IndexIntroductionINTRODUCTIONMaterials and MethodsPreparation of Trypsinized ErythrocytesHemagglutination (HA) AssayHapten Inhibition AssayEffect of pHAmmonium Sulfate PrecipitationSDS-PAGEProtein EstimationResultsCGL lectin is strongly inhibited come on mucin glycoproteins.Discussion ConclusionAltered expression of cell surface glycans can act as markers of various diseases including cancer and AIDS. Identification of these altered glycans can be easily achieved using glycan binding proteins, particularly antibodies and lectins. Therefore, it is always important to identify and isolate new lectins with varying carbohydrate specificity that can be used as diagnostic markers of different diseases. The present study describes the isolation and specificity of lectin carbohydrates from Calotropis gigantea seeds. The lectin Calotropis gigantea (CGL), has shown blood group nonspecificity and is strongly inhibited by glycans of the mucin glycoprotein. Precipitation of the crude extract of Calotropis gigantean with ammonium sulphate results in a concentration of the haemagglutination activity at a saturation of 30-60%. The lectin maintained its activity when exposed to temperatures up to 50°C for 1 hour. Since Calotropis gigantean is commonly used as a medicinal plant, the lectin contained in this plant can be exploited for hematological applications and to purify glycoproteins. Say no to plagiarism. Get a tailor-made essay on "Why Violent Video Games Shouldn't Be Banned"? Get an original essayIntroductionINTRODUCTIONVarious key biological processes, including cell-cell interactions, cell migration, induction of apoptosis, molecular trafficking, receptor activation, signal transduction and endocytosis are invariably mediated by ligands of carbohydrates (Zeng et al. 2012). Understanding the qualitative and quantitative expression of these glycans that tend to change under various conditions of the cell provides useful information on whether the cell is normal or diseased along with their mechanisms. Among the different molecules that recognize carbohydrates both qualitatively and quantitatively are lectins (Sharon and Lis 2004). Lectins are carbohydrate-binding proteins of nonimmune origin that recognize glycans specifically expressed on the cell surface or free in solutions. This glycan recognition property of lectins has been exploited in several fields of life sciences (Sharon and Lis 2004). Some lectins specifically bind to tumor-associated carbohydrates and therefore have the potential to serve as biomarkers to distinguish between normal and cancerous condition of mammalian cells. Many of these specific glycans are considered disease markers and are targets for both diagnosis and therapy (Brockhausen I. 2006). Plant-derived lectins were the first proteins of this class to be studied, and to date most of the lectins studied so far come primarily from plant sources. Since the discovery of the first lectin from castor bean by Stillmark in 1888, many lectins have been reported from almost all parts of plants (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, many hypothesized roles have been assigned to plant lectins "as storage proteins", "as defense molecules", in symbiosis. A number of lectins have been isolated from plant storage tissues (seeds or vegetative storage tissues) where they constitutea very high percentage of the total protein content in the tissue (Van Damme et al. 1995). Some plant lectins have been implicated in plant defense mechanisms (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 has been undertaken to screen weeds for the presence of lectin activity and to isolate them from the same source. The study describes the isolation and partial purification of the lectin from Calotropis gigantea and its specificity in carbohydrates. Materials and methods Seeds of Calotropis gigantea were collected during the month of March from botanical garden, Karnatak University, Dharwad. The seeds were separated and used for the extraction of lectin, EDTA, trypsin, bovine serum albumin (BSA), ammonium sulfate, Folin-Ciocalteau reagent, sodium dodecyl sulfate, acrylamide, N,N1-methylene-bis-acrylamide, N,N,N1, N1-tetra methyl ethylenediamine (TEMED) and Commassie brilliant blue were from Sisco Research Laboratory or Himedia Laboratory, India. Sugars used for hapten inhibition studies were from Sigma Chemicals, USA. All other chemicals, plastic items and glassware are analytical grade unless 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. Subsequently, the seeds were homogenized (5 g 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 (phenylmethylsulfonylfluoride). The extraction procedure was carried out overnight at 4ºC. The extract was filtered through a muslin cloth and clarified by centrifugation at 8000 RPM for 15 minutes at 4ºC. The supernatant was stored at 4°C until further analysis. A similar procedure has also been adopted for other plant seeds. Preparation of trypsinized erythrocytes Human blood with different blood groups (A, B, and O) was collected in 1 ml of 4% sodium citrate solution. The erythrocytes were separated by centrifugation at 1500 rpm for 5 minutes. Erythrocytes were washed three times with saline and finally in PBS and adjusted to an OD of 2.5 at 660 nm. The total volume is measured and the final concentration of 0.025% trypsin was added and incubated at 37 ºC for 1 hour. Excess trypsin was removed by repeated washing in saline and finally adjusted to OD 3.5 at 660 nm and used for hemagglutination assay and inhibition assays. Hemagglutination (HA) test To perform the hemagglutination test, 96-well U-bottom microtiter plates were used. Initially, 50 µl of saline solution was added to all wells of the respective rows. Subsequently, 50 ul of the analysis solution was added to the first well of each row and a double serial dilution was carried out up to the eleventh well. 50 ul were discarded from well 11. Trypsinized erythrocytes of each blood group were added (50 µl per well) to each row of the plate. For each blood group and sample, wells containing only saline and erythrocytes were included as negative controls. Plates were incubated at room temperature for 1 hour and visualized. The plates have been photographed and aregeometric mean titers (GMT) were calculated. The highest dilution of the extract causing visible agglutination was arbitrarily regarded as the “titer” and the minimum protein concentration required for agglutination was regarded as MCA which is equivalent to “one unit of haemagglutinating activity (1 HAU ). The specific hemagglutination activity was expressed as activity in 1 mg (unit mg -1) of protein. Hapten Inhibition Assay Inhibition tests were performed by incubating the lectin sample in serially diluted sugar/glycoprotein prior to the addition of erythrocytes in 25 µl of test solution. The lowest concentration of the sugar/glycoprotein that inhibited agglutination was taken as the hapten inhibitory titer. In the tenth well, saline solution is added in place of sugar/glycoprotein solutions, while in the eleventh well, saline solution is added in place of the lectin. These wells served as positive and negative controls for the inhibition studies, respectively. The 12th well served as a regular control and had received only 50 ul of saline and erythrocyte suspension. The wells were mixed and incubated for 1 hour at room temperature, then 50 µl of erythrocyte suspension was added and further incubated for 1 hour at room temperature. Finally, the inhibition of lectin activity was visualized and photographed as previously described, and for each sugar/glycoprotein the minimum inhibitory concentration (MIC) was determined, defined as “the lowest concentration of sugar/glycoprotein, which has inhibited agglutination". Effect of pHIn To know the optimal pH for lectin activity, lectin was extracted in a different buffer with different pH. For the extraction, the same procedure described above was followed, containing appropriate protease inhibitors and sodium chloride. Various buffer systems used to achieve 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 lectin activity using trypsinized erythrocytes. Precipitation with ammonium sulphate The crude extract was subjected to precipitation with ammonium sulphate [(NH4)2SO4] at 0-30, 30-60 and 60-90%. Ammonium sulfate was added at room temperature, and the precipitated proteins were separated by centrifugation at 8000 rpm for 30 minutes. The supernatant was stored while the precipitate (residue) was dissolved again in 2 ml of PBS. Both the precipitate and supernatant were extensively dialyzed against PBS, and hemagglutination activity was determined in all fractions. SDS-PAGE protein samples from the crude extract and ammonium sulfate precipitations were separated on 15% acrylamide gels. The protein sample was treated with 6x SDS buffer and boiled for 5 minutes at 100℃. Cooled and the protein was loaded into the wells and electrophoresed at 80 V for 4 hours. After completion of electrophoresis, gels were stained with Comassie brilliant blue R-250. A standard protein ladder with molecular weight ranging from 14.3 to 97.4 kDa was also processed and electrophoresed under similar conditions. Estimation of proteins The protein content in various phases, including crude extracts, was estimated according to the protocol described by Lowry et al., (LOWRY et al. 1951). Results Among the different weed seeds, only the seeds of Calotropis gigantea showed the highest hemagglutination activity (Titer-16) as determined by double serial dilution technique using rabbit erythrocytes (Table 1). In addition to Calotropis gigantean, Lantana camara seeds also showed activityhaemagglutinating but with a lower titer (04). Since the highest hemagglutination activity was observed in the Calotropis gigantean plant, further studies were conducted using this plant for lectin isolation, hapten inhibition test, etc. Calotropis gigantean lectin (CGL) recognized all erythrocytes of the blood group equally. Since the lectin agglutinated rabbit erythrocytes, the next human erythrocytes of blood groups A, B and O were used for the test and it was found that CGL did not discriminate between erythrocytes of blood groups A, B and O. However, the lectin bound with variable intensity and recognized the erythrocytes of the blood group “O” with maximum titer (64) while the erythrocytes of the blood group “B” with minimum titer (08). These results are presented in Figure 1. For further studies, blood group O erythrocytes were used due to the easy availability of red blood cells. The lectin CGL is strongly inhibited by mucin glycoproteins. To determine the carbohydrate specificity of the lectin, various monosaccharides, disaccharides, and glycoproteins have been used. to perform the hapten inhibition test. The list of different sugars and glycoproteins used for this assay is given in Table 2. As presented in Figure 2, the hemagglutination activity of CGL lectin was strongly inhibited by mucin followed by fetuin. Lectin activity was not inhibited by any of the monosaccharides and disaccharides tested. These results indicate that the lectin is not specific for simple sugars but recognizes complex sugars present in mucin or fetuin glycoproteins. This may be another reason why this lectin is not blood type specific. The lectin is stable at different temperatures. To determine the stability of lectin activity at different temperatures, the lectin was extracted and incubated at different temperatures for 1 h, and then the hemagglutination activity was determined. As illustrated in Fig. 3, the lectin showed constant stability in its activity from 40°C to 60°C. Although the titer decreased in the 40°C-60°C treatments, the same activity remained for several days. This could be due to the inactivation of the proteases present in the extract. Furthermore, lectin activity remained stable for at least 7 days when kept at room temperature. The maximum hemagglutination activity of CGL was found to be 30-60% of ammonium sulfate saturation. Subsequently, precipitation of the crude extract was performed with ammonium sulfate to fractionate the proteins. The results of ammonium sulfate precipitation are presented in Figure 4. The results indicate that the lectin concentration increased in 30-60% of the ammonium sulfate precipitated fraction, as evidenced by increased hemagglutination activity (titer-64) . It is evident from Fig. 4 that some of the contaminated proteins can be removed in this step. The 0-30% fraction showed some hemagglutination activity with the 08 titer. This could be due to the residual presence of lectin in this fraction. Although a good amount of protein was precipitated in the 60–80% fraction, it did not show any hemagglutination activity. SDS-PAGE analysis of the partially purified lectin was performed. SDS-polyacrylamide gel electrophoresis of the crude sulfate- and ammonium-precipitated fractions was performed to analyze the number of proteins present in the samples. As shown in Figure 5, after 30–60% ammonium sulfate precipitation, the number of protein bands was significantly reduced (Lane-3) compared to the raw sample (Lane-1)...