Mucin (porcine stomach, type III), fetuin (fetal calf serum), Cyanogen bromide, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) were procured from Co, St. Louis, USA. Sepharose 4B was obtained from Pharmacia Fine Chemicals, Uppsala, Sweden. Standard protein molecular weight marker was procured from Merck, India. Amicon Ultrafiltration centrifugation tubes 10 kDa were procured from Millipore, USA. Human blood samples were collected from German hospital, Dharwad. For cell culture, Dulbecco's Modified Eagle’s Medium F12 (DMEM F12) and foetal bovine serum (FBS) were obtained from Gibco Invitrogen (Paisley, UK), with Costar 96-well plates obtained from Corning Incorporate (Corning NY, USA). Bovine serum albumin (BSA), Fluorescein isothiocyante (FITC) were obtained from Sigma Chemical Co. (St. Louis MO, USA). ELISA kits for IL-6 (#MBS261259), IL-8(#MBS2886709) were obtained from My Biosources. All other reagents used were of analytical grade.
Asialofetuin, Asialomucin were prepared from fetuin as described by Spiro and Bhoyroo [18]. Mucin-Sepharose 4B affinity matrix was prepared by coupling fetuin to cyanogen bromide activated Sepharose 4B, according to the method of March et.al [19].
2.2 Cell culture
Immortalized human corneal epithelial cells (HCECs) were procured from ATCC and were grown and maintained in DMEM/F-12 media (Invitrogen) containing 10% FCS, at 37 ºC with 5% CO2 and used for the study.
2.3 Media and Growth conditions
Purpureocillium lilacinum was procured from Postgraduate Institute of Medical Education and Research, Chandigarh, India. Strains were maintained on potato dextrose agar plates containing agar concentrate 1.7% (w/v), stored at 4 ± 1ºC until further use. A loop full of fungal spores from the mother culture were inoculated on to freshly prepared potato dextrose broth in 500mL Erlenmeyer’s flasks and grown for different time points and were screened for the presence of lectin activity on different days at room temperature. The highest cell-associated hemagglutinating titer was observed with human erythrocytes on 5th and 6th day of post inoculation of the culture. Hence mycelial mat was harvested on 6th day post inoculation and used for lectin purification.
2.4 Lectin purification and characterization
Mycelial mat was harvested on 6th day of post inoculation and homogenized overnight at 4 ºC with phosphate buffered saline (0.05 M, pH 7.2) using motor and pestle. After extraction, centrifuged at 10,000 rpm for 20 mints at 4ºC and supernatant of the crude extract was tested for the presence of lectins/adhesins. The crude extract with lectin activity was then subjected for ammonium sulphate precipitation (70%) and kept for 1-2 hrs at 40C. The precipitates were centrifuged (10,000rpm, 20 min, 40C), dissolved in PBS and dialyzed against the same buffer. The dialysate was loaded on to Mucin Sepharose 4B affinity column pre-equilibrated with PBS. Unbound proteins were washed with PBS till absorbance of eluting fractions read zero at 280 nm. Affinity bound protein was eluted with 100mM Glycine-HCl buffer pH 2.0, containing 500mM NaCl. Fractions with high lectin activity were pooled and dialyzed extensively against PBS, pH 7.2. Lectin-positive fractions were concentrated using Amicon Ultrafiltration centrifugation tubes (10 kDa) and stored at -20 °C for further use. All purification steps were carried out in a cold room at 40C. At each step of purification, lectin activity was estimated by performing hemagglutination assay.
2.5 Preparation of trypsinized erythrocytes
Trypsinized human blood erythrocytes required for the determination of lectin activity by hemagglutination assay were prepared as described by E. Liener 1953 [20].
2.6 Hemagglutination Assay
Hemagglutination activity of PCL during various stages of purification was determined by the serial two fold dilution method using trypsinized human erythrocytes in a 96 well “U” bottom micro titer plates [20]. The highest dilution of the extract causing visible hemagglutination was regarded as the titre and the minimum concentration of the protein was required for agglutination (MCA) as one unit of hemagglutinating activity. The specific hemagglutination activity was expressed as unit mg-1protein.
2.7 Hapten Inhibition Studies
The sugar specificity of the purified lectin was determined by a hapten inhibition assay. Inhibition assays were carried out by incubating the lectin sample showing 4 titre with serially diluted sugar/glycoprotein in a total volume of 50 μL prior to the addition of 50μL of erythrocytes in PBS and the hemagglutination was visually observed. The minimum concentration of the sugar/glycoprotein required for complete inhibition was taken as the inhibitory titre of the hapten (MIC).
2.8 Estimation of protein:
Protein concentrations in the extract at different stages of purification were determined by Lowry’s method [21].and total sugar content of the lectin was estimated by phenol-sulphuric acid method using D-glucose as standard [22].
2.9 Preparation of FITC conjugated PCL
Fluorescein isothiocyanate conjugated lectin (FITC-PCL) required for the binding studies was prepared as described by Goldman [23].
2.10 SDS PAGE
SDS–PAGE of purified PCL and crude extracts was carried out using 15% gel as described by LaemmLi [24]. Molecular mass of the purified PCL was determined by comparison of its electrophoretic mobility with standard molecular weight marker proteins both under reducing and non-reducing conditions. The gel was stained both with Coomassie brilliant blue and silver staining [25].
2.12 Periodic Acid Schiff staining of PCL
In order to know the presence of carbohydrate in the lectin the gel after electrophoresis was stained with periodic acid Schiff reagent by the method of Zacharius et al [25]. Briefly, the gel was immersed in 12.5 % TCA for 30min. After rinsing the gel with water, it was immersed in 1% periodic acid prepared in 3% acetic acid and allowed to stand for 50 min. Then, excess periodic acid was removed by repeated washing of the gel with water. The gel was then transferred to fuschin sulphite stain and kept in dark for 50min.The gel was washed with sodium metabisulphite (0.5%) for 30min with three changes. Finally, the gel was destained by washing it several times with water and stored in acetic acid.
2.13 Effect of temperature and pH on PCL
In order to determine the thermo stability of the lectin , PCL (200 µg/mL) in PBS pH- 7.2 was incubated at different temperatures (0°C to 90°C) for 20 min in temperature controlled water bath. The stability of the PCL at various pH was determined by evaluating the hemagglutination activity of the lectin incubated in buffers of different pH (2.0 to 12.0). PCL (200µg/mL) was incubated with an equal volume of buffers (100 mM) of pH 2.0 (Glycine-HCl), pH 4.3 (Acetate buffer), pH 7.0-8.0 (Tris-HCl buffer) and pH 9.0-12.0 (Carbonate buffer) for 24h at 4°C. After incubation lectin samples were neutralized to pH 7.0 by adding 0.1N NaOH or 0.1N HCl before testing hemagglutination activity.
2.14 Effect of metal ions on PCL activity
The effect of divalent metal ions on lectin activity was assessed by extensive dialysis of PCL against 10 mM EDTA for 48 h at 4°C followed by deionized water for 48h [26]. The haemagglutination activity was tested before and after addition of 40 mM Ca2+, Mn2+, Mg2+, Al2+ and Fe2+ ions. Haemagglutination assay was performed at 0, 2, 4 and 24 h of incubation. Activity was expressed as percentage relative activity as compared to control (lectin incubated with 0.05 M phosphate buffered saline, pH 7.2 at 4ºC).
2.15 Cell Surface binding of PCL to HCECs by flow cytometry
Immortalized HCECs (0.2 x 106) were treated with 3% BSA in PBS for 1 h at 4 ºC for blocking the non-specific binding sites. HCECs were then washed and treated with lectins and lectin-hapten mix and allowed to bind for 1 h at 4 ºC. Lectin-hapten mix required for inhibition studies was prepared by incubating lectin (2µg/mL) with mucin (100µg/mL) and Chitin (200mM/mL) in PBS for 1 h at 37 ºC. After incubation, HCECs were washed twice with PBS and then processed for flow cytometry using Beckman Coulter FC500. Data was acquired for 10,000 events using lectin untreated cells as control and analysed using CXP Software.
2.16 Effect of PCL on viability of HCECs by MTT assay
To study the effect of PCL on HCECs, cells were seeded in 96-well plates (density 5 × 104 cells/mL) and grown in complete DMEM F12 medium for 48h prior to lectin treatment. Medium was replaced with serum-free DMEM F12 after 48h, then treated with PCL at different concentrations (0.15–10 μg/mL) and maintained in humidified atmosphere (37°C, 5% CO2) for 24h and 48h. At each time point, 50 μL of MTT (5 mg/mL) was added to each well followed by lysis with 100 μL dimethyl sulfoxide (DMSO). To observe effects of competing glycans, PCL (5μg/mL) was pre-incubated for 1h with Chitin (200mM/mL), before its addition to cells, and processed at 24h and 48h as mentioned above. Cell viability was quantified by measuring absorbance at 570 nm using a micro-plate reading spectrophotometer. Percentage viable cell number was calculated, by comparing with untreated controls, considered as 100%.
2.17 Quantification of interleukins secreted in PCL treated HCECs by ELISA
In order to monitor the expression of IL-6 and IL-8 in immortalized HCECs following PCL treatment for 24 and 48 h, spent media from lectin-treated cells were collected and further used for the quantification of interleukins. ELISA kit (MyBioSources) for detection of interleukins using different dilutions of the spent media was prepared by using the dilution buffer provided in the kit and IL-6 and IL-8 were estimated following the manufacturer’s instructions. Spent media from the lectin untreated cells were used as negative control. Data were represented as picogram of IL-6 and IL-8 per 100 µl of spent media.
Statistical analysis
Results were expressed as mean ± SD. Statistical comparisons were performed using the Student’s t-test in order to determine statistical significance. Microsoft Excel was used to perform statistical analysis.