From the Zentrum für Molekulare Biologie (ZMBH), Universität Heidelberg, Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany
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ABSTRACT |
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Avian hepatitis B virus infection is initiated by
the specific interaction of the extracellular preS part of the large
viral envelope protein with carboxypeptidase D (gp180), the primary cellular receptor. To functionally and biochemically characterize this
interaction, we purified a soluble form of duck carboxypeptidase D from
a baculovirus expression system, confirmed its receptor function, and
investigated the contribution of different preS sequence elements to
receptor binding by surface plasmon resonance analysis. We found that
preS binds duck carboxypeptidase D with a 1:1 stoichiometry, thereby
inducing conformational changes but not oligomerization. The
association constant of the complex was determined to be 2.2 × 107 M Hepatitis B viruses are a group of small enveloped hepatotropic
partially double-stranded DNA viruses that cause acute and chronic
infections in humans, mammals, and birds (1). In case of the human
hepatitis B virus (HBV)1
chronic infections dramatically increases the risk for the development of primary hepatocellular carcinomas, and HBV therefore represents a
major health problem to the world population (2). While many details of
hepadnaviral genome replication are understood in considerable detail
(reviewed in Refs. 3 and 4), our knowledge of the early events in HBV
infection, namely the identity of the cellular receptor for HBV, is
poor, and reflects the lack of a suitable infection system (5).
However, promising results have been achieved in the duck hepatitis B
virus (DHBV) model system where systematic infection studies using
primary duck hepatocytes can reproducibly be performed (6). In this
system infections can be suppressed by the simultaneous application of
nucleocapsid-free, non-infectious subviral particles which are produced
in an about 1000-fold excess over virus during infection (7). Both
subviral particles and virions contain the same two envelope proteins, namely the large viral surface protein (L-protein) and the small viral
surface protein (S-protein). Both proteins are transcribed from a
common open reading frame and share the hydrophobic S moiety which is
responsible for membrane anchoring. The L-protein additionally contains
the 161-amino acid amino-terminal hydrophilic preS sequence which, on
its own, inhibits the DHBV infection of primary duck hepatocytes. A
subsequent deletion analysis showed that an 85-amino acid preS element
constitutes the receptor-binding domain within preS (8).
In an attempt to identify receptor candidates for DHBV, Kuroki et
al. (9) and independently Tong et al. (10) identified a
glycoprotein of 170-180 kDa (gp180/p170) which binds DHBV particles and Escherichia coli-derived GST-preS polypeptides. Sequence
comparisons of gp180/p170 cDNA with known sequences suggested that
it represents the prototype of a new family of membrane bound
carboxypeptidases (10, 11). Independently, several mammalian homologues
of gp180/p170 were discovered in bovine, rat, mouse, and humans, and
have been classified as metallocarboxypeptidase D (CPD) (12-15). The
primary sequence analyses indicate that CPDs consist of three
luminal/extracellular carboxypeptidase B-like domains (called A, B, and
C), a hydrophobic transmembrane anchor and a highly conserved
cytoplasmic tail. While in the A and B domains all essential amino
acids for the enzymatic activity of CPDs investigated so far are
conserved, the C domains lost most of them, despite their high overall
sequence homology. It has therefore been hypothesized that this domain of CPD is catalytically inactive and serves a different, as yet unknown
function (15). This assumption was recently confirmed for duck CPD,
whose A and B domains displayed CPD activity, while the C-domain
contains the DHBV preS-binding site (16).
CPDs from rat and duck have been shown to be resident
trans-Golgi network membrane proteins which cycle between
the trans-Golgi network and the plasma membrane (17, 18).
Their localization, enzymatic activity, and the broad tissue
distribution support the notion that CPDs are involved in processing of
a variety of polypeptides that traverse the secretory pathway of
various tissues (19). However, evolutionary conservation of an
enzymatically inactive C-domain, recycling from the cell surface to the
trans-Golgi network, and the uptake of DHBV particles
strongly implies a yet unidentified function of CPDs, possibly
involving binding of a natural ligand.
We have recently characterized the receptor-binding domain within the
preS region of the DHBV L-protein and demonstrated that duck
carboxypeptidase D is the primary receptor for avian hepatitis B
viruses (8, 18). In the present study we have extended these results by
the detailed biochemical analysis of the DHBV preS-duck
carboxypeptidase D interaction. Using a set of preS deletion mutants,
we investigated binding and dissociation rates of the preS·receptor
complex by real time surface plasmon resonance spectroscopy. We defined
a receptor-binding domain, comprising about one-half of preS, which
consists of a short receptor attachment site and a conformation
dependent stabilizing element. Binding of dCPD to this domain is strong
and, considering the presence of multiple binding sites in viral
particles, implies that DHBV binding to hepatocytes is probably
irreversible. Together with findings from earlier studies our results
allow a model to be proposed for the early steps in hepadnaviral
infection with several implication regarding the mechanisms of
hepadnaviral infection.
Plasmid Construction and Generation of Recombinant
Baculoviruses--
The baculovirus transfer vector pVL-sdCPD was
constructed by ligating the NcoI/XhoI fragment of
plasmid pBKRSV-gp180 (kindly provided by K. Kuroki, Kanasawa, Japan)
into a modified version of plasmid pVL1393 (Fig. 1A). This
vector contains an additional NcoI site as a part of the
start codon and a polylinker at the 3' end which introduces an
artificial stop codon. The NcoI/XhoI fragment of
pBKRSV-gp180 encodes the signal sequence of duck CPD and the three
carboxypeptidase-like domains but lacks the carboxyl-terminal transmembrane anchor and the cytosolic tail.
Recombinant baculoviruses were obtained by co-lipofection of
Spodoptera frugiperda (Sf9) cells with a mixture of
100 ng of linearized Baculo-Gold DNA (Pharmingen) and 5 µg of the
baculovirus transfer vector pVL-sdCPD using the manufacturer's
protocol for lipofection with DOTAP (Boehringer-Mannheim). Two hours
after lipofection, the medium was changed and recombinant viruses were collected after 5 days. The virus was purified according to the protocol of O'Reilly et al. (20), amplified in
Sf9 cells by two additional rounds of infection and used for
infection of High Five cells as described below.
SDS-PAGE, Silver Staining, and Immunological
Techniques--
Protein samples for SDS-PAGE were dissolved in sample
buffer (200 mM Tris/Cl, pH 6.8, 6% SDS, 20% glycerol,
10% dithiothreitol, 0.1 mg/ml bromphenol blue, 0.1 mg/ml Orange G),
boiled for 5 min, and subjected to electrophoresis in 7.5 or 13%
polyacrylamide-SDS gels (21). After electrophoresis, proteins were
either silver-stained according to the method described by Heukeshoven
and Dernick (22) or transferred to a nitrocellulose filter for
immunological analysis. As a primary antibody we used 1:5000 dilutions
of a polyclonal rabbit antiserum raised against a recombinant
polypeptide of the COOH-terminal third of dCPD (kindly provided by K. Breiner). As a secondary antibody we used a horseradish
peroxidase-conjugated goat anti-mouse antibody (Dianova). Detection was
done by enhanced chemoluminescence (ECL, Amersham) according to the
manufacturer's instructions.
Synthesis of
4-Amino-benzoyl-arginine-Sepharose--
4-Amino-benzoyl-arginine
(PABA) was synthesized by a combination of the methods described by
Hitchcock and Smith (23) and Plummer and Hurwitz (24). The product was
recrystallized and its structure verified by 3H,
13C NMR, and mass spectrometry. Coupling of PABA to
activated CH-Sepharose 4B (Pharmacia) was performed according to the
suppliers standard protocol.
Purification of sdCPD--
5.4 × 107 High Five
insect cells (three T175 flasks), grown in 100 ml of Express Five
serum-free medium (Life Technologies, Inc.) were infected with 9 ml of
culture supernatant of AcNPV-sdCPD infected Sf9 cells
(multiplicity of infection = 100). Cells were incubated at
27 °C for 72 h to allow protein expression. The culture supernatant was centrifuged (5000 × g, 15 min) to
remove cells, passed through a 0.22-µm nitrocellulose filter,
adjusted to pH 5.5 with acetic acid and applied to a
4-amino-benzoyl-arginine-Sepharose column (15-ml bed volume, flow rate
of 0.5 ml/min) equilibrated with 20 mM NaAc, 1 M NaCl, pH 5.5. Due to the absence of the transmembrane domain, detergent was not required during purification. All
purification steps were performed at 4 °C on a FPLC system
(Pharmacia). After extensive washing (15 bed volumes), the buffer was
changed to 10 mM NaAc, pH 5.5 (5 bed volumes), and sdCPD
was eluted with 50 mM L-arginine in 20 mM Tris/Cl, pH 8.0. Protein containing fractions were
dialyzed against 25 mM NaPi, pH 7.0, and
concentrated to a final volume of 2-4 ml using a Centriplus 30 concentrator (Amicon). Concentration was determined by measuring the
extinction at 280 nm as described by Gill and von Hippel (25) based on the molar extinction coefficient of 159,400 calculated from the primary
sequence using the program Protean (Lasergene).
Infection Competition Assay with sdCPD--
Primary duck
hepatocytes were prepared and cultivated as described previously (26).
For infection, competition assays 8 × 105 cells were
cultivated for 3-8 days in 12-well plates, and infected with 4 × 107 DNA-containing DHBV particles (determined by DNA dot
blot) in the absence or presence of increasing concentrations of sdCPD. To this aim DHBV-containing duck serum was mixed with appropriate stock
solutions of sdCPD, completed to a final volume of 500 µl with
maintenance medium, and applied to a 12-well plate of primary duck
hepatocytes. After infection for 14 h at 37 °C, cells were washed twice with phosphate-buffered saline and cultured for 6 additional days. Intracellular viral DNA was prepared, using the QIAamp
blood kit (Qiagen) and subjected to DNA dot-blot analysis as described
by Rigg and Schaller (26). All assays were quantified using a Molecular
Dynamics PhosphoImager.
Gel Filtration--
Isolation of monomeric sdCPD and
determination of apparent molecular weights of proteins and protein
complexes were achieved by exclusion chromatography on a calibrated
Superdex 200 column (1.6 × 60 cm; Pharmacia), connected to a FPLC
system (Pharmacia) and equilibrated in 5% sucrose, 150 mM
NaCl, 25 mM NaPi, pH 7.0. All chromatographic
steps were performed at 4 °C with a flow rate of 2.2 ml/min. Sample
volumes were 0.5 ml for analytical and up to 2 ml for preparative
purposes. Eluted proteins were collected in fractions of 2.2 ml,
subjected to SDS-PAGE, and analyzed by silver staining or
immunoblotting. The column was calibrated with thyroglobulin (670 kDa),
fraction 12; Binding Assays--
PreS polypeptides were prepared as described
previously (8) and covalently immobilized on covalink immunoplates
(Nunc) via COOH groups by NHS/EDC activation chemistry. Each well
consisted of 15-30 µg of preS polypeptide in 100 µl of 25 mM NaPi, pH 6.3, and were mixed with 50 µl of
N-hydroxysulfosuccinimide (3.48 mg/ml in aqua bidest.) and
50 µl of EDC (3.07 mg/ml in aqua bidest.). Coupling was allowed to
proceed for 30 min at room temperature with gentle agitation. To remove
uncoupled polypeptide, plates were washed 3 times with 300 µl of
water and 3 times with 300 µl of phosphate-buffered saline, 0.2%
Tween 20. After blocking with 200 µl of 2% BSA in phosphate-buffered
saline for 30 min, CPD containing protein samples were added in either
standard binding buffer (1% Triton X-100, 50 mM Tris/Cl,
150 mM NaCl, pH 7.4) for solubilized membrane fractions or
detergent-free binding buffer (50 mM Tris/Cl, 150 mM NaCl) for recombinant soluble dCPD. Binding assays at
different pH values were performed in sodium acetate buffer, pH
3.5-5.5, sodium phosphate buffer, pH 6.0-8.0, and Tris buffer, pH
8.5-10.0. Binding was allowed to occur for at least 4 h at
4 °C. Unbound proteins were removed and the plate was washed 3 times
with binding buffer at 4 °C. Bound proteins were eluted with 50 µl
of SDS sample buffer at 80 °C and analyzed by PAGE and Western blotting.
Surface Plasmon Spectroscopy--
Surface plasmon resonance
analysis (BIAcore-Upgrade, BIAcore-System) of DHBV preS protein binding
to sdCPD was done at 37 °C in 1 × HBS buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 0.005% Surfactant P-20 (Pharmacia)) at flow rates of 5-30 µl/min according to standard protocols provided by the manufacturer. DHBV preS proteins
were coupled to a CM5 sensor chip via standard NHS/EDC activation
chemistry (BIAcore amine coupling kit) in amounts that yielded
2,200-3,500 response units. Monomeric and oligomeric sdCPD at the
indicated concentrations were injected for 5 min followed by 3 min
elution with HBS buffer. The sensor chip was cleaned of sdCPD bound to
immobilized DpreS by injection of 30 µl of regeneration solution (20 mM HCl). Binding constants of the sdCPD-DpreS interaction were calculated with the BIAevaluation program version 2.1 (Pharmacia). The baselines for the curves shown in Fig. 4 were adjusted to zero
before calculation. Calculation of Kd was done by fitting the data to the equation R = R0e Purification of Soluble Duck Hepatitis B Virus Receptor from a
Baculovirus Expression System--
To facilitate biochemical studies
on the interaction of duck hepatitis B virus (DHBV) with duck
carboxypeptidase D (dCPD), we constructed recombinant baculoviruses
that encode the three extracellular carboxypeptidase-like domains of
dCPD and the amino-terminal export signal but lack the COOH-terminal
transmembrane anchor and the highly conserved cytoplasmic tail (Fig.
1A). Recombinant baculoviruses
were amplified in S. frugiperda 9 (Sf9) cells and used for infection of the High Five insect cell line (see
"Experimental Procedures"). Omission of the transmembrane anchor
and the cytoplasmic part resulted, as intended, in the secretion of a
soluble dCPD variant of 170 kDa (sdCPD), as shown by Western blot
analysis of culture supernatants using an antiserum against gp180 (Fig. 1B). Treatment with endoglycosidases H and F led to a
decrease of the molecular weight indicating that sdCPD had been
modified by both complex and high mannose-type glycosylation (Fig.
1B).
Purification of sdCPD from the culture supernatant of infected High
Five cells was achieved by affinity chromatography on a PABA-Sepharose
column (Fig. 1C) as described previously (12). Silver
staining of proteins from pooled fractions eluting from the PABA column
revealed a single band at about 170 kDa with only minor contaminants.
Western blot analysis using an anti-dCPD specific antibody verified the
identity of the 170 kDa as dCPD (Fig. 1D) as does analysis
by Edman degradation. The NH2-terminal sequence AHIKKAEAA ... indicated cleavage of the leader sequence at
position 25 in insect cells, and was found to be identical to
previously published data for endogenous duck CPD (11) and bovine CPD
(19). In addition, detection of a second sequence KAEAA ... , lacking 4 amino acids, suggests that processing can also occur after
lysine 28 in insect cells. The comparison of the yields of recombinant sdCPD obtained from Sf9 cells with the yields obtained from High Five cells revealed an about 8-fold higher expression rate in the High
Five cell line (25 µg of sdCPD per 107 Sf9 cells
in contrast to 200 µg/107 High Five cells).
Further analysis of affinity purified sdCPD on a calibrated Superdex
200 gel filtration column (Fig.
2A) resulted in two forms with
different apparent molecular weights, which were, however, indistinguishable in SDS-PAGE (Fig. 2B) and Western blot
analysis (data not shown). About 35% of sdCPD eluted in the void
volume and represent oligomers with molecular masses of approximately 3,600 kDa as determined by electron microscopy (data not shown). 65%
of sdCPD eluted at 248 kDa and represents monomeric sdCPD eluting at a
slightly higher molecular mass than expected. Since proteins were
initially purified by affinity chromatography on PABA-Sepharose it can
be assumed that both forms have carboxypeptidase activity but differ
considerably in their binding properties to DHBV preS, as shown
below.
sdCPD Efficiently Inhibits the Infection of Primary Duck
Hepatocytes with Duck Hepatitis B Virus--
To investigate whether
recombinant sdCPD exhibits receptor function comparable to the cell
surface bound molecule on hepatocytes, we performed infection
inhibition assays essentially as described previously for DHBV preS
(DpreS) polypeptides (8). Primary duck hepatocytes were infected with
DHBV in the presence of increasing amounts of monomeric or oligomeric
sdCPD, and viral markers (intracellular viral DNA or secreted DHBV
e-antigen) were quantified 6 days post-infection. As shown in Fig.
3A, both forms of sdCPD
efficiently inhibit DHBV infection. About 7 molecules of monomeric
sdCPD per viral particle were sufficient for 50% inhibition of
infection (18). In comparison, oligomeric sdCPD, despite its inability
to bind recombinant DHBV preS, as shown below, competed DHBV infection
also remarkably well; with about 15 molecules needed for 50%
inhibition. In these calculations we assumed that an approximately
1,000-fold excess of non-infective subviral particles is present in the
infective serum. Taking into account that one particle consists of
approximately 20 L-protein molecules with 50% having an inverse
topology and therefore their receptor-binding domain is located inside
the viral particle (27), we conclude that every single sdCPD molecule is able to bind viral particles. Ratios of 100 sdCPD molecules/particle almost completely block DHBV infection, indicating that viral particles
bound to sdCPD are unable to infect hepatocytes. Insect cell-derived
soluble duck carboxypeptidase D therefore constitutes a suitable tool
for virus-receptor interaction studies at the molecular level.
Competitive inhibition of infection was also performed with the
E. coli-derived preS polypeptides used in the binding
studies described below. As shown in Fig. 3B, full-length
DpreS and the DHBV preS fragment DpreS30-115, representing the
receptor-binding domain, inhibit DHBV infection equally well in a
concentration-dependent manner. Under the conditions used,
the IC50 was determined to be 0.4-0.8 µM,
corresponding to approximately 3000 DpreS molecules per viral particle.
Recombinant sdCPD Binds DHBV PreS Polypeptides Similarly to
Authentic dCPD--
To confirm that the biological activity of sdCPD
in infection competition experiments correlates with its physical
binding properties to DHBV preS polypeptides, we performed binding
assays with the solid phase bound preS polypeptides depicted in Fig. 4A and compared the results
with those obtained for authentic dCPD from duck liver lysates. As
shown in Fig. 4B, authentic dCPD binds DpreS and the DpreS
fragment consisting of amino acids 30-115 (DpreS30-115). The deletion
mutant DpreS
In Fig. 5 the pH dependence of the
DpreS-sdCPD interaction is shown. Binding was observed between pH 5.5 and 10.0 with an optimum at pH 6.5. Binding still occurred at pH 5.0. However, further protonation abolished binding completely.
Determination of Binding Constants of the DHBV preS·sdCPD Complex
by Real Time Surface Plasmon Resonance Spectroscopy--
To determine
association and dissociation rates of the DHBV preS·sdCPD complex and
accordingly deduce the affinity of DHBV to its cellular receptor we
followed complex formation and dissociation by real time surface
plasmon resonance analysis (BIAcore). DHBV preS polypeptides were
covalently immobilized to CM5 sensor chips (see "Experimental
Procedures") and three different concentrations (0.06, 0.15, and 0.29 µM) of monomeric sdCPD were injected onto the surface at
37 °C (Fig. 6A). The
kinetics of binding (100-400 s) and release (400-575 s) of sdCPD were
calculated from the slopes of the curves. The association rate
ka was determined to 4.0 × 104
M
Consistent with our observation in the qualitative binding assay (Fig.
4B), the deletion mutant DpreS
Despite its ability to bind the substrate PABA, most likely via the two
enzymatically active A and B domains, oligomeric sdCPD was found to
interact only weakly with DpreS (Fig. 6C). Thus
oligomerization restricts DpreS binding.
The Receptor-binding Domain of DHBV-preS Consists of an Essential
Attachment Site and a Conformation Stabilizing Element--
We
extended surface plasmon resonance analysis to investigate the
contribution of particular sequence elements within DpreS to receptor
binding, by testing the same set of deletion mutants (Fig.
7A) that have been
characterized in infection competition experiments (8) for sdCPD
interaction (Fig. 7, B-D). Deletions outside the
receptor-binding domain (mutants DpreS Binding of DHBV preS to Monomeric sdCPD Induces Conformational
Changes but Not Complex Oligomerization--
To determine the
stoichiometry of the DpreS·sdCPD complex in solution and to address
the question whether binding of the ligand induces conformational
changes in the receptor that lead to oligomerization, we performed a
calibrated gel filtration analysis. To this aim, full-length DHBV preS
or the fragment DpreS30-115 were incubated with sdCPD at 4 °C to
allow complex formation and applied to a calibrated Superdex 200 column. At this temperature the complex stability is greatly enhanced
and complex dissociation could be prevented. Eluted fractions were
analyzed for the presence of DpreS polypeptides by Western blot
analysis and apparent molecular weights of the corresponding peaks in
the elution profiles were determined. In a control experiment,
monomeric sdCPD and DpreS were analyzed separately (Fig.
8, A and B).
Monomeric sdCPD did not form oligomers under the chosen conditions and
eluted with an apparent molecular mass of 248 kDa (as described above).
DpreS alone eluted at 46 kDa with an apparent molecular mass more than 2-fold higher than expected for the monomer (19,822 Da as determined by
mass spectrometry (data not shown)). This behavior is probably not
caused by dimerization (8), but reflects particular structural elements
in the polypeptide that stabilize a non-globular conformation.
As shown in Fig. 8, C and D, both DpreS and
DpreS30-115 co-elute in complex with sdCPD, with apparent molecular
masses of 254 and 215 kDa, respectively. Consequently, binding of
full-length DpreS or DpreS30-115 to sdCPD neither requires nor induces
oligomerization of the dCPD ectodomain. While the DpreS·sdCPD complex
runs at an only slightly greater molecular mass compared with sdCPD
alone (254 kDa), the DpreS30-115·sdCPD complex eluted at an even
lower apparent molecular mass than free sdCPD (215 kDa) in gel
permeation experiments. Hence, binding of DpreS to its receptor induces
conformational changes that cause a significant reduction of the
apparent molecular weight. The size of both of the complexes suggests
that a single molecule DpreS binds to one molecule sdCPD in a 1:1
stoichiometry. This assumption was confirmed by a quantitative binding
assay shown in Fig. 9. DpreS that has
been applied in a molar ratio of 1:1 to covalently immobilized
monomeric sdCPD is completely absorbed by sdCPD (right two
lanes). An increase in the molar ratio of DpreS/sdCPD, from 1:1 to
1:2 or 1:4, did not lead to further preS-binding, but resulted in
additional amounts of DpreS remaining in the supernatant (Fig. 9,
left four lanes).
We have investigated the interaction of the extracellular portion
of the avian hepatitis B virus receptor, purified from a baculovirus
expression system, with DHBV preS-polypeptides representing different
parts of the ectodomain of the large viral envelope protein. We have
found that the COOH-terminal truncated and thus soluble dCPD binds DHBV
preS in a similar manner as to the full-length membrane-bound
carboxypeptidase D, and therefore provides a valuable tool for studies
of avian hepatitis B virus-receptor interactions. In particular,
utilization of sdCPD had several advantages when compared with the
authentic dCPD: (i) sdCPD could be purified in detergent-free buffers
and consequently be used in cell culture assays. (ii) sdCPD becomes
secreted into the culture medium and has by-passed all control elements
of the secretory pathway. (iii) High level protein expression in
serum-free culture medium is feasible in High Five insect cells and
facilitates subsequent purification steps.
Recombinant sdCPD was produced in a monomeric and an oligomeric
form. Monomeric sdCPD eluted at a slightly greater apparent molecular
mass than expected (248 kDa instead of 170 kDa), which may be
attributed to its three-domain structure rendering the protein an
ellipsoidal overall shape. The monomeric fraction, comprising about
two-thirds of total sdCPD, showed PABA as well as preS binding activity
and was remarkably active in infection competition assays. In contrast,
the remaining one-third of sdCPD had an apparent molecular mass greater
than 1 MDa and was impaired in DpreS binding. Our initial assumption
that this fraction consists of inactive aggregates, seems unlikely
because (i) electron microscopic analysis revealed that the majority of
molecules had a defined size of approximately 3,600 kDa as calculated
from the Stokes radii (data not shown), (ii) oligomeric sdCPD was able
to bind PABA and therefore probably possesses carboxypeptidase
activity, (iii) infection competition activity was, interestingly, only slightly reduced when compared with monomeric sdCPD, indicating that,
at least in the presence of natural viral particles and living cells,
oligomeric sdCPD can be converted to an active state (Fig.
3A). Nevertheless, it remains to be investigated if
oligomerization of dCPD also occurs in vivo or is just an
artifact of our expression system.
It has recently been shown that DpreS binds to the enzymatically
inactive C-domain of dCPD (16). With respect to this observation, the
impaired preS binding capability of oligomeric sdCPD suggests that it
may also be the C-domain of dCPD that is involved in oligomerization. This could either mean that the preS-binding site is directly involved
in homomeric contacts, or that the preS-binding site within the dCPD
C-domain is inaccessible in the oligomer.
Despite the evolutionary loss of most essential amino acids required
for carboxypeptidase activity, the C-domains of all CPDs investigated
so far have conspicuously conserved primary sequences, implying that
they have preserved a still unknown function (15). One of these
hypothetical functions could be binding to a yet unknown natural
ligand. With respect to this idea and with respect to our observation
of a ligand-independent formation of oligomers, binding of this natural
component as well as of DHBV might also cause oligomerization and, as a
consequence, might activate intracellular signal transduction pathways.
The recently observed enhancement of DHBV infection by subviral
particles at very low multiplicity of infections (28) is presumably
achieved by utilizing this CPD-mediated signaling pathway, as the
essential region responsible for enhancement of infection coincides
with the primary receptor attachment site defined here.
Purified sdCPD from insect cells binds DHBV preS in a comparable manner
to the authentic full-length dCPD from liver lysates. The dissociation
constant for the interaction of a single preS moiety with a single
receptor molecule at 37 °C was found in the range of
10 Using a set of DHBV preS insertion mutants in an in vitro
dCPD binding assay, Ishikawa et al. (30) mapped the viral
determinants required for dCPD binding to amino acids 43-108 within
preS. In a similar approach but using terminally deleted preS
mutants, Tong et al. (10) mapped the dCPD-binding
site to amino acids 87-102. These conflicting results are resolved by
the kinetic analysis presented in this study. As the preS fragment from
amino acids 87 to 102 overlaps with the essential receptor-binding site defined here, we assume that this polypeptide binds dCPD with a
drastically reduced association constant when compared with full-length
DpreS. This low affinity interaction was presumably measured by Tong
et al. (10) and led them to conclude that amino acids
87-102 represent the complete dCPD-binding domain. In contrast, our
data, however, indubitably demonstrate that amino acids preceding this
primary attachment site also contribute substantially to dCPD
interaction. This stabilization is conformation dependent explaining
why insertions in this region of preS destabilize the preS·dCPD
complex (30). In addition, insertions, in contrast to deletions, might
alter the tertiary structure of preS in such a way that even low
affinity interaction could be disrupted. Thus, the apparently
conflicting data in fact support our conclusion that the
receptor-binding domain within DHBV preS consists of an essential
primary binding site which, on its own, interacts with low affinity and
a more NH2-terminal located region which enhances receptor affinity.
1 at 37 °C, pH 7.4, with an association rate of 4.0 × 104
M
1 s
1 and a dissociation rate
of 1.9 × 10
3 s
1, substantiating high
affinity interaction of avihepadnaviruses with their receptor
carboxypeptidase D. The separately expressed receptor-binding domain,
comprising about 50% of preS as defined by mutational analysis,
exhibits similar constants. The domain consists of an essential
element, probably responsible for the initial receptor contact and a
part that contributes to complex stabilization in a conformation
sensitive manner. Together with previous results from cell biological
studies these data provide new insights into the initial step of
hepadnaviral infection.
INTRODUCTION
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Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
-globulin (158 kDa), fraction 16; ovalbumin (44 kDa),
fraction 20; myoglobin (17 kDa), fraction 23 and vitamin B-12 (1.3 kDa), fraction 28.
Kd(t
t0).
RESULTS
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Fig. 1.
Purification of sdCPD from a baculovirus
expression system. A, the coding sequence of the three
carboxypeptidase-like extracellular domains of dCPD, preceded by the
secretory signal sequence, was inserted into the baculovirus transfer
vector pVL1393. The vector was modified by introduction of a
XhoI/XbaI/PstI stop linker
(boxed sequence with underlined amber codon), and
a NcoI site, downstream from the vector encoded
BamHI site, containing the start codon. The
NH2-terminal methionine and the COOH-terminal valine of
sdCPD are numbered. Numbers 25 and 29 indicate
the cleavage sites of the signal peptidase as determined by
NH2-terminal sequencing. B, sdCPD is secreted as
a glycoprotein into the culture supernatant of infected insect cells.
Cell culture media of AcNPV (WT) and AcNPV-sdCPD
(AcNPV-sdCPD) infected Sf9 cells were analyzed by
Western blot for the presence of dCPD specific proteins in the absence
( ) or presence of endoglycosidase H (Endo H) and
endoglycosidase F (Endo F). The position of undigested sdCPD
is indicated by an arrow. C, elution profile of
sdCPD on a PABA affinity column. Culture supernatants of AcNPV-sdCPD
infected High Five insect cells were applied to the column
(start) and sdCPD (pool) was eluted with 50 mM
arginine (elution). D, silver-stained SDS-gel
electrophoresis and dCPD-specific Western blot analysis of the pooled
fractions eluted from the affinity column, as shown in
C.
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Fig. 2.
Separation of monomeric and oligomeric sdCPD
by size exclusion chromatography. A, elution profile of
affinity purified sdCPD on a calibrated Superdex 200 column.
Vo and the elution volumes of thyroglobulin (670 kDa), -globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa),
and cobalamin (1.3 kDa) used for calibration are indicated at the
top. Fraction numbers correspond to lane numbers
in B. The apparent molecular mass of monomeric sdCPD (248 kDa) was calculated from standard curves as a mean value of three
independent measurements. B, silver-stained SDS gel of
fractions applied to (load = pool of the PABA affinity column) and
eluted from the Superdex 200 column, as shown in A. sdCPD is
indicated by an arrow. Lane numbers correspond to fraction
numbers in A.
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Fig. 3.
Competitive inhibition of DHBV infection by
recombinant sdCPD (A) and the DHBV preS polypeptides
DpreS and DpreS30-115 (B). A, 8 × 105 primary duck hepatocytes were infected with DHBV at
a multiplicity of infection of 100 in the presence of increasing
amounts of monomeric (straight line) or oligomeric
(dotted line) sdCPD. Cells were harvested 6 days
post-infection and intracellular viral DNA was quantified by DNA dot
blot. Mean values of two independent measurements are shown and given
as percentage of viral DNA compared with an uncompeted infection. The
ratios of sdCPD molecules to viral particles were calculated on the
basis of sdCPD concentrations determined by the method of Gill and von
Hippel (25) and viral particle titers quantified by dot blot analysis
assuming 103 noninfectious subviral particles per virion.
B, primary duck hepatocytes (8 × 105) were
infected with 4 × 107 DHBV particles in the presence
of increasing concentrations of full-length DHBV preS (straight
line) or the DHBV preS fragment DpreS30-115 (dotted
line). Virus replication between day 5 and day 9 post-infection
was determined by immuno dot blot analysis of DHBeAg secreted into the
culture medium and is presented as a percentage of the value for an
uncompeted infection. Each point represents the average of three
independent experiments, bars indicate the standard
deviation.
85-96, inactive in DHBV infection competition (8), was
used as a control and showed no binding. Likewise, recombinant sdCPD
eluted from the PABA column binds DpreS but not DpreS
85-96 (Fig.
4C). However, even at a large excess of DpreS we did not
observe complete binding of sdCPD (Fig. 4C, left frame, left
lane) and therefore assumed that a fraction of sdCPD might be
inactive in DpreS binding. This interpretation was confirmed in a
quantitative BIAcore analysis showing that oligomeric sdCPD does not
bind DpreS (see below).
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Fig. 4.
Recombinant sdCPD binds DHBV preS
polypeptides similarly to authentic dCPD. A, schematic
drawing of DHBV preS polypeptides used in the qualitative binding assay
shown in B and C. Numbers at the
top of the scale mark relevant amino acid positions within
the DHBV preS region. DpreS represents the complete ectodomain of the
large viral surface protein, DpreS30-115 the receptor-binding domain
and DpreS 85-96 the internal deletion mutant which served as a
control. B, the three preS polypeptides were covalently
immobilized on microtiter plates and incubated with duck liver extract
as described under "Experimental Procedures." Bound dCPD was
detected by Western blot using a polyclonal antiserum directed against
a COOH-terminal fragment of recombinant dCPD. dCPD, runs at 180 kDa,
and is indicated by an arrow. The slightly reduced binding
of dCPD to DpreS30-115 in this qualitative solid phase binding assay
was not taken to be significant as it could not be reproduced in the
more elaborated BIAcore analyses shown in Fig. 6. C, binding
of recombinant sdCPD to DpreS. DpreS and deletion mutant DpreS
85-96
were covalently immobilized on covalink plates and incubated with sdCPD
containing cell culture supernatants of AcNPV-sdCPD infected insect
cells. Bound and free sdCPD were detected by Western blot using the
same antibody as in B. sdCPD running at 170 kDa is indicated
by an arrow.
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Fig. 5.
pH dependence of DpreS-sdCPD
interaction. Covalently immobilized DHBV preS was incubated with
purified sdCPD at pH values between 3.5 and 10.0 (indicated at the
top of each lane). DpreS-bound sdCPD was
quantitatively eluted and relative amounts of sdCPD were determined by
Western blotting using a dCPD-specific antibody.
1 s
1, the dissociation rate
kd to 1.9 × 10
3
s
1, corresponding to a half-life for the complex of about
6.0 min at 37 °C. From these data we calculated the dissociation
constant Kd to 4.6 × 10
8
M. Using the DHBV preS fragment from amino acid 30 to 115 (DpreS30-115), which has been identified by infection competition
experiments as the receptor-binding domain (8), we observed similar
constants: ka, 7.1 × 104
M
1 s
1; kd,
2.7 × 10
3 s
1;
t1/2, 4.3 min; Kd, 3.8 × 10
8 M (Fig. 6B). Consequently
full-length DpreS and DpreS30-115 are indistinguishable in both
infection competition and dCPD interaction.
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Fig. 6.
Determination of binding constants of
sdCPD·DHBV preS complexes by surface plasmon resonance
spectroscopy. A, overlay of sensorgrams for the
interaction of immobilized full-length DpreS with monomeric sdCPD at
three different concentrations as indicated (0.06, 0.15, and 0.29 µM). At 100 s monomeric sdCPD was injected onto
immobilized DpreS for 300 s. At 400 s the injection was
stopped and dissociation followed in running buffer for 150 s. The
association rate ka, the dissociation rate
kd and the dissociation constant
Kd given in the inserted frame, were calculated from
three independent measurements using the BIAevaluation program version
2.1. Note that sudden changes in refractive indices for all 4 sensorgrams shown are due to differences in the composition between
buffer and protein solution. B, overlay of sensorgrams for
the interaction of Dpres30-115 with monomeric sdCPD at three different
concentrations as indicated. Injection of sdCPD and determination of
constants was as described in A. C, overlay of sensorgrams
for the interaction of full-length Dpres with oligomeric sdCPD. At
100 s oligomeric sdCPD was injected for 300 s and
subsequently replaced by buffer. As neither significant binding nor
release was observed, calculations of kinetic constants could not
reasonably be performed. D, overlay of sensorgrams for the
interaction of Dpres 85-96 with monomeric sdCPD at the three
different concentrations indicated. Injection of sdCPD was as described
in A.
85-96 showed no obvious interaction with sdCPD in the BIAcore experiment (Fig. 6D).
This observation led us to conclude that amino acid residues 85-96 of
DpreS contain absolutely essential elements for receptor interaction. Interestingly, anti-preS antibodies which are capable of blocking DHBV
infection have been mapped to bind epitopes within this
region2,3
indicating their surface exposure.
22-30 and DpreS
128-139) showed kinetics of association and dissociation similar to full-length DpreS (Fig. 7B). This indicates that the part of preS that
binds the receptor folds independently of the deleted flanking amino acids and constitutes a distinct domain within the viral L-protein. In
contrast, DpreS
85-96 and deletion mutant DpreS
101-109 were completely impaired in sdCPD binding (Fig. 7D). This region
of preS (amino acids 85-109) therefore contains elements which are absolutely required for receptor interaction. An intermediate phenotype
was found for deletion mutants DpreS
67-70 and DpreS
74-84. They
were able to interact with sdCPD but displayed drastically diminished
binding and dissociation rates (Fig. 7D). This was confirmed
using the terminal deletion mutants depicted in Fig. 7C.
While DpreS30-115 binds sdCPD as strongly as full-length DpreS, mutants with successive deletions beyond amino acid 30 (DpreS38-115, DpreS43-115, and DpreS52-130) were affected by increasing alteration of association and dissociation constants. Taken together, two regions
in the receptor-binding domain of DpreS could be distinguished as
summarized in Fig. 7E. One, comprising amino acids 85-109, defines the essential attachment site for the receptor, and a second,
comprising amino acids 30-85, contributes to a conformation dependent
stabilization of the complex. For both functions formation of a defined
three-dimensional structure is important, since even short internal
deletions throughout the whole domain drastically influenced
interaction.
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Fig. 7.
The receptor-binding domain of DpreS consists
of an essential attachment site and elements that contribute to recep tor complex stabilization. A, schematic drawing
of terminal and internal DpreS deletion mutants used for the surface
plasmon resonance analysis of DpreS-sdCPD interaction shown in B,
C, and D. Numbers indicate positions of amino acids
deleted (e.g. DpreS 22-30 lacks amino acids 22-30 within
DpreS). Bars in light gray correspond to
polypeptides that bind sdCPD with comparable binding constants as
full-length DpreS (DpreS, DpreS
22-30, DpreS
128-139, and
DpreS30-115). Bars in black represent
polypeptides that did not show a detectable interaction with sdCPD
(DpreS
85-96 and DpreS
101-109). Mutants with reduced binding
constants are illustrated by the shading of the bars
(DpreS38-115, DpreS43-115, DpreS 52-130, DpreS
67-70, and
DpreS
74-84). B, sensorgram overlays for the interaction
of sdCPD with full-length Dpres (red), DpreS
22-30
(blue), and DpreS
128-139 (green). DpreS
polypeptides were immobilized on a CM5 sensor chip as described under
"Experimental Procedures." At 100-s monomeric sdCPD was injected at
a concentration of 0.15 µM for 300 s. At 400 s
the sample was replaced with buffer and dissociation followed for
150 s. Changes in refractive indices (visible as jumps in the
curves) represent differences in solution composition between the
buffer and the protein solution. C, sensorgram overlays for
the interaction of sdCPD with deletion mutants DpreS30-115
(blue), DpreS38-115 (red), DpreS 43-115
(green), and DpreS 52-130 (yellow). Sample
application was performed as described in B. D,
sensorgram overlays for the interaction of sdCPD with the internal
deletion mutants Dpres
67-70 (blue), DpreS
74-84
(red), DpreS
85-96 (yellow), and
DpreS
101-109 (green). Sample application was performed
as described in B. Note that despite the changes in
refraction indices caused by differences between buffer and protein
solutions, Dpres
67-70 (blue) and DpreS
74-84
(red) in contrast to DpreS
85-96 (yellow) and
DpreS
101-109 (green) clearly show time dependent binding
and release of sdCPD. E, DpreS consists of an internal
receptor-binding domain comprising amino acids 30-115
(bracket) and two flanking regions (1-29 and 116-161)
which are non-essential with respect to receptor binding and serve
other functions. The receptor-binding domain contains an essential
(amino acids 85-109) and a stabilizing (amino acids 30-85)
element.
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Fig. 8.
Size exclusion chromatography of
sdCPD·DpreS complexes. A, elution profile of
monomeric sdCPD on a Superdex 200 column. The void volume
(V0) and the elution volumes of thyroglobulin
(670 kDa), -globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa), and cobalamin (1.3 kDa) used for calibration are indicated above
the profile. The apparent molecular mass of sdCPD (248 kDa) was
determined as a mean value of three independent calibrations.
B, elution profile of recombinant DpreS on Superdex 200. The
apparent molecular mass of 46 kDa was determined as described in
A. Note that DpreS elutes exclusively at 46 kDa without
forming higher molecular mass species. C, calibrated gel
filtration of the sdCPD·DpreS complex. sdCPD was incubated overnight
at 4 °C with an 10-fold excess of DpreS and applied to a Superdex
200 column. The upper part of the figure shows the elution
profile with size markers, the apparent molecular mass of the
sdCPD·DpreS complex (254 kDa) and the surplus free DpreS eluting at
46 kDa. The lower part displays a DpreS-specific Western
blot of fractions indicated by the numbers below
the elution profile. D, calibrated gel filtration of the
sdCPD·DpreS30-115 complex. Gel filtration and Western blot analysis
of fractions were performed as described in C. DpreS30-115
elutes at an apparent molecular mass of 19 kDa without forming higher
molecular mass complexes (not shown).
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Fig. 9.
DpreS binds monomeric sdCPD with a 1:1
stoichiometry. 1 pmol of monomeric sdCPD was covalently
immobilized on a covalink immunoplate and incubated with 1, 2, and 4 pmol of DpreS (1:1, 1:2, and 1:4). The relative amounts of bound and
free DpreS were determined by Western blot using the DpreS-specific
monoclonal antibody 4F8. Note that the amount of bound DpreS does not
change with increasing concentrations of free DpreS indicating
saturation of binding.
DISCUSSION
8 M, indicating high affinity interaction
at the molecular level. Taking into account that DHBV exhibits several
receptor-binding sites on its surface and hence probably interacts with
more than a single dCPD molecule, binding of virions to the hepatocyte
is likely to be strong and irreversible. This conclusion, at first sight, appears to be incompatible with particular observations regarding in vitro DHBV infections of primary duck
hepatocytess as well as in vivo infections of ducklings. For
example, the question arises why in vitro infections of
primary duck hepatocytess with serum-derived DHBV requires high
multiplicity of infections and long incubation times (26). By contrast,
in vivo infection has been described to be extremely
effective (29), although dCPD is expressed in various other organs of
the duck at much higher levels than in the liver (data not shown and
Ref. 19). With regard to the low in vitro infection
efficiency, one possible partial explanation is given by the
observation that only a very limited number of receptor molecules
(<100) are accessible on the surface of
hepatocytes.4 Thus saturation
of cell-surface exposed dCPD with viral particles could be achieved
already at very low multiplicity of infections (assuming an at least
1000-fold excess of non-infectious subviral particles), and the rate of
receptor cycling, as has been shown to occur for dCPD (18) and rat CPD
(17), rather than kinetics of binding would determine infection efficiency.
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ACKNOWLEDGEMENTS |
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This work was performed in the laboratory of Heinz Schaller, whom we thank for intellectual and material support. We are especially grateful to Kazuyuki Kuroki for providing dCPD cDNA. We thank Bärbel Glass for the preparation of primary duck hepatocytes, Christa Kuhn for advise and anti-DpreS antibodies, Rainer Frank for protein sequencing, Almuth Liebich for contributing work to synthesize PABA, Hans Will for providing the internal DpreS deletion mutants, and Klaus Michael Breiner for contribution in the preparation of DpreS polypeptides, the supply of anti-dCPD antibodies, and many helpful discussions. We also thank Oliver Gallay and Michael Nassal for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by Deutsche Forschungsgemeinschaft Grants SFB 229 (to H. Schaller), SFB 317 (to G. M.), and by the Fonds der Chemischen Industrie through Konrad Beyreuther.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.
To whom correspondence should be addressed: Zentrum für
Molekulare Biologie (ZMBH), Universität Heidelberg, Im
Neuenheimer Feld 282, D-69120 Heidelberg, Germany. Tel.:
49-6221-546887; Fax: 49-6221-545893; E-mail:
s.urban{at}zmbh.uni-heidelberg.de.
2 C. Kuhn, personal communication.
3 L. Cova, personal communication.
4 S. Urban and H. Schaller, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: HBV, hepatitis B virus; DHBV, duck hepatitis B virus; CPD, carboxypeptidase D; dCPD, duck carboxypeptidase D; sdCPD, soluble duck carboxypeptidase D; DpreS, duck hepatitis B virus preS polypeptide; Sf, Spodoptera frugiperda; PABA, 4amino-benzoyl-arginine; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbo-diimide hydrochloride; AcNPV, Autographa californica nuclear polyhedrosis virus.
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REFERENCES |
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