of transmission efficiency of vCJD in the order MM > MV > VV. It has been reported that human PrP protein with Val129 prevents the transmis- sion of bovine spongiform encephalopathy-derived prions using transgenic mice [15,16]. Almost all those with vCJD in the UK were found to have the methio- codon 129 nine homozygous genotype [17,18], although one vCJD case with the M ⁄ V geno- type [19] and another possible vCJD case with the V ⁄ V genotype have been reported [20]. Comparing Met and Val, the Van der Waal’s volume of Met is only slightly larger than that of Val (124 A˚ 3 for Met and 105 A˚ 3 for Val) but the side-chain hydrophobicity of Val is greater than that of Met ()3.10 kcalÆmol)1 for Val and )1.41 kcalÆmol)1 for Met) [21]. Why a sin- gle residue difference affects the seeding barrier in bovine-to-human transmission so greatly and whether there are other residues involved in this seeding dis- crimination remain elusive.
Structural conversion from the normal cellular form (PrPC) to the disease-causing form (PrPSc) is the key
Recently, based on the nucleation-dependent poly- merization model, we developed a seed-titration method to study the seeding efficiency of amyloids formed from hamster and mouse PrP peptides by com- paring the minimum seed amount required to skip the lag time of amyloidogenesis as monitored by time- resolved CD spectroscopy [12]. The minimum required seed amount for skipping the lag time is inversely pro- portional to the monomer concentration. When com- paring the seeding efficiencies of different monomers seeded by the same seed, a monomer with a higher seeding efficiency should need less seed. We have tried to apply the seed-titration method to the full-length protein. Unfortunately, CD ellipticity at 218 nm increased rather than decreased during the observation time, suggesting that the amyloid fibrils formed of recombinant PrP protein sank to the bottom of the sample cuvette too quickly and hampered measure- ment. Synthetic PrP peptides corresponding to hamster PrP sequence 108–144 were used as a simple system to quantify seeding efficiency. The reason for choosing this region is that previous observations have shown that it is the probable segment in which the conforma- tional change takes place and the structural conversion of this peptide simply incubated in buffer has been reported [3,4,9,27–30]. Here, we used the same peptide segment to examine whether M ⁄ V variation in the human PrP peptide shows seeding discrimination in this simple system. Bovine PrP peptide 111–147 (bPrP) and human PrP peptide 108–144 with different amino acids at residue 129 (huPrP129M and huPrP129V) were synthesized chemically (Table 1). To mimic the situation in heterozygous individuals, huPrP129M and huPrP129V were mixed in a 1 : 1 molar ratio and the In addition, mixture denoted as huPrP129M ⁄ V.
FEBS Journal 278 (2011) 4351–4361 ª 2011 The Authors Journal compilation ª 2011 FEBS
4352
T.-Y. Liao et al. Seeding discrimination of codon-129 M ⁄ V
Table 1. Comparison of the amino acid sequences of human prion protein (huPrP) (sequence 108–144) and bovine prion protein (bPrP) (sequence 111–147). Sequence differences (corresponding to the human prion sequence 112, 138, and 143) are underlined. Residue 129 in huPrP is shown boxed.
112 129 138 143 NMKHMAGAAA AGAVVGGLGG Y M LGSAMSRP IIHFGSD huPrP (108–144) NMKHVAGAAA AGAVVGGLGG YMLGSAMSRP LIHFGND bPrP (111–147)
because three residues are different between bPrP and huPrP129M at positions 112, 138 and 143 (human sequence number), three mutant peptides, V112M, L138I and N143S, were constructed in which a single residue in the bovine PrP peptide was replaced with the corresponding residue from the human sequence. Our aim was to determine whether these residues are involved in the differential seeding efficiencies of codon 129 polymorphism.
Results and Discussion
Spontaneous amyloidogenesis of the PrP peptides
Fig. 1. Time course of amyloidogenesis for the different prion pep- tides. The peptides (50 lM) were dissolved in 20 mM NaOAc, 140 mM NaCl, pH 3.7, and incubated at 25 (cid:2)C. At the indicated time, the CD negative ellipticity at 218 nm (A) and the fluorescence emis- sion of ThT at 487 nm (B) were measured. The fluorescence intensity of the huPrP129V sample was much higher than that of the other samples and is shown on the right axis. bPrP, black; huPrP129M, red; huPrP129V, green; huPrP129M ⁄ V, blue. Inset in (B): time course normalized to the maximum fluorescence emission.
To determine whether the Val ⁄ Met substitution chan- ged the conformational energy barrier of the monomer for structural conversion and thus led to differential spontaneous amyloidogenesis of seeding efficiency, peptides bPrP, huPrP129M, huPrP129M ⁄ V and hu- PrP129V was monitored. These PrP peptides were ran- dom coil when dissolved in buffer and then gradually went through structural conversion to form a b-struc- ture which has characteristic negative ellipticity at 218 nm in the CD spectrum. The amount of amyloid fibril formation was measured by CD spectroscopy and the thioflavin T (ThT) binding assay, in which the time course of amyloidogenesis is monitored by the emitted fluorescence of ThT bound to the amyloid structure (Fig. 1). The time courses obtained using these two methods are consistent. It should be noted that the emitted ThT fluorescence was particularly high for the huPrP129V peptide (note the different scale for the y axis in this peptide). We surmise that the big dif- ference in ThT fluorescence emission might be because: (a) position 129 is in the ThT-binding site and Val129 favors ThT binding; (b) position 129 is involved in the ThT-binding interface and Val129 increases ThT fluo- rescence quantum yield; and (c) Val129 affects fibril assembly which affects ThT binding.
Despite the three-residue difference between bPrP four-residue difference
and huPrP129M and the
between bPrP and huPrP129V, the lag times for these three peptides were very similar, suggesting that the sequence difference between bovine and human PrP and human 129M ⁄ V polymorphism did not signifi- cantly affect the nucleation step. The huPrP129M ⁄ V mixture had a lag time almost twice that of the others, highlighting the homology requirement in nucleation. Our data agree well with the statistical observation of the occurrence of sporadic CJD (sCJD) (Table 2) [31,32]. As shown in Table 2, both the Met and Val homozygotes have a higher risk of acquiring sCJD when the number of sCJD cases is normalized to the proportion of each genotype in the UK population. Although the case number of sCJD in M ⁄ V heterozygotes
FEBS Journal 278 (2011) 4351–4361 ª 2011 The Authors Journal compilation ª 2011 FEBS
4353
T.-Y. Liao et al. Seeding discrimination of codon-129 M ⁄ V
Table 2. Codon 129 genotype distribution in patients with sporadic Creutzfeldt–Jakob disease in the UK and in the total UK population.
Genotype
M ⁄ M M ⁄ V V ⁄ V
a Data taken from Head et al. [31]. b Data taken from Palmer et al. [32].
110 37 297 31 51 61 24 12 200 Number of casesa UK populationb (%) Cases ⁄ population
electron microscopy (Fig. 2). No clear difference in morphology can be concluded based on these images except that huPrP129V fibrils did not favor lateral association compared with the other three. FTIR was used to compare the structural difference among the fibrils of bPrP, huPrP129M and huPrP129V (Fig. 3). After deconvolution, all samples showed a common peak at 1624 cm)1 which is typical of cross-b-amyloid aggregates. The bPrP and huPrP129M fibrils had the same absorption bands in their deconvoluted FTIR spectra, whereas the absorption bands of the hu- PrP129V fibrils were different. Our results suggested the huPrP129M fibrils had a similar amyloid that structure to the bPrP fibrils, whereas the huPrP129V fibril structure might have some differences. The structural difference might be related to the high ThT fluorescence emission mentioned above.
is more than that in the V ⁄ V homozygotes, after nor- malization to the proportion of each genotype, the risk of acquiring sCJD for the M ⁄ V heterozygotes is actu- ally the lowest for the three groups. Moreover, the hu- PrP129V peptide had the slowest elongation rate, as shown by the slope of the line in the inset, consistent with the longer disease duration in sCJD patients with the V ⁄ V genotype at codon 129 [33].
Evaluation of bPrP seeding efficiency by the seed-titration method
Comparison of the amyloid structures of bPrP, huPrP129M and huPrP129V
According to the results in Fig. 1, the lag times of all peptides are much longer than the time window (60 min) used in the seed-titration experiments, indicat- ing that all signals detected in the seed-titration experi- ment come from fibril elongation on the supplied seed.
The amyloid fibrils formed from bPrP, huPrP129M, huPrP129M ⁄ V and huPrP129V are shown by transmission
A
B
D
C
FEBS Journal 278 (2011) 4351–4361 ª 2011 The Authors Journal compilation ª 2011 FEBS
4354
Fig. 2. Electron microscopy images of the bPrP, huPrP129M, huPrP129M ⁄ V and huPrP129V fibrils. The bars represent 100 nm.
T.-Y. Liao et al. Seeding discrimination of codon-129 M ⁄ V
In order
to control
different volumes of seed solution, made up to the same final volume, were compared in the absence of spontaneous nucleation. the amount of the seed, we prepared homogeneous seed by ultrasonication of amyloid fibrils [12]. The initial amy- loid elongation rate was fitted according to the ellip- ticity at 218 nm using time-resolved CD spectroscopy. The minimal required seed amount was arbitrarily and empirically determined as that giving an initial elonga- tion rate > 1 · 10)4 mdegÆs)1. (The slope in the time- resolved CD spectrum of the peptide solution without fibril formation is always < 1 · 10)4 and the slope increase is proportional to the seed volume increase only when the slope is > 1 · 10)4.) For a particular seed, its seeding efficiency for samples A, B and C is normalized as A : B : C = [seed]A ⁄ [seed]A : [seed]A ⁄ [seed]B : [seed]A ⁄ [seed]C, where [seed]x is the minimum amount of seed needed for efficient seeding sample x.
Fig. 3. FTIR analysis of bPrP (A), huPrP129M (B) or huPrP129V (C) fibrils. The original curve (dotted line) was fitted to the Lorentzian curve. The peak positions of the amide I band components were deduced from the second derivative spectra. The sum of the fitted curves (gray curves) is shown as a continuous line.
Amyloid fibrils formed from the bPrP peptide were sonicated into small fragments and the same batch of sonicated fragments was used to seed the various PrP peptides. The kinetics of amyloidogenesis was moni- tored by time-resolved CD spectroscopy (Fig. 4A). The initial amyloid elongation rate was plotted against the seed amount for each seeding reaction (Fig. 4B). As the minimal bPrP seed amount shown in Fig. 4B, required to efficiently seed the bPrP, huPrP129M, hu- PrP129M ⁄ V and huPrP129V peptide solutions was 1, 2, 10 and 20%, respectively. Clearly, bPrP amyloid was more efficient at seeding the huPrP129M peptide than the huPrP129V and huPrP129M ⁄ V peptides. According to our peptide model, the normalized seeding efficiency of the bPrP seed was 1 ⁄ 1 : 1 ⁄ 2 : 1 ⁄ 10 : 1 ⁄ 20 or 1 : 0.5 : 0.1 : 0.05 for bPrP, huPrP129M, huPrP129M ⁄ V and huPrP129V. The Met129 fi Val substitution resulted in a 10-fold lower seeding effi- ciency for bPrP seeds. Interestingly, although the hu- PrP129M peptide concentration in the huPrP129M ⁄ V mixture sample was only half that in the huPrP129M sample, the seeding efficiency of the bPrP fibrils for the huPrP129M ⁄ V mixture was fivefold lower (rather than twofold lower) than that for the pure huPrP129M peptide. We used another peptide mixture composed of 50 lM huPrP129M and 25 lM huPrP129V to test the bPrP seeding efficiency, and its seeding efficiency was also lower than that for the pure huPrP129M pep- tide (50 lM) (Fig. S1). Our data strongly support that the decrease in seeding efficiency resulted from compe- tition between the two heterologous molecules for the same binding site in the structural conversion process, as suggested by the inhibitory effect of heterologous recombinant PrP on the accumulation of PrPres in mouse neuroblastoma cells [34].
To evaluate the sequence dependency of the seeding efficiency, the kinetics of amyloidogenesis of samples concentrations and containing identical monomer
FEBS Journal 278 (2011) 4351–4361 ª 2011 The Authors Journal compilation ª 2011 FEBS
4355
T.-Y. Liao et al. Seeding discrimination of codon-129 M ⁄ V
A
B
Fig. 4. bPrP seeding experiment monitored using time-resolved CD spectroscopy. (A) Kinetic traces of the bPrP seeding experi- ment. Different volumes of bPrP seed were added to the bPrP, huPrP129M, huPrP129M ⁄ V and huPrP129V peptide solutions and the kinetics of amyloidogene- sis monitored using time-resolved CD spectroscopy at 218 nm. The monomer peptide and the used seed volume are indicated in each panel. (B) Plot of initial amyloid elongation rate versus seed volume. The initial elongation rate was averaged from two independent experiments. The fibril elongation rate was obtained by linearly fitting the data within 2000 s. The minimal bPrP seed amount required to efficiently seed the bPrP, huPrP129M, huPrP129M ⁄ V and huPrP129V peptide solutions was 1, 2, 10 and 20%, respectively. The normalized seeding efficiency of the bPrP seed was 1 ⁄ 1 : 1 ⁄ 2 : 1 ⁄ 10 : 1 ⁄ 20 = 1 : 0.5 : 0.1 : 0.05 for bPrP, huPrP129M, huPrP129M ⁄ V and huPrP129V.
Estimation of huPrP129M-to-huPrP129(M, V or M ⁄ V) seeding efficiency using the seed-titration method
huPrP129M, huPrP129M ⁄ V and huPrP129V (Fig. 5A). The relative seeding efficiency was the same as that when pure huPrP129M fibrils were used as seed (Fig. 5B). This suggested that the free ends of the [hu- PrP129M]bPrP fibrils have the same binding surface as the free ends of pure huPrP129M fibrils.
Bovine Leu138 is responsible for the interference effect of huPrP129V
In the bPrP seeding experiment, the seeding efficiency for the huPrP129M peptide is 10 times higher than that for the huPrP129V peptide. Why and how can one residue change lead to such a dramatic difference? In our study, a similar lag time for spontaneous amy- loid formation of huPrP129M and huPrP129V was
In the bPrP seeding experiment, residue 129 of the human PrP peptide being M or V greatly affected the seeding efficiency. In order to compare whether the ([huPrP129M]bPrP) bPrP-seeded huPrP129M fibrils retain the same seeding discrimination for huPrP129M [huPrP129M]bPrP and pure hu- and huPrP129V, PrP129M fibrils were used as seed in the seeding exper- iment. When [huPrP129M]bPrP was used as seed, the amount of seed required for efficient seeding of the huPrP129M, huPrP129M ⁄ V and huPrP129V peptide solutions was 2, 4 and 8%, respectively, giving a normalized seeding efficiency of 1 : 0.5 : 0.25 for
FEBS Journal 278 (2011) 4351–4361 ª 2011 The Authors Journal compilation ª 2011 FEBS
4356
T.-Y. Liao et al. Seeding discrimination of codon-129 M ⁄ V
The seeding difference is not so great when the supplied seed is huPrP129M fibrils or [huPrP129M]bPrP. This difference suggested that huPrP129V interferes with the fibrillization of huPrP129M only when the seed is bPrP fibrils. The interference effect of huPrP129V in the huPrP129M ⁄ V sample must come from its interac- tion with the bPrP seed. The FTIR data suggested that the structure of the bPrP fibrils is similar to that of the huPrP129M fibrils (Fig. 3). Hence, we propose that human PrP peptide with Val at position 129 might pre- fer to bind to a different surface on the bPrP seed in the association step, thus preventing the subsequent conversion step. If so, at least one of the three residues that differ between bPrP and huPrP129M is involved in this incorrect binding surface.
To examine which residue in the bPrP peptide might be involved in the association interference, three bPrP mutant peptides were synthesized in which Val112, Leu138 or Asn143 of bPrP (residue number according to the number of the human PrP protein sequence) was replaced by Met, Ile or Ser, respectively. The fibrils formed from these peptides were then used to seed huPrP129M and huPrP129V. The mutation on the key residue of bPrP should have the same ratio of seeding efficiencies for huPrP129M and huPrP129V as that of the huPrP129M seed.
Fig. 5. Plots of initial amyloid elongation rate versus seed volume in huPrP129M seeding experiment. Different volumes of the indi- cated seed were added to the huPrP129M, huPrP129M ⁄ V and hu- PrP129V peptide solutions and the kinetics of amyloidogenesis monitored using time-resolved CD spectroscopy at 218 nm. (A) bPrP-seeded huPrP129M fibrils, denoted as [huPrP129M]bPrP, were used as the seed. (B) Pure huPrP129M fibrils were used as the seed. For these two types of seed, the seed volume required for efficient seeding of the huPrP129M, huPrP129M ⁄ V and huPrP129V peptide solutions was 2, 4 and 8%, respectively. The normalized seeding efficiency was 2 ⁄ 2 : 2 ⁄ 4 : 2 ⁄ 8 = 1 : 0.5 : 0.25 for hu- PrP129M, huPrP129M ⁄ V and huPrP129V.
(Fig. 3)
The seeding efficiencies of the V112M, L138I and N143S seeds for the bPrP, huPrP129M, huPrP129M ⁄ V and huPrP129V monomer are shown in Fig. S2. For clarity, the data are summarized and compared with the bPrP seeding results in Table 3. The normalized seeding efficiency of the V112M seed was 1 : 0.5 : 0.1 : 0.05 for bPrP, huPrP129M, huPrP129M ⁄ V and huPrP129V. This result is the same as that of the bPrP seed. These data suggested that the fibrils formed from V112M are very similar to those formed from bPrP and that the Val112 fi Met mutation is not involved in the interference effect.
observed (Fig. 1). Because lag time is the period required for nuclei formation, our data suggested that the conformational energy barriers for the structural conversion of these two peptides and their nucleation rates are similar. Interestingly, the seeding efficiency of huPrP129M was 10 times higher than that of huPrP129V only when the supplied seed is bPrP fibrils.
Table 3. Comparison of the seeding efficiencies using bovine prion peptide (bPrP) fibrils or the fibrils formed of the bPrP mutants (V112M, L138I and N143S) as seeds.
Seed
bPrP V112M L138I N143S
Seed amount (%) Normalized seeding efficiency Seed amount (%) Normalized seeding efficiency Seed amount (%) Normalized seeding efficiency Seed amount (%) Normalized seeding efficiency Monomer
FEBS Journal 278 (2011) 4351–4361 ª 2011 The Authors Journal compilation ª 2011 FEBS
4357
bPrP huPrP129M huPrP129M ⁄ V huPrP129V 1 2 10 20 1 0.5 0.1 0.05 2 4 20 40 1 0.5 0.1 0.05 2 2 4 8 1 1 0.5 0.25 2 4 6 6 1 0.5 0.33 0.33
T.-Y. Liao et al. Seeding discrimination of codon-129 M ⁄ V
Interestingly,
the normalized seeding efficiency of the L138I seed was 1 : 0.5 : 0.25 for huPrP129M, hu- PrP129M ⁄ V and huPrP129V. This result is the same as that of the huPrP129M seed. When L138I fibrils were used as seeds, huPrP129V had no interference effect in the huPrP129M ⁄ V mixture. These data suggested that Leu138 in the bovine PrP peptide is the residue responsible for the interference effect of huPrP129V in the huPrP129M and huPrP129V mixture and for the large seeding barrier in seeding huPrP129V.
subsequent ‘locking’ step, and thus prevent fibril elon- gation. The interference effect of the huPrP129V pep- tide in the 1 : 1 peptide mixture (huPrP129M ⁄ V) seeded with bPrP probably arises from incorrect sur- face association between bPrP and huPrP129V, which interferes with the normal binding between the bPrP seed and the huPrP129M monomer. Therefore, in Fig. 5, [huPrP129M]bPrP has the same efficacy in seed- ing as pure huPrP129M fibrils, because Leu138 is no longer at the fibril ends to interact with the coming monomer.
surmised that
When the N143S fibrils were used as seed, surpris- ingly, the difference in the seeding efficiency of hu- PrP129M and huPrP129V was markedly decreased. The normalized seeding efficiency of the N143S seed was 0.5 : 0.33 : 0.33 for huPrP129M, huPrP129M ⁄ V and huPrP129V. We the bovine Asn143 fi Ser substitution might dramatically affect the resulting amyloid structure. The kinetic trace of amyloidogenesis indeed showed that N143S formed fibrils at a much slower rate than the others (Fig. S3). The lag time of N143S is almost four times of that of bPrP.
In conclusion, one single Met fi Val substitution in the human PrP peptide increased the seeding barrier in the bPrP seeding experiment by 10 times. Moreover, huPrP129V impeded the association between the bPrP seed and huPrP129M when presented in the peptide mixture huPrP129M ⁄ V. According to the results of the bPrP mutants, we suggested that Leu138 in the bPrP seed is the key residue in this interference effect. How- ever, our results can not be directly applied to full- length prion protein because it is hard to assume that the C-terminal helices will not affect the seeding. For example, Priola et al. have reported that homology at residue 155 is also related to hamster prion transmis- sion [35]. Yamaguchi et al. have reported that helix 2 might be involved in the ultrasonication-induced fibril formation of the full-length mouse PrP [36]. Because of the limitation of applying time-resolved CD mea- surement in the seeding experiment of full-length PrP, as mentioned earlier, protein misfolding cyclic amplica- tion reaction with the same seed titration idea could be
Based on the bPrP mutation studies, we concluded that Leu138 is also a key residue leading to the 129M ⁄ V seeding discrimination in this peptide seeding experiment. To explain this phenomenon, we propose a ‘surface competition hypothesis’ (Fig. 6). We propose that Val129 in huPrP129V might be prone to bind to a surface formed from residues including at least Leu138 in the bPrP seed. Unlike the association in the ‘dock- ing’ step, this incorrect binding cannot go through the
FEBS Journal 278 (2011) 4351–4361 ª 2011 The Authors Journal compilation ª 2011 FEBS
4358
Fig. 6. Proposed ‘surface competition hypothesis’. There is no structure solved for the amyloid fibrils formed of the prion pep- tide sequence 108–144. The ‘b-helix’ model was adopted to present the fibril structure. Val129 in huPrP129V might be prone to bind to a surface formed from residues including at least Leu138 in the bPrP seed. This incorrect binding cannot go through the subsequent ‘locking’ step and thus prevent fibril elongation.
T.-Y. Liao et al. Seeding discrimination of codon-129 M ⁄ V
a solution to estimate the transmission barrier of the full-length PrP protein in vitro [37].
Materials and methods
Peptide synthesis and characterization
175 mM NaCl, pH 3.7. Typically, up to 50 lL of sonicated seed solution (all made to a final volume of 50 lL with dis- tilled water) was added to 200 lL of peptide solution to give a final monomer concentration of 50 lM. When more seeds were required, concentrated seed solution was used. The samples were placed in 1-mm quartz cuvettes and the kinetics of fibril formation was recorded every 5 s at 218 nm for 60 min. The trace within 2000 s was fit linearly to get the amyloid propagation rate and the rate was aver- aged from two or three independent experiments.
FTIR
The various PrP peptides were prepared using the batch Fmoc-polyamide method. The N-terminal end of the pep- tide was acetylated and the C-terminal end amidated in order to mimic the configuration and charge state in the full-length protein. After synthesis and purification, pep- tides were characterized by mass spectroscopy, lyophilized and stored at )20 (cid:2)C.
CD spectroscopy
The peptides (50 lM) were dissolved in 20 mM NaOAc, 140 mM NaCl (pH 3.7) and incubated at 25 (cid:2)C for amyloid fibril formation. At different incubation times, the samples were placed in 1-mm quartz cuvettes and the CD spectra between 200 and 250 nm recorded on a JASCO J-715 spec- trometer (JASCO, Tokyo, Japan). The band width was set to 2 nm and the step resolution was 0.05 nm. Two scans were averaged for each sample. To avoid contamination, a differ- ent quartz cuvette was used for each sample, and the same cuvette was used for the same sample for all time points.
ThT-binding assay
Peptide samples (50 lM) were dissolved in D2O containing 20 mM 1-13C-NaOAc and 140 mM NaCl (pH 3.7) and incu- bated at 25 (cid:2)C for amyloid fibril formation. The amyloid fibrils were collected by centrifugation and resuspended in 0.25 volumes (compared with the buffer volume used in the incubation) of D2O. The fibril samples were placed between a pair of CaF2 windows separated by a 0.2-mm spacer. FTIR spectra were recorded using a Bruker TENSOR 27 spectrometer purged with a continuous flow of nitrogen gas. Two hundred and fifty-six scans were accumulated at a spectral resolution of 2 cm)1. The spectra were analyzed using OPUS software (version 6.5; Bruker, Ettlingen, Ger- many). The D2O spectrum was recorded under the same conditions and subtracted from the sample spectra. After water compensation, the peak positions in the raw spectra were identified from the second derivative of the raw spec- tra, then the raw spectra were fitted to a series of Lorentz- ian peaks with the identified absorbance maxima.
Transmission electron microscopy
The samples were deposited on carbon-coated 300-mesh copper grids and incubated for 3 min for absorption. Nega- tive staining was carried out by staining with 2% uranyl acetate for 3 min. After drying, the samples were viewed using a Hitachi H-7000 electron microscope.
Acknowledgements
A 5 mM stock solution of ThT was prepared by dissolving the dye (2 mg) in 1.25 mL of 100 mM phosphate buffer, 140 mM NaCl, pH 8.5, and filtered through a 0.22-lm Mil- lipore filter. A fresh working solution of ThT was prepared by adjusting the final dye concentration to 200 lM. A 30-lL aliquot of each sample was mixed with 30 lL of the 200 lM ThT dye solution, then, after 1 min incubation at room temperature, the fluorescence emission at 487 nm was measured in a 3-mm pathlength rectangular cuvette on a FP-750 spectrofluorometer (JASCO) with excitation at 442 nm.
Seed-titration experiments
We thank Mr Tai-Lang Lin and the Core Facility of the Institute of Cellular and Organismic Biology, Aca- demia Sinica, Taiwan for assistance in transmission electron microscopy. This work was supported by the Academia Sinica and the National Science Council, Taiwan, R.O.C (grant Nos. NSC 98-2321-B-001-019, NSC 99-2321-B-001-014 and NSC 99-2321-B-001-038).
The amyloid fibrils were spun down by centrifugation, resuspended in distilled water and fragmented with 20 cycles of intermittent pulses (five pulses of 0.5 s each; 5-s interval between cycles) using an ultrasonic processor (UP100H; Hielscher, Ringwood, NJ, USA) equipped with a 1-mm microtip and a power setting of 40%.
References
1 Prusiner SB (1998) Prions. Proc Natl Acad Sci USA 95,
13363–13383.
For seed-titration experiments, the kinetics of fibril for- mation were measured by time-course measurement on a JASCO J-715 spectrometer. The peptide solution was pre- pared at a concentration of 62.5 lM in 25 mM NaOAc, and
FEBS Journal 278 (2011) 4351–4361 ª 2011 The Authors Journal compilation ª 2011 FEBS
4359
2 Prusiner SB (1982) Novel proteinaceous infectious parti-
cles cause scrapie. Science 216, 136–144.
valine 129 prevents expression of variant CJD pheno- type. Science 306, 1793–1796.
16 Asano M, Mohri S, Ironside JW, Ito M, Tamaoki N & Kitamoto T (2006) vCJD prion acquires altered viru- lence through trans-species infection. Biochem Biophys Res Commun 342, 293–299.
3 Supattapone S, Bosque P, Muramoto T, Wille H, Aag- aard C, Peretz D, Nguyen HO, Heinrich C, Torchia M, Safar J et al. (1999) Prion protein of 106 residues cre- ates an artificial transmission barrier for prion replica- tion in transgenic mice. Cell 96, 869–878.
4 Kaneko K, Peretz D, Pan K, Blochberger T, Wille H,
17 Zeidler M, Stewart G, Cousens SN, Estibeiro K & Will RG (1997) Codon 129 genotype and new variant CJD. Lancet 350, 668.
Gabizon R, Griffith O, Cohen F, Baldwin M & Prusin- er S (1995) Prion protein (PrP) synthetic peptides induce cellular PrP to acquire properties of the scrapie isoform. Proc Natl Acad Sci USA 92, 11160–11164.
18 Zou S, Fang CT & Schonberger LB (2008) Transfusion transmission of human prion diseases. Transfus Med Rev 22, 58–69.
19 Kaski D, Mead S, Hyare H, Cooper S, Jampana
5 Supattapone S, Bouzamondo E, Ball HL, Wille H,
R, Overell J, Knight R, Collinge J & Rudge P (2009) Variant CJD in an individual heterozygous for PRNP codon 129. Lancet 374, 2128.
20 Mead S, Joiner S, Desbruslais M, Beck JA,
Nguyen HO, Cohen FE, DeArmond SJ, Prusiner SB & Scott M (2001) A protease-resistant 61-residue prion peptide causes neurodegeneration in transgenic mice. Mol Cell Biol 21, 2608–2616.
6 Baskakov IV, Aagaard C, Mehlhorn I, Wille H, Groth D, Baldwin MA, Prusiner SB & Cohen FE (2000) Self- assembly of recombinant prion protein of 106 residues. Biochemistry 39, 2792–2804.
O’Donoghue M, Lantos P, Wadsworth JDF & Collinge J (2007) Creutzfeldt–Jakob disease, prion protein gene codon 129VV, and a novel PrPSc type in a young British woman. Arch Neurol 64, 1780– 1784.
21 Creighton TE (1984) Proteins: Structures and Molecular
Properties. Freeman, New York.
7 Harrison PM, Bamborough P, Daggett V, Prusiner SB & Cohen FE (1997) The prion folding problem. Curr Opin Struct Biol 7, 53–59.
8 Huang Z, Prusiner SB & Cohen FE (1996) Scrapie prions: a three-dimensional model of an infectious fragment. Fold Des 1, 13–19.
22 Come J, Fraser P & Lansbury P Jr (1993) A kinetic model for amyloid formation in the prion diseases: importance of seeding. Proc Natl Acad Sci USA 90, 5959–5963.
9 Vanik DL, Surewicz KA & Surewicz WK (2004) Molec- ular basis of barriers for interspecies transmissibility of mammalian prions. Mol Cell 14, 139–145.
23 Harper JD & Lansbury PT Jr (1997) Models of amyloid seeding in Alzheimer’s disease and scrapie: mechanistic truths and physiological consequences of the time- dependent solubility of amyloid proteins. Annu Rev Biochem 66, 385–407.
24 Jarrett JT & Lansbury PT Jr (1993) Seeding ‘one-
10 Jones EM & Surewicz WK (2005) Fibril conformation as the basis of species- and strain-dependent seeding specificity of mammalian prion amyloids. Cell 121, 63– 72.
11 Kocisko DA, Priola SA, Raymond GJ, Chesebro B,
dimensional crystallization’ of amyloid: a pathogenic mechanism in Alzheimer’s disease and scrapie? Cell 73, 1055–1058.
Lansbury PT Jr & Caughey B (1995) Species specificity in the cell-free conversion of prion protein to protease- resistant forms: a model for the scrapie species barrier. Proc Natl Acad Sci USA 92, 3923–3927.
25 Krebs MRH, Morozova-Roche LA, Daniel K, Robin- son CV & Dobson CM (2004) Observation of sequence specificity in the seeding of protein amyloid fibrils. Protein Sci 13, 1933–1938.
26 Bocharova OV, Breydo L, Salnikov VV, Gill AC &
12 Lee LY & Chen RP (2007) Quantifying the sequence- dependent species barrier between hamster and mouse prions. J Am Chem Soc 129, 1644–1652.
Baskakov IV (2005) Synthetic prions generated in vitro are similar to a newly identified subpopulation of PrPSc from sporadic Creutzfeldt–Jakob Disease. Protein Sci 14, 1222–1232.
27 Zhang H, Kaneko K, Nguyen JT, Livshits TL, Baldwin
13 Raymond GJ, Hope J, Kocisko DA, Priola SA, Ray- mond LD, Bossers A, Ironside J, Will RG, Chen SG, Petersen RB et al. (1997) Molecular assessment of the potential transmissibilities of BSE and scrapie to humans. Nature 388, 285–288.
14 Bishop MT, Hart P, Aitchison L, Baybutt HN,
MA, Cohen FE, James TL & Prusiner SB (1995) Conformational transitions in peptides containing two putative a-helices of the prion protein. J Mol Biol 250, 514–526.
28 Chen PY, Lin CC, Chang YT, Lin SC & Chan SI
Plinston C, Thomson V, Tuzi NL, Head MW, Ironside JW, Will RG et al. (2006) Predicting susceptibility and incubation time of human-to-human transmission of vCJD. Lancet Neurol 5, 393–398.
15 Wadsworth JD, Asante EA, Desbruslais M, Linehan
JM, Joiner S, Gowland I, Welch J, Stone L, Lloyd SE, Hill AF et al. (2004) Human prion protein with
(2002) One O-linked sugar can affect the coil-to-b struc- tural transition of the prion peptide. Proc Natl Acad Sci USA 99, 12633–12638.
FEBS Journal 278 (2011) 4351–4361 ª 2011 The Authors Journal compilation ª 2011 FEBS
4360
T.-Y. Liao et al. Seeding discrimination of codon-129 M ⁄ V
hamster PrP is determined by homology at amino acid residue 155. J Virol 75, 4673–4680.
29 Chabry J, Caughey B & Chesebro B (1998) Specific inhibition of in vitro formation of protease-resistant prion protein by synthetic peptides. J Biol Chem 273, 13203–13207.
36 Yamaguchi K, Matsumoto T & Kuwata K (2008) Criti- cal region for amyloid fibril formation of mouse prion protein: unusual amyloidogenic properties of the helix 2 peptide. Biochemistry 47, 13242–13251.
37 Fernandez-Borges N, de Castro J & Castilla J (2009) In vitro studies of the transmission barrier. Prion 3, 220–223.
30 Peretz D, Williamson RA, Kaneko K, Vergara J, Lecl- erc E, Schmitt-Ulms G, Mehlhorn IR, Legname G, Wormald MR, Rudd PM et al. (2001) Antibodies inhi- bit prion propagation and clear cell cultures of prion infectivity. Nature 412, 739–743.
31 Head MW, Bunn TJR, Bishop MT, McLoughlin V,
T.-Y. Liao et al. Seeding discrimination of codon-129 M ⁄ V
Supporting information
Lowrie S, McKimmie CS, Williams MC, McCardle L, MacKenzie J, Knight R et al. (2004) Prion protein het- erogeneity in sporadic but not variant Creutzfeldt–Ja- kob disease: U.K. cases 1991–2002. Ann Neurol 55, 851–859.
32 Palmer MS, Dryden AJ, Hughes JT & Collinge J (1991) Homozygous prion protein genotype predisposes to sporadic Creutzfeldt–Jakob disease. Nature 352, 340– 342.
33 Alperovitch A, Zerr I, Pocchiari M, Mitrova E, de
The following supplementary material is available: Fig. S1. The interference effect of huPrP129V in the bPrP seeding experiment. Fig. S2. Plot of initial amyloid elongation rate versus seed amount in the V112M, L138I and N143S seeding experiments. Fig. S3. Time course of amyloidogenesis for V112M, L138I and N143S.
This supplementary material can be found in the
online version of this article.
Pedro Cuesta J, Hegyi I, Collins S, Kretzschmar H, van Duijn C & Will RG (1999) Codon 129 prion protein genotype and sporadic Creutzfeldt–Jakob disease. Lancet 353, 1673–1674.
34 Priola SA, Caughey B, Race RE & Chesebro B (1994)
Heterologous PrP molecules interfere with accumulation of protease-resistant PrP in scrapie-infected murine neuroblastoma cells. J Virol 68, 4873–4878.
35 Priola SA, Chabry J & Chan K (2001) Efficient conver-
Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.
sion of normal prion protein (PrP) by abnormal
FEBS Journal 278 (2011) 4351–4361 ª 2011 The Authors Journal compilation ª 2011 FEBS
4361
