Recombinant Neural Protein PrP Can Bind with Both Recombinant and Native Apolipoprotein E In Vitro

Original Paper

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Acta Biochim Biophys

Sin 2006, 38: 593-601

doi:10.1111/j.1745-7270.2006.00209.X

Recombinant Neural Protein PrP

Can Bind with Both Recombinant and Native Apolipoprotein E In Vitro

Chen GAO¹, Yan-Jun

LEI^1,2, Jun HAN¹, Qi SHI¹, Lan CHEN^1,3,

Yan GUO^1,4, Yong-Jun GAO¹, Jian-Ming CHEN¹,

Hui-Ying JIANG¹, Wei ZHOU¹, and Xiao-Ping DONG¹*

1

State Key Laboratory for Infectious Disease Prevention and Control, National

Institute for Viral Disease Control and Prevention, Chinese Center for Disease

Control and Prevention, Beijing 100052, China;

2

School of Medicine, Xi’an Jiaotong University, Xi’an 710061, China;

3

National Laboratory of Medical Molecular Biology, Institute of Basic Medical

Science, Chinese Academy of Medical Sciences and Peking Union Medical College,

Beijing 100005, China;

4

College of Science and Veterinary Medicine, Northwest Agriculture and Forest

University, Yangling 712100, China

Received: April 10,

2006       

Accepted: June 20,

2006

This work was

supported by the grants from the National Natural Science Foundation of China

(30130070, 30571672 and 30500018), the National High Technology Research and

Development Program of China (2001AA215391), the National Science and

Technology Task Force Project (2003BA712A04-02) and the EU Project (QLRT 2000

01441)

*Corresponding

author: Tel/Fax, 86-10-83534616; E-mail, [email protected]

Abstract        The most essential and crucial step during the pathogenesis

of transmissible spongiform encephalopathy is the conformational change of

cellular prion protein (PrPC) to pathologic isoform (PrPSc). A lot

of data revealed that caveolae-like domains (CLDs) in the cell surface were the

probable place where the conversion of PrP proteins happened. Apolipoprotein E

(ApoE) is an apolipoprotein which is considered to play an important role in

the development of Alzheimer’s disease and other neurodegenerative diseases by

forming protein complex through binding to the receptor located in the

clathrin-coated pits of the cell surface. In this study, a 914-bp cDNA sequence

encoding human ApoE3 was amplified from neuroblastoma cell line SH-SY5Y. Three

human ApoE isomers were expressed and purified from Escherichia coli.

ApoE-specific antiserum was prepared by immunizing rabbits with the purified

ApoE3. GST/His pull-down assay, immunoprecipitation and ELISA revealed that

three full-length ApoE isomers interact with the recombinant full-length PrP

protein in vitro. The regions corresponding to protein binding were

mapped in the N-terminal segment of ApoE (amino acid 1–194)

and the N-terminal of PrP (amino acid 23–90).

Moreover, the recombinant PrP showed the ability to form a complex with the

native ApoE from liver tissues. Our data provided direct evidence of molecular

interaction between ApoE and PrP. It also supplied scientific clues for

assessing the significance of CLDs on the surface of cellular membrane in the

process of conformational conversion from PrPC to PrPSc and

probing into the pathogenesis of transmissible spongiform encephalopathy.

Key words        prion disease; PrP; apolipoprotein E; protein interaction;

caveolae-like domain

PrP is a cell surface glycoprotein that exists in neurons and other tissues

in mammals. Numerous evidences implied­ that PrP plays an important role in

copper metabolism, signal transduction and other biological processes in the

central nerve system [1]. In a group of rare and fatal neuro­degenerative

diseases, such as transmissible spongiform encephalopathy (TSE) or prion

diseases, the normal cellular­ membrane protein PrPC

conformationally changes to its abnormal pathogenic form PrPSc by exposure to extraneous­ PrPSc or

other unknown pathways [2]. Prion diseases have been described in numerous mammalian­ species,

including sheep and goat scrapie, bovine spongiform encephalopathies (BSE) and

human Creutzfeldt-Jakob disease (CJD). The main neuropathology­ changes of

these diseases are spongiform degeneration and the abnormal deposit of PrP in

central nerve tissues [3]. Although it has been widely recognized that the most

essential step is the conformational conversion from normal­ PrPC to abnormal PrPSc, the exact sites in cells and

the mechanisms­ still remain unknown. Several reports indicated­ that the

conformational change of prion protein might take place in cell membrane.

Recent studies suggested­ that the pathogenic conformational change possibly­

occurs in the CLDs within the plasma membrane [4].Apolipoprotein E (ApoE) is a lipoprotein that exists widely in

various tissues. Three isoforms have been mapped in the population, ApoE2, E3

and E4, which are encoded by e2, e3 and e4 alleles, respectively, differing only in their amino acid

sequences at positions 112 and 158 [5]. ApoE forms a protein complex through

binding to its low-density­ lipoprotein receptor (LDLR), located in the

clathrin-coated pits and CLDs of the cell surface. The ligand-receptor complex

can be taken up by the cells via clathrin-mediated endocytosis, mediating the

clearance of cholesterol particles­ from blood [6]. Some other physiological

functions­ of ApoE have also been described, such as signal­ transduction,

cellular nutrition, cell generation and deve­lop­ment­ [7].ApoE is considered to play an important role in the pathogenesis­ of

some neurodegenerative diseases, such as Alzheimer’s disease (AD) and

Parkinson’s disease [8,9]. In AD, ApoE can affect the clearance and deposit of b-amyloid by

binding with it [10]. Some studies suggested that ApoE is related to prion

diseases. In a squirrel monkey­ TSE animal model, ApoE has been found to be

co-localized­ with PrP in brain tissue [11]. Increased transcription of the

specific ApoE mRNA was observed in mouse brains infected by TSE agent

[12]. In the cerebral spinal fluids of BSE infected cattle, remarkable

increases in ApoE were repeatedly found [13]. These evidences highlighted that

ApoE might somehow participate in the pathogenesis of TSE. In order to address the possible molecular interaction between PrP

and ApoE, the two proteins were employed into the assays for protein-protein

interaction. We found that recombinant PrP was able to form complexes with both

recombinant and native ApoE in vitro. Our findings supplied scientific

clues to the hypothesis that the molecular­ interaction between PrP and ApoE

may help PrP, even PrPSc, enrich in CLDs, where the PrP

pathological conformational change may take place.

Materials and Methods

Cell culture and RNA

extraction

Human neuroblastoma cell line SH-SY5Y was maintained in Dulbecco’s

modified Eagle’s medium (DMEM; Gibco, Grand Island, USA) containing 10% fetal

cattle serum, 50 U/ml penicillin and 50 mg/ml streptomycin. Total

cellular RNA was extracted with a commercially supplied Trizol agent

(Invitrogen, Carlsbad, USA) and stored at –70 ?C.

Plasmid construction

To obtain cDNA of human ApoE, 1 mg SH-SY5Y cellular­ RNA

was mixed with 5 U AMV reverse transcriptase (Invitrogen), 10 U RNasin, 20 mM

dNTP and 20 pM oligo(dT) in a total volume of 20 ml at 42 ?C for 1 h. Two mi­cro­liters

of product was mixed with 2.5 U LA Taq polymerase (TaKaRa, Dalian,

China), 20 mM dNTP, 2?GC buffer

II, and human ApoE gene specific primers ApoE-F (5‘-AGGATCCAAGGTGGAGCAAGCG-3‘,

BamHI site underlined) and ApoE-B (5‘-AGAATTCGTGATTGTCGC­TGGG-3‘,

EcoRI site underlined) for PCR at following conditions: 94 ?C for 30 s,

58 ?C for 30 s, 72 ?C for 60 s, 30 cycles. The 914 bp PCR product was ligated with

commercially supplied pMD18-T vector (TaKaRa) generating­ pT-ApoE3. After being

verified by sequencing, the insert was cleaved from pT-ApoE3 with BamHI

and EcoRI, and cloned into a His-tag fusion expression vector pET32a

(Novagen, San Diego, USA) generating pET-ApoE3. The plasmids pET32-E2 and

pET32-E4 containing full-length human ApoE2 and E4 cDNA respectively were

kindly provided by Prof. K. H. WEISGRABER [14].To obtain cDNA of human ApoE, 1 mg SH-SY5Y cellular­ RNA

was mixed with 5 U AMV reverse transcriptase (Invitrogen), 10 U RNasin, 20 mM

dNTP and 20 pM oligo(dT) in a total volume of 20 ml at 42 ?C for 1 h. Two mi­cro­liters

of product was mixed with 2.5 U LA Taq polymerase (TaKaRa, Dalian,

China), 20 mM dNTP, 2?GC buffer

II, and human ApoE gene specific primers ApoE-F (5‘-AGGATCCAAGGTGGAGCAAGCG-3‘,

BamHI site underlined) and ApoE-B (5‘-AGAATTCGTGATTGTCGC­TGGG-3‘,

EcoRI site underlined) for PCR at following conditions: 94 ?C for 30 s,

58 ?C for 30 s, 72 ?C for 60 s, 30 cycles. The 914 bp PCR product was ligated with

commercially supplied pMD18-T vector (TaKaRa) generating­ pT-ApoE3. After being

verified by sequencing, the insert was cleaved from pT-ApoE3 with BamHI

and EcoRI, and cloned into a His-tag fusion expression vector pET32a

(Novagen, San Diego, USA) generating pET-ApoE3. The plasmids pET32-E2 and

pET32-E4 containing full-length human ApoE2 and E4 cDNA respectively were

kindly provided by Prof. K. H. WEISGRABER [14].To construct the expression recombinant plasmids for N- and

C-terminal of ApoE, the segment encoding amino acid 1–194 of ApoE was amplified

with the primers ApoE-F and ApoE194-B (5‘–AAGCTTTCAAGTGGCG­G­C­C­­CGC-3‘,

HindIII site underlined), and the segment encoding ApoE peptide from

amino acid 195 to 299 was amplified with the primers ApoE299-F (5‘–GGATCCACTG­T­­G­­G­G­CTCCCTG-3‘,

BamHI site underlined) and ApoE-B, both using pT-ApoE3 as the templates.

The amplified ApoE fragments were separately cloned into expressing plasmid

pQE30-GST containing both His-tag and GST-tag [15], generating pQEG-ApoE-N and

pQEG-ApoE-C. To generate C-terminus truncated human prnp gene that

encodes 68 amino acids (amino acid 23–90), PCR was carried out with primers HuPrP-F

(5‘–GGATCCATGAAGAAGCGGCCAAAGCCTGG-3‘, BamHI site

underlined) and HuPrP-B-90 (5‘–GAATTCCTGACTGTGGGTGCCACCTTATTGA-3‘,

EcoRI site underlined) at following conditions: 94 ?C for 50 s, 58 ?C

for 50 s and 72 ?C for 60 s, 30 cycles, using pT-HuPrP [16] as the template.

The 216 bp fragment of PCR product was ligated to pMD18-T vector generating

pT-HuPrP23–90, and then subcloned into a GST fusion expression vector pGEX-2T

(Amersham Pharmacia, Uppsala, Sweden) generating pGST-HuPrP23–90.

Protein expression and

purification

Three isoforms of His-ApoE, GST-ApoE-N and GST-ApoE-C, as well as

HuPrP23–90, HuPrP91–231 and HuPrP23–231 [16] were expressed in Escherichia coli strain BL21(DE3)

or JM109, respectively. Briefly, transformed bacteria were grown to an A600 of 0.5–0.6 and induced by isopropyl-b–D-thiogalactoside

at final concentration of 0.5 mM. Cells were harvested by centrifugation. Then

cells were resuspended in PBS (pH 7.4) containing 1 mM phenylmethylsulfonyl

fluoride (PMSF) as protease inhibitor for His-tagged protein expression; or

resuspended in PBS containing 1 mM EDTA, 300 mM NaCl and 30 mM Tris-HCl, pH

8.0, 1 mM PMSF for GST-fusion protein­ expression. Lysozyme was added to a

final concentration of 20 mg/ml, and cells were lysed by incubation for 30 min and sonication

for 24?10 s with a 10 s interval at 400 W. The

His-tagged proteins were purified with Ni-NTA agarose (Qiagen, Hilden,

Germany), and GST-fusion proteins were purified with glutathione-Sepharose 4B

(Amersham Pharmacia), according to the manufacturers’ protocols. Protein

concentrations were determined using the BCA kit (Qiagen).

Western blot

Various purified ApoE proteins, ApoE N- and C-terminal­ proteins

were separated by 12% SDS-PAGE and transferred­ to nitrocellulose membranes.

After blocking with 5% defatted­ milk in PBST (phosphate buffered saline, pH

7.6, containing 0.05% Tween-20) overnight at 4 ?C, the membranes­ were

incubated with 1:2000 rabbit anti-ApoE antibody (Santa Cruz, Santa Cruz, USA)

for 2 h at room temperature and then further incubated with 1:2000 horseradish

peroxidase (HRP)-conjugated anti-rabbit IgG (Santa Cruz). The protein bands

were visualized by ECL kit (PE Applied Biosystems, Foster City, USA).

Antibody preparation

Five hundred micrograms of purified ApoE protein was mixed with

complete Freund’s adjuvant and injected hypodermically­ into SPF-level rabbits

at multi-points. Ten days later, the rabbits were boosted by 200 mg of ApoE

protein mixed with incomplete adjuvant. Total five boosting­ were done at a 10

d interval. Two weeks after the fifth boosting, rabbits blood was collected using

a carotid intubation­ under anesthesia with ether. For the purification­ of

IgG, the collected sera were precipitated with 50% and 33% ammonium sulfate in

sequence, and furthermore, purified with Sepharose G chromatography.

ELISA

Polyclonal antibodies of Doppel [17] and Tau [18] were described

elsewhere. Anti-GST polyclonal antibodies were purchased from Santa Cruz. An

ELISA protocol was established­ to screen the potential interactions between

ApoE and other proteins. His-ApoE in 0.05 M sodium bicarbonate­ buffer, pH 9.6,

was coated onto a 96-well plate at 100 ng/well at 4 ?C overnight. All wells

were blocked with 5% bovine serum albumin (BSA) in PBST at room temperature for

2 h. Various testing proteins of the same molar concentration in PBS containing

2% BSA were transferred­ to the wells. After 2 h incubation, the plates were

washed with PBST three times, and polyclonal antibodies­ against the

corresponding proteins were used at a dilution of 1:4000 and incubated for 45

min. Bound antibodies were detected using horseradish peroxidase conjugated­

secondary antibody and developed with 3,3‘,5,5‘-tetramethylbenzidine

(Sigma, St. Louis, USA). Absorbance­ at 450 nm was measured using a microplate

reader after the reaction was terminated by addition of 2 M H2SO4. An equal amount of GST protein was used as

the control.To screen the potential interactions of various PrP segments­ with

different isoforms of ApoE, 50 ng of each PrP protein was coated onto wells of a

96-well microplate and subsequently incubated with various ApoE proteins. The

bound ApoE was measured with the same protocol described above.

Immunoprecipitation

Ten micrograms of three isoforms of ApoE were respectively­ mixed

with 5 mg of HuPrP23–231 or HuPrP91–231 in binding buffer (50 mM Tris-HCl, 100 mM NaCl, pH 8.0) in a

volume of 500 ml at 4 ?C for 2 h. After incubation with 1:2000 diluted monoclonal

antibody 3F4 (DakoCytomation, Cambridgeshire, UK) for 2 h, 10 ml of protein G

Sepharose beads pre-equilibrated with binding­ buffer were introduced into the

reaction mixture and incubated for another 2 h with vibrant shaking. The

Sepharose beads were precipitated by centrifugation at 500 g for 5 min

and washed with 500 ml of washing buffer (50 mM Tris-HCl, 200 mM NaCl, pH 8.0) for three

times. The bound antibody-antigen complexes were separated by 12% SDS-PAGE and

transferred to nitrocellulose membranes. The bound ApoE proteins were detected

with 1:2000 diluted anti-ApoE polyclonal antibodies. To address the interaction

between ApoE and PrP N-terminal segments, 10 mg of each of three

isoforms of ApoE protein was incubated with 5 mg of HuPrP23–90 respectively,

and subsequently precipitated with anti-PrP polyclonal antibodies [19]. The

bound ApoE proteins were detected according to the protocol­ described above.

GST fusion protein pull-down

assay

To identify interactions between HuPrP23–231 and ApoE N- or

C-terminal fragment, 5 mg of purified HuPrP23–231 protein was incubated with 10 mg of ApoE N- or

C-terminal fragment in 500 ml of binding buffer containing­ 20 mM Tris-HCl, 200 mM NaCl, 10 mM

aprotinin, pH 8.0, at 4 ?C for 4 h, while an equal amount of GST protein was

used as a control. Fifteen microliters of glutathione agarose beads were added

to the reaction solution­ and incubated at 37 ?C for 30 min with end-over-end

mixing. After centrifugation at 500 g for 2 min, the supernatants were

discarded and beads were washed three times with 500 ml of binding buffer. The

complex was separated on 12% SDS-polyacrylamide gel and transferred­ to

nitrocellulose membranes. To visualize the bound PrP protein, a Western blot

assay was carried out, using 3F4 antibody at 1:2000 as the primary antibody and

HRP-conjugated anti-mouse IgG (Santa Cruz) at 1:4000 as the secondary antibody.One gram of liver tissue from healthy hamsters was prepared to 10%

homogenates in lyses buffer [20]. The homogenate was centrifuged at 20,000 g

for 90 min, removing­ the debris of the tissue. Ten microgrammes of HuPrP23–90 (with GST-tag)

protein was added to the homogenate in a volume of 2 ml at 4 ?C for 4 h. The

bound ApoE was detected as described above.

His-tagged protein pull-down

analysis

Five microgrammes of HuPrP23–231 (with His-tag) and 2 ml

of 10% hamster liver homogenate were incubated at 4 ?C for 4 h. Ni-NTA agarose

(10 ml) pre-equilibrated with binding buffer were introduced into the

mixture and incubated­ for 2 h with vibrant shaking. The mixture was

centrifuged at 500 g for 2 min, and the supernatant was discarded and

beads were washed three times with 500 ml of binding buffer. The

complexes were separated on 12% SDS-polyacrylamide gel and transferred to

nitrocellulose membranes. The bound ApoE was detected as described above.

Results

Expression of various ApoE

proteins in E. coli

A 914 bp cDNA fragment corresponding to the full length human ApoE

was amplified from cell line SH-SY5Y, with A mutated to G at nt 787. Sequence of

Cys112/Arg158 indicated that it was the E3 isoform. Using affinity

chromatography­ of Ni-NTA agarose, an approximately 54 kDa His-ApoE fusion

protein was purified from the lysate­ of E. coli BL21(DE3) cells

transformed with the recombinant plasmid pET-ApoE3 [Fig. 1(A)]. Western

blot analysis­ revealed that the 54 kDa protein was specifically recognized by

the commercial anti-ApoE antibody [Fig. 1(D), lane 2]. N- and C-truncated ApoE proteins containing GST were expressed in

the E. coli strain JM109 and purified by the affinity chromatography of

Ni-NTA agarose. As expected, two fusion proteins, at approximately 48 kDa

(ApoE-N) [Fig. 1(B)] and 37 kDa (ApoE-C) [Fig. 1(C)], were

specifically recognized by anti-ApoE antibody in Western blot [Fig. 1(D),

lanes 1 and 3].

Three ApoE isoforms interacted

with PrP protein in vitro

It has been described that ApoE is involved in the growth and other

activities of neuron cells [21]. To demonstrate the interactions of ApoE3 and PrP

proteins, an ApoE3-coated ELISA was established to capture the possibly bound

protein, using GST protein as a negative control. At the same mole ratios as

coated ApoE, the full-length human PrP (HuPrP23–231) showed obvious binding

activity (Fig. 2, column 3), while the truncated PrP (HuPrP91–231) did not

show any binding capacity with the coated ApoE compared­ with the negative

control (Fig. 2, column 4). To address the potential interactions of

ApoE and other neuroproteins, recombinant Tau and Doppel were tested in the

ApoE-coated ELISA. Both Tau and Doppel did not show any activity in binding

with the coated ApoE (Fig. 2, columns 1 and 2) compared with the

negative control. It implied that ApoE might specially interact with PrP. Immunoprecipitation tests revealed that all ApoE2, E3 and E4 could

be precipitated with anti-PrP antibody in the presence of HuPrP23–231 (Fig. 3),

whereas none of the three ApoE proteins showed any detectable interaction with

HuPrP91–231 (Fig. 3). Quantitative analyses of the immunoblot images

did not show a remarkable difference between the three ApoE proteins and

HuPrP23–231 (data not shown). These results implied a molecular interaction

between ApoE and PrP in vitro, probably in the N-terminal region of PrP.To test whether PrP protein had different binding activities­ with

various ApoE isoforms, ApoE2, E3 and E4 were incubated in the wells coated with

HuPrP23–231 respectively, and GST was used as a negative control. Obviously,

with the increasing amounts of ApoE in the preparations, the A values

increased, showing a dose-dependant­ manner (Fig. 4). No notable

difference in binding­ activity of HuPrP23–231 was observed among

three isoforms, when mole ratios of ApoE to PrP were 2:1, 1:1, 1:2 and 1:4.

Only in the preparations with more ApoE molecules (ApoE to PrP was 5:1 and

10:1), did ApoE3 show relatively stronger binding ability.

Binding position of PrP to

ApoE located at amino acid 23–90 of PrP

The failure for HuPrP91–231 to bind with ApoE in immuno­precipitation

and ELISA indicated that the region in PrP that interacts with ApoE might

locate in its N-terminal. To confirm this possibility, a 204-bp human prnp

sequence that encodes amino acid 23–90 was inserted into pGEX-2T and transformed

into E. coli BL21(DE3). An approximately 30-kDa protein in GST-fusion

form was purified by the affinity chromatography of glutathione agarose­ and

verified by Western blot with anti-PrP polyclonal antibodies. An

immunoprecipitation test showed that all ApoE isoforms formed detectable

complexes with HuPrP23–90, and the bound ApoE was recognized by anti-ApoE antibody in

Western blot [Fig. 5(A)]. Furthermore, full-length (HuPrP23–231), N-terminal

(HuPrP23–90) and C-terminal (HuPrP91–231) PrP segments­ were tested in ApoE3-coated

ELISA, in which GST-coated wells were used as negative controls in parallel. Fig.

5(B) showed that HuPrP23–231 and HuPrP23–90 have remarkable binding­ activities with the fixed ApoE3, whereas

HuPrP91–231 failed. At molar ratios of 1:1 and 1:2 (ApoE3 vs. PrP), HuPrP23–90 showed

comparable binding ability as the full-length­ HuPrP23–231. These results

indicated that the interacting­ region of PrP with ApoE is located at the

N-terminal.

N-terminal of ApoE binds with PrP protein

To map the region within ApoE interacting with PrP protein, same

mole amount of full-length ApoE protein, ApoE N- and C-terminal fragments were

incubated with the HuPrP23–231 and precipitated with anti-PrP-specific antibody respectively. The

bound ApoE molecules were visualized by Western blot with anti-ApoE specific

antibody. The protein complexes were detected clearly in the reactions­

containing full length and N-terminal ApoE proteins and HuPrP23–231 [Fig.

6(A), lanes 2 and 3], but not in the reactions of C-terminal ApoE (lane 4)

and GST protein (lane 5). Since the expressed ApoE N- and C-terminal proteins­

had GST-tag, GST pull-down tests were also conducted­ with the same amount of

HuPrP23–231. After eluted from glutathione-Sepharose 4B, the bound PrP was

detected by Western blot with anti-PrP-specific antibody. A very clear PrP

signal was detected in the preparation of ApoE N-terminal protein [Fig. 6(B),

lane 2], but not in that of ApoE C-terminal protein (lane 3), the control GST

(lane 4) or in that of GST-CAT (lane 5), indicating that ApoE N-terminal

peptide formed a complex with the input of PrP protein. It also indicated that

the region within ApoE responsible for interaction with PrP might locate at

N-terminal region.

PrP proteins interact with the

native ApoE from liver tissues

ApoE is remarkably expressed in liver and brain tissues. To find out

whether PrP protein could form a complex with native ApoE in vitro,

hamster liver homogenates were prepared. After incubation with recombinant

HuPrP23–231, the mixture was incubated with Ni-NTA agarose and the possible

bound ApoE signals were visualized by Western­ blot with anti-ApoE antibody. Fig.

7(A) showed a 34 kDa ApoE-specific band in the reaction of HuPrP23–231 with liver

tissue extracts (lane 1), whereas there was no positive­ signal in the

preparation containing only HuPrP23–231 or liver extracts (lanes 2 and 3),

indicating that the recombinant­ PrP was able to react with the native ApoE in

the liver homogenate. Furthermore, GST fusion protein HuPrP23–90 was mixed

with liver extracts and GST pull-down assay was conducted. Subsequent

immunoblot with anti-ApoE antibody revealed that a 34 kDa band in the

preparation of HuPrP23–90 with liver extract [Fig. 7(B), lane 1], but not in GST

control (lane 2) or in the preparations either containing only HuPrP23–90 (lane 3) or

liver extracts (lane 4). The results suggested that the N-terminal­ PrP peptide

could bind the native ApoE.

Discussion

The data in this study provided direct evidence that recombinant­

PrP can bind to both recombinant and native ApoE proteins in vitro.

ApoE, a 299-amino acid protein (34 kDa), plays a significant role in

lipoprotein metabolism, as it is the major ligand in receptor-specific

lipoprotein uptake. ApoE is a ligand for all members of the LDLR family and a

constituent of lipoprotein particles that transport­ lipids throughout the

circulation and between cells. In the nervous system, non-neuronal cell types,

most notably astroglia and microglia, are the primary producers of ApoE, while

neurons preferentially express the receptors­ for ApoE [22]. Increased

transcription of ApoE mRNA, remarkable co-deposits of ApoE with PrP and disease

progressive­-related increase of ApoE in the brain tissues from naturally and

experimentally infected animals indicate­ that ApoE might participate in the

pathogenesis of TSE [12]. Our study suggested a novel molecular basis that

other proteins in nerve tissues, i.e., ApoE may participate in the pathogenesis

of prion diseases. In humans, ApoE exists in three major isoforms, E2, E3 and E4. Among

them, E4 isoform is at greater risk for developing late-onset Alzheimer? disease [23]. A French research

group has even suggested that the ApoE alleles are major susceptible factors

for CJD, in which e4 allele of the ApoE gene is taken as a risk factor [24]. However,

subsequent researches with more samples proved that the difference is not

statistically significant [25,26]. Our protein­ interaction tests in vitro

did not reveal a significant dif­ference in the binding activity with PrP among

the three ApoE isoforms. Although the influence of different ApoE isoforms on

TSE sensitivity and pathogenesis still remains unclear, similar binding

activities of ApoE proteins to PrP suggest that ApoE isoforms may not have

differences, at least, in recognizing PrP molecules.It is generally accepted that the conformational change of prion is

the most important and crucial step in TSE pathogenesis. The region responsible

for interaction with ApoE within PrP protein was assigned to residue 23–90 at the

N-terminus. There are four proline/glycine rich octarepeats (PHGGGWGQ) between

amino acid residues 51 and 90. Structural analyses of PrP protein reveal that

the N terminal is highly flexible and lacks identifiable secondary­ structure

under the experimental conditions. Several biological activities have been

confirmed in this region, including binding Cu2+,

interacting with sGAG proteoglycan and several neuron proteins [27]. However,

the region correlating with neural toxic lies in the middle region of PrP

protein (amino acid 106–126), while C terminus­ segment corresponds to the conformational

change [4,28]. It indicates that PrP protein may bind to target proteins or

receptors through its N-terminal segment, and afterwards, displays its

physiological or pathological activities through exposing its middle and C

terminal domains.Our results showed that the fragment of residue 1–194 at

N-terminus within ApoE protein region is responsible­ for interaction with PrP

protein. Human ApoE N-terminal domain (amino acid 1–191) bears low-density

lipoprotein receptor-binding sites, which locates at the domain­ of amino acid

136–158.

Its C-terminal domain (amino acid 210–299) is a lipoprotein-binding site with

supercoil structure. ApoE is the ligand for several receptors, including the

apolipoprotein low-density lipoprotein receptor (LDL receptor),

lipolysis-stimulated receptor (LSP) and human ApoE receptor 2, which

participated in the signal transduction, cell nutrition during brain

development [7]. One might think that the interaction between PrP and

ApoE, especially accumulation of PrPSc during the pathogenesis

of TSE, would block cell nutrition and signal transduction processes, leading

to neuron death. Actually, in AD, the direct binding of ApoE with amyloid

peptide impairs ApoE receptor-dependent protective signals that promote

neuronal­ survival and synaptic plasticity that may influence­ the amyloid

clearance and fibril formation [10,29].Our research only proposes the data of molecular interaction between

ApoE and PrP protein, however, the biological­ significance is still unknown.

The floating characteristic­ of ApoE in body fluids and between the cells, wide

distribution of ApoE receptors among various tissue cells make it possible to

be a carrier for extraneous PrPSc transferring from peripheral

tissues to central nerve system. Wide distribution of various ApoE receptors in

caveolae-like domains on the surface of neuron cells correspond well with the

newly proposed domain in which conversion­ from PrPC to PrPSc occurs. It is reasonable to hypothesize that PrP, even PrPSc, is enriched in caveolae-like domains through interacting with

ApoE. Highly concentrated PrP molecules in the special room may help PrPSc contact with its normal isoform PrPC, leading

to conformational conversion. In fact, presence of potential receptors of PrPC in CLDs has been already supposed that it might be a trans-membrane

protein recognizing PrP with its extracellular­ portion [30]. More detailed

studies are needed to clarify whether the receptors of ApoE correlate with or

even are the hypothesized receptors for PrP.

Acknowledgements

We thank Prof. K. H. WEISGRABER (Gladstone Institute­ of

Neurological Disease, University of California, San Francisco, USA) for kindly

providing plasmids pET32-E2 and pET32-E4 and Prof. Cai-Min XU and her team

(Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing,

China) for very valuable guidance. We are indebted to Mr. Bao-Yun ZHANG for

protein purifications.

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