From the Department of Virus and Cell Biology, Merck
Research Laboratories, West Point, Pennsylvania 19486, the
¶ Department of Bioprocess and Bioanalytical Research, Merck
Research Laboratories, Rahway, New Jersey 07065, and the
Department of Biological Sciences, Lehigh University,
Bethlehem, Pennsylvania 18015
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ABSTRACT |
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The L1 major capsid protein of human
papillomavirus (HPV) type 11, a 55-kDa polypeptide, forms particulate
structures resembling native virus with an average particle diameter of
50-60 nm when expressed in the yeast Saccharomyces
cerevisiae. We show in this report that these virus-like
particles (VLPs) interact with heparin and with cell-surface
glycosaminoglycans (GAGs) resembling heparin on keratinocytes and
Chinese hamster ovary cells. The binding of VLPs to heparin is shown to
exhibit an affinity comparable to that of other identified
heparin-binding proteins. Immobilized heparin chromatography and
surface plasmon resonance were used to show that this interaction can
be specifically inhibited by free heparin and dextran sulfate and that
the effectiveness of the inhibitor is related to its molecular weight
and charge density. Sequence comparison of nine human L1 types revealed
a conserved region of the carboxyl terminus containing clustered basic
amino acids that bear resemblance to proposed heparin-binding motifs in
unrelated proteins. Specific enzymatic cleavage of this region eliminated binding to both immobilized heparin and human keratinocyte (HaCaT) cells. Removal of heparan sulfate GAGs on keratinocytes by
treatment with heparinase or heparitinase resulted in an 80-90% reduction of VLP binding, whereas treatment of cells with laminin, a
substrate for Papillomaviruses are non-enveloped, double-stranded DNA viruses
containing a circular genome of approximately 8,000 base pairs. The
viral capsid is composed of a major and minor capsid protein, both
products of late gene expression and termed L1 and L2, respectively (1). The L1 protein has a molecular mass of 55-60 kDa by
polyacrylamide gel electrophoresis and is well conserved across types.
In the virion it accounts for 80-90% of total viral protein (2, 3). The viral capsid is built up from pentameric capsomers of L1, with 72 such structures arranged in a T = 7 icosohedral array (4, 5). The localization of L2 in this ordered structure is not known
at present nor is the mechanism of assembly, although recent reports
suggest disulfide bonding may play a role (6, 7). Over 80 types of
human papillomavirus are currently identified, and many have been shown
to be associated with various forms of warts occurring on the surface
epithelia of skin and mucous membranes. HPV1 types can be broadly
grouped into those that cause benign lesions such as condyloma
acuminata and others that show the potential for malignant
transformation. HPV types 11 and 6 are representative of the former and
are the etiological agents responsible for approximately 90% of all
benign warts associated with the anogenital tract and respiratory
mucosa (8). Members of the second group include HPV types 16 and 18, which cause cervical intraepithelial neoplasia (9-11) that may
progress to squamous cell carcinoma (12).
Progress has recently been made producing either L1 alone or L1 and L2
proteins recombinantly in a number of expression systems (13-16). It
has been shown that newly synthesized L1 is translocated to the nucleus
where it self-associates to form capsid-like structures that resemble
the native virus but lack viral nucleic acid (14, 17, 18). These
virus-like particles (VLPs) are capable of eliciting neutralizing
antibodies and can competitively block infection by live virus (18,
19). Because native virus has proved exceedingly difficult to propagate
in vitro, the ability to produce large amounts of
recombinant VLPs has greatly aided cell-based binding studies aimed at
identifying candidate HPV receptors.
Although papillomaviruses can bind to a wide variety of cell types (20,
21), their productive tropism is generally limited to epithelial
keratinocytes and fibroblasts (20). The fact that most HPVs can bind to
cells of epithelial origin in vitro but cannot be propagated
suggests that intracellular events following uptake of virus are
crucial for establishment of successful infection and that host
specificity is not linked to the presence of a given cell-surface
receptor (20). This is particularly true in keratinocytes where
establishment of infection has been linked to cellular differentiation (22). The ability of HPV to bind to a wide range of nonpermissive tissue- and species-specific cell lines argues for a receptor that is
either highly conserved or of low specificity. Recently, Evander
et al. (23) have proposed the
Alternatively, HPVs may exhibit broad binding ranges as a result of a
multiple receptor mechanism. In this model a receptor of relatively low
specificity causes interaction of virus with the cell followed by
binding to a specific protein component resulting in internalization
and infection. One group of molecules able to serve as putative
receptors is cell-surface glycosaminoglycans. This mode of binding has
been established for herpes simplex virus 1 (HSV-1) (24) and human
herpesvirus 7 (25) in which binding of viral glycoproteins to
cell-surface heparan sulfate molecules provides initial contact. Recent
reports have suggested that GAGs may play a similar role in binding of
varicella-zoster virus to human embryonic lung fibroblasts (26).
Similarly, infection of cells by human immunodeficiency virus 1 can be
blocked by polyanions such as dextran sulfate (27) although
proteoglycans are not implicated as viral receptors.
Our laboratory has been involved in the purification and
characterization of various HPV VLP types (13, 28). In this report we
have made a sequence comparison based on the method of Pearson and
Lipman (29) to identify a conserved region in the L1 carboxyl terminus
containing amino acid sequences of the general type
XBBBBXB where B is either Arg or Lys. These are
similar to the XBBXBX and
XBBBXXBX sequences identified by
Cardin and Weintraub (30) as putative heparin-binding motifs. We
therefore investigated the ability of L1 VLPs to bind to heparin and
characterized the nature of this interaction with regard to charge and
polymer size. Finally, we examined the role of cell-surface
glycosaminoglycans in binding VLPs. The HaCaT human keratinocyte cell
line was chosen for binding studies because it represents a cell type
that serves as an in vivo infection target, and its ability
to internalize HPV VLPs has been established (31). We show in this
study that the carboxyl-terminal portion of HPV 11 L1 interacts with
heparin and that this interaction displays a certain degree of
specificity. Furthermore, this region is shown to be crucial for
interaction with the cell surface.
Cell Culture
HaCaT cells were maintained as monolayer cultures at 37 °C,
5% CO2 in Dulbecco's modified Eagle's medium (DMEM; Life
Technologies, Inc., Rockville, MD) supplemented with 10% fetal calf
serum (FCS; Life Technologies, Inc.) and 1% penicillin/streptomycin
(Life Technologies, Inc.). For sulfate-free medium, the DMEM
formulation was the same except that magnesium sulfate was omitted and
dialyzed FCS (Sigma) was used. CHO-K1 and pgsA-745 cell
lines were obtained from American Type Culture Collection (ATCC;
Rockville, MD) and were grown as monolayer cultures at 37 °C, with a
5% CO2 atmosphere in F-12K medium (ATCC) supplemented with
10% FCS and 1% penicillin/streptomycin. Cells were routinely
subcultured every 14-21 days.
Expression and Purification of VLPs
Recombinant HPV type 11 L1 VLPs were expressed in
Saccharomyces cerevisiae using the pGal110-11 vector as
described previously (32). VLPs were purified from clarified yeast cell
lysate by ion exchange chromatography using a modification of
previously described methodology
(13).2 The presence of VLPs
was confirmed by electron microscopy, and purity was determined by
denaturing polyacrylamide gel electrophoresis (PAGE) and
immunoblotting. VLPs were quantitated using a commercial bicinchoninic
protein assay (Pierce) and a specific radioimmunoassay described
elsewhere (28).
Polyacrylamide Gel Electrophoresis and Immunoblotting
Aliquots (100 µg) of purified VLP preparations were
concentrated by precipitation with 10% trichloroacetic acid at 0 °C
for 1-2 h. The pellets were resuspended at a concentration of 1.0 mg/ml in 2× Laemmli buffer containing 200 mM
dithiothreitol and heated at 100 °C for 15 min. Sufficient volume to
give 2.5-20 µg of total protein was loaded on pre-cast 4-20%
polyacrylamide gels (NOVEX, San Diego, CA) utilizing a Tris glycine
buffer system. The gels were electrophoresed at a constant current of
30 mA/gel for 1 h. Protein bands were fixed with 12%
trichloroacetic acid and visualized with a commercial colloidal
Coomassie reagent (Integrated Seperation Systems, Natick, MA) according
to manufacturer's recommendations. Quantitation of bands was performed
using a Molecular Dynamics Personal Densitometer model SI (Sunnyvale,
CA). For immunoblotting, proteins were electrophoretically transferred
to polyvinylidene difluoride membranes at a constant voltage of 25 V
for 1 h. Type 11 L1 was detected using a polyclonal goat antiserum
against an HPV 11 L1/TrpE fusion protein as primary antibody and
alkaline phosphatase-conjugated rabbit anti-goat antiserum (Pierce) as secondary antibody. Visualization was by nitro blue tetrazolium chloride/5-bromo-4-chloro-3'-indolyphosphate Electron Microscopy
Electron microscopy was performed by Advanced Biotechnologies
Inc. (Columbia, MD). Briefly, an aliquot of diluted VLPs was placed on
a 300-mesh carbon-coated copper grid and air-dried. A drop (20 µl) of
2% phosphotungstic acid, pH 7.0, was placed on the grid for 30 s.
The grid was allowed to air-dry prior to transmission electron
microscopy examination. All microscopy was performed using a Hitachi
HU-12A transmission electron microscope with micrographs taken of
random sections at various magnifications.
Heparin Affinity Chromatography
Chromatography was performed on a Waters 2690XE Alliance system
(Milford, MA) at ambient temperature using 1- or 5-ml cartridges of
Hi-Trap® heparin-Sepharose (Amersham Pharmacia Biotech) equilibrated with 50 mM MOPS, pH 7, and varying concentrations of NaCl.
Flow rates were 1 or 4 ml/min for the small and large columns. Purified VLPs were diluted to the appropriate NaCl concentration in buffer and
applied to the column. For most studies, the column was washed with 5 volumes of equilibration buffer, and proteins were eluted with a
10-volume linear salt gradient to 2.0 M NaCl. Absorbance was monitored using either a Waters 990 or 996 photodiode array detector. For inhibition studies, VLP preparations were diluted to 100 µg/ml in MOPS buffer containing 0.4 M NaCl. Compounds
tested as inhibitors were prepared as 10 mg/ml stock solutions in MOPS buffer and added to diluted VLPs. The samples were held at 4 °C for
16-20 h and brought to ambient temperature prior to chromatography.
Enzymatic Degradation of L1
VLPs (300 µg) were treated with bovine factor Xa (bFXa)
(Hematological Technologies, Essex Junction, VT) to remove the carboxyl terminus of the L1 molecule as described
elsewhere.3 Briefly,
digestions were performed by blowing argon saturated with water over
the surface of a 1.0-ml VLP sample in a sealed vial for 45-60 min in a
25 °C circulating water bath. The appropriate volume of bFXa or
buffer (control) was added to give an enzyme/substrate weight ratio of
1/20. The sample was mixed and incubated at 25 °C for 16 h. To
remove bFXa, soybean trypsin inhibitor-agarose resin (50 µl) was
added, and the sample was mixed on a rotary shaker for 1 h at room
temperature. Resin was pelleted by centrifugation at 2,000 × g for 15 s, and the supernatant was recovered.
Denaturing PAGE was performed to determine the extent of digestion.
Reversed phase identification of released peptide, intact L1, and
truncated L1 was performed using a high pressure liquid chromatography
assay described elsewhere.3
Surface Plasmon Resonance
The BIAcore system, CM5 sensor chip, amine coupling kit,
ethanolamine, and surfactant P20 were obtained from Pharmacia Biosensor (Amersham Pharmacia Biotech). Running buffer (HBS) was 10 mM HEPES, pH 7.4, 0.15 M NaCl. Heparin was
oxidized and immobilized to the CM5 chip according to BIApplications
handbook (Pharmacia Biosensor). Heparin was dissolved in 100 mM sodium acetate buffer, pH 5.5, at 1 mg/ml. A 1/50 volume
of freshly prepared sodium metaperiodate solution was added to give a
final concentration of 1 mM. The sample was incubated on
ice for 20 min, and the reaction was stopped by dialysis against 10 mM sodium acetate buffer, pH 4, at 4 °C. The sensor chip
surface was prepared by equilibration with HBS, 0.05% (v/v) surfactant
P20 at 5 µl per min, followed by successive injections of (i) 35 µl
of a mixture of equal volumes of 0.1 M N-hydroxysuccinimide, 0.1 M
N-ethyl-N-(dimethylaminopropyl)carbodiimide; (ii)
35 µl of 5 mM carbohydrazide; (iii) 35 µl of 1 M ethanolamine hydrochloride, pH 8.5; and (iv) 35 µl of
oxidized heparin at 1 mg/ml. The flow rate was lowered to 2 µl per
min, and 40 µl of 0.1 M sodium cyanoborohydride in 0.1 M sodium acetate buffer, pH 4, was injected. The
immobilized heparin was treated with two 5-µl pulses of 20 mM HCl to remove noncovalently attached ligand. VLP
preparations were diluted to the appropriate concentration of NaCl in
HBS and injected (20-µl volume) onto the heparin sensorchip at a
constant eluent flow rate of 5 µl/min. The chip was regenerated by a
7-µl injection of 20 mM HCl. Data points corresponding to initial binding rates were routinely collected manually except during
quantitative free heparin inhibition analysis where collection was
automatic. For inhibition studies, samples of VLPs were prepared as
above at constant protein concentration, and inhibitors were added from
concentrated stocks in 50 mM MOPS buffer, pH 7. Samples were allowed sufficient time to reach equilibrium prior to analysis.
Cell Binding Assays
VLP Binding Enzyme-linked Immunosorbent Assays--
The standard
assay employed to study VLP binding to cells and the effects of various
inhibitors on binding was a sandwich enzyme-linked immunosorbent assay.
Cells were seeded on 96-well plates at a density of 5 × 10 4 (HaCaT) or 1 × 10 4 (CHO-K1 and
pgsA-745) cells/well. Plates were maintained in standard media for 48-72 h until Inhibition Assays--
Compounds previously determined to
inhibit VLP binding to immobilized heparin were added from concentrated
stock solutions to a fixed concentration of VLPs, and the samples were
held for 16-20 h at 4 °C. Treated or control VLPs were diluted to
the appropriate concentrations and added to fixed cells as described above.
Glycosaminoglycan Modification Assays--
HaCaT cells were
treated with a variety of reagents known to modify or inhibit synthesis
of cell-surface glycosaminoglycans. For most experiments, cells were
grown to Heparinase Treatment--
HaCaT cells were grown to confluence
on 96-well plates. Culture medium was removed, and cells were washed
once with PBS. Heparinase (forms I and II) or heparitinase (heparinase
III) was diluted in digestion buffer (20 mM Tris-HCl, 50 mM NaCl, 4 mM CaCl2, pH 7.5, containing 0.01% BSA) and added to cells at a final concentration of 1 or 3 units/well. Control wells consisted of buffer without enzyme.
Duplicate plates were prepared and incubated at room temperature or
37 °C for 1 h. Plates were washed once with PBS. Cells were fixed and used for assay as described above. All enzymes were purchased
from Sigma.
Characterization of Purified VLPs--
Recombinant HPV 11 L1 VLPs
were isolated from yeast cells as described under "Experimental
Procedures." Fig. 1A shows
the results of SDS-PAGE analysis on 20 µg of purified VLPs. The major protein band migrating at 55,000 Da (p55) is confirmed to be HPV L1 by
immunoblotting and accounts for 84% of all L1-reactive material. In
addition to p55, approximately 6% of L1-reactive bands appear as high
molecular weight multimers, probably representing non-denaturable aggregates, whereas the remaining 10% migrate at molecular weights below p55 and represent proteolytic degradation products of L1. Such
degradates have been previously observed in this expression system
(15). Purity of the VLP preparation was determined to be 96% by
densitometric analysis. Fig. 1B shows an electron micrograph of a purified preparation. VLPs routinely exhibited an average diameter
of 50-60 nm and were present as individual, well defined particles
with minimal aggregation.
Interaction of VLPs with Immobilized Heparin--
The
carboxyl-terminal sequence (residues 447-501) of HPV 11 L1 is shown in
comparison with other HPV L1 types in Fig.
2. This alignment was generated using the
"FastA" and "Pileup" programs contained in Intelligenetics
version 8.0 software (Genetics Computing Group Inc., Madison, WI).
"FastA" was used to identify sequences similar to the complete HPV
11 L1 query sequence. Out of more than 50 hits, human sequences
containing at least 64% amino acid identity are shown. The carboxyl
terminus is fairly well conserved among HPV types and is characterized
by a cluster of 6-8 basic amino acids within the final 15 residues and
a shorter 3-4 basic amino acid cluster upstream from this. Eight of
nine types contain the putative heparin-binding motif BBBXB
where B is a basic residue (Lys or Arg).
To explore the ability of HPV 11 L1 to interact with heparin, VLPs were
applied to a column of immobilized heparin-Sepharose. Preliminary
experiments showed that binding could be achieved at salt
concentrations up to 0.5 M NaCl. The VLPs bound
quantitatively as judged by the lack of a significant breakthrough
peak. VLPs were eluted with porcine mucosal high molecular weight (HMW)
heparin of average molecular mass
( Inhibition of VLP Binding--
A variety of compounds with
structural similarities to heparin were tested as inhibitors of VLP
binding by heparin-Sepharose chromatography. For these experiments, the
concentration of VLPs was held constant while inhibitor concentration
was varied. Mixtures of VLPs and inhibitor were held at 4 °C for
various times (1-24 h) prior to analysis (data not shown). It was
found that inhibition was relatively rapid, occurring within 1 h,
but in order to ensure that equilibrium had been obtained, samples were
typically incubated 16-20 h prior to chromatography. The total amount
of p55 was calculated as the sum of retained and breakthrough peak
areas, and recoveries were typically 90-95% that of control VLPs.
This was done for each sample in order to account for slight variations
in injection amount and recovery. The percent p55 bound was then
calculated from the area of the retained peak with the same retention
time as untreated controls (average of triplicate injections). The effect of various inhibitors is shown in Fig.
4. Binding data were fit to two sigmoidal
dose-response models and a one-site competition model. The best fits
were achieved using a sigmoidal dose-response model with variable
slope, and this was used to calculate IC50 values. Binding
was inhibited by HMW heparin with a calculated IC50 of 14.9 µM. By contrast, a commercial low molecular weight (LMW)
preparation (
The importance of sulfation was evident in that chemical
N-desulfation of glucosamine residues in heparin led to a
complete loss of the inhibitory effect at all concentrations tested.
Furthermore, inhibition by dextran sulfate was dependent on the anionic
nature of the polysaccharide as non-sulfated dextrans of low or high Effect of Polymer Molecular Weight on Inhibition--
The
difference in IC50 values exhibited by HMW and LMW heparins
suggested a dependence of inhibition strength on molecular weight. This
was tested using a series of sized dextran sulfate preparations with
Dependence of VLP Binding on an Intact L1 Carboxyl
Terminus--
Treatment of HPV 11L1 VLPs with bFXa releases a
28-residue carboxyl-terminal peptide, generating a truncated L1 with a
molecular mass of approximately 53,000 Da (p53) as determined by
denaturing PAGE analysis. Following removal of enzyme by soybean
trypsin inhibitor-agarose treatment, the reaction products were
analyzed by heparin-Sepharose chromatography. Fig.
6A shows the profiles of
control (VLP without enzyme) and digested VLPs. A characteristic VLP
peak (I) accounting for 92% of
A220 nm area is present in the control sample.
Approximately 94% of retained peak area is lost and recovered in the
column breakthrough upon treatment with enzyme. The small amount of
peak I remaining after bFXa treatment (6%) correlates well with the
5% intact p55 observable by gel electrophoresis of the digest. Reverse
phase analysis (data not shown) was used to identify the species
present in each peak from the heparin-Sepharose column. The
carboxyl-terminal peptide was recovered in peak III and truncated L1 in
peak II. Peak II was re-injected in order to determine if p53 did not
bind to heparin as a result of inhibition by the highly basic free
peptide. An identical elution profile was obtained, confirming that
competition by free carboxyl-terminal peptide did not inhibit p53 from
binding. To study further the effect of free peptide, a synthetic
15-mer carboxyl-terminal peptide was analyzed by heparin-Sepharose
chromatography. The peptide showed lower affinity for the resin
compared with intact VLPs. When peptide or VLPs were injected on a
column equilibrated in 0.36 M NaCl, the 15-mer was retained
but eluted at a lower ionic strength than VLPs (data not shown). When
the initial concentration of NaCl was raised to 0.5 M, the
peptide interacted weakly, eluting isocratically in the wash portion of
the run (Fig. 6B, top). Co-injection of peptide and protein
confirmed this difference in affinity and revealed that the 15-mer was
unable to inhibit VLP binding even at a 67-fold molar excess (Fig.
6B, bottom). The study was repeated with a 28-mer peptide
which corresponded more closely to the region removed by bFXa. Although
this peptide eluted at higher ionic strength than the 15-mer, it was
also less retained than VLPs and did not inhibit VLP binding (data not
shown). Given the weak affinity of the synthetic peptides, the failure
of the enzymatically generated carboxyl-terminal fragment to bind the
heparin-Sepharose column (Fig. 6A) may have been a result of
chromatographic displacement by p53.
Analysis of Heparin Binding by Surface Plasmon
Resonance--
Surface plasmon resonance performed on a BIAcore system
was used to characterize further the nature of VLP binding to heparin. This technique was chosen since binding has been shown to be directly proportional to the change in reflection units (RU) (33) and because it
eliminates some of the uncertainty associated with column
chromatography, notably nonspecific interactions with the resin matrix.
Heparin was immobilized to a carboxymethylated dextran surface
following the manufacturer's suggested protocol, and this biosensor
chip was used for subsequent studies. For inhibition studies, percent
residual binding in the presence of inhibitor was calculated from RU
values measured during the dissociation phase. The RU measured in the
absence of inhibitor was taken as 100% bound, and inhibition samples
were calculated relative to this value. A typical sensorgram of VLPs in
the absence of inhibitor is presented in Fig.
7A. The initial vertical rise
in RU was due to introduction of sample containing higher
concentrations of salt than the running buffer. Antigen bound well to
the heparin surface at 0.5 M NaCl, characterized by a
further RU increase occurring from 800 to 1020 s. Equilibrium
binding was not achieved at the protein concentrations (4.3 µM) used in this experiment. At 1020 s, the wash
step was initiated, resulting in an initial sharp decrease of RU, a
refractive index artifact caused by the change in NaCl concentration
between sample and wash buffer. A flat dissociation phase was then
observed, indicating that under these conditions VLPs bind with a very
high affinity, and no koff constant could be
measured. At 1180 s, a pulse of 20 mM HCl was used to
strip VLPs and regenerate the surface.
Fig. 7A also shows that VLPs did not bind at 0.7 M NaCl, as indicated by the flat binding phase (130-380 s)
and immediate return of RU to base line as wash buffer is introduced.
The higher absolute value of RU initially attained was reflective of
the higher salt concentration of the sample.
Inhibition by HMW heparin was assessed using three different
experimental conditions (Fig. 7B). In the first trace,
inhibitor was injected immediately following injection of VLPs (340 s). The sensorgram shows a pronounced decrease in RU during the wash phase
(580-990 s) corresponding to 72% inhibition. In the second case
inhibitor was added after VLPs had bound and been washed with running
buffer (1590 s). Here, a slower dissociation was observed, reaching an
equilibrium value of approximately 40% inhibition. When VLPs were
preincubated with inhibitor as in column experiments (2500-3000 s),
inhibition approached 98%. The preincubation method was used for
subsequent studies to generate inhibition curves for heparin and HMW
dextran sulfate in an analogous manner to heparin-Sepharose studies.
The observed IC50 of 4.91 nM for dextran sulfate was in good agreement with that obtained by chromatography; however, HMW heparin gave a much lower value of 75.9 nM.
This discrepancy may be explained in part to differences in the way VLPs are able to interact with the immobilized ligand in a given system. Analysis of bFXa-treated VLPs is shown in Fig. 7C.
Injection of reaction control (VLPs without enzyme) (0-840 s) showed
typical high avidity binding of uncleaved p55 VLPs. Digested VLPs
(840-1450 s) in which >90% of the L1 had been converted to p53
failed to interact with heparin as indicated by the flat association
curve from 900 to 1200 s. Analysis of a reaction blank (enzyme
without VLPs) (1450-2040 s) showed no measurable interaction of enzyme with the heparin surface at the concentration used. The ability of the
15-mer carboxyl-terminal peptide to compete with VLPs was examined.
Even at 1000-fold molar excess of peptide, less than 30% inhibition
was observed, suggesting that a structural feature or the multiply
charged nature of intact VLP contributes to high affinity binding (data
not shown).
Interaction of VLPs with HaCaT Cells--
For initial binding
studies VLPs were added to fixed cells in the standard assay and
allowed to bind for 1-3 h at ambient temperature. Fig.
8A shows that when VLP
titrations were performed using low or high ionic strength buffers,
saturation was achieved at approximately 80-160 ng of L1 protein,
which was similar to previously reported results describing HPV 33 L1
VLP binding to HeLa cells (21). The interaction exhibited some
dependence on ionic strength in the linear region of the curve since
the absorbance values at a given concentration were lower for VLPs
diluted in 0.5 M as opposed to 0.15 M NaCl. At
low VLP concentrations, approximately 65% reduction in binding was
observed when the ionic strength was raised. The effect of bFXa
treatment is shown in Fig. 8B. Denaturing gel
electrophoresis and densitometry of reaction products confirmed that
89% of p55 was converted to p53. Replicates of reaction control and
digest samples were assayed for their ability to bind HaCaT cells. The
A450 nm values for control replicates were
averaged, and this value was taken as 100% bound. Individual replicates were calculated relative to the average. In all cases, binding of treated VLPs was nearly quantitatively reduced with an
average decrease of 93%. As was observed for heparin-Sepharose chromatography and SPR, reduction in binding correlated well with the
percent degradation measured by densitometry of Coomassie-stained gels.
Binding of VLPs to HaCaT Cells Is Inhibited by Sulfated
Polysaccharides--
The ability of HMW heparin and high or low
molecular weight dextran sulfate to inhibit VLP binding to HaCaT cells
was assessed by preincubating a constant concentration of VLPs with
increasing amounts of inhibitor prior to assay. Controls in which (i)
untreated VLPs were added to cells or (ii) high concentrations of
inhibitor were added during the primary antibody incubation step were
run on the same plate. These showed that the polysaccharides did not affect binding of the monoclonal detector antibody to VLPs. Inhibition curves for the three polysaccharides are shown in Fig.
9. All exhibited a
dose-dependent effect similar to that observed with immobilized heparin-Sepharose and SPR. Significant dissociation of
VLP·inhibitor complexes did not occur upon dilution in binding buffer
as judged by good agreement of IC50 values with those
derived from non-cell-based assays. The IC50 for HMW
heparin (293 nM) agreed closely with the value determined
by SPR but was lower than that observed with column chromatography.
Similarly, the 5,000 Effect of Laminin on VLP Binding--
A previous study (23)
proposing the Binding of VLPs to HaCaT Cells Is Reduced by Heparinase and
Chlorate--
In order to probe more directly the role of cell-surface
anionic polysaccharides in VLP binding, cells were treated with a variety of agents designed to reduce or alter glycosaminoglycan content. For all analyses untreated controls were included on every
plate, and binding of VLPs to treated cells was expressed relative to
the amount bound to control cells. In all experiments VLPs were added
at various concentrations, and quantitative data were calculated using
those concentrations that gave the best signal above background.
Treatment of GAGs with heparinases (Fig.
11A) produces
oligosaccharides of varying length containing an unsaturated uronic acid residue. Heparin lyases (heparinase I and II) cleave the polysaccharide chain at GlcNSO3-L-iduronic
(2-O-sulfate) sequences whereas heparitinase (heparinase III) cleaves
less sulfated regions containing L-glucuronic-hexosaminidic
linkages. These enzymes are specific for heparin and heparan sulfate,
respectively (34). Confluent cells were treated with 1 or 3 units/well
of enzyme at 25 or 37 °C for 1 h and subsequently fixed.
Control cells incubated at either temperature showed no difference in
VLP binding, confirming that the digestion buffer had no effect on
cell-surface properties. No significant differences in the data were
observed between cells treated with 1 or 3 units of enzyme; for
clarity, data results were averaged. At 25 °C, all enzymatic
digestions led to a decrease in VLP binding. The heparin lyases
provided 70-85% inhibition, which was slightly more efficient than
the 60-75% decrease in VLPs bound observed using heparitinase. At
elevated temperatures, a more significant difference was noted. The
activity of heparin lyases was slightly enhanced to give 80-90%
inhibition, whereas heparitinase treatment reduced it by only 20-30%.
This result was not due to temperature-induced inhibition of enzyme
since the optimum for heparitinase is 43 °C but may reflect
differences in the type and accessibility of surface proteoglycans.
Treatment of cells with sodium chlorate (Fig. 11B) was used
to produce undersulfated GAG chains. Confluent cells maintained in
standard medium were washed and cultured for 48 h in sulfate-free
medium containing sodium chlorate. At the highest concentrations used
for this experiment cell morphology was somewhat altered, and at
concentrations above 100 mM acute toxicity was observed.
Cells treated with increasing concentrations of reagent stained less
intensely with Alcian blue, indicative of a loss of anionic
cell-surface components (data not shown). The effect of undersulfation
was comparable to removal of GAG chains enzymatically. At 20 and 50 mM sodium chlorate, less than 25% of VLPs bound relative
to controls. Interestingly, cells treated with 100 mM
chlorate bound approximately 50% as many VLPs as controls. At this
concentration of chlorate some morphological changes were noted,
particularly in loss of cells from the surface. If a significant
portion of fixed cells had been lysed, VLP binding might be enhanced
due to nonspecific binding to intracellular components.
Inhibition of GAG Synthesis Reduces VLP Binding--
Substituted
In order to determine if N-linked glycosylation played a
significant role in VLP binding cells were cultured in the presence of
tunicamycin for 20 h (Fig. 12D). No true
dose-dependent effect was seen, although at higher
concentrations approximately 10-20% inhibition was observed at some
VLP concentrations. No changes in cellular morphology or growth rate
were observed.
Binding of VLPs to CHO-K1 and pgsA-745 Cells--
Several
well characterized mutants of CHO cells have been identified in which
GAG production is hampered to various degrees. The cell line
pgsA-745 is deficient in the enzyme
UDP-D-xylose:serine-1,3-D-xylosyltransferase and does not produce glycosaminoglycans (35). Cultures of CHO-K1 and
pgsA-745 cells were grown under standard conditions and used for VLP binding studies (Fig. 13). Both
lines grew at a comparable rate under the conditions employed, although
wild type cells presented a more typically striated morphology and
tended to shed less cells into the medium than did the mutant. Initial
experiments (Fig. 13A) were performed using conditions
developed for HaCaT binding assays, and these indicated that wild type
CHO-K1 cells bound fewer VLPs than did HaCaT cells as evidenced by the
higher concentrations of protein required to obtain signals above
background. For HaCaT cells, saturation was reached at VLP
concentrations of 1600 ng/ml, whereas linear response was still evident
when 12,800 ng/ml VLPs was added to CHO-K1 cells. Below approximately
3200 ng/ml, the specific VLP response was not above background. It was
found that VLP binding to pgsA-745 cells was reduced by
75-80% relative to wild type. Experiments in which primary or
secondary antibody was omitted suggested that the primary VLP-specific
monoclonal antibody exhibited a degree of nonspecific binding to both
cell lines (data not shown). To alleviate this, a low percentage
(0.1%) of Tween 20® was included in the antibody buffers,
and higher VLP concentrations were tested (Fig. 13B). Under
these conditions the linear range of the assay corresponded to
approximately 3,200-12,800 ng/ml VLPs. Within this range VLP binding
was reduced by 75-90% for pgsA-745 cells.
In this report we have identified a heparin-binding region on the
carboxyl-terminal portion of human papillomavirus L1 protein, and we
have shown that this region plays an important role in binding of VLPs
to cell surfaces. An overwhelming majority of "heparin-binding"
proteins share at least one motif in common, that being one or more
regions of clustered basic amino acids which provide the site of
interaction with negatively charged polysaccharides. However, more
stringent efforts designed to identify definitive sequence requirements
have met with only marginal success (36). Sequence comparison of nine
HPV types identified a conserved region in the final 15 amino acid
residues of the L1 protein of the general type
XBBBBXB where B is Lys, Arg, or His. This pattern is similar to the XBBXBX and
XBBBXXBX consensus sequences
identified through molecular modeling of known heparin-binding proteins
(30). Although tight groupings of basic residues are somewhat atypical, the heparin-binding region of extracellular superoxide dismutase (37,
38) contains the sequence XBBBB(X)BBX,
which is similar to that proposed for L1. Consensus sequence searching
can be misleading when investigating interactions between proteins and
anionic polysaccharides. The high affinity interaction of heparin and
antithrombin III has been well characterized, leading to the
identification of a unique polysaccharide sequence required for binding
(39); however, for other proteins such as those of the fibroblast
growth factor family (40), binding to heparin and related
glycosaminoglycans appears to require less stringency. Still, for
most heparin-binding proteins the specificity of interaction generally
surpasses that expected from simple cation exchange chromatography.
HPV 11 L1 VLPs could be eluted from heparin-Sepharose columns using
free heparin or NaCl. The ionic strength required for elution (0.8 M) was similar to that seen for HSV glycoprotein C (24) as
well as non-viral proteins such as lactoferrin (41), thrombin (42), and
the insulin-like growth factor-binding proteins (43). By contrast, high
affinity heparin-binding proteins such as basic fibroblast growth
factor (44) and antithrombin III (45) typically require 1 M
NaCl or greater. Enzymatic removal of the HPV L1 carboxyl terminus
completely abolished binding to immobilized heparin. The high affinity
interaction displayed by HPV L1 is most likely due to the presence of
multiple exposed carboxyl termini on the surface of the particle. This
is suggested by synthetic 15-mer peptide binding studies in which the
free peptide elutes at lower ionic strength and does not effectively compete with intact VLPs for binding sites. An alternative explanation is that a secondary structural motif present in the intact particle may
confer higher affinity on the VLP in a manner analogous to basic
fibroblast growth factor (44). This possibility was explored using a
longer peptide of 28 residues with the assumption that this might more
easily adopt a secondary structure in solution. Although some
enhancement of binding was observed, most likely due to the
contribution of a second short grouping of basic residues (Arg477-Arg482), the degree of interaction was
less than that exhibited by VLPs.
Inhibition of VLP binding by polyanions revealed a dependence on
both mass and charge density of the polymer. The heparin and dextran
sulfate preparations used in these studies contained an average sulfate
content of 2.5 to 2.7 mol/mol disaccharide monomer unit and effectively
competed for binding to immobilized heparin. The strength of inhibition
was dependent on average polymer size as evidenced by the 50-fold
higher IC50 value of LMW heparin. Carlson et al.
(46) showed a similar effect of polymer weight on the activation of a
"very high" affinity isoform of antithrombin III. The molecular
mass effect for VLPs was more definitively shown by analysis of a
series of sized dextran sulfates. The dramatic decrease in
IC50 observed between polymers of
Inhibition studies using chondroitin sulfates illustrate that, in
addition to molecular weight, sulfate content and possibly saccharide
composition are factors in binding. Chondroitin sulfate A and C, both
of which were less effective inhibitors compared with HMW heparin,
contain D-glucuronic acid ( The binding of VLPs to keratinocytes closely coincided with most of the
results observed with immobilized heparin, suggesting an interaction
with cell-surface GAGs. Preincubation of VLPs with heparin or dextran
sulfate resulted in a dose-dependent inhibition of binding
that was dependent on the molecular mass of the polymer preparation.
The IC50 for HMW heparin (293 nM) was lower
than that determined by heparin-Sepharose chromatography (14.9 µM) but comparable to the SPR results (76 nM). This value corresponded to a concentration of 5.1 µg/ml that was very comparable with values of 2.5 and 1.2 µg/ml
measured for heparin binding of PrV gIII (56) and HSV gC (24) viral
glycoproteins, respectively. The elevated value calculated from
chromatographic studies could be the result of secondary nonspecific
interactions between VLPs and the resin matrix, i.e. mixed
modal binding mediated by regions distinct from the carboxyl terminus.
For HMW dextran sulfate, good agreement was noted among all three
methods. Given the relationship between polymer mass and charge with
regard to inhibitory effectiveness, it is not surprising that this
compound was the most effective identified in this study.
Of course, binding of inhibitor might have masked an unrelated site
which was the true receptor target. If so, the bFXa studies indicate
that this region is also localized to the last 28 amino acids since
removal of these residues eliminated binding. Treatment of VLPs with a
combination of reducing and chelating agents had only a marginal effect
on reducing binding. This indicates that either the treatment was
ineffective for type 11 L1 VLPs under the experimental conditions
employed or that capsomers also bind to cells and retain the
conformational epitope recognized by the monoclonal antibody. Based on
similar gel filtration profiles exhibited by treated and control VLPs,
the former explanation is more likely and suggests possible stability
differences among various VLP types.
Specific enzymatic removal of heparin-like GAGs was very effective at
reducing VLP binding as was reduction of sulfate content by chlorate
treatment. Previous studies have shown that heparan sulfate GAGs
account for 60-70% of proteoglycan on HaCaT cells (49). Although
heparan sulfate was not tested specifically as an inhibitor, the
question was addressed through the use of specific heparin lyases.
Interestingly, heparinase was a more effective enzyme than
heparitinase, especially at 37 °C, suggesting that the heparan
sulfate chains of HaCaT cells may contain increased regions of tri- and
tetrasulfated di- and tetrasaccharides and thus more closely resemble
heparin. This has been demonstrated for BALB/c 3T3 fibroblasts (50),
and recent studies have indicated that keratinocytes produce more
highly sulfated glycosaminoglycans in confluent, high density cultures
than under proliferative conditions (51). Similarly, treatment of cells
with The evidence for VLP interaction with cellular GAGs was further
supported using the CHO cell pgsA-745 mutant. This cell line lacks the xylosyltransferase enzyme that mediates the initial step in
glycosaminoglycan synthesis and as a result produces no cell-associated
GAGs (35). VLPs were found to bind to CHO cells with lower affinity
than they bound to HaCaT cells, which is consistent with previous
reports (21). This suggests that CHO cells may carry less of the
specific receptor molecule for HPV. Alternatively, if GAGs are involved
in binding, the lower affinity may be related to the proportion and
composition of heparan sulfate chains on the CHO cell surface. Binding
to pgsA-745 cells was significantly reduced relative to wild
type, demonstrating that cells that do not produce GAGs show a markedly
reduced capacity to bind HPV VLPs.
The search for a putative VLP receptor has focused on a protein
constituent based in part on the observation that trypsinization of
cells eliminates binding. This observation is not inconsistent with a
proposed role for GAGs since the majority of these are present
covalently bound to core proteins and are also removed by mild
trypsinization (50). Although Evander et al. (23) reported
that laminin was effective in preventing binding of type 6b L1 VLPs to
the The current study establishes the ability of recombinant HPV VLPs to
interact with heparin and cellular glycosaminoglycans in
vitro. It does not identify a defined class of GAGs as high specificity receptors for papillomaviruses, but it does demonstrate that these cell-surface molecules could be involved in initial recognition and association of virus in vivo. Furthermore,
this work does not preclude the possibility that an integrin may also be involved in binding and uptake. In fact, the close association of
proteoglycan, integrin, and laminin in basement membranes offers a
feasible scenario for a multiple receptor mechanism for HPV binding.
6 integrin receptors, provided minimal
inhibition. Cells treated with chlorate or substituted
-D-xylosides, resulting in undersulfation or secretion
of GAG chains, also showed a reduced affinity for VLPs. Similarly,
binding of VLPs to a Chinese hamster ovary cell mutant deficient in GAG
synthesis was shown to be only 10% that observed for wild type cells.
This report establishes for the first time that the carboxyl-terminal
portion of HPV L1 interacts with heparin, and that this region appears
to be crucial for interaction with the cell surface.
INTRODUCTION
Top
Abstract
Introduction
References
6
4 integrin as a candidate receptor based
on the ability of HPV 6b VLPs to immunoprecipitate proteins with
molecular weights corresponding to the integrin subunits and the
ability of laminin to block VLP binding.
EXPERIMENTAL PROCEDURES
-toluidine reagent (Pierce).
90% confluency was reached. All subsequent steps were done at ambient temperature. Cells were washed once manually
with phosphate-buffered saline (PBS) and fixed with 4% aqueous
paraformaldehyde for 15-30 min at ambient temperature. All subsequent
washes were done in Tris-buffered saline containing 0.1% Tween 20®
(TTBS) using a Denley WellWash 5000 plate washer. Blocking buffer for
VLP and antibody incubations was PBS containing 1% non-fat milk. VLPs
at the proper dilutions were added for 1-2 h. Bound VLPs were detected
with the conformational-specific monoclonal antibody 8740 (Chemicon,
Tecumla, CA). Mouse IgG was detected with goat anti-mouse IgG
conjugated to horseradish peroxidase (Pierce) utilizing a
3,3',5,5'-tetramethylbenzidine in hydrogen peroxide buffer detection
system (Pierce). Absorbance at 450 nm was read with a Bio-Tek EL312
microplate reader. A modified form of the assay was used for certain
studies. In this protocol, confluent cells were washed with PBS but not
fixed. Antibody and VLP dilutions were made in PBS containing 1%
bovine serum albumin, and all washes were done manually in PBS.
Incubation and wash steps were performed on ice at 4 °C to minimize
VLP internalization.
90% confluence on 96-well plates as described. Growth
medium was removed, and fresh or sulfate-free medium containing
appropriate concentrations of reagent was added. Cells were maintained
for a given time at 37 °C after which they were washed and used for
assay. See individual figure legends for details specific to each reagent.
RESULTS
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Fig. 1.
Characterization of purified VLPs.
A, purified VLPs (20 µg) were analyzed by denaturing PAGE
on 4-20% Tris glycine gels with colloidal Coomassie (lane
1) or immunoblot (lane 2) detection as described under
"Experimental Procedures." B, electron micrograph of a
purified VLP preparation. The sizing bar represents 100 nm.
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Fig. 2.
Alignment of HPV L1 carboxyl-terminal
sequences. Sequence alignment of the carboxyl-terminal regions of
nine HPV types was performed using Intelligenetics® version 8.0 software as described in text. The carboxyl-terminal portions from
residues 447 to 501 (numbering based on HPV type 11) are shown.
Boxed residues indicate the sequence BBXB where B
represents Arg or Lys.
) 17,500 using a series of step
gradients of increasing concentration. Pools collected during elution
were analyzed by denaturing PAGE (Fig.
3A.). Approximately 93% of
bound VLPs could be eluted with free heparin, with 60% eluting at 1.0 mg/ml heparin and an additional 33% being recovered at concentrations
up to 10 mg/ml. The remaining 7% of applied protein was recovered in a
2 M NaCl strip of the resin and most likely represents
aggregated species. VLPs could also be eluted from immobilized heparin
using a linear NaCl gradient in which they eluted at approximately 0.8 M NaCl (Fig. 3B). The broadness and asymmetry of
the chromatographic peak further indicates microheterogeneity with
regard to affinity for the resin. All subsequent studies of inhibition
of VLP binding were performed using NaCl gradient elution.
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Fig. 3.
Heparin-Sepharose chromatography of
VLPs. A, VLPs (150 µg) were injected onto a 5-ml
column of Hi Trap® heparin equilibrated in 50 mM MOPS, pH
7, 0.5 M NaCl. The column was washed with equilibration
buffer and sequentially eluted with 1-ml volumes of free heparin
followed by a 2 M NaCl wash. Peaks collected during elution
were analyzed by denaturing PAGE on 4-20% Tris glycine gels with
Coomassie staining. Lanes 1-4, 1, 2, 5, and 10 mg/ml
heparin; lane 5, 2 M NaCl; lane 6, 2.0 µg of L1 protein. B, VLPs (100 µg) were injected on
a 1-ml column of Hi Trap® heparin equilibrated in 50 mM
MOPS, pH 7, 0.36 M NaCl. The column was washed with
equilibration buffer and eluted with a linear gradient to 2.0 M NaCl.
6,000) gave 50%
inhibition at a concentration of 762 µM. Furthermore,
whereas VLP binding was completely eliminated by HMW heparin at
concentrations above 60 µM, approximately 15-20%
residual binding was observed for the LMW species at concentrations up
to 1.7 mM. Since chemical depolymerization may be
accompanied by some degree of desulfation, enzymatically depolymerized
heparin was used for these experiments. Independent charge analysis and
size exclusion experiments (data not shown) confirmed that the
depolymerized heparin populations retained charge densities similar to
HMW heparin. HMW sulfated dextran
(
500,000) exhibited a
dose-dependent inhibitory effect similar to heparin,
although its IC50 (11.9 nM) was dramatically lower.
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Fig. 4.
Inhibition of VLP binding to
heparin-Sepharose. VLPs were incubated in MOPS-buffered 0.5 M NaCl with increasing concentrations of inhibitor for
16-20 h prior to chromatography on heparin-Sepharose as described in
Fig. 3. Compounds tested for their ability to inhibit binding
were HMW heparin, 17,500 (
);
LMW heparin,
6,000 (
);
N-desulfated heparin,
17,500 (×); and HMW dextran sulfate,
500,000 (
). The
abscissa shows the percent of VLPs which bound the column
relative to control VLPs incubated under same conditions in absence of
inhibitor.
(40,000 and 2,000,000, respectively) had no effect on binding (data not shown).
ranging from 5,000 to 500,000. The molecular weights used were those estimated from low angle laser light scattering by the manufacturer. Independent assessment of these
preparations was made by high performance size exclusion chromatography
(data not shown). Although some polydispersity was evident in all
preparations, the gel filtration peaks were fairly symmetrical and
eluted in the predicted order. Fig.
5A shows that HMW
polyelectrolytes are much better at inhibiting VLP binding to heparin
than LMW ones, but the surprising conclusion was how abrupt the
difference was between dextran sulfate of
8,000 and 10,000. When these
polymers were examined using a narrowed concentration range, the
IC50 values for LMW and HMW polymers were found to be 69 and 0.82 µM, respectively. Inhibition by the glycosaminoglycans chondroitin sulfate A and C (Fig. 5B)
also gave significantly different profiles. Chondroitin sulfate C
(
50,000) exhibited a
dose-dependent inhibitory effect similar to HMW heparin,
although its IC50 (159 µM) was 10-fold
higher. By contrast, chondroitin sulfate A
(
20,000) showed no inhibition at
the highest concentrations tested (500 µM). These results
suggest that molecular weight alone does not determine inhibition
ability, but that charge distribution and possibly the type of glycan
residues present in a given structure play a role. Sulfated
monosaccharides such as glucose 6-sulfate and galactose 6-sulfate were
tested as inhibitors at concentrations up to 27 mM. No
reduction in binding was provided by these low molecular weight
molecules (data not shown).
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Fig. 5.
Molecular weight effects of inhibitors.
VLP and inhibitor samples were prepared as described in Fig. 4 and
analyzed by heparin-Sepharose chromatography. A, the effect
of sized dextran sulfates of
5,000 (
); 8,000 (
); 10,000 (×); 50,000 (
); and
500,000 (
). B, inhibition by the glycosaminoglycans
chondroitin sulfate A,
20,000 (
); and chondroitin sulfate C,
50,000 (
).
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Fig. 6.
Role of L1 carboxyl terminus in VLP binding
to heparin-Sepharose. A, VLP binding to heparin was
inhibited by treatment with bFXa. VLPs (300 µg) were digested with 15 µg of enzyme for 16 h at 20 °C as described under
"Experimental Procedures." After removal of free enzyme, digest and
control samples were diluted to 0.5 M NaCl in MOPS buffer
and chromatographed on heparin-Sepharose. B, binding of a
synthetic carboxyl-terminal peptide. A sample of the 15-mer peptide
(150 µg) was prepared in MOPS-buffered 0.5 M NaCl and
injected on heparin-Sepharose. The peptide was eluted with a linear 0.5 to 2 M NaCl gradient. A co-injection of VLPs (140 µg) and
peptide (300 µg) was performed under the same conditions. The
arrow indicates the start of the salt gradient.
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Fig. 7.
Analysis of VLP binding to heparin by surface
plasmon resonance. A, ionic strength requirement for
binding. VLPs were diluted to 50 µg/ml and various final NaCl
concentrations in 10 mM HEPES, pH 7.4, 0.15 M
NaCl (running buffer), and 20 µl was injected onto a heparin
biosensor chip equilibrated in running buffer. Representative
sensorgrams of VLPs in 0.7 M NaCl (100-600 s) and 0.5 M NaCl (750-1300 s) are shown. B, for heparin
inhibition studies VLPs were diluted to 0.36 M NaCl in
running buffer at 50 µg/ml. A 20-µl aliquot was injected followed
by a 20-µl injection of 10 mg/ml heparin using the instrument
"Co-inject" (0-1000 s) or "Queue" (1100-2000 s) sequence. In
the Co-inject mode, heparin was added immediately following completion
of VLP injection; in Queue mode, the bound VLPs were washed for 2 min
with running buffer before heparin addition. Alternatively, VLPs were
diluted to 0.36 M NaCl in running buffer and incubated with
10 mg/ml heparin at ambient temperature for 1 h prior to injection
(2500-3000 s). Arrows indicate time of heparin addition.
C, analysis of bFXa-treated VLPs was performed by diluting
samples of reaction control (0-750 s), digest (900-1350 s), and blank
(1470-1940 s) to 50 µg/ml and 0.36 M NaCl in running
buffer and performing 20-µl injections.
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Fig. 8.
Interaction of VLPs with keratinocytes.
A, the effect of ionic strength on binding of VLPs to HaCaT
cells was determined by diluting increasing concentrations of VLPs in
0.15 M ( ) or 0.5 M (
) NaCl buffer and
performing the standard binding assay described under "Experimental
Procedures." For all assays, the reported absorbance (450 nm) values
have been corrected for background absorbance of cells in the absence
of VLPs. B, treatment of VLPs with bFXa was performed as in
Fig. 6. Control and digested VLPs were assayed for their ability to
bind HaCaT cells and by denaturing PAGE on 8% Tris glycine gels with
Coomassie staining. The average absorbance for five control replicates
was taken as 100% VLPs bound, and the individual control and digest
absorbances were expressed relative to this value.
dextran
sulfate preparation gave an IC50 of 21.7 µM,
which was comparable with that determined for the 8,000
preparation during dextran sizing
experiments on heparin-Sepharose. The best agreement between all
methods was obtained with HMW dextran sulfate, most likely because the
extended, highly charged polymer offers more flexibility and less
heterogeneity than smaller sized dextran sulfates or heparins.
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Fig. 9.
Inhibition of VLP binding by heparin and
dextran sulfate. VLPs were mixed with increasing concentrations of
HMW heparin, 17,500 (
), LMW
dextran sulfate,
5,000 (
), or
HMW dextran sulfate,
500,00 (×), in 50 mM MOPS, pH 7, 0.5 M NaCl and held at 4 °C for 18 h. Samples were
diluted to 800 ng/ml VLPs and added to confluent cells for binding
assay.
6
4 integrin as a candidate
receptor for papillomavirus showed that HPV 6b VLP binding to HaCaT
cells was completely inhibited by laminin when added at 500 ng/well in
a 96-well assay format. VLP binding in this study was performed on
cells that had been trypsinized, washed, and held in suspension. Since
keratinocytes in vivo are normally polarized and associated
with complex basement membranes, we examined the effect of laminin on
VLP binding to HaCaT cells maintained as adherent monolayers. For these
experiments live cells were used in order to eliminate any artifacts
caused by fixation, and all manipulations were performed on ice to
minimize VLP internalization. Both mouse and human laminin were tested
over a wide concentration range (250-10,000 ng/well) and in different
binding buffers. Initial experiments examined pretreatment of cells,
with the expectation that laminin would bind to the integrin and
prevent binding of VLPs. Fig.
10A shows that VLP binding
to cells treated with 500 ng/well human laminin was 95-99% that of
control cells. Concentrations of laminin 10-20-fold higher showed a
variable 30-50% decrease in VLP binding when added in PBS. When human
laminin was added to cells in growth medium no effect on binding was
seen, most likely due to adsorption of the protein by components of
bovine serum present in the medium. Mouse laminin failed to inhibit VLP binding when added in either PBS or growth medium. It would seem unlikely for the inhibitory effect to be narrowly species-specific, so
the reason for this result is unclear. A second experiment (Fig.
10B) examined whether inhibition of binding was dependent on
the time of laminin addition. A fixed amount of laminin (5000 ng/well)
was preincubated with cells as in the initial study or added at the
time of VLP addition. VLP titrations were performed, and the
concentrations chosen for analysis were those giving the highest signal
above background. Results of preincubation were similar to the initial
experiment. Human laminin co-added with VLPs inhibited binding by 20%
compared with approximately 35% when preincubated. A surprising
30-40% enhancement of binding was observed when mouse laminin was
co-added with VLPs, although the mechanism for this effect is not
known. The similar effectiveness of pretreatment and co-addition
observed with human laminin stands in contrast to inhibition by heparin
as measured by SPR where co-addition was much less effective at
preventing binding.
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Fig. 10.
Effect of laminin on VLP binding.
A, confluent HaCaT cells were washed with PBS and incubated
with increasing concentrations of laminin in PBS or cell medium for
2 h at 0 °C. The cells were washed, and VLPs (80 ng) were added
using the modified assay described under "Experimental Procedures."
VLP and antibody incubations were performed in PBS with 1% non-fat
milk or DMEM with 10% FCS. , human laminin in PBS;
, human
laminin in DMEM;
, mouse laminin in PBS;
, mouse laminin in DMEM.
B, in a second experiment HaCaT cells were either incubated
with 5 µg of laminin for 1 h at 0 °C prior to VLP addition or
VLPs were mixed with 5 µg of laminin and added to cells in the
modified binding assay. The percent VLPs bound were calculated relative
to duplicate controls at each VLP concentration. Data bars
represent averages for 80, 40, and 20 ng of VLP/well.
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Fig. 11.
Alteration of glycosaminoglycans affects VLP
binding. A, confluent HaCaT cells were washed with PBS
and incubated with PBS ( ) or 1 unit/well of heparinase I (
),
heparinase II (
), or heparitinase (
) in PBS at 25 (solid
line) or 37 °C (dashed line) for 1 h. Cells
were washed with PBS, fixed, and incubated with VLPs using standard
binding assay. B, HaCaT cells grown to confluence in
serum-containing DMEM were transferred to sulfate-free medium
containing 0 (
), 20 (
), 50 (×), or 100 (
)
mM sodium chlorate. Cells were maintained for 48 h
before they were washed, fixed, and used for binding assays. Each data
point represents the average of duplicate determinations.
-D-xylosides serve as artificial acceptors of nascent
GAG chains, resulting in a large increase in the percentage of
glycosaminoglycans which are secreted into the medium. Although not
covalently linked to core proteins, the highly charged structure of the
GAGs can cause them to remain non-covalently associated with the cell
through ionic interactions. Commercially available
-nitrophenyl
(
NP) and 4-methylumbelliferyl (4MU) xylosides were employed for this
study (Fig. 12, A-C).
Reagents were added to cells in sulfate-free medium in order to
directly compare results with those observed using chlorate treatment.
Medium was replenished every 2-4 days through the course of the
experiment to avoid build-up of potentially toxic hydrolysis products
of the xylosides. VLP binding was most effectively reduced by treatment
with
NP-Xyl (Fig. 12A) which showed a typical
dose-dependent effect at all concentrations tested. This
effect was enhanced when cells were washed with PBS containing 0.6 M NaCl instead of PBS alone (data not shown), and the high
salt wash was used for all subsequent assays. It is reasonable to
assume that high ionic strength can remove non-covalently associated
GAGs which would be expected to retain their ability to bind VLPs. The
4MU-Xyl analog provided a somewhat lower degree of inhibition in the
concentration range of 0.2 to 1 mM. When higher
concentrations of each reagent were tested an effect similar to that
which occurred at high chlorate concentrations was observed (Fig.
12B) for 4MU-Xyl. VLPs bound approximately 40% better to
treated cells than to controls. This effect was not noted for
NP-Xyl, which provided greater than 70% inhibition at elevated
concentrations. No adverse morphological changes were evident in cells
treated with either reagent, suggesting that the 4MU-Xyl effect may be
specific to the form of
-xyloside used and not to cellular damage.
The effect of xylosides on actively growing cultures as opposed to
confluent, contact-inhibited cells was examined in a second experiment
(Fig. 12C). The xyloside concentration selected was one
which provided significant inhibition in initial studies. Reagents were
added to cells that were 30-40% confluent, 16 h after seeding.
Total growth time in the presence of xyloside was 6 days, after which
the cells were washed with high salt, fixed, and assessed for VLP
binding. Treated cells were found to bind approximately 80% as many
VLPs as untreated controls, and no difference was noted between
NP
or 4MU substrates. The degree of inhibition provided by xylosides under
these conditions was approximately 50% less than when confluent cells
were treated. Light microscopy indicated that cells reached confluency
much slower in medium containing xylosides suggesting that intact
proteoglycan structure may be a requirement for proper spreading and
growth of keratinocytes.
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Fig. 12.
Inhibition of glycosaminoglycan
synthesis. The effect of substituted -D-xylosides
(A-C) or tunicamycin (D) on VLP binding was
investigated. A, HaCaT cells grown to confluence in
serum-containing DMEM were transferred to sulfate-free medium
containing no inhibitor (×),
NP-Xyl (0.27 mM
(
), 0.80 mM (
)), or 4MU-Xyl (0.27 mM
(
), 0.80 mM (
)). Cells were grown for 4 days in
inhibitor after which they were washed with 0.6 M NaCl in
PBS, fixed, and used for standard binding assay. B, cells
were treated with no inhibitor (×),
NP-Xyl (3.32 mM (
), 6.65 mM (
)), or 4MU-Xyl (3.32 mM (
), 6.65 mM (
)) and processed as in
A. C, HaCaT cells were grown to 40-50%
confluence in normal medium at which point they were switched to
sulfate-free medium containing no inhibitor (×),
NP-Xyl
(0.2 mM (
), 1.0 mM (
)), or 4MU-Xyl (0.2 mM (
), 1.0 mM (
)). Cells were grown for 6 days in inhibitor and processed as in A. D,
confluent HaCaT cells were washed twice with normal growth medium, and
fresh medium containing 0 (
), 1.0 (
), 3.0 (
), or 5.0 (
)
µg/ml tunicamycin was added. Cells were grown for 20 h in
inhibitor, washed with PBS, fixed, and used for standard binding
assay.
View larger version (12K):
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Fig. 13.
Binding of VLPs to CHO cells and a
GAG-deficient CHO mutant. Wild type CHO-K1 ( ) and
xylosyltransferase-deficient pgsA-745 cells (
) were grown
to confluence on 96-well plates. Cells were washed, fixed, and used for
standard binding assays. VLP titrations were performed in PBS
containing 1% milk V (A) or TBS containing 1% milk and
0.1% Tween 20® (B).
DISCUSSION
8,000 and 10,000 indicates that a
critical size requirement was attained in this range. It is generally
accepted that highly charged hydrophilic polymers exist in solution as
extended, flexible rod-like structures, and it is reasonable to assume
that these would tend to bind particles carrying multiple positively
charged protein chains in a cooperative manner. Interestingly, Abbott
et al. (47) showed that a critical dependence of the
partitioning coefficient existed for the two-phase aqueous
poly(ethylene oxide)-dextran system at poly(ethylene oxide)
below 10,000. Models were
proposed for different degrees of interaction between protein and
polymer chains, and it was shown that when the attractive forces were
strong (i.e. ionic binding) the polymer tended to form an
encapsulated "shell" surrounding the globular protein. In the case
of very large, rigidly spherical particles such as a VLP, higher
molecular weight chains would be able to effectively cover more surface
area at lower concentrations. Such strong, multiple site attachments
can be viewed in a sense as being irreversible and offer a likely
explanation why dissociation constants could not be measured under the
binding conditions used for SPR measurements. This mechanism also can
account for the SPR results showing different degrees of inhibition
exhibited by HMW heparin depending on its time of addition.
Preincubation of VLPs and free ligand would result in a uniformly
coated particle carrying a negative surface charge and unable to bind
to immobilized heparin. Alternatively, free VLPs bound to the biosensor
chip strongly with no measurable dissociation and could not be
quantitatively displaced using the same IC50 concentration
of heparin calculated from preincubation experiments.
1-3)
N-acetylgalactosamine (
1-4) core disaccharides instead
of the L-iduronic acid (
1-4) N-acetylglucosamine (
1-4) unit found in heparin. The
presence of iduronic acid in heparin is postulated to offer a greater
degree of flexibility to the GAG chain, allowing more possible
conformations for binding to proteins (36). Chondroitin sulfates are
monosulfated on either C4 or C6 of the GalNAc
residue and thus possess a lower sulfate to carboxyl ratio than
heparin. Fromm et al. (48) showed that synthetic peptides of
the form BB(X)nBB and BBB(X)nB
bound more effectively to heparin when n = 0 or 1 and
more effectively to heparan sulfate when n = 1-3.
Furthermore, chemically desulfated heparin and non-sulfated dextrans
were completely unable to inhibit VLP binding, even when very high
molecular weight preparations were used. These data demonstrate that
VLP binding to heparin was electrostatic in nature but not of
sequence-defined high specificity, which explains why dextran sulfates
of equal or higher
than heparin
were more effective inhibitors.
NP-Xyl reduced VLP binding at all concentrations tested,
whereas the 4MU-Xyl analog caused a 40% enhancement in binding at high
concentrations. The efficiency of GAG priming by substituted xylosides
has been shown to depend on the nature of the aglycone used (52); this
is further defined by the cell type chosen for analysis. In most cases
chondroitin sulfate is primarily transferred to the xyloside, but at
high enough concentrations heparan sulfate synthesis is also affected. Because GAG chains can be associated with membranes in the absence of
protein cores and because this interaction does not appear to be ionic
(50), some cell-surface GAG may still be present under the conditions
used for this assay. Furthermore, the degree of sulfation can be
increased in the presence of xylosides resulting in chains containing
higher contents of trisulfated disaccharides (53). It can be postulated
that 4MU-Xyl is more efficient at producing structures of these types
in keratinocytes. Both of these studies suggest that heparan sulfate
may serve as a less effective inhibitor than heparin.
6 integrin, this result was not observed in the
current study except at very high laminin concentrations. This
discrepancy may be in part due to differences in assay methods. In the
former, cell monolayers were disrupted with trypsin and suspended prior
to binding assays, whereas in this report all assays were performed on
intact cell monolayers. In vivo, the
6
integrin is localized to hemidesmosomes on HaCaT cells and since
cultured keratinocytes produce rudimentary, poorly formed structures of
this type (54), the amount of integrin available for binding may be
reduced. Inhibition by laminin may also reflect the fact that the long
arm of the laminin A chain can bind to heparan sulfate (55). Finally,
VLP binding to the CHO cell mutant was reduced by the same amount as
that observed for heparinase and chlorate treatment of HaCaT cells. To
date, no evidence has suggested that the levels of
6
integrin are different between wild type CHO and pgsA-745 cells.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Hugh George and Wayne Herber for providing yeast fermentations, Robert Hepler and Dr. Charlotte Ip for assay support, Dr. Robert Lowe for assistance in setting up cell binding assays, and Dr. Norbert Fusenig for providing HaCaT cells. We also extend thanks to Drs. Linda Lowe-Krentz and Michael Behe for helpful discussion and critical reviewing of the manuscript.
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FOOTNOTES |
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* 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: Dept. of Virus and Cell Biology, Merck Research Laboratories, Sumneytown Pike, West Point, PA 19486. Tel.: 215-652-5617; Fax: 215-652-2142.
2 J. C. Cook, manuscript in preparation.
3 C. T. Przysiecki, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are:
HPV, human
papillomavirus;
VLP, virus-like particle;
HSV, herpes simplex virus;
MOPS, 3-(N-morpholino)propanesulfonic acid;
SPR, surface
plasmon resonance;
GAG(s), glycosaminoglycan(s);
bFXa, bovine Factor
Xa;
RU, reflection unit;
NP-Xyl,
-nitrophenyl-
-D-xyloside;
4MU-Xyl, 4-methylumbelliferyl-
-D-xyloside;
HMW, high molecular
weight;
LMW, low molecular weight;
CHO, Chinese hamster ovary;
RU, reflection units;
PBS, phosphate-buffered saline;
DMEM, Dulbecco's
modified Eagle's medium;
FCS, fetal calf serum.
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REFERENCES |
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