Báo cáo khoa học: The propagation of hamster-adapted scrapie PrPSc can be enhanced by reduced pyridine nucleotide in vitro pdf

We found that reduced pyridine nucleotide NADPH pro-moted PrPSc-propagating activity in PMCA using PrPC from normal hamster brains as the substrate, and that this was a dose-dependent re

enhanced by reduced pyridine nucleotide in vitro

Song Shi, Chen-Fang Dong, Chan Tian, Rui-Min Zhou, Kun Xu, Bao-Yun Zhang, Chen Gao,

Jun Han and Xiao-Ping Dong

State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Viral Disease Control and Prevention, Beijing, China

Transmissible spongiform encephalopathies (TSEs),

also known as prion diseases, are lethal

neurodegener-ative diseases, including Creutzfeldt–Jakob disease,

Gerstmann–Straussler–Scheinker syndrome and fatal

familial insomnia in humans, bovine spongiform

encephalopathy in cattle, scrapie in sheep and goats,

and chronic wasting disease in deer and elk [1,2] TSEs

are caused by a proteinaceous infectious agent, termed

a prion, which is considered to consist of a misfolded

and aggregated protease-resistant isomer of a

host-encoded glycoprotein (PrPC) [3,4] The pathological

isomer present in the tissues of infected individuals is

called PrPSc Although the clinical and pathological

characteristics of TSEs have been recognized for a long

time, the mechanisms underlying prion conversion are

only partially settled

Recently, some endogenous factors encoded by prion hosts have been proposed as essential during the propagation of prions in studies in animal models and cell-culture systems, i.e protein X and some chaperons [5–10] Polyanions and sulfated glycans are also thought to be involved in converting PrPC into the abnormal isomer in vitro [11–14] Moreover, pyridine nucleotides, a group of coenzymes ubiquitous in bio-synthesis and metabolism, are associated with the aggregation of recombinant prion protein (rPrP) Following incubation with reduced pyridine nucleo-tides, e.g NADPH, rPrP can accumulate in fibrils and acquire weak proteinase resistance [15,16] Several studies have shown that the amount of NADPH-diaphorase increases during the early stage of prion disease [17], but there is no direct molecular evidence

Keywords

PMCA; prion; propagation; pyridine

nucleotide; transmissible spongiform

encephalopathies

Correspondence

X.-P Dong, State Key Laboratory for

Infectious Disease Prevention and Control,

National Institute for Viral Disease Control

and Prevention, Chinese Center for Disease

Control and Prevention, Ying-Xin Rd 100,

Beijing 100052, China

Fax: +86 10 63532053

Tel: +86 10 83534616

E-mail: dongxp238@sina.com

(Received 16 September 2008, revised 15

November 2008, accepted 22 December

2008)

doi:10.1111/j.1742-4658.2009.06871.x

Transmissible spongiform encephalopathies (TSEs), or prion diseases, are fatal neurodegenerative disorders caused by an infectious agent termed a prion, which can convert normal cellular prion protein (PrPC) into a patho-logically misfolded isoform (PrPSc) Taking advantage of protein misfolding cyclic amplification (PMCA), a series of experiments was conducted to investigate the possible influences of pyridine nucleotides on the propaga-tion activities of hamster-adapted scrapie agents 263K and 139A in vitro using normal hamster brain homogenates and recombinant hamster PrP as the substrates The results showed that PrPSc from both scrapie agent 263K- and 139A-infected brains propagated more efficiently in PMCA with the addition of reduced NADPH, showing an obvious dose-dependent enhancement Reduced NADH also prompted PrPSc propagation, whereas NADP, NAD and vitamin C failed Moreover, following incubation with NADPH, recombinant hamster PrP could be efficiently converted into the proteinase K-resistant form when exposed to the trace of PrPSc from infected hamsters Our data provide evidence that the reduced pyridine nucleotide plays an important role in the propagation of prion and this process seems to target PrPCmolecules

Abbreviations

NBH, normal brain homogenate; PK, proteinase K; PMCA, protein misfolding cyclic amplification; rPrP, recombinant PrP; ScBH, scrapie-infected brain homogenate; SHa, Syrian hamster; TSEs, transmissible spongiform encephalopathies.

to clarify the mechanism by which reduced pyridine

nucleotides affect PrP molecules

A novel technology, named protein misfolding cyclic

amplification (PMCA) has been described in recent

years [18], providing an efficacious, unique and

conve-nient experimental approach to evaluating the

replica-tion and infectivity of newly formed PrPScin vitro[19]

Following incubation of PrPScfrom TSE-infected

ani-mals with homologous PrPCfrom normal brains, large

quantities of PrPSccan be achieved after a short cycle of

alternate sonication and incubation [20,21] This

tech-nology is considered the most appropriate approach to

understanding the fundamental mechanisms involved in

PrPSc propagation, aggregation and neuroinvasion

in vivo[22] In this study, possible influences of pyridine

nucleotides on the propagation of PrPSc from

scrapie-infected hamsters in PMCA were investigated We

found that reduced pyridine nucleotide NADPH

pro-moted PrPSc-propagating activity in PMCA using PrPC

from normal hamster brains as the substrate, and that

this was a dose-dependent response Other reduced

ana-logues of pyridine nucleotides also showed enhanced

capacity, whereas an oxidized analogue failed

Further-more, we proposed that after incubation with NADPH,

recombinant hamster PrP could be converted to the

proteinase K (PK)-resistant isoform in the presence of

scrapie PrPScin PMCA

Results

The propagating activities of PrPScin PMCA are

remarkably enhanced in the presence of NADPH

To see the influence of NADPH on the ability of PrPSc

to propagate in PMCA, a serial PMCA protocol

con-sisting of seven rounds was conduced (Fig 1A) Prior

to mixing with a 10)2dilution of ScBH prepared from

scrapie 263K-infected hamsters (SHa-263K), hamster

NBH was mixed with 10 lm NADPH (referred as

NBH + NADPH, lanes 3–9) and subjected to 12

PMCA cycles Thereafter, 10 lL of PMCA product

was added to 90 lL of fresh NBH with 10 lm

NADPH and the next round was performed This was

repeated several times, leading to the original ScBH

being diluted to 10)8 Meanwhile, mixture containing

only NADPH and ScBH in the conversion buffer, but

without NBH (referred as NADPH control, lanes 3–9),

or one containing NBH and ScBH but without

NADPH (referred as NBH control, lanes 3–9), was

prepared as above Each PMCA product was digested

with PK and subjected to western blotting

PK-resis-tant PrP signal was detected only in the first NADPH

control preparation (upper gel), representing the initial

input PrPSc (Fig 1A, lane 2 in all gels) In the NBH control, the PrPScsignal was strongest during round 1, weakened gradually and vanished from round 4 (mid-dle gel), indicating lower PMCA-propagating activity under these conditions By contrast, in the presence of NADPH, obvious PrPScsignals were seen in all prepa-rations from round 1 to round 7 (Fig 1A, lower gel), highlighting a more actively propagating capacity for PrPSc This suggests that NADPH can prompt the propagation of scrapie agent 263K In addition, no sig-nificant propagation of PrPSc was observed in seeded preparations without sonication (Fig S1A) or in PMCA samples without seeding ScBH (Fig S1B),

Fig 1 NADPH induced more efficient propagation of PrP Sc from scrapie agents in PMCA Ten microliters of a 100-fold dilution of ScBH of SHa-263K or SHa-139A were used as the seed in each prep-aration PMCA was conducted over 12 cycles per round, with seven rounds in total (A) Propagation of PrP Sc from hamster-adapted scra-pie agent 263K in PMCA The NADPH control (upper gel) contained

10 l M NADPH in conversion buffer and ScBH, but without NBH The NBH control (middle gel) contained NBH and ScBH, but without NADPH NBH plus NADPH (lower gel) contained NBH, ScBH and NADPH (B) Propagations seeded with hamster-adapted scrapie agent 139A underwent the same PMCA procedure containing NBH alone (upper gel) or NBH plus NADPH (lower gel) In all gels, NBH was loaded directly in the first lane without proteolysis as a refer-ence for comparison of electrophoretic mobility (PrP C PK )) All other samples were treated with 50 lgÆmL)1PK (PK+) 0 indicates reac-tions containing ScBH before PMCA process Round numbers are given on top The molecular markers are indicated on the right.

implying that NADPH can neither enhance PrPSc

propagation without PMCA nor induce spontaneous

conversion of PrPCinto PrPScwith PMCA in vitro

To test whether NADPH-related enhancement was

also appropriate to another scrapie agent,

hamster-adapted scrapie strain 139A (SHa-139A) was used in

PMCA following the same procedure In order to

avoid possible environmental contamination, all

mate-rials and reagents were freshly prepared, including the

homogenizer, chemicals and conversion buffer NBH

and ScBH (SHa-139A) were prepared in another

labo-ratory that had never been exposed to prions Like

SHa-263K, PK-resistant signals were detected in

reac-tions from round 1 to round 7 in the presence of

NADPH (Fig 1B, lower), but only in the first three

rounds without NADPH (Fig 1B, upper) These

results indicate that propagation of SHa-139A in vitro

can also be prompted by NADPH, and therefore this

enhancement of PrPSc propagation in PMCA is not

limited to only one prion strain

NADPH increased the sensitivity for detection of

PrPScin brain homogenates by PMCA

To test the potential of using NADPH as an assistant

chemical in increasing the detection sensitivity of

PMCA, comparative analysis of serial PMCA, with or

without NADPH, was conducted using serially diluted

ScBH from agent 263K as the seed Dilutions of the

original ScBH ranged from 10)4 to 10)12 by 100-fold

serial dilution One round of PMCA consisted of 24

cycles (24 h), which was considered to be more

effi-cient at increasing PrPScaggregation [19] and allowed

low levels of PrPScto be detected [23] After one round

of PMCA, 10 lL of product was mixed with 90 lL of

fresh NBH for the next round, up to seven rounds

Figure 2 shows that PrPSc signals were detected at

dilutions of 10)6 and 10)8 in the second round in the

presence of 10 lm NADPH (second gel, lanes 2 and

3), whereas PrPScsignals could be observed at a

dilu-tion of 10)6 in the third round (third gel, lane 7) and

10)8in the fifth round (fifth gel, lane 8) in the absence

of NADPH After seven rounds of PMCA, clear PrPSc

signals were observed at ScBH dilutions of 10)4 to

10)10, and even at a 10)12-fold dilution a weak band

was observed in the presence of NADPH (lower gel,

lane 5) However, no PrPSc signal was detected at

dilutions of 10)10 and 10)12 without NADPH (lower

gel, lanes 9 and 10) This indicates that NADPH can

increase, by at least 102-fold, the sensitivity for the

detection of PrPSc in brain homogenates under these

experimental conditions At the concentration of PrPSc

in ScBH used in this study (see Materials and

methods), a 1· 10)10 dilution of ScBH contained

 7.265 · 10)19gÆlL)1PrPSc, or 12.5 molecules of ini-tial PrPSc per lL Thereby, one can speculate that

 12.5 monomeric PrPSc molecules can successfully induce detectable amplification in a seven-round PMCA with NADPH, whereas in the absence of NADPH at least 1250 molecules of PrPScare required for the same amplification

PrPSc generated from PMCA in the presence of NADPH had further propagation ability

To determine whether newly generated PMCA PrPSc

by addition of NADPH could further propagate in the PMCA system in the absence of NADPH, 10 lL of PMCA product (PMCA-productNADPH) from a 10)10 dilution in the seventh round (Fig 2, lane 4) was diluted in 90 lL of NBH and subjected to one round

of PMCA (24 cycles) without NADPH (i.e eight rounds in total) Obvious PrPSc signals were detected

Fig 2 Comparative evaluation for serial PMCA detecting the sensi-tivity of PrP Sc in ScBH in the presence or absence of NADPH Aliquots of SHa-263K ScBH were 100-fold serially diluted with ham-ster NBH, reaching dilutions of 10)4, 10)6, 10)8, 10)10and 10)12 Each dilution was divided equally into two PCR tubes, one mixed with 10 l M NADPH and the other added to an equal volume of NaCl ⁄ P i After 24 cycles PMCA as the first round, 10 lL of product was mixed with 90 lL of fresh NBH for the next round of PMCA The five left-hand lanes are fresh NBH plus NADPH (NADPH+) and the five right-hand lanes are without NADPH (NADPH-) After seven rounds, the digested PMCA products of each round were analysed

by western blotting ScBH dilutions are shown on top PMCA round numbers (1–7) are shown on the left All samples were digested with 50 lgÆmL)1PK Molecular markers are indicated on the right.

in all samples from rounds 1–8, without significant

alterations in signal intensity (Fig 3) This indicates

that PMCA-productNADPH is able to utilize native

PrPC as a substrate to replicate in PMCA in the

absence of NADPH

Enhancement of NADPH for PrPSc propagation

in PMCA was dose–response and

oxidation/deoxidation related

To estimate the influence of the NADPH

concentra-tion on promoting propagaconcentra-tion of PrPScin vitro,

two-fold serially diluted NADPH from 160 to 1.25 lm

was mixed with NBH, and seeded with 1000-fold

diluted ScBH of 263K Preparations were subjected

to PMCA for 24 cycles (24 h) followed by PK

treat-ment Western blot analysis showed that the

effi-ciency of PrPSc propagation was enhanced by the

addition of NADPH, which was closely related to

increasing NADPH concentrations (Fig 4A, compare

lane 1 and lanes 2–9) After densitometric

quantifica-tion of the PrPSc signals from three independent

assays, the relationship between the concentration of

NADPH and PrPSc propagation was analysed We

found that PrPSc propagation was gradually

enhanced with increasing NADPH concentration

(from 1.25 to 10 lm), and reached a plateau at

10 lm NADPH (Fig 4A, lane 5; Fig 4B)

Calculat-ing the signal intensity of the reactions of 0 and

10 lm NADPH revealed that the efficiency of PMCA

was increased by  2.59-fold in the presence of

NADPH To evaluate whether the effect of NADPH

on PrPSc propagation was due to its reduced state, NADPH oxidized by overnight incubation at room temperature was serially diluted and amplification was performed as above Interestingly, although PrPSc signals could be detected in all preparations, there was no promotion effect of PrPSc formation in PMCA compared with the preparation without NADPH (Fig 4C, compare lane 1 and lanes 2–9) This suggests that oxidized NADPH cannot enhance the propagation of PrPSc, and only its reduced state

is enhances PrPSc propagation in vitro

The reduced, but not oxidized, structural analogue of NADPH possessed similar promoting ability on PrPScpropagation as NADPH

As a pyridine nucleotide, NADPH is believed to be an electron donor in some biochemical reactions [24] To

Fig 3 NADPH-induced PMCA product (PMCA-product NADPH )

prop-agated efficiently in further PMCA in the absence of NADPH Ten

microliters of PMCA product from the 10)10dilution in the seventh

round with NADPH (Fig 2, lane 4 in the lower gel) was mixed

90 lL of hamster NBH without NADPH PMCA was conducted

over 24 cycles per cycle, eight rounds in total, the dilution of the

original ScBH reaching 10)18in the final round The PMCA products

of each round were digested with 50 lgÆmL)1PK and analysed by

western blotting NBH without PK treatment was loaded directly

onto the gel as a reference for comparison of electrophoretic

mobil-ity (PrP C PK )) PMCA round numbers (0–8) are shown along top.

PK+ represented the preparations digested with PK Molecular

markers are indicated on the right.

Fig 4 The enhancement of NADPH for PrP Sc propagation in PMCA was dose dependent and oxidation ⁄ deoxidation related (A) Samples seeded with a 1000-fold dilution of ScBH (SHa-263K) were mixed with different concentrations of NADPH (0, 1.25, 2.5, 5, 10,

20, 40, 80 and 160 l M ) PMCA was conducted over 24 cycles (B) Quantitative analyses of each gray numerical value of PrP Sc PrP Sc

signals from each preparation were quantified densitometrically Relative gray values of the PrP Sc signals in each experimental con-dition were normalized by division with that of the respective reac-tion without NADPH [0 l M , lane 1 in (A)] The average values were calculated from three independent experiments and presented with

as mean ± SD (C) Oxidized NADPH, rather than fresh NADPH, was added into seeded preparations to undergo the PMCA proce-dure as in Fig 4A All samples were treated with 50 lgÆmL)1PK NADPH concentrations are shown on top of the gels Molecular markers are shown on the right.

address whether other pyridine nucleotides or an

elec-tron donor also help PrPSc to propagate in PMCA,

some NADPH structural analogues, including NADP,

NADH and NAD, were selected and subjected to

serial PMCA using 100-fold diluted ScBH of

SHa-263K as the seed In total, six rounds were performed,

each consisting of 12 cycles (Fig 5, lanes 3–8) NADH

enhanced PrPSc propagation in the reactions from

rounds 1–5 (fourth gel, lanes 3–7), comparable with

NADPH (second gel) which induced PrPSc

propaga-tion in all reacpropaga-tions However, compared with the

NBH control (upper gel), both NADP (third gel) and

NAD (fifth gel) failed to promote PrPSc propagation

in PMCA In addition, ascorbate, which is also

believed to be an electron donor in some biological

reduction–oxidation reactions, was applied as a

func-tional analogue of NADPH in this experiment

Nota-bly, ascorbate did not significantly enhance PrPSc

propagation in PMCA (lower gel) These results

indi-cate that only reduced pyridine nucleotides, i.e

NADPH and NADH, promote PrPSc propagation

in vitro

rSHaPrP could be converted into the PK-resistant isoform by addition of NADPH in PMCA

To investigate whether NADPH helps native PrPSc to induce the conformation change in recombinant PrP, purified recombinant hamster PrP23–231 and human PrP23–230 (rSHaPrP and rHuPrP) were incubated with 10 lm NADPH in PMCA conversion buffer After mixing with 10)3 to 10)6-fold diluted ScBH, the preparations were subjected to PMCA (24 cycles) Each product was exposed to PK digestion and wes-tern blotting (Fig 6) Compared with the PK-resistant signal of preparations treated without NADPH, in which only a faint signal at Mr 25 kDa appeared in the 10)3dilution (lane 2, upper gel), more PK-resistant bands were observed in all reactions with NADPH, among them a  17 kDa PK-resistant band was pre-dominant (lanes 6–9, upper gel), which may represent the PK-resistant fragment of rSHaPrP Unseeded assays revealed that rSHaPrP could not be induced spontaneously by NADPH and alternative sonication⁄ incubation to PK-resistant forms (lane 10, upper gels), even serial PMCA failed to induce the PK-resistance of rPrP spontaneously (Fig S2) This indicates that rSHaPrP can be used as a substrate for PrPSc propagation in the presence of NADPH using PMCA

Fig 5 Influence of other analogues of NADPH and ascorbic acid

on PrPSc propagation in PMCA ScBH of SHa-263K were mixed

with NBH at 1 : 100 dilutions by the addition of 10 l M of NADPH,

NADP, NADH, NAD or ascorbate, respectively NBH control seeded

with ScBH was also prepared (upper gel) PMCA was conducted

with 12 cycles per round, for a total of six rounds Identical aliquots

of each PMCA product of each round were treated with

50 lgÆmL)1PK and subjected to western blotting NBH without PK

treatment was directly loaded into gel as a reference for

compari-son of electrophoretic mobility (PrP C PK )) PMCA round numbers

(0–6) are shown at the top PK+ represents the preparations

digested with PK Individual chemicals are indicated on the left.

Molecular markers are shown on the right.

Fig 6 rSHaPrP, but not rHuPrP, could be converted into a PK-resistant isoform in the presence of NADPH Aliquots of ScBH

of SHa-263K were serially diluted into solutions of rSHaPrP or rHu-PrP, with final ScBH dilutions of 10)3, 10)4, 10)5and 10)6 Each preparation was divided equally into two PCR tubes, one incubated with 10 l M NADPH and the other with NaCl ⁄ P i PMCA was con-ducted with 24 cycles per round Identical aliquots of each PMCA product were treated with PK and subjected to western blotting rPrP was the directly loaded recombinant protein without PK diges-tion as a reference for comparison of electrophoretic mobility (lane 1 in all gels) 0 represents the preparation of rPrP with NADPH, but without ScBH (lane 10 in all gels) Arrow indicates the PK-resistant fragment of rPrP ScBH dilutions are shown at the top PK+ represents preparations digested with 20 lgÆmL)1PK Mole-cular markers are indicated on the right.

One of the hallmarks of prions is their species

speci-ficity in propagation [24] Our previous experiments

confirmed that PrPScfrom scrapie agent 263K-infected

hamsters could not utilize PrPC from mice or rabbits

as a substrate to replicate in PMCA (data not shown)

To test the possible species barrier when recombinant

PrPs were used as the substrate in PMCA, especially

in the presence of NADPH, rHuPrP was subjected to

PMCA using the above protocol Almost no

PK-resis-tant signals were detected any preparation, regardless

of the presence or absence of NADPH (Fig 6, lower

gel) These results correspond well with the

pheno-menon that PrPScfrom hamster cannot convert

recom-binant mouse PrP into the PK-resistant form [25],

showing clear species specificity at the level of

recom-binant PrP in PMCA, while NADPH fails to break

this limitation

Discussion

The crucial event in prion infection is the

conforma-tional change of PrPCinto PrPSc It has been reported

that several compounds or chemicals can enhance the

conversion of PrPC into aggregations in vitro [26,27]

In this study, we have proposed that in the presence of

reduced pyridine nucleotide chemicals, NADPH and

NADH, two scrapie agents propagate more efficiently

in PMCA This implies that endogenous reduced

pyri-dine nucleotide chemicals may play an important role

in the infection process of prions in vivo Our

observa-tions also indicate that reduced pyridine nucleotide

chemicals as ubiquitous agents may convert normal

PrPC into PrPSc more easily when PrPC is exposed to

exotic PrPSc Because both native PrPCin brain

homo-genates and recombinant PrP can be converted more

efficiently into PK-resistant isoforms by PrPScwith the

help of NADPH, enhancing the conversion from PrPC

to PrPSc in PMCA seems to target PrPC molecules

Our study has also shown that NADPH-enhanced

PrPSc propagation in PMCA increases the detection

sensitivity of PrPScby ‡ 102-fold and does not lead to

any false positives This suggests the potential use of

this chemical as an accelerant in PMCA to detect trace

levels of prions in biosamples

Reduced pyridine nucleotides have been shown to be

involved in many reduction–oxidation reactions

NADPH and NADP+ can bind the active site of

glucose phosphate dehydrogenase competitively to

regulate the speed of the pentose phosphate pathway

[28] and NADPH also participates in the synthesis of

cholesterol [29] Recent studies have revealed that

NADPH-diaphorase, which is known to catalyse

NADPH to transfer electrons to their targets, is

associ-ated with neuronal death [30] Increasing hippocampal NADPH-diaphorase has been observed at early stages

in the murine model of scrapie agent ME7, however, the level of NADPH-diaphorse decreases in the late stages of TSEs in animal models [17] Interestingly, we also find that the presence of NADPH enhances

de novo PrPSc propagation in PMCA PMCA-derived PrPSc maintains a stable propagating capacity in nor-mal brain homogenates after NADPH is removed Because the increased level of NADPH-diaphorase may correlate with the active synthesis of NADPH, one may think that in the early stage of TSE more NADPH in brain tissue helps the trace levels of exotic PrPSc replicate more efficiently Although NADPH synthesis may decrease along with the loss of neurons and spongiform degeneration during the pathogenesis

of TSE, PrPScmaintains its replication

Our data showed that oxidized NADPH will lose its enhancing activity for PrPSc propagation in PMCA, indicating that the role as an electron donor might contribute to this enhancement This enhancement is also observed in other reduced structural analogues, such as NADH Neither oxidized structural analogue, such as NADP and NAD, nor ascorbate, which merely works as an electron donor, possess this activity Therefore, it outlines that this enhancement depends

on both electron transfer and structural similarity Nevertheless, the presence of oxidized pyridine nucleo-tides does not influence the efficacy of PrPSc propaga-tion compared with the reacpropaga-tion without pyridine nucleotides It is clear that pyridine nucleotides are not absolutely necessary during prion propagation Because a large amount of NADPH is oxidized during the long incubation time in PMCA, it also will be interesting to know whether there is a competitive rela-tionship between reduced pyridine nucleotide and its oxidized counterpart

Our study illustrates that in the presence of NADPH, hamster rPrP is successfully converted to the PK-resistant form when seeded with native PrPSc from experimental scrapie hamsters Similarly, many other chemicals or compounds may function as co-factors to facilitate the propagation of PrPSc

in vitro, including sulfated glycan, RNA molecules, DNA molecules and chaperons [6] More strikingly, it has been proposed that polyanions, especially poly(A) RNA, can induce normal hamster PrPC to convert into infectious agents directly without any prion in PMCA, which may mimic the process of Creutzfeldt– Jakob disease [31] NADPH has the ability to bind with recombinant PrP and induce PrP aggregation when the rPrP are refolded by some ions, such as

Cu2+, Zn2+ and Mn2+ [32,33] We cannot exclude

the possibility that the NADPH-induced formation of

PK-resistant rPrP in PMCA is due to interaction with

other unknown cellular components of the ScBH

This implies that reduced pyridine nucleotides mainly

act on the PrP molecules directly, because farthing of

ScBH (10)6 dilution) used in PMCA is able to

pro-duce large quantity of PK-resistant rPrP Therefore,

we speculate that certain binding domains of pyridine

nucleotides may exist within the PrP molecule

Through transferring electrons, the energy class of

PrP may change, leading to an unstable status

Another aspect may be the potential metal-catalysed

oxidation of PrP Reduced Cu+ from Cu2+ by

NADPH can react with H2O2 and produce .OH

in vivo, which contributes to the structural alternation

seen in a variety of diseases [34] NADPH may cause

copper-bound PrP to be a transient reduced state,

which might result in uncertain structural changes in

PrPCor rPrP, leading to the protein being more easily

converted to PrPSc The level of extracellular NADPH

or NADH has not been determined [35], so it is

unclear whether PrPC on the cell surface can be

influenced by this chemical However, PrPC is not

exclusively a cell-surface protein Detectable cytoplasm

PrP in neurons [36] leads us to presume that the

abundant NADPH in cytoplasm [37] may affect PrPC

molecules during the protein trafficking in

endo-plasmic reticulum or Golgi These processes may

result the PrPCmisfolds to PrPScspontaneously

Materials and methods

Preparation of brain homogenates

Frozen scrapie agents 263K- and 139A-infected brains [38]

were homogenized in PMCA conversion buffer, containing

1· NaCl ⁄ Pi, 1% Triton X-100, 5 mm EDTA, 150 mm NaCl

and protease inhibitor cocktail tablets (Roche Applied

Sci-ence, Basel, Switzerland) as described elsewhere [23] Crude

homogenates were centrifuged for 30 s at 5000 g, aliquots

of the supernatant (10% scrapie-infected brain homogenate,

indicated as ScBH) were serially diluted with PMCA

con-version buffer by 10-fold dilution to reach a 10)12 dilution

as the stock seeds These homogenates were used

immedi-ately in subsequent experiments

Whole normal brains were removed surgically from

purchased 5-week-old male hamsters Brains were washed

thoroughly in cold NaCl⁄ Pi containing 50 mm EDTA to

remove as much blood as possible After homogenization

in PMCA conversion buffer, 10% (w⁄ v) NBH were

centrifuged for 30 s at 5000 g, and aliquots of the

super-natant were immediately frozen at )80 C for subsequent

experiments All processes of experiments, including

anaesthetic and surgical procedures, as well as animal man-agement, have been reviewed and approved, and were performed in accordance with the relevant China national legislations and regulations

Recombinant protein expression and purification

The recombinant plasmid pQE-haPrP 23–231 expressing hamster PrP residues 23–231 and pQE-huPrP 23–230 expressing human PrP residues 23–230 were generated as described previously [39] The expression and purification

of the recombinant His-tagged PrP proteins (rPrP) were performed by using Ni-NTA affinity chromatography (Qia-gen, Hilden, Germany) as described previously [40]

Refolding of the recombinant PrP

After filtering with a 0.22 lm filter membrane (Millipore, Bedford, MA, USA), the concentration of the purified pro-tein solution was estimated by measuring A280 Each recom-binant PrP was refolded by a 10-fold molar ratio of Cu2+ oxidation of the disulfide bond at room temperature for

3 h Thereafter, the protein solution was dialysed into a 100-fold volume of NaCl⁄ Pi, pH 6.0, containing 10 mm EDTA for 4 h The protein solution was transferred into fresh dialysis buffer (NaCl⁄ Pi, pH 6.0) without EDTA and this procedure was repeated five times as described else-where [25,41] After filtering the pooled fractions, the con-centration of oxidized rPrP was determined by measuring

A280 Estimates of rPrP purity were made by SDS⁄ PAGE and Coomassie Brilliant Blue staining All final protein preparations were diluted to 0.2 mgÆmL)1 in PMCA con-version buffer and frozen at)80 C Solutions were kept at

4C once thawed

In vitro amplification procedure

PMCA was performed on a water-bath sonicator (Misonix sonicator 3000; Misonix, Farmingdale, NY, USA), which had a microplate horn for PCR tubes Ninety microliters of NBH or 0.2 mgÆmL)1rPrP were mixed with 10 lL of vari-ous concentrations of ScBH in a volume of 100 lL in 0.2 mL PCR tubes The tubes were positioned on the soni-cator and amplification cycles were programmed as described elsewhere [20,21] One PMCA cycle consisted of sonication at 60% potency ( 140 W) for 40 s and fol-lowed by incubation at 37C for 59 min 20 s In this study, one round of PMCA contained 12 or 24 cycles After each round, 10 lL of amplified product was added to 90 lL of fresh normal homogenates (10-fold dilution), or 1 lL of product was added to 99 lL of fresh normal homogenates (100-fold dilution) New mixtures were subjected to another round This was repeated several times to reach an ideal amplification

Treatment of chemicals

Chemicals, including NADPH, NADP, NADH, NAD and

ascorbate, were purchased from Sigma-Aldrich (St Louis,

MO, USA) (The structures of the chemicals are shown in

Table S1.) All chemicals were freshly prepared as 10·

stock solutions in NaCl⁄ Pi before each experiment

Vari-ous concentrations of chemicals were added to NBH or

rPrP solutions and gently shaken at room temperature for

10 min, followed by mixing with ScBH and subjecting to

PMCA

PK digestion and western blotting assay

PMCA products were digested with PK at 37C for 1 h

The concentration of PK was 50 lgÆmL)1 for brain

homo-genates or 20 lgÆmL)1 for recombinant PrP solutions

Reactions were stopped by the addition of an equal

vol-ume of 2· SDS loading buffer and boiled for 10 min

Samples were separated in 0.75 mm, 15% SDS⁄ PAGE and

electronically transferred to poly(vinylidene diflouride)

membranes (Immobilon-P; Millipore) at 10 V for 1 h For

immunoblotting, the membrane was blocked with 5%

non-fat milk in NaCl⁄ Pi-T and incubated with 1 : 5000 diluted

PrP mAb 3F4 (Dako, Glostrup, Denmark) in 0.5% nonfat

milk in NaCl⁄ Pi-T at room temperature for 2 h After

washing three times in NaCl⁄ Pi-T, the membrane was

immersed in a 1 : 5000 diluted horseradish peroxidase

con-junct anti-mouse IgG (Boehringer, Ingelheim, Germany) in

NaCl⁄ Pi-T and incubated at room temperature for 2 h

Signal detection was performed with an ECL detection kit

(GE Healthcare Bio-Sciences, Piscataway, NJ, USA)

Immunoblot images were scanned and quantified by

densi-tometry using a Gel-Pro analyser (Binta 2020D; Binta,

Beijing, China)

PrPScquantitation

To estimate the concentration and number of PrPSc

mole-cules, serial dilutions of scrapie-infected brain homogenate

were analysed by western blots in the same gel as aliquots

of known concentrations of recombinant PrP (Fig S3A)

according to the admitted protocol [18,23] Signal intensities

were evaluated by densitometry, and the concentration of

PK-treated PrPScin these samples was calculated by

extrap-olation of the calibration curve calculated with recombinant

PrP (Fig S3B) To minimize errors due to saturated or

weak signals, several dilutions were analysed and the

experi-ment was repeated three times for each dilution In this

way, the average concentration of PrPScin scrapie-infected

brain homogenate in this study was measured to be

 7.265 ngÆlL)1 The number of molecules of PrPScwas

cal-culated by a mathematical method, i.e for 1 lL of ScBH,

no of molecules = 7.265· 10)9⁄ monomeric PrP molecular

mass 35 000· Avogadro’s number = 1.25 · 1011

Acknowledgements

This work was supported by National Science and Technology Task Force Project (2006BAD06A13-2) National Basic Research Program of China (973 Pro-gram) (2007CB310505), Institution Technique R&D Grand (2008EG150300) and Chinese National Natural Science Foundation Grants 30571672, 30500018,

30771914 and 30800975

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Supporting information

The following supplementary material is available:

Fig S1 (A) Evaluation the possible influence of

NADPH on PrPSc without sonication (B) Evaluation

of the possible influence of NADPH on NBH during PMCA

Fig S2 Evaluation of the possible influence of NADPH on rSHaPrP during PMCA

Fig S3 Quantification of PrPSc in scrapie-infected brain homogenate

Table S1 Structure of chemicals

This supplementary material can be found in the online version of this article

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