Novel hemagglutinating, hemolytic and cytotoxic activities of the intermediate subunit of Entamoeba histolytica lectin

Galactose and N-acetyl-D-galactosamine (Gal/GalNAc) inhibitable lectin of Entamoeba histolytica, a common protozoan parasite, has roles in pathogenicity and induction of protective immunity in mouse models of amoebiasis. The lectin consists of heavy (Hgl), light (Lgl), and intermediate (Igl) subunits. Hgl has lectin activity and Lgl does not, but little is known about the activity of Igl. In this study, we assessed various regions of Igl for hemagglutinating activity using recombinant proteins expressed in Escherichia coli. We identified a weak hemagglutinating activity of the protein. Furthermore, we found novel hemolytic and cytotoxic activities of the lectin, which resided in the carboxy-terminal region of the protein. Antibodies against Igl inhibited the hemolytic activity of Entamoeba histolytica trophozoites. This is the first report showing hemagglutinating, hemolytic and cytotoxic activities of an amoebic molecule, Igl.

ECOS TM competent BL21(DE3) cells (Nippon Gene Co.) were transformed with plasmids containing a DNA fragment coding NM-Igl (aa 14 to 753 of Igl) ligated in a pET19b vector (Novagen). The primer sets are shown in Table 1. The gene transformed BL21(DE3) cells were cultured in 2 ml of LB medium containing ampicillin (100 µg/ml medium) in a multi-shaker incubator at 37°C overnight. The turbid culture of E. coli was transferred to a 2×YT medium and incubated at 37°C until the optical density (OD 600 ) reached 0.6.
Isopropyl-β-D-thiogalactopyranoside (IPTG, 0.25 mM) was added to the culture, incubated at 22°C for 20 h, and centrifuged at 6500×g for 15 min. The pellet of E. coli was washed with 1×IB Wash Buffer (20 mM Tris-HCl pH 7.5, 10 mM EDTA, 1% Triton X-100) from a Protein Refolding Kit (Novagen) and resuspended in the same buffer supplemented with Benzonase (Sigma, 1 µl/100 ml culture). To the sample, 100 µg/ml of Lysozyme (Thermo) was added and incubated at 30°C for 15 min. Sonication of the sample was conducted using a Sonifier SLPe Digital Ultrasonic Homogenizer (Branson) and the sample was centrifuged at 10,000×g for 10 min at room temperature after it was no longer viscous. The pellets of inclusion bodies of the recombinant protein were suspended with Solubilization Buffer (50 mM CAPS, pH 11.0, supplemented with 0.3% N-lauroylsarcosine and 1 mM DTT), incubated at room temperature for 15 min, and centrifuged at 10,000×g at room temperature for 10 min. The supernatant containing the solubilized protein was dialyzed in a cellulose membrane tube (20/32, Sanko Junyaku Co., Ltd., Tokyo, Japan) against a dialyzing buffer (20 mM Tris-HCl, pH 8.5) supplemented with 0.1 mM of DTT) at 4°C overnight through two changes of the buffer. Dialysis was continued in a dialyzing buffer without DTT for 6 h (3 h each) and then in redox refolding buffer (dialysis buffer supplemented with 0.2 mM oxidized glutathione and 1 mM reduced glutathione) at 4°C overnight to refold the protein and to promote disulfide bond formation. The sample was further dialyzed for a further 3 h at room temperature to enhance disulfide exchange rates. These processes were conducted according to the instructions in the Protein Refolding Kit (Novagen).

Ni column purification of a recombinant NM-Igl protein
His-tagged refolded NM-Igl was purified using a Ni column. The protein solution (5 ml) was batched with 1 ml of gel slurry of Ni-NTA agarose (Qiagen) at 4°C overnight. The mixture was loaded onto hand-made columns. After collecting the flow through fraction, the column was washed three times with 1 ml of PBS containing 10 mM imidazole.
Recombinant NM-Igl protein was eluted with PBS containing 200 mM imidazole (1 ml/fraction). Five eluted fractions from each run were collected and applied to SDS-PAGE gels. Fractions containing the recombinant protein were pooled and buffers were changed to PBST (PBS with 0.05% Tween 20) with Amicon Ultra Ultracel-10K (Millipore) before use in further studies. Protein concentrations were measured using BioRad protein assay reagent.

SDS-PAGE and Coomassie Brilliant Blue staining of purified recombinant proteins
Recombinant proteins (1 µg each) were mixed with a one-third volume of SDS sample buffer (Invitrogen) and subjected to SDS-PAGE using NuPAGE Novex Bis-Tris (4-12% gradient) gel in reducing condition. Electrophoresis was conducted for 40 min at 200 V.
For Coomassie staining, the gel was treated with SimplyBlue Safe stain solution (Invitrogen) and incubated until blue bands appeared on the gel. The gel was washed with distilled water and photographs were taken.
Hemolytic assay using recombinant lectins NM-Igl or C-Igl (2 µM) in PBST (total volume 50 µl) was loaded on to a 96U Nunclon microwell plate (Nunc). HoRBCs in PBS (2% v/v) were prepared as described above and 50 µl/well of the RBC solution was added to the Igl-preloaded wells at room temperature.
Hemolytic activities of the Igls were evaluated after RBC loading and images were taken at several time points.

Detection of released hemoglobin concentration
A Hemoglobin B Test Kit (Wako, Osaka, Japan) was used to quantify the hemolytic activity of each recombinant protein. The concentration of hemoglobin released by hemolysis in the supernatants of RBCs and in the recombinant protein mixtures described above were measured after incubation for 8 h. The results are expressed as a mean of 5 experiments with the standard deviation (SD).

Trypan blue exclusion assay of F-Igl treated Caco-2 cells
Caco-2 cells (ATCC, HTB-37) were cultured in MEM basic medium (Gibco) supplemented with Earle's salts, L-glutamine and 20% fetal bovine serum. After detachment with 0.25% Trypsin-EDTA (Gibco), the cells were cultured in a 96-well plate at approximately 2×10 4 cells/100 µl/well at 37°C under 5% CO 2 for 24 h. Then the medium was changed to 100 µl of medium with 1 µM F-Igl or PBST containing 200 units/ml penicillin G and 200 µg/ml streptomycin (Wako, Japan). The cells were incubated for an additional 1 or 12 h under the same conditions. After collecting the culture supernatants, 100 µl of fresh medium with 5 µl of 0.4% trypan blue was loaded to each well and the viability of attached cells was assessed. One microliter of 0.4% trypan blue was added to the collected culture supernatant and the viability of detached cells was also assessed.
Those samples were observed under EVOS XL microscopy.

Lectin activity assay using neoglycoprotein coated plate
Tn Antigen-HSA, human serum albumin conjugated with the average of 19 GalNAc residues per a molecule (Dextra Laboratories, UK), was coated to a F96 MAXISORP plate (Nunc, Denmark) at 10 pmol/well at 4°C overnight. HSA (Sigma Aldrich, St. Louis, MO) was used as a control. After washing with TBST for 3 times, the plate was blocked with 3% BSA at 4°C overnight. Two-fold serial diluted recombinant Igls in TBST (from 50 pmol/ well) were loaded on to the plate after washing the plate with TBST and the plate was incubated at 4°C overnight. Bound recombinant Igls were detected after incubated with a mouse anti-His tag antibody (Novagen, San Diego, CA; 1: 1000 dilution in TBST), a HRP conjugated rabbit anti-mouse IgG (H+L) (abcam, Tokyo, Japan; 1: 5000 dilution in TBST) and 1-step ultra TMB ELISA solution (Thermo, Rockford, IL). The reaction was stopped by adding 50 µl of 1 M H 2 SO 4 and absorbance of the samples at 450 nm was measured.
Two-fold serial diluted recombinant Igls (from 10 pmol/well) were coated to a F96 MAXISORP plate and detected by the same antibodies to confirm that the same concentrations of the Igls were equivalently detected in this assay.