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* Department of Biological Sciences and
Department of Microbiology, National University of Singapore, Singapore 117543
1Correspondence: Department of Biological Sciences, National University of Singapore, 10, Kent Ridge Crescent, Singapore 117543. E-mail : dbsdjl{at}nus.edu.sg
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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and hIL-8 by THP-1 and human peripheral blood
mononuclear cells with greater potency than polymyxin B. SSCrFCES is
noncytotoxic, with a clearance rate of 4.7 ml/min. The
L.D.90 of SSCrFCES for LPS lethality is achieved at 2 µM.
These results demonstrate the endotoxin-neutralizing capability of
SSCrFCES in vitro and in vivo and its
potential use for the treatment of endotoxin-induced septic
shock.—Tan, N. S., Ho, B., Ding, J. L. High-affinity LPS binding
domain(s) in recombinant factor C of a horseshoe crab neutralizes
LPS-induced lethality.
Key Words: Carcinoscorpius rotundicauda • novel secretory signal • endotoxin binding and neutralization
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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. LPS is a
mixture of glycolipids consisting of a variable polysaccharide domain
covalently bound to a conserved glucosamine-based phospholipid known as
lipid A. LPS directly stimulates host monocytes and macrophages to
secrete a wide array of inflammatory cytokines that includes tumor
necrosis factor (TNF-), interleukins-1 (IL-1), and interleukin-8
(IL-8) (2)
. Excessive release of these cytokines by host
macrophages almost assuredly contributes to organ failure and death
that occur after episodes of gram-negative bacterial sepsis
(3)
. The proinflammatory bioactivities exhibited by LPS
are known to reside in the lipid A (4)
.
LPS from gram-negative bacteria induce the amoebocytes of horseshoe
crabs to aggregate and degranulate. Presumably, LPS-induced coagulation
cascade represents an important defense mechanism used by horseshoe
crabs against invasion of gram-negative bacteria (5)
. The
amoebocyte lysate has been used for decades as a tool to detect trace
concentrations of LPS in solution: Limulus amoebocyte lysate
(LAL) test (6
, 7)
. The molecular mechanism of coagulation
in horseshoe crab has been established and involves a protease cascade.
This cascade is based on three kinds of serine protease
zymogens—factor C, factor B, proclotting enzyme—and one clottable
protein, coagulogen (8)
. Being the initial activator of
the clotting cascade, factor C functions as a biosensor that responds
to LPS. Since factor C can be activated by femtograms of LPS, it is
conceivable that it has an LPS binding region that exhibits exceptional
high affinity for LPS. Consequently, this LPS binding domain can be
used to detect and remove pyrogenic contaminants in pharmaceutical
products intended for parenteral administration as well as for in
vivo immunohistochemical determination of endotoxin localization
(9)
.
Our laboratory has cloned the homologous factor C zymogen cDNAs from
one of the four extant species of horseshoe crab, Carcinoscorpius
rotundicauda (CrFC) (10)
. Initial attempts to express
CrFC and its truncated forms in Escherichia coli resulted in
a nonactive enzyme (11)
. Subsequently, CrFC was cloned and
expressed in methylotropic yeast, Pichia pastoris. However,
neither the native nor the Saccharomyces cerevisiae
mating factor signal sequences were capable of directing secretion of
this mosaic protein into the culture media for easier purification
(12)
. Furthermore, the full-length CrFC was enzymatically
nonactive although it retains weak endotoxin binding properties
(13)
. Here, for the first time, we report the expression
and secretion of a functional LPS binding domain of C.
rotundicauda factor C (SSCrFCES) via a novel secretory signal. The
secreted protein was purified to homogeneity and subjected to
amino-terminal sequencing. The biological functions of the recombinant
SSCrFCES were assessed by measuring the ability of the SSCrFCES to bind
lipid A using an ELISA-based lipid A binding assay as well as surface
plasmon resonance interaction. We measured the SSCrFCES-mediated
inhibition of endotoxin-induced Limulus amoebocyte lysate
coagulation with a sensitive LAL kinetic-QCL assay. The SSCrFCES
protein was also tested for its ability to suppress LPS-induced
cytokines (TNF- and IL-8) production by THP-1 and normal human
peripheral blood mononuclear cells (hPBMC). SSCrFCES is noncytotoxic
and has a clearance rate of 4.7 ml/min. Finally, preliminary analysis
shows that a low dose of SSCrFCES protein protects
galactosamine-sensitized mice from LPS-induced lethality.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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and IL-8 were purchased from PharMingen (San Diego, Calif.).
Cell Titer 96 AQueous for cytotoxic assay was from Promega (Madison,
Wis.). EZ-Link PEO-maleimide activated biotein was purchased from
Pierce (Rockford, Ill.). Oligonucleotides were synthesized by Genosys
Biotechnologies, Inc. (The Woodlands, Tex.). Enzymes for DNA
manipulation and polymerase reactions were purchased from NEB
(Beverly, Mass.) and Boehringer Mannheim (Mannheim, Germany). DNA
purification kits used were from Qiagen (Chatsworth, Calif.).
Pyrogen-free water for making buffers was from Baxter (Morton Grove,
Ill.).
Construction of a secreted LPS binding domain of C.
rotundicauda factor C gene (SSCrFCES)
A novel secretory signal, SS (patent filed), was isolated via
polymerase chain reaction (PCR) performed in a 100 µl reaction using
100 ng of template DNA, 10 µl of 10 x Thermol buffer, 200 µM
each of dNTPs, 1 U of VentR (exo-) polymerase,
and 200 µM of T3 forward primer (5'-ATTAACCCTCACTAAAGGGA-3') and
reverse primer (patent filed). The PCR cycles consisted of an initial
denaturation at 94°C for 3 min, followed by 29 cycles of 94°C for
45 s, 52°C for 30 s, and 72°C for 30 s, with a final
extension step at 72°C for 3 min. The PCR product was purified using
Qiaquick PCR Purification Kit and subsequently digested with
SacI. This 131 bp SS fragment was subcloned into pEGFP-N1
vector (Clontech; Palo Alto, Calif.) at the SacI and
SmaI sites. To reduce the length of 5' UTR, the SS was
digested with EcoRI and BglII, blunted with
Klenow enzyme, and religated. The sequences at the junction were
determined by Taqtrack Sequencing System (Promega) using EGFP-reverse
primer (5'-CCCTCGCCGGACACGCTGA-3'). The modified vector containing the
SS was designated pSSEGFP. The full-length CrFC was digested with
BglII and NotI and subcloned into the
BamHI and NotI of pSSEGFP. The LPS binding region
of CrFC was released by Eco 47III and SalI and
inserted into pAc5/V5-His Drosophila expression vector
backbone. The vector coding for a secreted LPS binding domain of CrFC
was designated pAc5/SSCrFCES-V5-His.
Stable expression of SSCrFCES in Drosophila S2
cells
To express SSCrFCES protein, the expression vector
pAc5/SSCrFCES-V5-His and selection vector, pCoHygro, were purified via
EndoFree plasmid kits. The vectors were introduced into
Drosophila S2 cells (14)
by calcium phosphate
coprecipitation method (15)
. Drosophila S2
cells were routinely maintained in DES expression medium supplemented
with 10% FBS. The cells were incubated at 25°C in a humidified
incubator. For transfection, 3–6 x 106
cells were introduced with 20 µg of plasmid DNAs at a ratio of 19
SSCrFCES:1 pCoHygro. After transfection, cells were maintained in
calcium phosphate solution for 24 h. Subsequently, cells were
washed twice with complete medium to remove the calcium phosphate
solution and allowed to recover for 2 days. Selection for stable cell
lines was performed by the addition of 350 µg/ml of insect
cell-tested hygromycin (Life Technologies, Inc.) over a period of 3 wk.
Stable cell lines expressing SSCrFCES were adapted to DES serum-free
medium by weaning over three or four passages. To test for SSCrFCES
expression, 30 µl of medium was electrophoresed on a 12% sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
proteins were electrophoretically transferred to PVDF membrane
(Millipore; Bedford, Mass.). The recombinant SSCrFCES was detected
using INDIA-HisProbe-HRP (Pierce) as described by the manufacturer.
Purification and amino-terminal amino acid sequencing of SSCrFCES
purified by affinity chromatography
A cell line expressing a high level of SSCrFCES was grown
without serum to generate conditioned medium containing SSCrFCES. The
recombinant protein used for amino-terminal sequencing was purified via
TALON metal affinity chromatography (Clontech) under denaturing
condition. The TALON resin was thoroughly resuspended and 2 ml of
homogeneous 50% suspension was packed by gravity in 10 ml disposable
column. The resin was preequilibrated with binding buffer (20 mM
NaH2PO4, 250 mM NaCl, and 4
M urea, pH 8.0). Typically, 300 ml of conditioned medium was desalted
and concentrated sevenfold via ultrafiltration through a 10 kDa
membrane. The concentrated medium was then adjusted to the same ionic
strength as the binding buffer. The medium was applied to the packed
resin. The filtrate was collected and reapplied to the column. The
resin–protein complex was then washed with 20x resin volume with wash
buffer (20 mM NaH2PO4, 250
mM NaCl, and 8 M urea, pH 8.0). The SSCrFCES was eluted by adding three
resin volumes of elution buffer (20 mM
NaH2PO4, 250 mM NaCl, and 4
M urea, pH 5.0). The eluate was collected and concentrated using
Microcon 10 (Millipore).
A 10% SDS-PAGE was cast and allowed to age overnight at 4°C. Prior to loading sample, the gel was pre-run in Tris-glycine-SDS buffer for 15 min at constant 50 mA. Two micrograms of Cobalt-column purified SSCrFCES was loaded and electrophoresed. PVDF membrane (Millipore) was prewetted by brief immersion in 100% methanol, rinsed with Milli-Q water, and equilibrated in transfer buffer (10 nM CAPS, 10% methanol, pH 11.0) for a minimum of 15 min. After electrophoresis, the gel was soaked in transfer buffer for 5 min. The gel and treated PVDF membrane were sandwiched, and electroblotting was carried out at constant 50 V for exactly 50 min. After the transfer was completed, the membrane was rinsed several times in Milli-Q water. The SSCrFCES protein was visualized by staining in Coomassie blue R-250 for 5 min and destained with several changes of destaining solution (50% methanol, 10% acetic acid). The membrane was rinsed again with several changes of water, air-dried, and the SSCrFCES band was excised for amino-terminal amino acid sequencing. Protein sequencing was performed on ABI 477 sequencer.
Purification of SSCrFCES by preparative isoelectric membrane
electrophoresis
Typically, two liters of conditioned medium were initially
subjected to successive ultrafiltration using a 100 kDa and 10 kDa
molecular mass cutoff with the Pellicon system (Millipore). The medium
was concentrated sevenfold. The enriched SSCrFCES was purified to
isoelectric homogeneity using preparative isoelectric membrane
electrophoresis (Hoefer IsoPrime, Pharmacia). A set of four membranes
with pHs of 6.5, 7.0, 7.25, and 7.5 was made. The concentration of
acrylamido buffers used for the membranes was calculated based on
information in Righetti and Giaffreda (16)
. The four
membranes were assembled in order, from acidic to basic, to delimit
five chambers. Each sample reservoir vessel was filled with 30 ml of
pyrogen-free water and pre-run at 4°C, 4 W constant power (3000 V
limiting, 20 mA maximum) for 2 h. After removing the pre-run
water, the protein sample was placed in sample reservoir vessel
corresponding to the chamber delimited by pH 7.0 and 7.25. The IsoPrime
was conducted under the same conditions for at least 48 h and the
content from each chamber was analyzed on a 12% SDS-PAGE. Purified
SSCrFCES was used for production of anti-SSCrFCES antibody in rabbits.
ELISA-based lipid A binding assay
A Polysorp 96-well plate (Nunc) was first coated with 100 µl
per well of various concentrations of lipid A diluted in pyrogen-free
phosphate-buffered saline (PBS). The plate was sealed and allowed to
incubate overnight at room temperature. The wells were aspirated and
washed six times with 200 µl wash solution (PBS containing 0.01%
Tween-20 and 0.01% thimerosal). Blocking of unoccupied sites was
achieved using a wash solution containing 0.2% BSA for 1 h at
room temperature. Subsequently, blocking solution was removed and the
wells were washed as described above. Varying concentrations of
SSCrFCES were allowed to interact with bound lipid A at room
temperature for 2 h. Bound SSCrFCES was detected by sequential
incubation with rabbit anti-SSCrFCES antibody (1:1000 dilution) and
goat anti-rabbit antibody conjugated with horseradish peroxidase (HRP)
(1:2000 dilution) (Dako; Carpentia, Calif.). Each antibody incubation
was for 1 h at 37°C with washing between incubations as
described above. In the final step, 100 µl of peroxide substrate ABTS
(Boehringer Mannheim) was added. Using a microtiter plate reader, the
absorbance of the samples was determined at 405 nm with reference
wavelength at 490 nm. The values were correlated to the amount of LPS
bound and SSCrFCES present. Quantitation of SSCrFCES was achieved from
a standard curve derived by immobilizing known amount of purified
SSCrFCES onto a Maxisorp plate. The detection was as described above.
Immobilization of lipid A and biospecific interaction with
SSCrFCES
Recognition of lipid A by secreted SSCrFCES was determined by
surface plasmon resonance (SPR), with BIAcore X biosensor instrument
using HPA (hydrophobic adsorption) sensor chip. Briefly, lipid A at 0.5
mg/ml in PBS was immobilized to a HPA sensor chip (Pharmacia) according
to the manufacturer’s specification. In all experiments, pyrogen-free
PBS was used as the running buffer at a flow rate of 10 µl/min.
Purified SSCrFCES at 4 µg/ml was injected into the flow cell at a
rate of 10 µl/min, and the binding response was measured as a
function of time. After injection of SSCrFCES, a solution of INDIA
HisProbe-HRP, diluted in PBS to 400 µg/ml, was also injected to cause
a shift in response unit in order to further confirm that SSCrFCES
binds to lipid A. For regeneration, the bound SSCrFCES was removed by
injection of 100 mM of NaOH solution for 5 min.
Limulus kinetic-QCL for determining the
ENC50 of SSCrFCES
The Limulus kinetic-QCL is a quantitative, kinetic
assay for the detection of gram-negative bacterial endotoxin. This
assay uses the initial part of LAL endotoxin reaction to activate an
enzyme, which in turn releases p-nitroaniline from a synthetic
substrate, producing a yellow color. The time required before the
appearance of a yellow color is inversely proportional to the amount of
endotoxin present. Throughout the assay, the absorbance at 405 nm of
each well of the microplate was monitored. Using the initial absorbance
reading of each well as its own blank, the time required for the
absorbance to increase 0.200 absorbance units was calculated as
reaction time. The 50% endotoxin-neutralizing concentration
(ENC50) reflects the potency of SSCrFCES; a low
ENC50 indicates high potency.
Briefly, 25 µl of endotoxin solution at 200 EU/ml was mixed with an equal volume of SSCrFCES at 1 µM in a series of twofold dilutions in LAL reagent water in disposable endotoxin-free glass dilution tubes (BioWhittaker) and incubated at 37°C for 1 h. The reaction mixtures were each diluted 1000-fold with LAL reagent water. The endotoxin activity was then quantified with Limulus kinetic-QCL. One hundred microliters of the diluted test mixture was carefully dispensed into the appropriate wells of an endotoxin-free microtiter plate (Costar; Cambridge, Mass.). The plate was then preincubated for >10 min in a temperature-controlled ELISA plate reader. Near the end of the preincubation period, 100 µl of freshly reconstituted kinetic-QCL reagent was dispensed into the wells using an 8-channel multipipettor. The absorbance at 405 nm of each well of the microtiter plate was monitored at time intervals of 5 min over a period of 2 h. A 5 s automix was activated prior to reading. In the Limulus kinetic-QCL, the assay was activated by 0.005 EU/ml of endotoxin. The high sensitivity of the assay allowed for very low levels of endotoxin to be detected. After incubation of endotoxin with SSCrFCES, a 1000-fold dilution was introduced to eliminate any potential effects of the SSCrFCES on the LAL enzyme system. A sigmoid curve is usually expected between relative reaction time and the logarithmic concentration of the SSCrFCES. The best fit curve was derived using SigmaPlot and the concentration corresponding to 50% relative increase in reaction time was designated ENC50. The mean values were obtained from three independent experiments.
LPS stimulation of THP-1 and hPBMC
All cell lines were grown at 37°C in a humidified environment
in the presence of 5% CO2. THP-1 cells were
grown in RPMI 1640 medium supplemented with 10% FBS, penicillin (100
U/ml), and streptomycin (0.1 mg/ml). The cells were maintained at a
density between 2.5 x 105 and 2.5 x
106 cells/ml. THP-1 cells were prepared for
experiment by addition of a concentrated stock solution of PMA (0.3
mg/ml in dimethyl sulfoxide) to cell suspension to give a final
concentration of 30 ng/ml PMA and 0.01% dimethyl sulfoxide
(17)
. PMA-treated cell suspensions were immediately plated
into 96-well microtiter plate at a density of 4 x
105 cells/ml and allowed to differentiate for
48 h at 37°C. Immediately before stimulation by 25 ng/ml LPS or
LPS preincubated with various concentrations of SSCrFCES, the culture
medium was removed and the cells were washed twice with serum-free RPMI
1640, then incubated at 37°C. At indicated times, the culture medium
was collected. Human TNF- and IL-8 concentrations in the
supernatants were assayed using ELISA as suggested by the manufacturer.
Heparinized venous blood drawn from healthy donors was subjected to
fractionation using Ficoll-Paque PLUS (Pharmacia) to obtain peripheral
blood mononuclear cells. PBMC were washed with PBS and suspended at a
cell density of 1.5 x 106 cell/ml with RPMI
1640 medium supplemented with 10% FBS. PBMC were incubated at 37°C
for 24 h at a density of 1.5 x 105 per
well. LPS stimulation and immunoassay of hTNF- and hIL-8 were
performed as described for THP-1 cells. In addition, the suppressive
effect of SSCrFCES on LPS-induced cytokine release was investigated in
the presence of 10% human serum. The difference between the test and
control groups was subjected to Student’s t test. The
values were obtained from at least three independent experiments.
Cytotoxicity of SSCrFCES in eukaryotic cells
2 x 104 THP-1 monocytes in 50 µl
of RPMI 1640 were mixed in a microtiter plate with 50 µl of an
increasing amount of twofold serial dilutions of SSCrFCES (0.004–4.0
mg/ml in PBS) and incubated for 60 min at 37°C. To determine
cytotoxicity induced by the SSCrFCES, 20 µl of CellTiter96
AQueous One Solution Reagent (Promega) was added
into each well for 90 min at 37°C.
[3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium
(MTS) is bioreduced by metabolically active cells into a colored
formazan product that is soluble in tissue culture medium (18
, 19)
. For detection, the absorbance was measured at 490 nm. To
determine the ratio of cell lysis induced by SSCrFCES, two controls
were used. Complete lysis (100%) was achieved by incubating cells in
PBS containing 0.2% Tween-20 instead of medium only. This absorbance
value corresponded to the background, as those cells could not
metabolize MTS. The second control representing 0% lysis was
determined by incubating cells in medium only. The
LD50 was calculated as the concentration of
SSCrFCES necessary to lyse 50% of the cells. The experiment was done
in triplicate.
Biotin labeling SSCrFCES and pharmacokinetic analysis
600 micrograms of SSCrFCES was labeled with biotin using EZ-Link
PEO-maleimide activated biotin (Pierce) according to the
manufacturer’s instructions. The excess biotin was subsequently
removed via ultrafiltration through Micron-10 (Milipore). Three
C57BL/6J mice were given a single intravenous (i.v.) bolus injection of
200 µg biotin-labeled SSCrFCES. Blood was collected in heparinized
capillary tubes from each of the three mice over a 5 h period. The
plasma was immediately treated with SDS-PAGE loading dye and boiled for
5 min. The mixture was resolved in a 12% SDS-PAGE and electroblotted
onto a PVDF membrane. Immunoblotting and hybridization were carried out
as described above except NeutrAvidin-HRP antibody (Pierce) was used.
Exposure time for chemiluminescence detection was extended to 1 h.
The signal on the X-ray film was quantitated via densitometric scan.
The clearance rate of biotin-labeled SSCrFCES was analyzed using NCOMP,
a Windows-based program for noncompartmental analysis of
pharmacokinetic data (20)
.
LPS-induced lethality in galactosamine-sensitized mice
Mice are typically resistant to endotoxin. However, the
sensitivity of mice to endotoxin can be enhanced > 1000-fold by
coinjection with a liver-specific inhibitor, galactosamine
(21)
. In our study, intraperitoneal (i.p.) injection of
2.5 ng of E. coli 055:B5 LPS together with 15 mg of
galactosamine hydrochloride in 0.2 ml of saline induced nearly 100%
lethality in 18–25 g C57BL/6J mice within 7 h. Various
concentrations of SSCrFCES (1, 2, and 4 µM) were injected i.v.
through tail vein 10 min after i.p. injection of the LPS-galactosamine
mixture. Lethality was observed for 3 days after injection. Statistical
analysis were performed using the Kaplan-Meier test (22)
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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. The Western blot revealed the presence of a protein with
an apparent molecular mass of ~38 kDa (Fig. 1B
). SSCrFCES
in medium represented > 90% of the total recombinant protein
expression. When stable cell line was cultured in serum-free medium
without hygromycin for a week in a 1L-Bellco spinner flask, a typical
yield ~1.6 mg/l of SSCrFCES was achieved.
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The secretion of CrFCES is directed by a novel 15 residue hydrophobic secretory signal. The precise cleavage point at the SS adjoining the mature fusion CrFCES protein was determined by NH2-terminal amino acid sequencing of TALON column-purified SSCrFCES, which indicated that SS was efficiently and accurately cleaved at a single site in recombinant Drosophila cells. Since sequence analysis of secreted CrFCES provided no evidence for frayed termini, we conclude that SS is processed properly in the insect cell system. The absence of multiple cleavage points suggests that SS allows for homogeneous production of secreted heterologous protein. The presence of SSCrFCES in the culture medium thus contributes to the ease of batch-continuous culture and purification. Most significantly, SSCrFCES expressed and secreted from insect cells was biologically active.
Purification of SSCrFCES using TALON column
Stable recombinant Drosophila cells secreting SSCrFCES
was grown in serum-free medium for easy purification. The medium was
initially concentrated and desalted via three rounds of ultrafiltration
using a 10 kDa cutoff membrane in an Amicon stirred cell (Millipore).
Affinity chromatography purification under denaturing conditions
yielded a ~38 kDa protein of interest in addition to a ~67 kDa
contaminating protein. Thus, this larger protein is likely due to
nonspecific adsorption to the resin. Several attempts to increase the
purity of SSCrFCES by increasing the NaCl concentration from 250 mM to
300–400 mM resulted in a drastic reduction in SSCrFCES yield. A
compromise was arrived between yield and purity. The SSCrFCES protein
represented ~85% of the total protein recovered. Consequently, prior
to amino-terminal amino acid sequencing, the two proteins were resolved
in a 10% SDS-PAGE. The ~38 kDa SSCrFCES protein band of interest was
excised for amino-terminal amino acid sequencing after electrotransfer
to a PVDF membrane.
Purification of SSCrFCES by preparative isoelectric membrane
electrophoresis
An initial attempt using 10 kDa concentrated medium for ISOPrime
resulted in the coprecipitation of SSCrFCES with minor high molecular
weight proteins. Thus, an additional 100 kDa cutoff ultrafiltration was
introduced that eliminated the coprecipitation problem. The 100 kDa and
10 kDa ultrafiltrated medium containing the protein was subsequently
purified to homogeneity using PI 8 IsoPrime Multi-Chambered Isoelectric
Focusing Unit. The pI of the SSCrFCES was determined to be 7.1 at
4°C. A Coomassie-stained SDS-PAGE of secreted SSCrFCES is shown in
Fig. 1A
. The relative ease with which SSCrFCES is purified
to apparent homogeneity from the culture medium illustrates the
advantage of using SS for the secretion and subsequent purification of
heterologous proteins that are otherwise not efficiently purified from
whole cell lysate. This method of purification yielded higher and purer
SSCrFCES with intact biological activity.
One SSCrFCES molecule binds cooperatively to more than two
molecules of lipid A
Results from the ELISA-based lipid A binding assay displayed a
biphasic curve (Fig. 2A
). This usually indicates the presence of multiple binding
sites for the ligand. SSCrFCES binds to lipid A with a stoichiometry of
one SSCrFCES to two or three lipid A molecules at saturation (Fig. 2B
). This observation of the LPS binding domain of factor C
interacting with two or three molecules of lipid A is novel. Scatchard
plots of the binding data (not shown) are very convex, indicating that
the binding of SSCrFCES to lipid A is highly cooperative. This is
confirmed by the slope of the line obtained from plotting the data
(Fig. 2C
) according to the Hill’s equation
(23)
, which gave a coefficient of 2.2.
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SPR studies of interactions between CrFCES and lipid A
The ability of SSCrFCES to bind LPS was also shown by SPR, using
the BIAcore X (Fig. 3
). Injection of purified SSCrFCES (400 ng/100 µl) over immobilized
lipid A resulted in an increase of ~200 relative response unit. This
represents a 92% saturation of lipid A. Subsequently, injection of
antibody (INDIA His-HRP Ab) against the poly-His tag of SSCrFCES
resulted in a further increase of relative response unit. The binding
of INDIA His-HRP Ab to SSCrFCES, which contains 6-His tag, further
confirms that only SSCrFCES was bound to the immobilized lipid A.
Surface plasmon resonance, in addition to INDIA His-HRP Ab, provided a
faster way to assess both quantitatively and qualitatively the
batch-to-batch variations in yield and potency of SSCrFCES.
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Inhibition of endotoxin-induced LAL reaction
The 50% endotoxin-neutralizing concentration
(ENC50) value of SSCrFCES against 200 EU of
endotoxin per milliliter (LPS, E. coli 055:B5) was
determined using kinetic-QCL. The time that is required before the
appearance of a yellow color (reaction time) is inversely proportional
to the amount of endotoxin present. A low ENC50
indicates high potency of endotoxin neutralization. The
ENC50 is taken as the concentration of SSCrFCES
that reduces the mean reaction time by 50%. A sigmodal curve was
obtained between relative reaction time and the logarithmic
concentration of SSCrFCES (Fig. 4
). ENC50 of SSCrFCES was determined to be
0.069 ± 0.014 µM. The low concentration of SSCrFCES required to
achieved ENC50 is not unexpected, since SSCrFCES
is derived from the factor C serine protease that activates the LAL
reaction.
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SSCrFCES inhibits LPS-stimulated hTNF and hIL-8 release
from THP-1
Results from our in vitro binding studies
suggested that SSCrFCES would be a potent inhibitor of the LPS
activation of monocytes. To test this prediction, we measured the
ability of SSCrFCES to inhibit hTNF- and hIL-8 production by THP-1
cells incubated with 25 ng/ml and 100 ng/ml of LPS in a serum-free
system containing various concentrations of SSCrFCES. As shown in
Fig. 5
, 0.5 µM of SSCrFCES potently inhibited >90% LPS-induced production
of TNF- and IL-8 in the presence of 100 ng/ml of LPS. At the 25
ng/ml LPS concentration tested, 0.7 µM of SSCrFCES is sufficient to
completely prevent LPS-induced TNF- production (Fig. 5A
).
At 100 ng/ml LPS, 1 µM of SSCrFCES reduced 90% IL-8 production as
compared to control (Fig. 5B
).
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SSCrFCES inhibits the production of TNF- and IL-8 by human PBMC:
effect of human serum on the anti-endotoxin potential of SSCrFCES
Purified human PBMC were used to test the suppression on
endotoxin-induced TNF- and IL-8 secretion by SSCrFCES under normal
physiological conditions. In the absence of human serum, addition of
only 0.1 µM of SSCrFCES completely inhibited the TNF- and IL-8
response to 10 ng/ml LPS by 50% (Fig. 6
). When SSCrFCES was added to human serum (final concentration, 10%)
before the addition of endotoxin, the suppressive effect of SSCrFCES
was attenuated. It required 17-fold more SSCrFCES to suppress TNF-
and IL-8 secretion by 50%. However, if the SSCrFCES was mixed with
endotoxin 5 min before the addition of serum, the effect of the serum
on the neutralization of endotoxin by SSCrFCES was greatly reduced,
requiring only fourfold more SSCrFCES for 50% inhibition (Fig. 6)
.
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Cytotoxicity of SSCrFCES to eukaryotic cells
The toxicity of SSCrFCES to mammalian cells was tested by
incubation with human monocytes and analyzed using MTS, a compound that
could only be metabolized by healthy cells. SSCrFCES had a minimal
effect on cell permeabilization (data not shown). At the highest
concentration of 4 mg/ml or 109 µM, only 20% cell lysis was
observed. This clearly indicates that SSCrFCES is a nontoxic
anti-endotoxin protein.
Pharmacokinetic analysis of biotin-labeled SSCrFCES in mice
Densitometric scan revealed that significant amounts of
circulating half-life of SSCrFCES is sufficiently long to allow easy
detection during the first 90 min postinjection. NCOMP, which provides
an interactive graphical environment for noncompartmental analysis of
pharmacokinetic
data by facilitating estimation of the zero and first moments of
concentration-time data, was used for analysis. The calculated
clearance rate of biotin-labeled SSCrFCES in C57BL/6J mice is 4.7
ml/min.
Effects of SSCrFCES on endotoxin-induced lethality in
galactosamine-sensitized mice
An i.p. injection of 2.5 ng of E. coli 055:B5 LPS per
mouse induced 100% lethality in the galactosamine-sensitized mice
within 7 h. As shown in Fig. 7
, this LPS-induced lethality was reduced by 20% when 1 µM of SSCrFCES
was injected i.v. 10 min after the i.p. injection of LPS. Higher
concentrations of SSCrFCES of 2 and 4 µM conferred 90% and 100%
protection, respectively. The data were analyzed using Kaplan-Meier
analysis and log rank pairwise test. The protection was correlated with
a reduction of the TNF- level in mouse serum (Table 1
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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and secreted
acetylcholinesterase (25)
, were successfully expressed,
few display the kind of complexity posed by the factor C LPS binding
domain. The LPS binding property of factor C resides in the
amino-terminal region spanning 333 amino acids. This short region
constitutes a signal peptide, a cysteine-rich region, followed by
epidermal growth factor-like domain, and finally three Sushi domains.
High LPS affinity, comparable to the native factor C, requires the
correct formation of 9 disulfide bonds (26)
. This obstacle
is compounded by the presence of a cysteine-rich region. Initial
attempts to express and direct secretion of 943 amino acid
NH2-end fragment in P. pastoris
resulted in intracellular accumulation of the protein
(12)
. This intracellular recombinant protein was later
shown to exhibit only weak endotoxin binding capacity. It was
speculated that either P. pastoris failed to fold the
protein correctly or that the reducing environment of the cytosol is
detrimental to the protein stability. Consequently, the LPS binding
domain(s) was expressed in Drosophila S2 cells and directed
secretion into the culture medium was achieved via a novel secretory
signal, SS. As starting material for purification work described in this study, we used a transfectant cell line that stably expresses and secretes recombinant SSCrFCES. We have developed two schemes to purify the SSCrFCES from the serum-free medium. The first scheme purifies SSCrFCES based on its carboxyl-terminal poly-His tag whereas the second scheme is based on its isoelectric value. To determine whether microheterogeneity exists during cleavage of SS, the first purification scheme was used. This allowed the purification of all recombinant proteins harboring the poly-His tag. The second scheme is unsuitable since it would result in the enrichment of a single isoform if present, and thus presents a biased interpretation. The SS is highly effective in directing secretion of SSCrFCES into the culture medium of Drosophila cells. Western analysis revealed the presence of a protein with an apparent molecular mass of ~38 kDa on SDS-PAGE. Practically all of SSCrFCES is secreted into the culture medium. The signal was cleaved at a single site as determined by amino-terminal amino acid sequencing, leaving behind six residues with SSCrFCES. Consequently, only a single species of SSCrFCES was expressed and accumulated in the medium. This is important for future crystallography studies. The six residues present in the mature SSCrFCES had no apparent influence on the LPS binding property of SSCrFCES.
Using the second purification scheme, the final SSCrFCES preparation is essentially an isoelectrically homogeneous protein species and is functional. The two key steps for the purification include 1) a concentration, desalting, and partial enrichment step using ultrafiltration through two molecular mass cutoff of 100 kDa and 10 kDa; and 2) preparative isoelectric membrane electrophoresis. The first step in purification uses tangential flow filtration technology and thus can be reliably scaled-up to industrial level. The second step can also be easily adapted to purify milligrams to grams of SSCrFCES. Recombinant SSCrFCES have been subjected to isoelectric focusing over a period of 3–4 days without detriment to the protein. The scheme reported here has been found to be reproducible in our laboratory throughout the course of more than 1 year. For the purification scheme, the overall recovery of SSCrFCES binding capacity is nearly 90%. This is attributable to its extreme stability conferred by the presence of 9 disulfide bonds.
This report also presents, for the first time, the expression and
secretion of a fully functional recombinant LPS binding domain of
factor C, SSCrFCES. We provide evidence that the amino-terminal 333
amino acids of factor C are sufficient to mediate LPS binding capacity.
The recombinant SSCrFCES, like other LPS binding proteins, binds to
lipid A with high affinity. However, unlike other LPS binding proteins
(27
28
29
30)
, SSCrFCES binds with a stoichiometry of one
SSCrFCES molecule for two or three lipid A molecules at saturation. The
binding is cooperative with a Hill’s coefficient (23)
of
2.2. This degree of cooperativity is comparable to hemoglobin for
oxygen (31)
and erythrocyte membrane tropomyosin for actin
(32)
. This phenomenon of multiple lipid A binding sites
has hitherto not been documented to exist in any known LPS binding
protein, with the exception of bactericidal/permeability-increasing
protein (BPI) (27)
. However, although BPI was reported to
bind > one lipid A molecule, it was not reported to exhibit
cooperativity in binding. This homotropic cooperativity for binding to
lipid A is thus novel and unique to SSCrFCES. The presence of multiple
lipid A binding sites that showed cooperativity assuredly confirm the
LPS binding domain of factor C, as well as full-length factor C, to be
the best candidate for removal and detection of endotoxin in solution.
Cooperative binding also contributed to factor C’s ability to detect
subpicogram levels of endotoxin (unpublished data; patent filed) as
well as a competitive binding advantage over Limulus
anti-LPS binding factor (LALF). Retrospectively, the degranulation of
amoebocytes in the presence of LPS would release a battery of
anti-bacterial/LPS binding factors, e.g., LALF, thus significantly
reducing the amount of free LPS. Nonetheless, factor C is capable of
capturing trace LPS to activate the coagulation cascade. Such
capability is attributed to its homotropic cooperativity as
demonstrated by SSCrFCES, viz, its LPS binding domain. Kinetic-QCL,
which uses the LAL reaction cascade, usually gives a good indication of
the pyrogenicity of the LPS. Only a very low concentration of SSCrFCES
(0.069 µM ± 0.014) is needed to achieve
ENC50. Comparatively, this value is 28- and
7.5-fold less than ENC50 of polymyxin B and LF-33
(33-mer peptide derived from lactoferrin) (33)
,
respectively. This shows that on a molar basis, much less SSCrFCES is
required to neutralize the same amount of LPS. It also indicates that
SSCrFCES is a potent anti-pyrogenic recombinant protein.
During gram-negative septicemia, the high concentration of LPS in the
blood leads to multiple organ failure syndromes. These adverse effects
are dependent on the generation of endogenous mediators. A multitude of
mediators have been implicated, including arachidonic acid metabolites,
PAF, cytokines such as TNF-, interferons, and various interleukins
(e.g., IL-1, IL-8, etc.), reactive oxygen metabolites, and components
of the coagulation cascade (1
2
3)
. Consequently, the
biological potential of SSCrFCES to bind and neutralize LPS-stimulated
production of cytokines in human promonomyelocytic cell line THP-1 and
normal human PBMC was investigated. Our findings indicate that 1 µM
of SSCrFCES effectively prevents the LPS-mediated induction of hTNF-
and hIL-8 production by THP-1 when these cells are incubated in the
presence of high endotoxin levels. It is important to note that the
concentrations of LPS (25 ng/ml and 100 ng/ml) used in these studies
are among the highest known concentrations reported for LPS-induced
cytokine production. On a molar basis, SSCrFCES appears to be more
potent than polymyxin B and LF-33 at suppressing LPS-induced LAL
coagulation and hTNF- or hIL-8 secretion by THP-1 cells under
serum-free conditions (33)
. This suggests that SSCrFCES
has a much greater intrinsic capacity to neutralize endotoxin than
polymyxin B. Again, it is attributable to its cooperative binding of
LPS. Under serum free condition, a similar profile was observed when
using human PBMC. In the presence of human serum, however, SSCrFCES
anti-endotoxin potency was attenuated by 17-fold. A similar effect of
human serum has also been observed with other cationic antiendotoxin
proteins such as LF-33 (33)
and LALF (34)
.
This is due to the interaction of these factors with serum proteins,
which effectively reduces their availability for binding to endotoxin.
Consistent with this explanation is our observation that mixing
SSCrFCES with serum before endotoxin treatment reduces the ability of
the SSCrFCES to suppress LPS-induced TNF- and IL-8 secretion,
requiring 17-fold more SSCrFCES to achieve 50% inhibition. This serum
attenuating effect is greatly alleviated if SSCrFCES is incubated for 5
min with LPS prior to addition of serum.
In addition to high specific LPS binding, important features when
using proteins for in vivo application to treat
gram-negative bacterial septic shock are their physicochemical
properties in biological systems. Problems that often arise in animal
experiments are due to toxicity, as in the case of polymyxin B, or a
short half-life in the circulating system—for example, BPI. To assess
these features, we investigated SSCrFCES for their ability to
permeabilize cultured cells. At the highest concentration of SSCrFCES
tested (4 mg/ml), only 20% cell lysis was observed. This represented a
109-fold excess of SSCrFCES required for complete inhibition of
cytokine secretion in THP-1 and PBMC cells. Comparatively, SSCrFCES is
less cytotoxic than polymyxin B, where 50% cell lysis occurred with
0.51 mg/ml polymixin B (35)
. The clearance rate of
SSCrFCES assessed via noncompartmental analysis was 4.7 ml/min. The
disappearance of SSCrFCES from the circulation was 2.7-fold slower than
BPI. Therefore, a lesser dose of SSCrFCES would be adequate to maintain
high enough circulating levels to compete with LBP for LPS. It is
conceivable that the clearance rate of SSCrFCES is significantly faster
than LBP because SSCrFCES is an exogenously administered recombinant
protein that is most likely metabolized faster in the liver.
Finally, the anti-endotoxin potency of SSCrFCES was also investigated
in C57BL/6J mice. An i.p. injection of 2.5 ng of E. coli
055:B5 LPS into D-galactosamine-sensitized C57BL/6J mice resulted in
100% lethality within 7 h. Whereas 1 µM of SSCrFCES was
sufficient to completely neutralize the LPS-induced TNF- and IL-8
secretion from THP-1 and human PBMC cells, it could reduce the
lethality of mice by only 20% (Fig. 7
). Higher concentrations of
SSCrFCES (2 and 4 µM) conferred >90% survival. Two main factors are
responsible for the requirement of this dose of SSCrFCES. First,
SSCrFCES will have to compete with many serum proteins (e.g., LBP and
albumin) for LPS. Second, the clearance rate of SSCrFCES with respect
to LPS, is an important factor, as it determines the actual effective
amount available for LPS binding. Nonetheless, i.v. injection of
SSCrFCES in our experiments blocked the rise in cytokine levels,
prevented liver damage, and thus significantly reduced LPS-mediated
mortality (Table 1)
. Thus we show for the first time a protective role
of SSCrFCES viz LPS binding domain of factor C in an i.p. murine sepsis
model. The mechanism by which SSCrFCES protects mice from LPS-induced
sepsis is presumably mediated through its high-affinity association
with lipid A moiety of LPS, which consequently reduces the secretion of
cytokines like TNF- and IL-8.
In conclusion, this report presents for the first time the expression
and localization of the functional LPS binding domain of factor C i.e.,
SSCrFCES, which resides in the amino-terminal 333 amino acid. Two
simple and rapid means of determining endotoxin binding were
established: ELISA-based and BIAcore biomolecular interactions. The
recombinant SSCrFCES has multiple lipid A binding sites and exhibits
strong positive cooperativity. Furthermore, a low concentration of
SSCrFCES is sufficient to completely inhibit the LPS-induced TNF-
and IL-8 secretion from THP-1 and PBMC cells. SSCrFCES, being only
weakly cytotoxic, can protect LPS-induced lethality in mice. This
report also clearly explains the remarkable sensitivity of factor C for
detection of trace endotoxin. Incidentally, the positive cooperativity
of LPS binding is also responsible to the issue of linearity of
LAL-based quantification of endotoxin. Additional investigations are
also being carried out to determine whether singly or multiply bound
lipid A are necessary to activate factor C. In view of the results
presented here, recombinant factor C with enhanced sensitivity for LPS
and better linear corelationship to endotoxin amount can be developed
and would represent the next generation of endotoxin detection and
quantification assay.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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