Chinese hamster ovary (CHO) cells were grown in Dulbecco’s modified Eagle’s minimum essential medium (DMEM; Gibco BRL Life Technologies, Rockville, MD) supplemented with 2 mM glutamine, 2 mM sodium pyruvate, and 10% heat-treated fetal calf serum. Cells were incubated at 37°C under 5% CO2 in air in a humidified atmosphere.
Reagents, Gangliosides and antibodies
Gangliosides, Methyl-b-cyclodextrin (MbCD) and Cholesterol were purchased from Sigma (St. Louis, MO). The presence of actin and VVH were detected using anti-actin monoclonal antibody clone C4 (Chemicon International Inc., Temecula, CA ) and anti-VVH polyclonal antibody, respectively.
Preparation of VVH
VVH was purified from the culture supernatant of the V. vulnificus K1 strain following the method of Oh et al. . The protein concentration of each fraction was checked by optical density at 280 nm, and fractions with a high concentration of protein were used for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The gel was stained with staining solution containing 0.5% Coomassie brilliant blue R-250. Purified VVH was observed as a single band. VVH-containing fractions were dialyzed in 10 mM glycine buffer (pH 9.8)–150 mM NaCl at 4°C for 16 h. The dialyzed fractions were pooled as the purified VVH. The specific activity of purified VVH was 70,000 hemolytic units/mg (HU/mg), which was confirmed by examining the hemolytic activity against mouse erythrocytes.
Preparation of ghost membrane
Bovine defibrinated blood was suspended in hemolysis buffer (5 mM Na2HPO4, pH 8.0) for 10 min, on ice. Cells were then centrifuged at 12,000 × g for 10 min. The pellet was washed several times until the color changed to white in hemolysis buffer. After hemolysis, the ghosts were kept in storage buffer (140 mM NaCl, 20 mM Tris-HCl pH 7.5), and were used for toxin binding assay and measurement of cholesterol contents.
Measurement of cholesterol contents
Cholesterol contents of both 10 mM MbCD treated- and untreated ghosts were assayed spectrophotometrically using a Cholesterol E-Test Wako. (Wako, Osaka, Japan). Briefly, after treatment with MbCD, the bovine erythrocytes were washed twice with 1 ml of cold PBS, and then lysed with lysis buffer. Six hundred fifty microliters of the cell lysate was mixed with 100 ml of the cholesterol assay kit buffer solution and then this mixture was further mixed with 750 ml of concentration enzyme mix solution. Samples were incubated for 5 min at 37°C prior to measuring absorbance at 600 nm. The cholesterol contents were determined as follows: (measured fluorescence of sample /fluorescence of standard cholesterol) ´ 200. The percentage of remaining cholesterol after pretreatment with MβCD was determined as follows: (measured fluorescence of treated cells obtained from a standard curve/total fluorescence of untreated cells) ´ 100.
Measurement of binding amount of VVH
The ghosts were treated by 10 mM of MβCD for 30 min at 37°C, and then washed twice with 1 ml of cold HBSS. After washing, the ghosts were incubated with 5 mg/ml of VVH for 30 min at 37°C. The ghosts were centrifuged at 8,000 × g, and washed twice with storage buffer. After washing, the ghosts were lysed by lysis buffer (24.7 mM Tris pH8.3, 192 mM glycine, 20% v/v methanol). The bound VVH and cellular actin were detected by dot blotting using antibodies against anti-VVH and anti-actin. The dot intensities of these proteins were measured using NIH Image J software. Amount of bound VVH was calculated by dividing the dot intensity of VVH by that of actin.
Cytotoxicity was determined by a Lactate dehydrogenase (LDH) release assay. Cells were seeded in 24-well tissue-culture plates at 1 ´ 105 cells/well. After 24 h, the cells were washed twice with HBSS, which was then replaced with pre-warmed DMEM. Indicated concentrations of VVH and various gangliosides were pre-incubated for 30 min at 37°C, and then inoculated into the wells and incubated for 2 hrs at 37 °C. After incubation, aliquots of medium samples (sample LDH) were assayed for LDH activity using pyruvate as a substrate. Cells treated with VVH vehicle only (control LDH) were used to assess background LDH activity, and cells lysed with 0.5% TritonX-100 were used to represent total LDH activity. The percentage LDH release was calculated as (sample LDH – control LDH) / (total LDH – control LDH) ´ 100
Prevention assay for binding of VVH on CHO cells
CHO cells were seeded in 6-well tissue-culture plates at 5 ´ 105 cells/well. After 48 h, the cells were washed twice with HBSS, and then replaced with DMEM. The mixture was pre-incubated with the VVH and GD1a or Gg4Cer at the indicated molar ratio, inoculated into the wells, and then incubated for 1 h at 37°C. During this incubation time, all the VVH that bound to CHO cells were oligomerized. After washing three times with HBSS, the cells were extracted with lysis buffer supplemented with 1% Triton X-100 and a protease inhibitor mixture. Bound VVH and cellular actin were detected by western blotting using antibodies against anti-VVH and anti-actin..
VVH were incubated with cholesterol, Gg4Cer or GD1a for 30 min at 37°C. The mixture of VVH and ganglioside was subjected to SDS-PAGE followed by western blotting using anti-VVH polyclonal antibody.
The glycolipid array assay was performed using a glycolipid array plate (Sumitomo Bakelite, Tokyo, Japan). VVH was adjusted to 100 μg/mL in a reaction buffer comprising 50 mm Tris/HCl, pH 7.5, 100 mm NaCl, 1 mm CaCl2, 1 mm MgCl2, 1 mm MnCl2 and 0.05% Tween‐20. The glycolipid array plates were incubated with VVH for 2 h at room temperature. After washing sequentially with a washing buffer (50 mm Tris/HCl, pH 7.4, 100 mm NaCl) and water, the plates were incubated with biotin conjugated anti‐VVH polyclonal IgG, and subsequently probed with streptoavidin-Cy5 (Jackson Immunoresearch). The fluorescent signal was measured using a ScanArray Express Version4.0 (Perkin‐Elmer, Waltham, MA, USA). The binding signal is measured by Cy5 fluorescence, and the data is expressed as signal / noise (S / N) values. The S / N values are calculated by dividing the fluorescence intensity of each spot by the background intensity three times and are expressed as the average intensity of those measurements. S/N values >3 were considered to indicate significant binding of the VVH to glycolipids.
Construction, expression, and purification of GST-fusion protein.
The V. vulnificus genome DNA was purified by Qiagen Genomic-tip (Qiagen, Hilden, Germany) as recommended by the manufacturer. VVH encoding gene, vvhA was amplified with signal sequence by PCR with the primers vvhA5’ (5’-GTGGGATCCATGAAAAAAATGACTCTGTTTACC-3’ ;the underline indicates an BamHI site) and the vvhA3’(5’-GTGGCATGCCTAGAGTTTGACTTGTTGTAATGT -3’ ; the underline indicates an SphI site), from V. vulnificus genome as the template. The amplified DNA was ligated to pGEM-T vector (Promega, Madison, WI) and the sequence was confirmed by DNA sequencing. GST-PD and GST-LD were amplified by using the following primer pairs from pGEMT vvhA as the template respectively. GST-PD FW; 5’-GGATCCGTGAAACAACGTATTCGCATCGAC-3’ (the underline indicates an BamHI site), GST-PD Rev; 5’-CTCGAGCTAGAGTTTGACTTGTTGTAATGT-3’ (the underline indicates an XhoI site), and GST-LD Fw; 5’-GGATCCCAAGAATATGTGCCGATTGTTGAG-3’ (the underline indicates an BamHI site), GST-LD Rev; 5’-CTCGAGCTAGGTACTGCTGGTTGACGAGCC-3’ (the underline indicates an XhoI site). The amplified each DNA was ligated to pGEM-T vector and the sequence was confirmed by DNA sequencing, and then ligated to pGEX4T3 (GE Healthcare Life Sciences, Chicago, IL) BamHI-XhoI site. Each plasmid was transformed to Escherichia coli DH5a. The bacteria were cultivated in Luria-Bertani (LB) broth containing 100 µg of Ampicilin/ml until OD 600 0.5 at 37 °C and then induced to produce the GST-fusion protein by adding 0.1mM isopropyl-b-D-thiogalactopyranoside (IPTG) at 20°C for 16hrs. After induction of the protein, the bacteria were suspended with the binding buffer (1 mM EDTA, 50 mM NaCl, 50 mM Tris-HCl pH 8.0). The bacterial suspension was sonicated using a Vibra Ultrasonic (model VCX-500, Sonics and Materials Inc, USA) and centrifuged at 21,000 × g at 4 °C for 20 min. The supernatant was used for purification of GST-fusion proteins (GST-PD and GST-LD). The GST-fusion protein was purified with Glutathione Sepharose 4B Resin according to the manufacturer’s instructions (GE Healthcare Life Sciences, Chicago, IL). After purification, each GST-fusion protein was dialyzed with phosphate buffered saline (PBS). The GST-PD protein (21-300 a.a. of VVH without 20 a.a. of signal sequence) and GST-LD protein (301-451 a.a. of VVH) were expressed and confirmed as the ca. 59kDa and the ca. 43kDa GST-fusion protein respectively by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
Pull-down assay by GM1-Sepharose
Pull-down assay by using GM1-Sepharose for the toxins were performed as previously described . Briefly, lyso-GM1 was coupled using NHS-activated Sepharose 4 Fast Flow (GE Healthcare, England) in 0.2 M NaHCO3 and 0.5 M NaCl (pH 8.3) at room temperature for 4 h with rotation. After the coupling reaction, non-reacted groups on the Sepharose were blocked by 0.5 M ethanolamine in the coupling solution. Lyso-GM1 Sepharose was then washed with 0.1 M Tris–HCl, 0.1 M acetate and 0.5 M NaCl and resuspended with PBS in a 1:1 (volume/volume) ratio. GST-LD, GST-PD, GST, or CTxB was incubated with lyso-GM1 or lyso-GM1 non-coupling Sepharose (control Sepharose) in PBS for 2 h at 4°C with rotation. After incubation, Sepharose was sedimented by centrifugation at 12,000 × g. The supernatant was discarded, and the remaining Sepharose was washed twice with PBS. The bound proteins were then solubilized with sample buffer (62.5 mM Tris–HCl, 2% SDS, 10% glycerol, 0.001% bromophenol blue and 100 mM dithiothreitol) and boiled for 5 min. The sample was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by 0.5% Coomassie Brilliant Blue R-250.