From the Department of Molecular Biology, Okayama
University Graduate School of Medicine and Dentistry, Okayama 700-8558, Japan, § Gastroenterological Center, Yokohama City
University Medical Center, Yokohama City University School of Medicine,
Yokohama 236-0004, Japan, and
Department of Pediatrics, Tsukuba
University School of Medicine, Tsukuba 305-8575, Japan
Received for publication, August 2, 2002, and in revised form, December 27, 2002
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ABSTRACT |
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Bovine and human lactoferrins (LF) prevent
hepatitis C virus (HCV) infection in cultured human hepatocytes; the
preventive mechanism is thought to be the direct interaction between LF
and HCV. To clarify this hypothesis, we have characterized the binding activity of LF to HCV E2 envelope protein and have endeavored to
determine which region(s) of LF are important for this binding activity. Several regions of human LF have been expressed and purified
as thioredoxin-fused proteins in Escherichia coli.
Far-Western blot analysis using these LF fragments and the E2 protein,
expressed in Chinese hamster ovary cells, revealed that the 93 carboxyl amino acids of LF specifically bound to the E2 protein. The 93 carboxyl
amino acids of LFs derived from bovine and horse cells also possessed
similar binding activity to the E2 protein. In addition, the amino acid
sequences of these carboxyl regions appeared to show partial homology
to CD81, a candidate receptor for HCV, and the binding activity of
these carboxyl regions was also comparable with that of CD81. Further
deletion analysis identified 33 amino acid residues as the minimum
binding site in the carboxyl region of LF, and the binding specificity
of these 33 amino acids was also confirmed by using 33 maltose-binding
protein-fused amino acids. Furthermore, we demonstrated that the 33 maltose-binding protein-fused amino acids prevented HCV infection in
cultured human hepatocytes. In addition, the site-directed mutagenesis to an Ala residue in both terminal residues of the 33 amino acids revealed that Cys at amino acid 628 was determined to be critical for
binding to the E2 protein. These results led us to consider the
development of an effective anti-HCV peptide. This is the first
identification of a natural protein-derived peptide that specifically
binds to HCV E2 protein and prevents HCV infection.
Hepatitis C virus (HCV)1
infection frequently causes chronic hepatitis (1, 2) and frequently
progresses to liver cirrhosis and hepatocellular carcinoma (3, 4). HCV
is an enveloped positive single-stranded RNA (9.6 kb) virus belonging
to the Flaviviridae (5-7). The HCV genome encodes
a large polyprotein precursor of about 3,000 amino acid (aa) residues,
which is cleaved by the host and viral proteases to generate at least
ten proteins: the core, E1 (envelope 1), E2,
p7, NS2 (nonstructural protein 2), NS3, NS4A, NS4B, NS5A, and NS5B (8-12). The most characteristic feature of the HCV genome is its remarkable sequence heterogeneities and variations, and to date at least six major HCV genotypes, which
have been further grouped into more than 50 subtypes, have been
identified (13-16). The genetic complexity of HCV is thus a major
hindrance to the development of the vaccines.
To date, interferon has been the sole effective antiviral reagent used
in the clinical therapy of hepatitis C, but its effectiveness is
limited to about 30% of the reported cases (17). Combined treatment of
interferon and ribavirin has been shown to be more effective than
treatment with interferon alone (18). The side effects of interferon
are also in some cases severe enough to lead to treatment cessation.
Although the entry mechanism of HCV, as well as that of hepatitis B
virus, remains unclear, it was reported recently that human CD81 (19)
and scavenger receptor class B type I (20) could be bound by a
truncated, soluble form of the E2 protein; such findings suggest that
these proteins may act as receptors for HCV on the cell surface. Low
density lipoprotein receptor (21) was also reported as a putative HCV
receptor in endocytosis experiments using isolated HCV-lipoprotein
complexes. However, because of the lack of a reproducible and efficient
HCV proliferation system, it is not known whether these candidate
receptors for HCV serve as the functional receptor on human hepatocytes
(22).
We previously reported that non-neoplastic human hepatocyte-derived
PH5CH8 cells supported HCV replication, although HCV proliferation was
at a fairly low level; in that study, we also demonstrated the
antiviral effects of interferon- LF has a molecular mass of 80 kDa and consists of two homologous
globular lobes (an N-lobe and a C-lobe), each with a single iron
(Fe3+) binding site. There is a notable degree of internal
homology between the two lobes, i.e. ~35% identical amino
acid residues have been identified in the corresponding portions (26).
The three-dimensional structures of human and bovine LFs have been clarified by crystallographic studies (27, 28). Although the overall
structure of LF is similar to that of transferrin (TF) (~60% amino
acid sequence homology to LF), LF has two distinct features that may be
functionally important. First, the association constant of LF for iron
is 300 times that of TF (29). Second, in contrast to TF, LF possesses
strong inhibitory activity against bacterial growth. The antimicrobial
activity of LF has been ascribed to the basic N-terminal region
("lactoferricin") (30). Lactoferricin (24 aa residues) shows
activity against a wide range of microorganisms including bacteria and
fungi (30). LF is present in the milk of most mammals, and the LF
content in milk changes substantially during the lactation period. The
concentrations of LF in mature milk are 0.1-0.4 mg/ml in bovines and
1-3 mg/ml in humans, and LF is especially enriched in the colostrum
(0.8 mg/ml in bovines and 10 mg/ml in humans) (31, 32). It is well
established that LF plays an important role in the newborn as the
primary nonspecific defense against pathogenic microorganisms (26).
Also, it has been reported that rats fed a 2% bovine LF diet displayed
no significant side effects (33). This low risk of severe side effects
presents a major clinical advantage of bovine LF; hence, clinical pilot studies have been performed recently. The results have shown that bovine LF was effective in some patients with chronic hepatitis C (34,
35).
A recent study by our group (36) suggested that the prevention of HCV
infection in these cells was because of interactions of LF with HCV
rather than with the cells themselves; our study demonstrated that LF
inhibited viral entry into the cells by interacting directly with HCV
(36). On the other hand, Yi et al. (37) independently
demonstrated that HCV envelope proteins (E1 and E2) could bind to human
and bovine LFs, although their binding specificities have not been
clarified. E2 protein expressed in mammalian cells specifically binds
to human target cells (38), and such binding is associated with HCV
particles binding to target cells in vitro, as well as with
HCV infection in vivo (38). In addition, the level of
antibody response to E2 protein has been shown to correlate with
protection against HCV in animal models (39) and with occasional
clearance of HCV in cases of natural infection (40), suggesting that
the E2 protein is the major receptor-binding protein. For these
reasons, we focused on the interactions between LF and E2 proteins to
understand the mechanism by which LF prevents HCV infection of target
cells. In this study, we have characterized the binding activity of LF to the E2 protein and have endeavored to determine which region(s) of
LF are important for this binding activity. Here, we report the finding
of 33 human LF-derived amino acids possessing binding activity to the
E2 protein of HCV, which leads to inhibition of HCV infection in target cells.
Far-Western Blot Analysis--
Far-Western blot analysis was
carried out according to a method described previously (37) with some
modifications. Briefly, 0.5 µg of human and bovine LFs (Sigma) and
various recombinant LF fragments were resolved by 10 or 12% SDS-PAGE
and were transferred to polyvinylidene difluoride membranes. The
membranes were then blocked in N-buffer (50 mM Tris, pH
7.5, 150 mM NaCl, 0.1% Triton X-100, 0.25% gelatin) with
2% bovine serum albumin (BSA) for 30 min at room temperature.
The binding reaction was carried out at room temperature in N-buffer
containing 2% BSA, and the secreted form of E2 protein (E2-681)
expressed in Chinese hamster ovary cells was used as a probe
(41). After 1 h of incubation, the membranes were washed with
N-buffer three times for 10 min at room temperature, blocked once again
with N-buffer with 2% BSA, and incubated at room temperature with rat
monoclonal antibody, MO-2 or MO-12, against E2 protein (42).
Normal rat serum (Invitrogen) was used as a control instead of
MO-2 or MO-12. After 1 h of incubation at room temperature, the
membranes were washed with 0.1% Tris saline three times for 5 min at
room temperature. Immunocomplexes on the membranes were detected by
enhanced chemiluminescence assay (Renaissance; PerkinElmer Life Sciences).
Deglycosylation of LF--
Human and bovine LFs (10 µg each;
Sigma) were denatured with 0.5% SDS, 1% 2-mercaptoethanol for 10 min
at 100 °C and were then treated with 2,000 units of
peptide-N-glycosidase F (PNGaseF; New England Biolabs) in 50 mM sodium phosphate, pH 7.5, 1% Nonidet P-40 for 4 h
at 37 °C. After incubation, the samples were immediately used for
the Far-Western blot analysis.
Isolation of LF, TF, and CD81 cDNAs--
Total RNAs (2 µg
each) from human cancer breast tissue (43), normal bovine breast
tissue, and normal horse peripheral blood mononuclear cells were used
as templates for reverse transcription (RT)-PCR to obtain the
full-length LF cDNAs. The total RNA (2 µg) from PH5CH8 cells was
also used as a template for RT-PCR to obtain the TF cDNA encoding
aa 587-679 and the CD81 cDNA encoding the large extracellular loop
(LEL; aa 113-201). Oligo(dT) was used to prime the cDNA synthesis
using Superscript II reverse transcriptase (Invitrogen). Amplification
by PCR with a highly efficient proofreading DNA polymerase, KOD-plus
(Toyobo) was performed for 20 cycles using each primer set (see
Table I) arranged from the nucleotide
sequences of human LF (X52941), bovine LF (M63502), horse LF
(AJ010930), human TF (S95936), and human CD81 (M33680) cDNAs. The
PCR product containing the coding region of full-length human LF was
cloned into the HindIII and BamHI sites of the
pHookTM-2 (Invitrogen), as described previously
(44). The PCR products containing the coding region of full-length
bovine and horse LFs were also cloned into the NotI and
HpaI sites of pCXbsr (45), as described previously (46). The
PCR products containing the human TF fragment (aa 587-678) and the
CD81 LEL were cloned into the BamHI and HindIII
sites of the pET32a (Novagen), respectively. The nucleotide sequences
of obtained cDNA clones encoding human, bovine, and horse LF, and
human TF and CD81 were determined by Big Dye terminator-cycle
sequencing on an Applied Biosystem 310 automated sequencer (Applied
Biosystems, Norwalk, CT). It was confirmed that these cDNAs had
identical nucleotide sequences to those in the databases.
Construction of Expression Plasmids for Escherichia
coli--
The pET32a was used for the production of LF, TF, or CD81
fragments as thioredoxin (TRX)-fused proteins in E. coli.
For the expression of human, bovine, and horse LFs, the inserts of
obtained cDNA clones were transferred into the BamHI and
HindIII sites of the pET32a. Based on these pET32a plasmids
containing the coding regions of the full-length LFs, the pET32a
expression plasmids encoding the various regions of LF were constructed
by inserting the PCR product amplified using the primer sets listed in
Table I into the BamHI-HindIII site of the
pET32a. The pMAL-c2X (New England Biolabs, Beverly, MA) was used for
the production of human LF fragment (aa 600-632) as maltose-binding
protein (MBP)-fused protein in E. coli. The DNA fragment
encoding the human LF fragment (aa 600-632), which was obtained from
the pET32a expression vector by digestion with BamHI and
HindIII, was transferred into the BamHI-HindIII sites of the pMAL-c2X. The
single-amino acid substitutions (i.e. changed to an Ala
residue) were introduced into pET32a containing the coding region of
human LF fragment (aa 600-632) by site-directed mutagenesis, as
described previously (47), using the mutation primer sets. The obtained
pET32a mutants were confirmed by nucleotide sequencing.
Expression and Purification of TRX-fused LF Fragments, TF
Fragment, and CD81 LEL--
Expression plasmids for TRX-fused human LF
fragments were transformed into the E. coli strain
AD494(DE3) (Novagen). The transformants were cultured in 10 ml of LB
medium containing ampicillin (50 µg/ml) and kanamycin (15 µg/ml) at
37 °C overnight and were then transferred to 200 ml of LB medium;
the culture period was then for 4 h at 37 °C. One
mM isopropyl- Expression and Purification of the MBP-fused LF
Fragment--
The expression plasmid for the MBP-fused human LF
fragment (aa 600-632) was transformed into the E. coli
strain JM109. The transformants were cultured in 100 ml of LB medium
containing ampicillin (100 µg/ml) and glucose (2 mg/ml) at 37 °C
until an optical density of 0.6 at 600 nm was reached. At this point, 1 mM of isopropyl- Enzyme-linked Immunosorbent Assay (ELISA)-based Binding
Assay--
A previously described ELISA-based binding assay was used
to examine the binding affinity between the E2 protein and MBP-fused human LF fragment (48). Briefly, 96-well microtiter immunoplates (Maxisorp; Nunc) were coated overnight at 4 °C with the secreted form of the E2 protein (E2-681) (50 µl/well, 10 µg/ml) in
HEPES-buffered saline (HBS) (10 mM HEPES, pH 7.0, 150 mM NaCl, 3.4 mM EDTA) (41). Both BSA and
human TF (each: 50 µl/well, 10 µg/ml in HBS) were also used as
negative controls. Rat monoclonal antibodies, MO-2 and MO-12, against
E2 protein were used to determine the appropriate concentration of the
E2 protein. 96-well microtiter plates were washed three times with HBS
containing 0.05% Tween 20 and treated with 0.15% BSA in HBS (100 µl/well) to reduce the incidence of nonspecific binding. Following
incubation on a shaking platform for 1 h at room temperature,
MBP-fused human LF fragment (aa 600-632) or MBP2 (each: 5, 25, and 50 ng, 50 µl/well) was added. Following further incubation on a shaking
platform for 3 h at room temperature, the plates were washed as
described above, and rabbit anti-MBP primary (New England Biolabs,
Beverly, MA) and horseradish peroxidase-conjugated anti-rabbit IgG
secondary (Amersham Biosciences) antibodies were used for the detection
of binding between MBP-fused protein and the E2 protein. After the
addition of the substrate, TMB One solution (Promega),
A450 was measured using a Benchmark microplate
reader (Bio-Rad).
Western Blot Analysis--
SDS-PAGE and immunoblotting analysis
were performed with polyvinylidene difluoride membranes as described
previously (9). The rabbit polyclonal antibody (A0186; DAKO)
against human LF and mouse polyclonal antibody (Pharmingen) against
human CD81 were used for the detection of LF or CD81 by Western blot
analysis. Rabbit anti-MBP antibody was also used for the detection of
MBP by Western blot analysis. Immunocomplexes on the filters were detected by enhanced chemiluminescence assay (Renaissance; PerkinElmer Life Sciences).
Anti-HCV Activity of the MBP-fused LF Fragment--
An assay for
anti-HCV activity of the MBP-fused LF fragment was performed by a
method described previously (36, 49) using LightCycler real-time PCR
technology. Briefly, 2 µl (2 × 104 HCV) of
HCV-positive serum 1B-2 (genotype 1b), described previously (23), and
the MBP-fused human LF fragment (aa 600-632) (final concentration,
0.5-2.0 mg/ml) were preincubated in 100 µl of medium for 60 min at
4 °C and then the mixture of HCV and the LF fragment was added to
the PH5CH8 cells (1.5 × 104 cells were cultured for 2 days at 37 °C before viral inoculation on a 96-well plate). The
mixture was then incubated for 90 min at 37 °C. The cells were
washed three times with 100 µl of PBS and cultured for 1 day at
32 °C. The cellular RNA was prepared using an ISOGEN extraction kit
(Nippon Gene Co., Toyama, Japan), and 0.5 µg of RNA was used for the
quantitative analysis of HCV RNA using LightCycler PCR (49). As the
positive and negative controls for anti-HCV activity, human LF and
MBP2, respectively, were used. All experiments in this assay system
were performed in triplicate at least three times.
Direct Interaction between Human and Bovine LFs and HCV E2 Envelope
Protein--
We found previously (23, 24) that human and bovine LFs
prevented HCV infection in human hepatocyte PH5CH8 cells (24) that were
susceptible to HCV infection (23). Using LightCycler real-time PCR
technology, we showed recently (49) that IC50 doses of
human and bovine LFs were 5 and 1.5 µM, respectively, for
HCV infection of hepatocyte cells. Our previous data (36) showed that
the HCV-inhibiting activity of LF is because of the direct interaction
between LF and HCV, suggesting that LF directly binds to HCV envelope
proteins. This hypothesis is compatible with a previous report by
another group (37) that HCV envelope proteins could bind human and
bovine LFs, although their binding specificities have not been
clarified. Therefore, we first examined the specificity of the direct
interaction between LF and the E2 protein, which is thought to play a
major role in HCV binding to target cells (38). Far-Western blot
analysis was performed using the secreted form (aa 384-681) of E2
protein expressed in Chinese hamster ovary cells as a probe (41). As
shown in Fig. 1, human and bovine LFs,
which show ~80% aa homology, bound to E2 protein with similar
intensity, whereas E2 protein binding activities of human TF showing
~60% aa homology to LF and BSA were not observed after a brief
exposure. However, the long period of exposure clarified that human TF
(see Fig. 4) and bovine TF (data not shown) did slightly bind to the E2
protein. This phenomenon suggests that the E2 protein binding activity
of LF is overwhelmingly greater than that of TF. To exclude the
possibility of cross-reactions between LF and anti-E2 antibody, we
performed a Far-Western blot analysis in the absence of the E2 protein,
as well as an analysis using normal rat serum instead of the anti-E2
antibody. As shown in Fig. 1, no significant bands were obtained in
these control experiments, indicating that the bands obtained in this
Far-Western blot analysis were derived from the interaction between LF
and the E2 protein. Although the E2 proteins used by our group and Yi
et al. (37) showed an ~14% amino acid difference because of the different HCV strains, bovine and human LFs were equally able to
bind to these E2 proteins. These results suggest that LF binds
specifically to the E2 protein, regardless of particular HCV
strains.
Deglycosylation of LF Strengthens the Interaction between LF and E2
Protein--
Both human and bovine LFs are glycoproteins possessing
three and five N-linked oligosaccharide chains,
respectively. These chains are composed of galactose, mannose, fucose,
N-acetylglucosamine, and sialic acid (50). To examine
whether these oligosaccharide chains reflect the E2 protein binding
activity of LF, human and bovine LFs were deglycosylated with PNGaseF,
which is preferred for the complete removal of the N-linked
oligosaccharide chain. As shown in Fig.
2A, the molecular masses of human and bovine LFs
decreased from 80 to 72 kDa, respectively, after treatment with
PNGaseF, thereby indicating that the N-linked
oligosaccharide chains of LF were removed. Using the deglycosylated and
natural forms of human and bovine LFs, we compared the binding
abilities of LFs to E2 protein. Far-Western blot analysis revealed that deglycosylation of human and bovine LFs resulted in intensified E2
protein binding activity (Fig. 2B). This result suggests
that the interaction between LF and the E2 protein is not mediated through the N-linked oligosaccharides of LF and that at
least one oligosaccharide interferes with the direct interaction
between LF and the E2 protein. These results led us to undertake the
preparation of various recombinant LF fragments using an E. coli expression system in which glycosylation does not occur.
Expression in E. coli and Purification of Human LF
Fragments--
We chose human LF for the preparation of the
recombinant LF to investigate which region(s) of LF is (are) important
for direct binding to the E2 protein, because the natural host of HCV
is human, and a human source would be advantageous in the context of
future medical applications. We obtained by RT-PCR a full-length LF
cDNA using the total RNA (43) from human breast cancer tissue. Sequence analysis of the obtained LF cDNA clone confirmed that the
deduced amino acid sequence was identical to the previously reported
human LF (X52941) sequence (51).
To identify the region(s) of LF that bind to the E2 protein (Fig.
3A), we divided LF into eight
fragments, namely, the N-lobe (N-terminal half without lactoferricin;
aa 48-340), N-s1 (aa 48-123), N-s2 (aa 146-255), N-s3 (aa 256-340),
the C-lobe (C-terminal half; aa 341-692), C-s1 (aa 341-486), C-s2 (aa
487-599), and C-s3 (aa 600-692), according to the well characterized
three-dimensional domain of human LF (27). These LF fragments were
successfully expressed as TRX-fused proteins in AD494 cells using the
pET32a expression vector; it is already known that the solubility of an
expressed protein increases in cells (52). Four LF fragments (N-s2,
N-s3, C-s2, and C-s3) were successfully expressed in the soluble form
in the cells, although the solubility of the remaining four LF
fragments (N-lobe, N-s1, C-lobe, and C-s1) was still at a low level.
All of the LF fragments expressed either as soluble or insoluble
fractions were purified by affinity column chromatography using
His-Bind resin, as indicated under "Experimental Procedures." As a
result, eight TRX-fused LF fragments were purified as a single staining
band with the expected molecular size after separation with SDS-PAGE
(Fig. 3B).
A C-terminal Fragment of Human LF Predominantly Interacts with E2
Protein--
Using the eight human LF fragments, we performed
Far-Western blot analysis to examine which region(s) of LF possess the
capacity to bind to E2 protein. The results revealed that two LF
fragments, i.e. N-lobe (aa 48-340) and C-s3 (aa 600-692),
specifically bound to the E2 protein, suggesting that at least two
regions of human LF are involved in the direct interaction with the E2
protein (Fig. 3C). We noticed that the binding activity of
C-s3 was similar to that of human LF, in contrast to the weak binding
activity of the N-lobe. To avoid bias in the reactivity of the MO-12
rat monoclonal antibody, used for the detection of the E2 protein in
the Far-Western blot analysis, the other rat monoclonal antibody, MO-2,
which recognizes a different epitope from that recognized by MO-12, was
also used. The obtained result was the same as that observed with MO-12
(data not shown). Because the C-s3 containing aa 600-692 of human LF
was considered to be a major binding region, we focused on C-s3 to
further characterize the E2 protein binding activity.
C-s3-relevant Fragments of Bovine and Horse LFs also Bind to E2
Protein--
It is already known that the aa sequences of mammalian
LFs show ~80% sequence homology with each other (53). We
demonstrated that human and bovine LFs bind equally well to E2 protein
(Fig. 1). Regarding C-s3, 74, 71, 58, and 53% aa sequence homology was demonstrated with the C-s3-relevant fragment of bovine and horse LFs
and human and bovine TFs, respectively. To evaluate the E2 protein
binding specificity of the C-s3, we examined the E2 protein binding
activities of C-s3-relevant fragments of bovine and horse LF and human
TF and compared their activities with that of C-s3. We first obtained
bovine and horse LF cDNAs from bovine breast tissue and horse
peripheral blood mononuclear cells, respectively, by RT-PCR. Human TF
cDNA was also obtained by RT-PCR from the cultured PH5CH8 human
hepatocytes. Using the obtained LF and TF cDNAs, three
C-s3-relevant fragments of bovine and horse LFs and human TF were
expressed as TRX-fused proteins in E. coli and were purified
along with TRX-fused C-s3. Far-Western blot analysis showed that the
C-s3-relevant fragments of bovine and horse LF also bound to the E2
protein, as well as C-s3, but the binding activity of the C-s3-relevant
fragment of human TF was very weak (Fig.
4). This result suggests that the
conserved amino acids among the three LF fragments were involved in the
binding to the E2 protein. Comparison with the amino acid sequences of
LFs and TFs of human, bovine, and horse revealed that LF-specific amino acids are mainly located in the first half of C-s3 (data not shown). This observation led us to carry out a homology search regarding the
first half of the C-s3 fragments using the amino acid sequence databases. We were unable to obtain any interesting proteins showing high homology to this region, although a number of LF- and TF-related proteins were obtained in this survey. However, during the course of
the survey, we did notice that the latter half (aa 146-201) of the LEL
of the putative HCV receptor CD81 (19) showed partial homology with the
first half (aa 600-647) of C-s3, although several gaps were necessary
to make an alignment (Fig.
5A). This observation prompted
us to compare the E2 protein binding activities of C-s3 and CD81
LEL.
E2 Protein Binding Activity of C-s3 Is Comparable with That of CD81
LEL--
To compare the binding activities of C-s3 (93 aa) and CD81
LEL (89 aa), we obtained a cDNA encoding a CD81 LEL from PH5CH8 cells by RT-PCR. We confirmed that the nucleotide sequences of the
obtained CD81 LEL cDNA were identical to that of previously reported human CD81 LEL (54). CD81 LEL was also expressed as a
TRX-fused protein in E. coli using a pET-32a expression
vector and was purified with the same efficiency as C-s3 (Fig.
5B). To confirm that the purified C-s3 and CD81 LEL are
actually parts of human LF and CD81, respectively, we examined their
immunological specificities by Western blot analysis. As shown in Fig.
5, C and D, anti-human LF and anti-human CD81
polyclonal antibodies specifically recognized TRX-fused C-s3 and CD81
LEL, indicating the successful preparation of recombinant C-s3 and CD81
LEL. Far-Western blot analysis revealed that both C-s3 and CD81 LEL
bound to E2 protein, as shown in Fig. 5E. Interestingly, the
binding activity of C-s3 was equal to or stronger than that of CD81
LEL. Because it appears that the E2 protein binding affinity of C-s3 is
comparable with that of CD81 LEL, we proceeded to narrow down the
potential binding domain to an area within the C-s3.
Identification of a Minimum E2 Protein Binding Domain within
C-s3--
To identify the E2 protein binding domain within C-s3, we
first generated several truncated forms of C-s3, as shown in Fig. 6A. Each truncated form of
C-s3 was expressed as a TRX-fused protein in E. coli and was
purified as described under "Experimental Procedures." Far-Western
blot analysis using these truncated C-s3 forms revealed that aa
600-652 retained the E2 protein binding activity (Fig. 6B).
In addition, the binding activity of the C-s3 fragment (aa 610-692)
was rather weak; the C-s3 fragment (aa 624-692) failed entirely at
binding to the E2 protein (Fig. 6B, lanes 4 and
5). These results indicated that the N-terminal half of C-s3
suffices for binding to the E2 protein. Moreover, it was suggested that aa 600-624 of human LF, overlapping with the CD81 homology region (aa
615-644), is necessary for binding to the E2 protein. To further narrow down the binding domain from the C-s3 fragment (aa 600-652), we
systematically created a series of truncated forms based on the C-s3
fragment (aa 600-652), as shown in Fig.
7A. These truncated forms were
then expressed as TRX-fused proteins and were purified along with the
other TRX-fused proteins. In this series of experiments, we created an
additional TRX-fused LF fragment consisting of aa 590-652 to clarify
whether aa 600 is an N-terminal limit for E2 protein binding activity.
As shown in Fig. 7B, the results showed that the E2 protein
binding activity of human LF (aa 590-652) was almost equal to that of
the C-s3 fragment (aa 600-652), suggesting that the region of aa
590-599 did not contribute to binding to the E2 protein. Although the
C-s3 fragment (aa 600-652) possessed such binding activity, the C-s3
fragment (aa 605-652) had almost no ability to bind to the E2
protein. Regarding the carboxyl-truncated forms of the C-s3 fragment
(aa 600-652), it appeared that the C-s3 fragment (aa 600-627) did not
bind to the E2 protein and that the C-s3 fragment (aa 600-632) bound
equally well to the E2 protein as the C-s3 fragment (aa 600-652) (Fig.
7B). Therefore, aa 600-632 of human LF appeared to be the
minimum region required for E2 protein binding activity. These results
suggest that both aa 600-604 and aa 628-632 contain critical amino
acid residues required for binding to the E2 protein.
MBP-fused C-s3 Fragment (aa 600-632) also Binds to the E2
Protein--
To exclude the possibility that the E2 protein binding
activity of the C-s3 fragment (aa 600-632) was an experimental
artifact in the presence of TRX, we prepared the MBP-fused C-s3
fragment (aa 600-632) using the E. coli expression system
and examined its ability to bind to the E2 protein. We confirmed the
purity of the MBP-fused C-s3 fragment (aa 600-632) with Coomassie
Brilliant Blue staining (Fig.
8A), and we demonstrated its
quality with Western blot analysis using anti-human LF antibody (Fig.
8B) and anti-MBP antibody (Fig. 8C). As shown in
Fig. 8D, it became apparent that the MBP-fused C-s3 fragment
(aa 600-632) and the TRX-fused C-s3 fragment (aa 600-632) bound
equally well to the E2 protein, whereas MBP2 (control protein without
LF) or TRX (control protein without LF) alone did not bind to the E2
protein. To verify the E2 protein binding activity of the C-s3 fragment
(aa 600-632), we performed an ELISA-based binding assay using the
MBP-fused C-s3 fragment (aa 600-632) and the E2 protein. As shown in
Fig. 9A, the MBP-fused C-s3
fragment (aa 600-632) bound to the E2 protein in a
dose-dependent manner, whereas the binding activity of MBP2 (control protein without LF) alone was at a significantly low level.
Furthermore, an ELISA-based binding assay was used to confirm the E2
protein binding specificity of the MBP-fused C-s3 fragment (aa
600-632). As shown in Fig. 9B, the MBP-fused C-s3 fragment (aa 600-632) strongly bound to the E2 protein, whereas the MBP-fused C-s3 fragment (aa 600-632) showed low binding affinity against human
TF and BSA. The binding levels of MBP2 to human TF and BSA, as well as
that to the E2 protein, were also fairly low. In addition, using this
ELISA assay, we confirmed that human LF also bound to the E2 protein in
a dose-dependent manner (data not shown). Taken together,
these results indicate that the C-s3 fragment (aa 600-632) is directly
involved in binding to the E2 protein. We observed that bovine LF (aa
597-629) corresponding to the C-s3 fragment (aa 600-632) and the C-s3
fragment (aa 600-632) were able to bind equally well to the E2 protein
(data not shown). It is also noteworthy that the region of the C-s3
fragment (aa 600-632) was located on a surface portion of the C-lobe
domain in the three-dimensional structure of human LF (27); the region of aa 606-622 has an The MBP-fused C-s3 Fragment (aa 600-632) prevents HCV infection in
the cells--
To evaluate whether the C-s3 fragment (aa 600-632),
identified as the minimum binding site to the E2 protein, can prevent HCV infection, we initially examined the anti-HCV activity of the
TRX-fused C-s3 fragment (aa 600-632) in our HCV infection system using
PH5CH8 cells. Although we carried out a quantitative analysis of HCV
RNA using LightCycler PCR, we did not successfully demonstrate the
anti-HCV activity of the TRX-fused C-s3 fragment (aa 600-632), because
TRX itself showed some cell toxicity in our viral infection system. As
regards this cell toxicity, we considered that it might have been
because of detergent remaining in the sample after the preparation of
the TRX-fused proteins. However, no detergents were used during the
process of the purification of MBP-fused protein; we next examined the
anti-HCV activity of the MBP-fused C-s3 fragment (aa 600-632) using
the same assay system described above. As shown in Fig.
11, the obtained results revealed that
the MBP-fused C-s3 fragment (aa 600-632) was able to inhibit HCV
infection in PH5CH8 cells in a dose-dependent manner, whereas MBP2 was not able to inhibit HCV infection of the cells. In
addition, the IC50 dose of the MBP-fused C-s3 fragment (aa 600-632) was estimated to be ~20 µM (1.0 mg/ml),
although the IC50 dose of human LF was 5 µM
(0.4 mg/ml). These results suggest that E2 protein binding activity of
the MBP-fused C-s3 fragment (aa 600-632) contributes to the inhibition
of HCV infection of these cells. Furthermore, the findings suggest that
aa 600-632 of human LF possess anti-HCV activity, although this type
of anti-HCV activity was somewhat weaker than that of human LF.
Cys on aa 628 Is Critical for the Binding to E2
Proteins--
Because both aa 600-604 and aa 628-632 of human LF
were estimated to be important for binding to the E2 protein, and five (aa 602, 604, 629, 630, and 631) of these 10 positions show aa differences between human LF and TF, we considered that the specificity of amino acids also provides E2 protein binding activity. To gain further insight into the roles of these aa sequences, a series of 14 point-substitution mutations (aa 600-605 and 625-632) of the C-s3
fragment (aa 600-632) were constructed by site-directed mutagenesis to
Ala. Far-Western blot analysis using these Ala mutants revealed that
the Cys of aa 628 is quite important for binding to the E2 protein,
because a Cys to Ala substitution on aa 628 completely abolished E2
protein binding activity (Fig. 12). In
addition, four Ala mutants (aa 626, 627, 629, and 630) increased
binding activity, suggesting that the binding affinity of the C-s3
fragment (aa 600-632) to the E2 protein was not optimal.
Based on the our studies (24, 36) and those of other groups (37),
we undertook observations regarding the direct interactions between LF
and HCV envelope proteins. In this study, we demonstrated the binding
specificity between LF and the E2 protein and identified 33 aa residues
from human LF that are primarily responsible for E2 protein binding
activity and inhibiting HCV infection of target cells.
We observed that the deglycosylation of human and bovine
LFs enhanced E2 protein binding activity. This observation suggests that a certain N-linked oligosaccharide chain interferes
with the interaction between LF and the E2 protein. Comparison with the
putative N-linked glycosylation sites between human LF (3 sites; aa 138, 479, and 624) and bovine LF (5 sites; aa 233, 281, 368, 476, and 545) revealed that only one glycosylation site (aa 479 for
human LF, aa 476 for bovine LF) is conserved in both LFs. Therefore, it
is likely that this conserved glycosylation site, which is located in
C-s1, weakens the E2 protein binding activity of human or bovine LF.
This may also explain why we were able to detect the strong binding
affinity of C-s3 for the E2 protein. However, the reason why the C-lobe
did not bind to the E2 protein remains unclear. One possibility would
be that the refolding of the TRX-fused C-lobe was not successful. Our
results indicate that at least two regions of human LF (the N-lobe and
C-s3) are involved in the interaction with the E2 protein, although we
could not identify the binding region in the N-lobe. Although N-s3
shows ~35% aa homology to C-s3, N-s3 is unable to bind to the E2
protein. In addition, 12 aa are shared in common between the N-s3
fragment (aa 256-287) and the C-s3 fragment (aa 600-632) identified
as a critical domain for binding to E2 protein. However, a Cys residue at aa 628 appeared to be essential for binding to the E2 protein; it is
of note that this Cys is not present in the corresponding position of
N-s3. Therefore, some boundary region between N-s1 and N-s2, or between
N-s2 and N-s3, may possess the binding ability to the E2 protein.
Interestingly, the C-s3 fragment (aa 600-632) was located close to the
boundary region between N-s2 and N-s3 in the three-dimensional
structure of human LF (27), suggesting that the E2 protein is able to
bind to both sites of human LF. To clarify this assumption, further
analysis will be needed.
We demonstrated that C-s3-relevant fragments (93 aa) of bovine and
horse LFs bound as well to the E2 protein as did C-s3. The
C-s3-relevant fragments of bovine and horse LFs show 74 and 71% aa
sequence homology to C-s3 of human LF, respectively. These values are
significantly higher than those (58 and 53%, respectively) of
C-s3-relevant fragments of human and bovine TFs, which possess little
binding activity to the E2 protein. The identified critical domain (aa
600-632) of human LF also shows 70% aa sequence homology to bovine LF
(aa 597-629), whereas it shows only 42% aa sequence homology to the
relevant regions of human or bovine TF (Fig. 10). These data suggest
that the binding activity to E2 protein is restricted to the LF family.
During the process of characterization of a C-s3 LF
fragment possessing E2 protein binding activity, we noticed that C-s3 showed partial aa homology with LEL of CD81, which can also bind to E2
protein and is considered as a candidate HCV receptor (19). The E2
protein binding activity of CD81 LEL has been well characterized in vitro (55) and in vivo (56), and the binding
specificity of CD81 to E2 protein has been clarified (57). However, it
has been reported that CD81 is not directly involved in the cell fusion caused by HCV (58). Because it has been shown that TRX-fused C-s3 (93 aa) has a comparable E2 protein binding ability with that of TRX-fused
CD81 LEL (89 aa), C-s3 may interfere with the binding of the E2 protein
to CD81. This may be one of the reasons why LF prevents HCV infection
in target cells. However, one major contradiction remains. Regarding
the interaction between human CD81 LEL and the E2 protein, it has been
shown that aa 186 (Phe) of the CD81 is the critical residue for binding
to the E2 protein (57). This Phe residue is conserved between human
CD81 and LF (aa 635) (Fig. 5A). However, in this study, it
appeared that the Phe at aa 635 of human and bovine LFs was unimportant
for binding to the E2 protein, because the C-s3 fragment (aa 600-632)
possessing the E2 protein binding activity does not contain this Phe
residue. In addition, it has been reported that the four Cys of CD81
LEL form two disulfide bridges, the integrity of which would be
necessary for CD81-E2 interaction (54). However, such a phenomenon was not observed in C-s3 containing five Cys residues, because the C-s3
fragment (aa 600-632) identified as the E2 protein binding domain
contains only one Cys. Therefore, these data suggest that the E2
protein region targeted by human LF and CD81 may differ. Preliminarily,
our experiment with Far-Western blot analysis showed that the C-s3
fragment (aa 600-632) preferentially bound to aa 411-500 of the E2
protein, which is one of two regions (aa 384-500 and 600-661)
identified previously (37) as regions binding to human LF; however, the
C-s3 fragment (aa 600-632) did not bind to aa 501-599 of E2 protein
(data not shown). Because it has been indicated that both aa 480-493
and aa 544-551 of the E2 protein are involved in the binding to CD81
(56), human LF and CD81 may recognize rather different sites on the E2
protein. Further analysis will be necessary to clarify this point.
Because aa 600-632 of human LF possesses only one Cys at
aa 628, it is unlikely that a disulfide bond is required for the E2
protein binding activity of the C-s3 fragment (aa 600-632). However,
we cannot exclude the possibility that Cys at aa 628 paired with other
Cys residues in TRX during the refolding process of the Far-Western
blot analysis; this could have provided E2 protein binding activity. To
exclude this possibility, we constructed the MBP-fused C-s3 fragment
(aa 600-632), because Cys was not present in the MBP portion including
the linker region. Although the MBP-fused C-s3 fragment (aa 600-632)
possesses only one Cys, this fusion protein showed similar E2 protein
binding activity with that of the TRX-fused C-s3 fragment (aa
600-632). Therefore, the present results suggest that a disulfide bond
is not required for binding to the E2 protein, in contrast to the case
involving CD81 LEL (55). However, it is of note that site-directed
mutagenesis to Ala in both terminal regions of the C-s3 fragment (aa
600-632) revealed that Cys at aa 628 is the most critical residue for
binding to the E2 protein. To clarify whether only the Cys residue at this position is necessary for binding to the E2 protein, further experiments (e.g. substitution of amino acids other than
Ala) will be needed.
Because it is well known that E1 and E2 proteins form a non-covalently
linked heterodimer, which probably represents the surface of infectious
virus particles (59), it is important to clarify whether the C-s3
fragment (aa 600-632) identified in this study binds to the
heterodimer of E1 and E2 proteins. To date, aa 441-500 of E2 protein
has been identified as the E1 protein heterodimeric binding region
(60). Although our preliminary results estimated that the C-s3 fragment
(aa 600-632) binds to aa 411-500 of the E2 protein, the E2 protein
binding activity of the C-s3 fragment (aa 600-632) may not be affected
by heteromeric complex formation between E1 and E2 proteins, because LF
prevents HCV infection by direct interaction between LF and HCV
(36).
We demonstrated the anti-HCV activity of the MBP-fused C-s3 fragment
(aa 600-632) in our HCV infection system using PH5CH8 cells (36, 49).
Although this result suggests that the E2 protein binding activity
contributes to the prevention of HCV infection, our results
revealed that the anti-HCV activity of the MBP-fused C-s3 fragment (aa
600-632) was severalfold weaker than that of human LF. However,
site-directed mutagenesis to an Ala residue within aa 600-605 and aa
625-632 of human LF revealed that several positions strengthened the
E2 protein binding activity. This result suggests that some peptides
possess stronger binding activity than that of the C-s3 fragment (aa
600-632) and that these peptides could be obtained by screening
peptide libraries, e.g. phage libraries. The antiviral
activities of such peptides will be evaluated using our cell culture
assay system (36, 49). Furthermore, such peptides may be useful for the
removal of circulating HCV. In any case, the present study broadens the
possibilities for developing anti-HCV peptides in the future.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
in HCV-infected PH5CH8 cells (23).
Using a PH5CH8 cell culture system, we found that bovine and human
lactoferrin (LF), a milk glycoprotein belonging to the iron transporter
family, specifically prevented HCV infection in the cells (24).
Recently, Matsuura et al. (25) also showed that bovine LF
specifically inhibited infection by the pseudotype vesicular stomatitis
virus possessing chimeric HCV E1 and E2 glycoproteins.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Oligonucleotides used for construction of expression plasmids
-D-thiogalactopyranoside was added to the culture and then continued for 4-5 h at 37 °C. After centrifugation at 3,400 × g for 10 min, the harvested
cells were suspended in 10 ml of B-PERTM (Pierce) by
pipetting until the cell suspension became homogenous and was incubated
for 15 min at room temperature. After centrifugation at 27,000 × g for 15 min, the supernatant obtained as the soluble fraction was used for the purification of LF fragments and CD81 LEL
using a His-Bind purification kit (Novagen) according to the manufacturer's protocol, because these TRX-fused proteins possesses His tag sequences. On the other hand, the pellets obtained as the
insoluble fraction were suspended in 10 ml of B-PERTM; 200 µl of lysozyme (10 mg/ml; Sigma) was then added, and the samples were
incubated for 5 min at room temperature. After centrifugation at
27,000 × g for 15 min, the pellets were resuspended in
30 ml of 10-fold diluted B-PERTM, and the treatment was
repeated two times. The pellets obtained by this method were then
dissolved in 10 ml of binding buffer (20 mM Tris-HCl, pH
7.9, 500 mM NaCl, 5 mM imidazole, and 6 M urea) as purified inclusion bodies. The inclusion bodies
thus obtained were also used for the purification of several LF
fragments using a His-Bind purification kit in the presence of 6 M urea. The purity of obtained LF fragments and CD81 LEL
was evaluated to be more than 95%, as determined by electrophoresis on
12% SDS-PAGE gels. The protein concentration was determined by using
Coomassie protein assay reagent (Pierce). TRX (22 kDa) produced from
the pET32a vector, with the linker sequence-derived 26 amino acids in
the C-terminal portion, was used as a control protein.
-D-thiogalactopyranoside was
added to the culture, and the culture was continued for 4 h at
37 °C. After centrifugation at 3,400 × g for 10 min, the harvested cells were resuspended in 5 ml of column buffer (20 mM Tris-HCl, pH 7.4, 200 mM NaCl, and 1 mM EDTA) and were then disrupted by sonication for 2 min with short pulses. Insoluble cellular debris was removed by
centrifugation at 10,000 × g for 30 min at 4 °C.
The supernatant obtained as the soluble fraction was applied onto an
amylose resin affinity column (New England Biolabs, Beverly, MA). The
column was washed with 10 column volumes of column buffer to remove the
unbound proteins. The bound protein was eluted with 10 mM
maltose under conditions recommended by the manufacturer. The purity of
the obtained MBP-fused protein was evaluated to be more than 95% by electrophoresis on 12% SDS-PAGE gels. The concentration of the purified MBP-fused protein was determined by using Coomassie protein assay reagent (Pierce). The MBP2 (43 kDa) produced from the pMAL-c2X with a stop codon inserted into the XmnI site was used as a
control protein.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Direct interaction between LF and HCV E2
envelope protein. Human and bovine LFs, and human TF and BSA (0.5 or 1 µg each; each sample was more than 90% pure) were resolved by
10% SDS-PAGE. Far-Western blot analysis using E2 protein expressed in
Chinese hamster ovary cells (41) as a probe was performed as described
under "Experimental Procedures." Rat monoclonal antibody MO-12 (42)
against E2 protein (Anti-E2 Ab) was used for the detection
of E2 protein bound to LF. Far-Western blot analyses in the absence of
the E2 protein and using normal rat serum instead of rat monoclonal
antibody MO-12 against E2 protein were also performed. The other rat
monoclonal antibody, MO-2, which recognizes a different epitope from
that recognized by MO-12, was also used for the detection, and the same
results were obtained as those observed with MO-12 (data not
shown).
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Fig. 2.
Deglycosylation of LF resulted in intensified
binding to E2 protein. A, human and bovine LFs
deglycosylated with PNGaseF (each 0.5 µg) were resolved by 12%
SDS-PAGE and were detected by Coomassie Brilliant Blue staining.
B, effect of deglycosylation of LFs on the E2 protein
binding ability. Far-Western blot analysis using E2 protein as a probe
was carried out as indicated in Fig. 1.
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Fig. 3.
Expression of human LF fragments in
E. coli and their E2 protein binding
activities. A, localization map of human LF fragments
expressed in E. coli. Numbers indicate the aa
positions of eliminated N-terminal signal sequences (19 amino acids) in
LF. NI-1, NII, NI-2, CI-1,
CII, and CI-2 indicate the three-dimensional
domain of human LF (27). B, purification of TRX-fused human
LF fragments. Each 0.5 µg of LF fragments was resolved by 12%
SDS-PAGE and was detected by Coomassie Brilliant Blue staining.
Lane M, molecular marker of proteins. C, E2
protein binding activities of human LF fragments. The purified
TRX-fused human LF fragments, human LF, and human TF were resolved by
12% SDS-PAGE and then Far-Western blot analysis using E2 protein as a
probe was performed as indicated in Fig. 1. MO-12, a rat monoclonal
antibody (42) against E2 protein, was used for the detection of E2
protein bound to LF or to LF fragments.
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Fig. 4.
Binding to E2 protein of C-s3 and
C-s3-relevant fragments of bovine and horse LFs and human TF.
Human LF and TF and purified TRX-fused C-s3 and C-s3-relevant fragments
of bovine LF, horse LF, and human TF (0.5 µg each) were resolved by
12% SDS-PAGE. Far-Western blot analysis was carried out as indicated
in Fig. 1.
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Fig. 5.
Comparison of E2 protein binding abilities of
C-s3 and CD81 LEL. A, amino acid sequence
alignment of the first half of C-s3 and C-s3-relevant regions of bovine
LF and the latter half of CD81 LEL. The same amino acids are
boxed in each case. Hyphens indicate gaps.
B, purification of TRX-fused human CD81 LEL. Human LF, human
TF, TRX-fused C-s3, and TRX-fused CD81 LEL (0.5 µg each) were
resolved by 12% SDS-PAGE and were detected by Coomassie Brilliant Blue
staining. C, immunological specificity of TRX-fused C-s3.
Western blot analysis using rabbit anti-human LF polyclonal antibody
was carried out using the same proteins as those shown in B. D, immunological specificity of TRX-fused CD81 LEL to E2
protein. Western blot analysis using anti-human CD81 polyclonal
antibody was carried out using the same proteins as those shown in
B. E, E2 protein binding activities of C-s3 and
CD81 LEL. Far-Western blot analysis using the E2 protein as a probe was
carried out using the same proteins as those shown in
B.
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Fig. 6.
Deletion analysis of C-s3 possessing E2
protein binding activity. A, localization map of the
C-s3 variants truncated from N-terminal or C-terminal portions of C-s3.
These LF fragments were expressed as TRX-fused proteins in E. coli and were purified for the Far-Western blot analysis.
Numbers indicate the aa positions of human LF. B,
E2 protein binding activities of C-s3 truncated variants. Far-Western
blot analysis using the E2 protein as a probe was carried out as
indicated in Fig. 1. To indicate that equal amounts of TRX-fused C-s3
variants were resolved by 12% SDS-PAGE, these LF fragments were
stained with Coomassie Brilliant Blue (CBB; lower
panel).
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Fig. 7.
Identification of a minimum E2 protein
binding domain in C-s3. A, localization maps of the
C-s3-truncated variants and a human LF (aa 590-652) were examined
regarding E2 protein binding activity. These LF fragments were
expressed as TRX-fused proteins in E. coli and purified for
Far-Western blot analysis. Numbers indicate the aa positions
of human LF. B, E2 protein binding activities of
C-s3-truncated variants and a human LF (aa 590-652). Far-Western blot
analysis using the E2 protein as a probe was carried out as indicated
in Fig. 1. To indicate that equal amounts of TRX-fused C-s3 variants
were resolved by 12% SDS-PAGE, these LF fragments were stained by
Coomassie Brilliant Blue (CBB; lower
panel).
-helix structure, and both terminal ends (five
aa each) of the C-s3 fragment (aa 600-632), thought to be important
for the E2 protein binding activity, are highly conserved between human
and bovine LFs (Fig. 10).
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Fig. 8.
E2 protein binding specificity of the C-s3
fragment (aa 600-632). The E2 protein binding activity of the
MBP-fused C-s3 fragment (aa 600-632) was examined by Far-Western blot
analysis as indicated in Fig. 1. Lane 1, human LF;
lane 2, human TF; lane 3, MBP-fused C-s3 fragment
(aa 600-632); lane 4, MBP2 without LF; lane 5,
TRX-fused C-s3 fragment (aa 600-632); lane 6, TRX without
LF. A, Coomassie Brilliant Blue staining after 12%
SDS-PAGE. B, Western blot analysis using rabbit anti-human
LF polyclonal antibody. C, Western blot analysis using
rabbit anti-MBP polyclonal antibody. D, Far-Western blot
analysis using the E2 protein as a probe.
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Fig. 9.
ELISA-based E2 protein binding activity of
the C-s3 fragment (aa 600-632). A, the MBP-fused C-s3
fragment (aa 600-632) bound to the E2 protein in a
dose-dependent manner. An ELISA-based binding assay was
performed to examine the E2 protein binding activity of the MBP-fused
C-s3 fragment (aa 600-632), as described under "Experimental
Procedures." MBP2 was used as a control protein without LF. 5, 25, and 50 ng of the MBP-fused C-s3 fragment (aa 600-632) or MBP2 were
used for binding to the E2 protein. B, binding specificity
of the MBP-fused C-s3 fragment (aa 600-632). To examine the E2 protein
binding specificity of the MBP-fused C-s3 fragment (aa 600-632), human
TF and BSA were also coated to immunoplates and were used as negative
controls in the ELISA-based binding assay. MBP2 was used as a control
protein without LF. 50 ng of the MBP-fused C-s3 fragment (aa 600-632)
or MBP2 were used for binding to the E2 protein, human TF, or
BSA.
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Fig. 10.
Amino acid sequence alignment of C-s3 and
C-s3-relevant fragments of bovine LF, human TF, and bovine TF.
Capital letters indicate amino acids that differed from
those in the sequences of aa 600-632 of human LF. The identical amino
acids are indicated with asterisks. The -helix structure
(aa 606-622) identified in human LF (22) is depicted by a thick
bar. Both the N-terminal five aa and C-terminal five aa, which are
considered to be critical for E2 protein binding, are shown in
boxes.
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Fig. 11.
Anti-HCV activity of the MBP-fused C-s3
fragment (aa 600-632) in PH5CH8 cells. PH5CH8 cells and inoculum
1B-2 were used for the HCV-inhibiting assay, as described under
"Experimental Procedures." The number in the
axis of the ordinate indicates the percent of the
amount of HCV RNA determined by quantitative RT-PCR using LightCycler
PCR (49). Approximately 2,000 copies of HCV RNA per µg of cellular
RNA was reproducibly obtained using this HCV infection system (36, 49).
In addition to the MBP-fused C-s3 fragment (aa 600-632), human LF and
MBP2 were also used for the assay as control materials.
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Fig. 12.
Site-directed mutagenesis of the TRX-fused
C-s3 fragment (aa 600-632). Site-directed mutagenesis to Ala was
carried out in aa 600-605 and 625-632, respectively. Lane
WT, TRX-fused C-s3 fragment (aa 600-632). Far-Western blot
analysis was performed using the E2 protein as a probe (upper
panel), as indicated in Fig. 1. The purified TRX-fused proteins
were stained with Coomassie Brilliant Blue (CBB) after 12%
SDS-PAGE to confirm their purities (lower panel).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. H. Kariwa (Hokkaido University) for providing normal bovine breast tissue and normal horse peripheral blood mononuclear cells. We thank Dr. K. Shimotohno (Kyoto University), Dr. K. Sugiyama (National Cancer Center Research Institute, Tokyo, Japan), and Dr. T. Tanaka (National Institute of Neurology and Psychiatry) for helpful suggestions and for discussions about the study. We thank A. Morishita for helpful experimental assistance.
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FOOTNOTES |
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* This work was supported by grants-in-aid for the Second-Term Comprehensive 10-Year Strategy for Cancer Control and for research on hepatitis and BSE from the Ministry of Health, Labor and Welfare and by grants-in-aid for scientific research from the Organization for Pharmaceutical Safety and Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Present address: The First Department of Internal Medicine, Gunma University School of Medicine, Maebashi 371-8511, Japan.
** To whom correspondence should be addressed. Tel.: 81-86-235-7385; Fax: 81-86-235-7392; E-mail: nkato@md.okayama-u.ac.jp.
Published, JBC Papers in Press, January 9, 2003, DOI 10.1074/jbc.M207879200
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ABBREVIATIONS |
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The abbreviations used are: HCV, hepatitis C virus; LF, lactoferrin; aa, amino acid; TF, transferrin; BSA, bovine serum albumin; PNGaseF, peptide-N-glycosidase F; RT, reverse transcription; LEL, large extracellular loop; TRX, thioredoxin; MBP, maltose-binding protein; ELISA, enzyme-linked immunosorbent assay; HBS, HEPES-buffered saline.
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