Isolation of Blood Group Non-specific Lectin from Calotropis Gigantean Seeds

Altered expression of glycans on the cell surface can act as markers of various diseases including cancer and AIDS. Identification of these altered glycans can be easily achieved by using glycan binding proteins, specifically antibodies and lectins. Therefore, it is always important to identify and isolate new lectins with varied carbohydrate specificity which can be used as diagnostic markers of different diseases. The present study describes the isolation and carbohydrate specificity of lectin from Calotropis gigantea seeds. Calotropis gigantea lectin (CGL), showed blood group non-specificity and strongly inhibited by glycans of mucin glycoprotein. Ammonium sulphate precipitation of Calotropis gigantean crude extract results in concentration of hemagglutination activity at 30-60% saturation. Lectin retained its activity when exposed to as high as 50°C for 1 h. Since Calotropis gigantean is commonly used as medicinal plant, lectin from this plant may be exploited for haematological applications and to purify glycoproteins.

IntroductionINTRODUCTION

Various key biological processes including cell-cell interactions, cell migration, induction of apoptosis, molecular trafficking, receptor activation, signal transduction and endocytosis are invariably mediated by carbohydrate ligands (Zeng et al. 2012). Understanding the qualitative as well as quantitative expression of these glycans which tend to change at various condition of cell, provides useful information on whether the cell is normal or diseased along with their mechanisms. Among different molecules that recognize carbohydrates both in qualitative and quantitative manner are lectins (Sharon and Lis 2004). Lectins are the carbohydrate binding proteins of non-immune origin which recognize glycans that are specifically either expressed on cell surface or free in solutions. This glycan recognition property of lectins has been exploited in different fields of life sciences (Sharon and Lis 2004). Some lectins bind specifically to tumor associated carbohydrates and therefore have the potential to serve as biomarkers to differentiate between normal and cancerous condition of mammalian cells. Many of these specific glycans are considered as disease markers and are targets for diagnosis as well as for therapeutics (Brockhausen I. 2006). Lectins from plant sources were the first proteins of this class to be studied and to date most of the lectins studied so far are mainly from plant sources. Since the discovery of the first lectin from castor bean by Stillmark in 1888, many lectins from almost all parts of plants have been reported (Goldstein and Poretz 1986). Although numerous plant lectins have been studied for their great structural detail, the physiological role of these proteins is still poorly understood. Recently, there are many speculated roles for plant lectins ‘as storage proteins’, ‘as defense molecules’, in symbiosis have been assigned. A number of lectins have been isolated from storage tissues in plants (seeds or vegetative storage tissues) where they make for a very large proportion of the total protein content in the tissue (Van Damme et al. 1995). Some plant lectins have been implicated in defence mechanism of plants (Mirelman et al. 1975). In contrast, some plant lectins are involved in cell wall extension and recognition (Barre et al. 1996).

Considering the application of lectins in various fields such as immunology (Ashraf and Khan 2003), cancer biology (Gastman et al. 2004), microbiology (Oppenheimer, Alvarez and Nnoli 2008), insect biology (Fitches et al. 2010), current research work was undertaken to screen weed plants for the presence of lectin activity and to isolate from the same source. The study describes the, isolation and partial purification of lectin from Calotropis gigantea and its carbohydrate specificity.

Materials and Methods

Calotropis gigantea seeds were collected during March month from botanical garden, Karnatak University, Dharwad. Seeds were separated and used for lectin extraction, EDTA, Trypsin, Bovine serum albumin (BSA), Ammonium sulphate, Folin-Ciocalteau reagent, Sodium dodecyl sulfate, Acrylamide, N,N1-Methylene-bis-acrylamide, N,N,N1,N1-tetra methyl ethylene diamine (TEMED), and Commassie brilliant blue were from either Sisco Research Laboratory or from Himedia Laboratory, India. Sugars used for hapten inhibition studies were from Sigma Chemicals, USA. All other chemicals, plastic wares, glassware are of analytical grade unless they are specified with company names.

Methods

Extraction of lectin from Calotropis gigantea seeds

To extract lectin, Calotropis gigantea seeds were collected, washed with distilled water and dried. Next, seeds were homogenized (5 gm in 25 ml) using mortar and pestle at room temperature with phosphate buffered saline (pH 7.2; 100 mM), containing 200 mM EDTA and 200 mM PMSF (Phenylmethylsulphonyl fluoride). The extraction procedure was carried out for overnight at 4ºC. The extract was filtered through muslin cloth and clarified by centrifugation at 8000 RPM for 15 min at 4ºC. The supernatant was stored at 4ºC till further analysis. Similar procedure was also adopted for other plant seeds.

Preparation of trypsinized erythrocytes

Human blood with different blood groups (A, B and O) were collected in 1 ml of 4% sodium citrate solution. The erythrocytes were separated by centrifugation at 1500 rpm for 5 min. Erythrocytes were washed three times with saline and finally in PBS and adjusted to an OD of 2.5 at 660 nm. Total volume is measured and final concentration of 0.025% trypsin was added and incubated at 37 ºC for 1 h. Excess trypsin was removed by repeated washing in saline and finally is adjusted to OD 3.5 at 660 nm and used for hemagglutination assay and inhibition assays.

Hemagglutination assay (HA)

To perform the hemagglutination assay, U-bottom 96-well micro titre plates were used. Initially, 50 ul of saline was added to all the wells of a respective rows. Next, to the first well of each row, 50 ul of assay solution was added and 2-fold serial dilution was made up to 11th well. From 11th well, 50 ul was discarded. Trypsinized erythrocytes of each blood groups were added (50 ul per well) to each row in the plate. For each blood group and sample, well containing only saline and erythrocytes were included as negative controls. The plates were incubated at room temperature for 1 h and visualized. Plates were photographed and the geometric mean titers (GMTs) were calculated. The highest dilution of the extract causing visible agglutination was arbitrarily considered as the “titre” and the minimum concentration of the protein required for agglutination was considered as MCA which equals to “one unit of hemagglutinating activity (1 HAU) The specific activity of hemagglutination was expressed as activity in 1 mg of (unit mg -1) protein.

Hapten inhibition assay

Inhibition assays were carried out by incubating the lectin sample in serially diluted sugar/glycoproteins prior to the addition of erythrocytes in 25 µl of assay solution. The lowest concentration of the sugar/glycoprotein, which inhibited the agglutination, was taken as the inhibitory titre of the hapten. To the 10th well, saline is added instead sugar/glycoproteins solutions, while in 11th well, saline is added instead lectin. These wells served as both positive and negative controls respectively for inhibition studies. 12th well served as regular control which had received only 50 ul of saline and erythrocyte suspension. Wells were mixed and incubated for 1hr at room temperature and then 50 ul of erythrocyte suspension was added and incubated further for 1 h at room temperature. Finally, Inhibition of lectin activity was visualized and photographed as described earlier and minimum inhibitory concentration (MIC) which is defined as “the lowest concentration of the sugar/glycoprotein, which inhibited the agglutination” was determined for each sugars/glycoprotein.

Effect of pH

In order to know the optimum pH for lectin activity, lectin was extracted in different buffer with varied pH. For extraction, same procedure was followed as described above containing appropriate protease inhibitors and sodium chloride. Various buffer system used for obtaining the desired pH are sodium acetate (pH 4.0), phosphate buffer (pH 7.2) and carbonate buffer (pH 9.5). After extraction, the clear extract was used to determine the lectin activity using trypsinized erythrocytes.

Ammonium sulphate precipitation

Crude extract was subjected to 0-30, 30-60 and 60-90% ammonium sulfate [(NH4)2SO4] precipitation. Ammonium sulfate was added at room temperature and precipitated proteins were separated by centrifugation at 8000 rpm for 30 minutes. Supernatant was saved while precipitate (residue) was re–dissolved in 2 ml of PBS. Both precipitate and supernatant were extensively dialysed against PBS and hemagglutination activity was determined in all fractions.

SDS-PAGE

Protein samples from crude extract, and ammonium sulfate precipitations were separated on 15% acrylamide gel. Protein sample was treated with 6x SDS buffer and boiled for 5 minutes at 100℃. Cooled and protein was loaded into the wells and electrophoresed at 80 V for 4 hours. After completion of electrophoresis, gels were stained with commassie brilliant blue R-250. A standard molecular weight protein ladder ranging from 14.3-97.4 kDa was also processed and electrophoresed under similar conditions.

Protein Estimation

The protein content in various steps including crude extracts were estimated according to the protocol described by Lowry et al., (LOWRY et al. 1951).

Results

Among different seeds from weed plants, only seeds of Calotropis gigantea exhibited highest hemagglutination activity (Titre-16) as determined by serial two-fold dilution technique using rabbit erythrocytes (Table 1). Apart from Calotropis gigantean, seeds of Lantana camara have also exhibited hemagglutination activity but with lower titre (04). Since maximum hemagglutination activity was observed in Calotropis gigantean plant, further studies were carried out using this plant for lectin isolation, hapten inhibition assay etc.

Calotropis gigantean lectin (CGL) recognized all the blood group erythrocytes equally.

Since lectin agglutinated rabbit erythrocytes, next human A, B and O blood group erythrocytes were used for the assay and found that CGL did not discriminate between A, B and O blood group erythrocytes. However, lectin did bind with varied intensity and recognized “O” blood group erythrocytes with maximum titre (64) while “B” blood group erythrocytes with least titre (08). These results are presented in Fig 1. For further studies, blood group O erythrocytes were used due to easy availability of RBCs.

CGL lectin is strongly inhibited by mucin glycoproteins.

To determine the carbohydrate specificity of lectin, various monosaccharides, disaccharides and glycoproteins were used to perform hapten inhibition assay. List of different sugars and glycoproteins used for this assay are given in table 2. As presented in Fig 2, hemagglutination activity of CGL lectin was strongly inhibited by mucin followed by fetuin. The lectin activity was not inhibited by any of the monosaccharides and disaccharides tested. These results indicate that lectin is not specific for simple sugars but recognizes complex sugars that are present in mucin or fetuin glycoproteins. This could be another reason why this lectin is blood group non-specific in nature.

Lectin is stable over different temperature.

In order to determine the stability of lectin activity over different temperature, lectin was extracted and incubated at different temperature for 1 h and then hemagglutination activity was determined. As depicted in Fig. 3, lectin exhibited steady stability in its activity from 40°C-60°C. Although the titre decreased in 40°C-60°C treatments, but same activity remained for several days. This may be due to inactivation of proteases that are present in the extract. Further, lectin activity was also stable for atleast 7 days when it was kept at room temperature.

Maximum hemagglutination activity of CGL was found in 30-60% of ammonium sulphate saturation.

Next, ammonium sulphate precipitation of crude extract was performed to fractionate the proteins. The results of ammonium sulphate precipitation are presented in Fig 4. Results indicate that lectin concentration has increased in 30-60% of ammonium sulphate precipitated fraction as evidenced by increased hemagglutination activity (titre-64). It is evident from the Fig. 4 that some of the contaminated proteins can be removed by this step. Fraction 0-30% showed some hemagglutination activity with titre 08. This may be due to residual presence of lectin in this fraction. Although good quantity of proteins was precipitated in 60-80% fraction, but it did not show any hemagglutination activity.

SDS-PAGE analysis of partially purified lectin.

SDS-Polyacrylamide gel electrophoresis of crude and ammonium sulphate precipitated fractions was performed to analysis the number of proteins present in the samples. As shown in Fig 5, after 30-60 % of ammonium sulphate precipitation, number of proteins bands were reduced significantly (Lane-3) compare to crude sample (Lane-1). The common protein bands that are present in all the fractions are near the molecular weight ranging from 40 to 50 kDa. These could be the proteins bands which may be associated with lectin activity.

Fold purification of lectin.

Since lectin activity was increased in ammonium sulphate precipitated fraction, fold increase in purification of lectin was calculated based on specific activity present in each step. Table. 3 summarizes the fold purification of lectin in each step. In accordance with SDS-PAGE, it is clear from the table 3 that there is removal of some of the contaminated proteins in 30-60% of ammonium sulphated fraction as evidenced by increase in specific activity by 5.7-fold.

Discussion

It has been well studied that different weed plants cause severe damage to economically important crops. There are many attempts to supress the growth of these weeds by different means. However, very few attempts were made to exploit these weed plants for beneficial purposes. In this context, we screened many weed plants for their presence of lectin activity and found that Calotropis gigantean exhibited maximum lectin activity with all the human blood group erythrocytes. Since Calotropis gigantean plant apart from being weed, it is widely used as medicinal plant (Kadiyala et al 2013), we carried out experiments on seeds of this plant to isolate and purify lectin from this plant.

Since crude extract of Calotropis gigantean did not agglutinate any of the human blood group specifically, it was assumed that lectin probably recognizing complex sugar that are present on the cell surface of erythrocytes. This prediction was confirmed by hapten inhibition assay which revealed that lectin indeed exhibited specificity towards O-linked glycans of mucin and fetuin. These results are in consistent with blood group non-specificity of lectin.

In order to know the stability of lectin, lectin extract was exposed to different temperature and found that lectin is stable up to 60°C and also when it was kept at room temperature for more than 7 days. This result suggested that lectin is less prone to protease attack making easy to operate during purification procedures. Many lectins are heat liable (Devi et al. 2011) however, CGL did not denatured at high temperature. Ammonium sulphate fractionation not only increased the lectin concentration, in addition it has also helped to remove most of the contaminated proteins that are present in crude extract. Electrophoretic pattern of crude and ammonium sulphate precipitated proteins revealed that, bands below 30 kDa are effectively removed during ammonium sulphate precipitation step.

Conclusion

The current study describes the isolation and partial purification of lectin from Calotropis gigantean seeds and its carbohydrate specificity. Given the fact that Calotropis gigantean is regularly used as medicinal plant, presence of lectin activity may have implication in its medicinal property. Although this prediction may be validated by further and detailed study, but current observation provides important information on lectin’s sugar specificity and its blood group non-specificity. Further, results from ammonium sulphate precipitation and SDS-PAGE steps also provides important information that this lectin could be purified by employing these steps coupled with other chromatographic techniques.