The axenic strain of Entamoeba histolytica (HM1: IMSS) which was grown in TY-I-S-33 medium was used in the study for preparation of Heparan Sulphate Binding Proteins (HSBPs). Before harvesting, the cells were inoculated in serum free medium for 48 hours. Trophozoites were harvested at 48 - 72 hour (mid-log phase) by chilling and after pelleting at 150× g for 5 min, the cell pellet was lysed in 10 mL of lysis buffer containing 150 mM NaCl, 50 mM Tris, 0.5% (v/v) Nonidet P-40 (Sigma, USA) and 20 µl protease inhibitor cocktail (Sigma, USA). The lysed amoebic trophozoites were microcentrifuged at 10,000 g for 10 min. The supernatant was stored at −20˚C and was used for further protein isolation by ammonium sulphate precipitation.

The proteins were precipitated from the culture supernatants with ammonium sulphate (NH₄)₂SO₄ at 40%, 60%, 80% and 100% saturation. The proteins precipitated with each concentration of ammonium sulphate were centrifuged (18,000 g, 30 min, 5˚C) and finally suspended in distilled water. The proteins were dialyzed against four changes of 0.01 M ammonium bicarbonate using Spectra/POR^Ò Membrane with molecular weight cut off 6 - 8 kDa. The measurement of protein content in each preparation was carried out by using bicinchonic acid protein assay [15] using protein estimation kit from Bangalore Genei Pvt. Ltd. India, as per the manufacturer’s instructions. All the preparations were stored at −20˚C until used.

The proteins were purified by Protein purify with Heparin Hi-Trap column (Amersham Biosciences Ltd., UK) as described [16] . The ligand in Heparin Sepharose High Performance is a naturally occurring sulfated glucosaminoglycan which is extracted from the native proteoglycan of porcine intestinal mucosa. Heparin consists alternating units of uronic acid and D-glucosamine, most of which are substituted with one or two sulfate groups. The molecular weight of the polymer is distributed over the range 5000 - 30,000. It is covalently coupled to highly cross-linked agarose beads. The coupling method gives high capacity and high performance. The medium is stable over the pH range 5 - 10, and tolerates all commonly used aqueous buffers.

Briefly, protein samples obtained with various concentrations of ammonium sulphate (40% - 100%) were filtered through a 0.45 μm membrane filter and diluted 1:1 with 0.1 M sodium acetate (pH 5.0). One mL of diluted sample was applied to a 5 mL Heparin Hi-Trap column, previously equilibrated with 0.1 M sodium acetate buffer (pH 5.0). Proteins lacking affinity for heparin were washed out with 0.1 M sodium acetate buffer (pH 5.0) at a flow rate of 1 mL/min. Adsorbed proteins were eluted with NaCl (0.25 and 0.5 M) over 30 min at the same flow rate and 1 mL fractions were collected. Finally, the column was washed with 0.01 M NaOH and then was regenerated with distilled water and 0.1 M sodium acetate buffer. The fractions collected were dialyzed extensively against 10 mM ammonium bicarbonate and stored at −20˚C until used.

After affinity chromatography, the protein preparations were electrophoresed [17] , using electrophoresis apparatus (Mini-PROTEAN^R II Electrophoresis System, Bio-Rad, USA). Protein samples were denatured before electrophoresis for 5 min at 100˚C in sample buffer (0.6 M Tris-HCl (pH 6.8), glycerol 10%, SDS 10%, β-metacaptoethanol 5% and bromophenol blue 0.05%). Samples containing 5 - 10 µg of total protein and molecular mass standard were loaded on to a discontinuous acrylamide gel (separating gel 12%, stacking gel 4%), electrophoresis was done at 80 V for 2 h and the gel was stained with Coomassie blue to visualize the proteins.

For immunoblotting, Heparan Sulphate (Sigma) was conjugated with Horseradish Peroxidase (HRP) by Periodate Oxidation Method [18] . In brief, 0.1 M solution of sodium periodate (NaIo₄) was prepared in freshly distilled water and 0.2 mL of this solution was added to 1 mL of 5 mg/mL HRP solution in distilled water. The solution was stirred gently for 20 minutes at room temperature. The pH of the solution was raised to 9.5 by addition of 25 μL of 0.2 M sodium carbonate. One mL of heparan sulphate (Sigma) solution (18 mg/mL) in 0.01 M Na₂Co₃, pH9.5 was added. The mixture was then put on an end to end shaker for 2 hours at RT. After 2 hours, 0.1 mL of sodium borohydride (4 gm/mL) was added and was kept at 4˚C for 2 hours. Finally the mixture was stored at −20˚C till use.

For immunoblotting, the purified proteins on SDS-PAGE gels were electro transferred onto nitrocellulose membrane by Mini Trans―Blot Electrophoretic Transfer Cell (Bio-Rad, USA). Immunoblotting was performed according to the method adopted by Bustos et al., 2000 using heparan sulphate―HRP conjugate (Sigma, USA). Conjugate bound to heparan sulphate binding proteins on nitrocellulose membranes was visualized with DAB (3,3’-diaminobenzidine) 2.5 mg and H₂O₂ (2.5 μl) in 10 mL of 0.1 M Sodium acetate, pH 5.0. The reaction was stopped with 0.1 M Sodium metabisulphite substrate solution and the results were interpreted on a Gel-Doc System (Uvi pro, UK) using UVI-Photo MW software.

1) 2-D Gel Electrophoresis

Separation of proteins in first dimension by isoelectric focusing was performed using the Ettan IPGphor 3 Isoelectric focusing system (GE Healthcare Bio-Sciences, UK). Fifty μL of HSBP antigen preparation, 20 μL of carbamylate marker and 60 μL Rehydration buffer was added to micro centrifuge tubes. Final volume of the mixture was adjusted to 130 μl using rehydration buffer containing 0.02% (w/v) Dithiothreitol as recommended. Rehydration buffer containing sample was added to the strip holder at the anode. The strip holder was placed on Ettan IPGphor 3 Isoelectric focusing system (GE Healthcare Bio-Sciences, UK). The running protocol was rehydration at 20˚C for 12 hours at 50 μA per strip followed by isoelectric focusing at 50 μA per strip as follows:

Step 1: 500 V for 5 hours,

Step 2: 4500 V for 1.5 hrs (gradient) and,

Step 3: 4500 V for 7 - 10 hrs till 14000 V hrs.

For the equilibration of the strip, 10 mL of fresh equilibration buffer with 100 mg DTT was prepared for each strip. Strip was incubated in equilibration buffer for 15 min on constant rocker.

For separation of proteins in second dimension based on molecular weight 15% separating acrylamide gel was prepared. The strip was placed across the top of the gel making sure that the strip is in touch with the gel and air bubbles between the strip and the top of the gel were removed. The warm 1% agarose made with 1X running buffer containing bromophenol blue was added. The agarose was allowed to cool and solidify, before moving to the electrophoresis apparatus. Running buffer (1X) was added to the upper and lower buffer chambers and the gel was placed inside the apparatus. Gel was run for 1.5 - 2 hrs at 15 - 20 mA. Gel was removed and stained with Coomassie Brilliant Blue G-250. The results were analyzed using PD Quest software (Bio-RAD).

2) MALDI TOF Analysis

Further characterization of these HSBPs was done by MALDI-TOF. As the spots in 40% (NH₄)₂SO₄ precipitated lysate showed the wider range in terms of pH (Figure 1(a)), these spots were further processed for MALDI-TOF. In brief, the proteins spots were excised from the 2D gel. The fragments were incubated with 50% acetonitrile (ACN) and dried in a vaccum centrifuge. Trypsinization of proteins was done with porcine trypsine and 5% ACN overnight at 37˚C. Gel fragments were sonicated to provoke the release of peptides. The organic phase containing the peptides was re-extracted. For MALDI-TOF mass spectrometry, samples were spotted on a MALDI target plate mixed with matrix (4x-cyano-4-hydroxycinnamic acid solution in 1 mL, 1:1 ACN:ethanol) and dried. Before analysis, the system was calibrated according to standards supplied by the company (Bruker Daltonics Flex). MALDI-TOF spectra was acquired on a Reflex IV in positive reflectron mode in the m/z 500 - 3000 range [19] .

Varying amounts of proteins were obtained from culture lysates precipitated with different concentration of ammonium sulphate i.e. with 40%, 60%, 80% and 100% followed by dialysis and protein purity with Heparin Hi trap column in E. histolytica (Table 1).

To get the high protein concentration, the two elutions (0.25 M NaCl and 0.5 M NaCl) of E. histolytica lysates precipitated with each concentration of ammonium sulphate were pooled for further analysis by SDS-PAGE and immunoblotting.

SDS PAGE analysis of purified eluted proteins revealed that in E. histolytica all the preparations except the protein precipitated with 100% ammonium sulphate showed protein bands capable of binding to heparin Hi-trap columns.

Immunoblotting analysis with heparan sulphate conjugated with horse radish peroxidase showed the presence of two proteins with heparan sulphate binding

^a: Ammonium Sulphate; ^b: Concentration; ^c: Sodium Chloride.

activity at 51.2 kDa and 61.0 kDa both in 40% and 80% preparations in E. histolytica.

PD Quest software (Bio-RAD) was used to analyze the isoelectric points of the protein spots obtained in the 2D-PAGE (Amersham Biosciences, Ltd., UK). On 2-D Gel electrophoresis analysis of HSBP obtained at 51.2 kDa in 40% (NH₄)₂SO₄ precipitated lysate, 3 spots were identified as indicated by arrows in Figure 1(a). These spots appeared in the region of pH between 3 and 4 which indicated three isoforms of this protein (Figure 1(a)). Another 2 spots were also seen at 61.0 kDa. These had isoelectric point between pH 4 and 7 (Figure 1(a)).

In 80% (NH₄)₂SO₄ precipitated lysate of E. histolytica, 51.2 kDa HSBP showed only one spot which had isoelectric point at pH 3 (Figure 1(b)). However, 61.0 kDa HSBP revealed 2 spots which appeared in the pH range of 4 and 5 (Figure 1(b)).

The mass spectrums obtained after MALDI-TOF analysis of 40% (NH₄)₂SO₄ precipitated HSBPs of 51.2 kDa (around pH 3 i.e. 3 spots) and 61.0 kDa (between pH 4 and 7 i.e. 2 spots) are represented in Figures 2-4 and Figure 5 and Figure 6 respectively.

The data of trypsinized protein mass fragments obtained from MALDI-TOF was analyzed with Matrix Science i.e. with MASCOT and was further searched on various protein search engines like Uniport, Swissprot and NCBI. The analysis revealed that peptide mass fragments of this protein were not available in the Data Basics. Thus, the current data of the proteins from all the five spots did not match with any previous data of available proteins from eukaryotes. Peptide mass fingerprinting of the Heparan Sulphate Binding Proteins of Entamoeba histolytica is not submitted to the above mentioned sites so far. The results indicated that it is a novel protein. This led to the conclusion that this protein is identified first time in our experiments.

However, immunogenicity and protective efficacy of this antigen is already reported by us [20] .