Original Paper |
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Acta Biochim Biophys |
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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
LEI1,2, Jun HAN1, Qi SHI1, Lan CHEN1,3,
Yan GUO1,4, 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 ºC.
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 ºC 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 ºC for 30 s,
58 ºC for 30 s, 72 ºC 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 ºC for 50 s, 58 ºC
for 50 s and 72 ºC 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 ºC, 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 ºC 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 ºC 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 ºC 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 ºC 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 ºC 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 ºC 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抯 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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