Departments of 1Biological Sciences and 2Microbiology, 14 Science Drive 4, National University of Singapore, 117543 Singapore
3 To whom correspondence should be addressed. e-mail: dbsdjl{at}nus.edu.sg
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Abstract |
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Keywords: endotoxin binding and neutralization/Factor C/limulus/S3 tandem repeats/Sushi3 peptide
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Introduction |
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LPS from Gram-negative bacteria induces the amoebocytes of limulus to aggregate and degranulate. This response underlies the important defense mechanism of limulus against invasion of Gram-negative bacteria (Ding et al., 1995). As a molecular biosensor, Factor C can be autocatalytically activated by femtograms of LPS to trigger the coagulation cascade (Ho, 1983
), suggesting that it contains high affinity LPS-binding domains. Recently, two regions of Factor C that exhibit exceptionally high LPS binding affinity were defined as the Sushi1 and Sushi3 domains (Tan et al., 2000a
). Two 34-mer synthetic peptides, S1 and S3, that span the 171204 and 268301 amino acid residues of Factor C (DDBJ/EMBL/GenBank accession No. S77063) are derived from Sushi1 and Sushi3 domains, respectively. Both peptides inhibit the LPS-induced limulus amoebocyte lysate (LAL) reaction and LPS-induced hTNF-
secretion (Tan et al., 2000b
). Thus, the S1 and S3 peptides are promising endotoxin antagonists. The application value of these two peptides would be boosted if they could be obtained by cost-effective and large-scale methods such as recombinant expression in prokaryotic systems. However, expression of small peptides tends to encounter technical difficulties (Le and Trotta, 1991
; Latham, 1999
). It is reported that some of these problems were resolved by multimerization of the small peptide followed by in vitro digestion to restore their activity (Mauro and Pazirandeh, 2000
). Besides Factor C, the tachylectin family members identified in circulating hemocytes and hemolymph plasma also contribute to the recognition of invading pathogens. Five types of lectins, named tachylectin-1 to -5, have different specificities for carbohydrates exposed on pathogens. Interestingly, all these lectins contain a different number of tandem repeats in their structure (Iwanaga, 2002
). Thus, studying the tandem repeats of S3 may provide explanations as to why these proteins adopt repetitive structures, and how they contribute strategically towards pathogen recognition. In this work, tandem repeats of S3 gene were cloned into a modified vector, which was subsequently transferred to an expression vector, pET22b. Induced expression of the most robust tetramer clone was scaled-up. Recombinant S3 tetramer (rS3-4mer) was purified and digested into monomers (rS3-1mer) by acid treatment, and both the recombinant peptides were tested for their endotoxin-binding and -neutralizing activities.
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Materials and methods |
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LPS from Escherichia coli 055:B5 was purchased from Sigma (St. Louis, MO). LAL kinetic-QCL kit was supplied by BioWhittaker (Walkersvile, MD). Human TNF- kit (OptEIA ELISA) was from Pharmingen (San Diego, CA). CellTiter 96 Aqueous One Solution Reagent for cytotoxicity assay was purchased from Promega (Madison, WI). Enzymes for DNA manipulation and polymerase reactions were purchased from NEB (Beverly, MA). DNA purification and extraction kits were from Qiagen (Chatsworth, CA). Pyrogen-free water for making buffers was from Baxter (Morton Grove, IL).
Construction of multimers of S3 gene
Using a cloned Factor C Sushi3 domain, pAC5.1Sushi3EGFP (Tan et al., 2000b), the LPS-binding motif, S3, was amplified by PCR. A cloning strategy, which allows for directional multimerization and cloning is shown in Figure 1. Briefly, the amplification vector pBBSI (Lee et al., 1998
) was modified to include an NdeI site containing the start codon adjacent to BbsI site. This modified vector was named pBC. Forward primer 5'-TCGAAGACGGCCCCAGGATCCCCATGCTGAACACAAGG-3' was designed with BbsI restriction site (GAAGAC) followed by GGCCCC in addition to the S3 flanking sequence. On the reverse primer, 5'-TAGAAGACCCGGGGGTCCA TCAAAGAAAGTAGTTA-3', a similar motif, was also introduced. Digestion of the PCR product by BbsI yielded fragments with a complementary overhang of CCCC on the sense strand and GGGG on the anti-sense strand, which can be used for directional multimerization and cloning. In addition, the GATCCC sequence, which codes for aspartate (D) and proline (P), was introduced into the forward primer. The peptide bond between D and P can be cleaved under acidic conditions (Szoka et al., 1986
), thus releasing single S3 units from the recombinant multimers. In this case, the PCR products of S3 were cloned into pBC vector, and the S3 gene was released by BbsI digestion and allowed to self-ligate first, before cloning into the pBC vector, which was previously linearized with BbsI. The multimers of S3 gene were selected and identified by enzyme digestion and sequencing.
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To construct expression vectors bearing tandem S3 genes under the control of T7 promoter, the fragments flanked by NdeI and HindIII (containing the multimeric S3 genes) were cloned into the vector pET22b, previously linearized with NdeI and HindIII. The constructs were transformed into the E.coli host, BL21 (DE3), for expression. The colonies were cultured overnight in LB medium with 100 µg/ml ampicillin at 37°C, then diluted 1:100 into fresh LB medium with 100 µg/ml ampicillin and grown to an OD600nm of 0.6 before induction with 0.5 mM IPTG (Promega). The cells were harvested every hour up to 12 h, and the expressed products were monitored by SDSPAGE.
Solubilization of inclusion bodies and purification of rS3-4mer
One litre cultures were pelleted at 5000 g for 10 min at 4°C and resuspended in 60 ml of lysis buffer containing 20 mM TrisCl, pH 8.0 and 0.5 mM DTT. The bacterial cells in the suspension were passed through a French Press (Basic Z 0.75KW Benchtop Cell Disruptor, UK) operated at 15 kpsi for four rounds in order to generate >90% cell disruption. The inclusion bodies were recovered by centrifugation at 12 000 g for 20 min at 4°C and washed with 20 mM TrisCl buffer containing 1 M urea and 0.5% Triton X-100. The inclusion bodies were denatured and solubilized in 20 mM TrisCl with 8 M urea at room temperature for 2 h. Insoluble materials were removed by centrifugation at 16 000 g for 20 min, and the supernatant was filtered and purified by anion exchange using ÄKTA explorer (Pharmacia). Briefly, 30 ml of solubilized proteins were applied to a Q-Sepharose column (26x300 mm) equilibrated with buffer A (4 M urea, 20 mM TrisCl pH 6.7). After washing with four column volumes of buffer A, the proteins were eluted with a linear gradient of 030% buffer B (4 M urea, 20 mM TrisCl pH 6.7, 1 M NaCl) and the fractions were collected for SDSPAGE analysis. The collected fractions were pooled and dialyzed in 10 kDa molecular weight cut-off (MWCO) pore size dialysis tubing (Snakeskin; Pierce, IL), against refolding buffer A containing 50 mM glycine, pH 9.5, 10% sucrose, 1 mM EDTA and 2 M urea at 4°C for 16 h, followed by buffer B containing 20 mM diethanolamine pH 9.5, 10% (w/v) sucrose and 1 mM EDTA at 4°C for another 8 h.
Monomerization of rS3-4mer into rS3-1mer by acid digestion
Two adjacent amino acids, aspartate and proline were added between the S3 units, so as to act as cleavable DP linkers. The renatured rS3-4mer was precipitated with nine volumes of ethanol, frozen at 80°C for 1 h or at 20°C overnight. The mixture was centrifuged at 16 000 g for 10 min and the pellet was washed with 90 % ethanol, dried, dissolved in digestion buffer (70% formic acid, 6 M guanidineCl) and digested at 42°C for 72 h. The final products were subjected to ethanol precipitation and dissolved in 20 mM TrisCl pH 7.3. The cleaved rS3 peptides were then dialyzed overnight against the same buffer using dialysis tubing of 1.5 kDa MWCO pore size (Sigma), thus removing the small linkers and residual salt. The endotoxin contaminant in rS3-4mer and rS3-1mer was removed by Triton X-114 phase separation ( Liu et al., 1997) followed by polymyxin B affinity chromatography (Detoxi-GelTM; Pierce).
Tricine SDSPAGE and western blot analysis
The recombinant proteins were resolved on tricine SDSPAGE, using 5% stacking gel and 15% separating gel, and detected by Coomassie blue staining (Schagger and von Jagow, 1987). Western blot analysis was performed according to the manufacturers instruction, using an ECL western analysis system (Pierce). The blot was probed with polyclonal rabbit anti-S3 antibody followed by goat anti-rabbit secondary antibody conjugated to horseradish peroxidase (HRP; Dako, CA). The blots were visualized using Supersignal West Pico Chemiluminescent Substrate and exposed to X-ray film.
ELISA-based LPS-binding assay
The polysorp 96-well plate (MaxiSorpTM; Nunc) was first coated with 100 µl per well of 4 µg/ml (1 µM) of LPS diluted in pyrogen-free phosphate-buffered saline (PBS). The plate was sealed and incubated overnight at room temperature. The wells were aspirated and washed four times with 300 µl wash solution (PBS containing 0.05 % Tween-20). The wells were blocked with wash solution containing 2% BSA for 1 h at room temperature. After washing twice, varying concentrations of peptides were allowed to interact with bound LPS at room temperature for 3 h. Bound peptides were detected by incubation with rabbit anti-S3 antibody and 1:2000 of goat anti-rabbit antibody conjugated with HRP. Each antibody was incubated for 2 h at 37°C. In the final step, 100 µl of substrate, ABTS (Boehringer Mannheim), was added. The absorbance was measured at 405 nm with reference wavelength at 490 nm.
Endotoxin neutralization assay based on anti-LAL test
The LAL KineticQCL kit utilizes the initial part of the LPS-triggered cascade in LAL to achieve an enzymatic reaction, which catalyzes the release of p-nitroaniline from a synthetic substrate, producing a yellow color, which is quantifiable by absorbance at 405 nm. The ENC50 (endotoxin neutralization concentration) refers to the peptide concentration required to neutralize 50% of a predetermined quantity of endotoxin. A low ENC 50 indicates high potency of the peptide for endotoxin neutralization.
In this assay, peptides of different concentrations were incubated for 1 h at 37°C with or without an equal volume of LPS in disposable, endotoxin-free borosilicate tubes. Fifty microliters of each mixture was then dispensed into wells of a sterile microtiter plate (NunclonTM surface; Nunc). Fifty microliters of freshly reconstituted LAL reagent was dispensed into each well. The absorbance at 405 nm of each well was monitored after 45 min, and the concentration of peptides corresponding to 50% inhibition of LAL activity was designated ENC50.
Suppression of LPS-induced hTNF- secretion in human THP-1 cells
THP-1 cells were cultured at 37°C in a humidified environment in the presence of 5% CO2. RPMI 1640 medium was supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml) and streptomycin (100 µg/ml). The cells were maintained at a density of 2.5x1056 cells/ml. THP-1 monocytes were transformed into macrophages by addition of phorbol myristic acid (PMA; Sigma) at a stock of 0.3 mg/ml in dimethyl sulfoxide to give a final concentration of 30 ng/ml and 0.01% dimethyl sulfoxide. PMA-treated cell suspensions were immediately plated into 96-well microtiter plate at a density of 4x105 cells/ml and allowed to differentiate for 48 h at 37°C. The culture medium was removed and the cells were washed twice with serum-free RPMI 1640. Thereafter, the macrophages were stimulated with 50 EU/ml LPS (a specific activity of LPS that has been standardized by LAL test against FDA-approved LPS standards), peptides alone or LPS (preincubated with various concentrations of peptides) and incubated at 37°C. After 6 h, the culture medium was collected and hTNF- concentration in the supernatants was assayed using ELISA.
Real time interaction analysis between peptides and LPS
Surface plasmon resonance (SPR) analysis of the real time interaction between peptides and LPS was performed with BIAcore 2000 (Pharmacia) using HPA chip (Tan et al., 2000b). The affinity constant was calculated using BIAevaluation software 3.0. The mean values were obtained from three independent experiments.
Cytotoxicity of peptides in eukaryotic cells
THP-1 monocytes in 50 µl of 2x104 cells/ml in RPMI 1640 were mixed in a microtiter plate with 50 µl of two-fold serial dilutions of peptides ranging in concentration, and incubated for 60 min at 37°C. To determine the cytotoxicity induced by the peptides, 20 µl of CellTiter 96 Aqueous One Solution Reagent was added into each well for 90 min at 37°C. MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium] is bioreduced by metabol ically active cells into a colored formazan product that is soluble in tissue culture medium. For detection, the absorbance was measured at 490 nm. To determine the ratio of cell lysis induced by the peptides, two controls were included by incubating cells in PBS containing 0.2% Tween-20 instead of medium only. This absorbance value corresponds to the background, as those cells could not metabolize MTS.
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Results |
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A 143 bp S3 gene fragment was obtained by PCR using pAC5.1Sushi3EGFP as the template. The S3 gene was cloned into pBC vector by digestion with BbsI. After multimerization, the clones containing one, two, four and eight copies of S3 were selected (Figure 2a) and named pBCS3-1, -2, -4 and -8mer, respectively. The NdeI and HindIII-flanking fragments of these clones were inserted into pET22b for expression of the multimeric S3 gene, and the expression levels were examined by SDSPAGE. Of all the expression cassettes, the tetramer yielded the highest expression level, giving the expected recombinant S3 tetramer (rS3-4mer) of 18.4 kDa, which represented 25% of the total cell proteins (Figure 2b). The monomer construct was not expression-competent, while the octamer construct expressed poorly.
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Samples from the total cell protein, purified rS3-4mer, partially digested rS3 polymers, rS3-1mer and synthetic S3 peptide were resolved on tricine SDSPAGE and subjected to western blot analysis against anti-S3 antibody. The rS3-1mer and its partially digested polymeric repeats (2, 3 and 4mers) were immunoreactive to the polyclonal rabbit anti-S3 antibody (Figure 3b). Thus, the antibody can be employed for the ELISA-based LPS-binding assay.
ELISA-based LPS-binding assay revealed different binding capabilities with rS3-1mer, rS3-4mer and synthetic S3. At 4 µg/ml, both recombinant peptides reached saturation of binding to LPS (Figure 4 ), while the synthetic peptide continued linearly and required 20 µg/ml to reach saturation of binding with LPS (data not shown). The EBC50 of the peptide, which achieves 50% of maximum binding to LPS on the ELISA plate, reflects the binding activity of peptide to LPS, with the lower EBC50 indicating higher potency. The rS3-4mer, rS3-1mer and synthetic S3 peptides displayed EBC50 at 0.41, 1.02 and 9.74 µg/ml, respectively. The kinetics of binding of peptides to LPS in 20 mM TrisCl pH 7.3, was also measured by SPR analysis with BIAcore 2000 using HPA chip, which was immobilized with LPS. The Kd values of synthetic S3, rS3-1mer and rS3-4mer are (7.80 ± 2.18)x107 M, (4.74 ± 2.34)x108 M, (1.71 ± 1.86)x108 M, respectively. Thus, both the results from ELISA and SPR suggest that rS3-4mer is most efficient at binding LPS.
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The ENC50 value of the peptides against 5 EU/ml of LPS was determined to be 5.4 µg/ml for rS3-4mer, 9.2 µg/ml for rS3-1mer and 11.2 µg/ml for synthetic S3 (Figure 5a). A lower ENC50 indicates higher potency of endotoxin neutralization. The binding isotherm of the two monomeric peptides, whether it is recombinant or synthetic, is similar, but rS3-4mer shows a 2-fold stronger LPS neutralization efficacy.
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The peptides show minimal cytotoxicity to eukaryotic cells
Both recombinant peptides had minimal effect on cell permeabilization (data not shown). At the highest concentration of 50 µM, rS3-4mer caused only 23% of cell lysis, indicating that the recombinant multimers of S3 would have negligible contraindications, although the LPS-binding activity is amplified significantly.
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Discussion |
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Many methods can be applied to construct the tandem repeats of a peptide (Dolby et al., 1999; Lee et al., 2000
; Mauro and Pazirandeh, 2000
). We chose the amplification vector that readily allows us to obtain various multimers of S3 gene. Furthermore, we designed the DP linker between the repetitive units, to afford convenient cleavage under mildly acidic buffer to release the monomers. The multimeric constructs exhibit different expression levels. No expression was observed with the pETS3-1mer. As the copy number increases, the expression level improved dramatically, especially with the S3 tetramer, where the expression level reached 25% of the total cell proteins. However, further doubling to 8mer reduced the expression level, suggesting that the copy number is not always proportional to the expression level for this peptide. The ELISA-based LPS-binding test and SPR results show differential binding efficiencies of rS3-4mer, rS3-1mer and the synthetic S3 for LPS, with highest binding achieved by rS3-4mer. Both the LAL inhibition test and suppression of TNF-
release in THP-1 cells showed that rS3-1mer works equally well as the synthetic S3 peptide to neutralize LPS, while rS3-4mer displayed a 2-fold higher anti-LPS activity. However, the rS3-1mer and synthetic S3 showed inconsistent results in ELISA and SPR tests.
Two major forces mediate the interaction between LPS and LPS-binding peptides. The positive charge on the peptides forms an electrostatic attraction with the negatively charged phosphate head groups of the LPS. The other is the hydrophobic interaction between them (Farley et al., 1988; Goh et al., 2002
). In fact, mutation of amino acid residues of S3 aimed at introducing positive charges, only achieved a slight increase in LPS-neutralizing activity (Tan et al., 2000b
). Besides charge modification, little effort has been taken to enhance the LPS-binding ability of such peptides. Herein, by creating tandem repeats of the LPS-binding units, instead of increasing the number of positive charges, we demonstrate a two-fold improvement in the activity of the tetramer compared to the original monomeric unit, thus providing an alternative strategy to improve the LPS-binding activity of similar peptides. The result of secondary structure analysis by the DNAMAN program (Version 4.15, Lynnon Biosoft) shows that both S1 and S3 have a distinctive structure of four regular ß-sheets alternately spaced by turns and coils. We presume that this structure may be important to the interaction with LPS, and in addition, the multiple ß-sheets in rS3-4mer, may form the ß-barrel structure to provide better shielding of hydrophobic acyl chains of LPS (Ferguson et al., 1998
). Further structure analysis by CD or NMR will help to explain the enhanced activity of the recombinant S3 tetramer.
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Acknowledgements |
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Received September 5, 2002; revised June 18, 2003; accepted June 20, 2003.