Isolation of Non-Blood Group Specific Lectin from Calotropis Gigantean Seeds -PAGEProtein estimationResultsCGL lectin is strongly inhibited by mucin glycoproteins. clusionAltered expression of cell surface glycans can act as markers for various diseases, including cancer and AIDS. Identification of these altered glycans can be easily accomplished using glycan-binding proteins, particularly antibodies and lectins. Therefore, it is still important to identify and isolate novel lectins with varied carbohydrate specificity that can be used as diagnostic markers for different diseases. The present study describes the isolation and carbohydrate specificity of the lectin from Calotropis gigantea seeds. Calotropis gigantea lectin (CGL) showed blood group non-specificity and is strongly inhibited by mucin glycoprotein glycans. Ammonium sulfate precipitation of the crude extract of Calotropis gigantean results in concentration of hemagglutination activity at 30-60% saturation. The lectin retained its activity when exposed to a temperature of up to 50°C for 1 h. As Calotropis gigantean is commonly used as a medicinal plant, the lectin of 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 should not be banned”?Get the 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 carbohydrate ligands (Zeng et al. 2012). Understanding the qualitative and quantitative expression of these glycans, which tend to change depending on the state of the cell, provides useful information indicating whether the cell is normal or diseased as well as their mechanisms. Among the different molecules that recognize carbohydrates qualitatively and quantitatively are lectins (Sharon and Lis 2004). Lectins are carbohydrate-binding proteins of non-immune origin that recognize glycans specifically expressed on the surface of cells or free in solutions. This glycan recognition property of lectins has been exploited in different fields of life sciences (Sharon and Lis 2004). Certain lectins bind specifically to tumor-associated carbohydrates and therefore have the potential to serve as biomarkers to differentiate between normal and cancerous states of mammalian cells. Many of these specific glycans are considered disease markers and are both diagnostic and therapeutic targets (Brockhausen I. 2006). Lectins from plant sources were the first proteins of this class to be studied, and to date, most lectins studied thus far come primarily from plant sources. Since the discovery of the first castor lectin by Stillmark in 1888, numerous lectins from almost all plant parts have been reported (Goldstein and Poretz 1986). Although many plant lectins have been studied for their structural details, the physiological role of these proteins is still poorly understood. Recently, much speculation has been attributed to plant lectins “as storage proteins”, “as defense molecules», in symbiosis. A number of lectins have been isolated from plant storage tissues (seed or vegetative storage tissues) where they represent a very large proportion of the total protein content of the tissues (Van Damme et al. 1995). Some plant lectins have been implicated in the plant defense mechanism (Mirelman et al. 1975). On the other hand, certain 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), ongoing research work has been undertaken to detect the presence of lectin activity in weed plants and to isolate them from the same source. The study describes the isolation and partial purification of Calotropis gigantea lectin and its carbohydrate specificity. Material and methods Calotropis gigantea seeds were collected during the month of March from the botanical garden of 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-tetramethylethylenediamine (TEMED) and Commassie brilliant blue were from either 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 of analytical grade unless specified by company names. Methods Lectin extraction from Calotropis gigantea seeds To extract lectin, Calotropis gigantea seeds were collected, washed with distilled water and dried. Then, the seeds were homogenized (5 g in 25 ml) using a mortar and pestle at room temperature with phosphate-buffered saline (pH 7.2; 100 mM), containing 200 mM of EDTA and 200 mM PMSF (phenylmethylsulfonyl fluoride). The extraction procedure was carried out overnight at 4°C. The extract was filtered through muslin cloth and clarified by centrifugation at 8,000 rpm for 15 min at 4°C. 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 of different blood groups (A, B and O) was collected in 1 ml of 4% sodium citrate solution. Erythrocytes were separated by centrifugation at 1,500 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. The total volume is measured and a final concentration of 0.025% trypsin was added and incubated at 37ºC for 1 h. Excess trypsin was removed by repeated washes in saline and finally adjusted to an OD of 3.5 at 660 nm and used for the hemagglutination test and inhibition tests. Hemagglutination (HA) test To perform the hemagglutination test, U-bottom 96-well microtiter plates were used. Initially, 50 μl of saline was added to all wells of a respective row. Then, to the first well of each row, 50 µl of test solution was added and a serial dilution of 2 was performed until the 11th well. From the 11th well, 50 ul were discarded. 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 onesaline and erythrocytes were included as negative controls. The plates were incubated at room temperature for 1 h and visualized. Plates were photographed and geometric mean titers (GMT) were calculated. The highest dilution of the extract causing visible agglutination was arbitrarily considered the "titer" and the minimum concentration of protein required for agglutination was considered MCA which is equivalent to "one unit of hemagglutinating activity ( 1 HAU). The specific hemagglutination activity was expressed as activity in 1 mg of (unit mg -1) protein. Hapten Inhibition Assay Inhibition assays were performed by incubating the lectin sample in serially diluted sugar/glycoproteins prior to the addition of erythrocytes in 25 µl of test solution. The lowest concentration of the sugar/glycoprotein, which inhibits agglutination, was taken as the hapten inhibitor titer. In the 10th well, saline is added in place of the sugar/glycoprotein solutions, while in the 11th well, saline is added in place of the lectin. These wells served as positive and negative controls, respectively, for the inhibition studies. 12th well serving as a regular control which had only received 50 μl of saline solution and erythrocyte suspension. 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, inhibition of lectin activity was visualized and photographed as previously described and the minimum inhibitory concentration (MIC), defined as “the lowest concentration of the sugar/glycoprotein, which inhibited agglutination” was observed. was determined for each sugar/glycoprotein. In order to know the optimal pH for lectin activity, lectin was extracted in different buffers with varied pH. For extraction, the same procedure was followed as described above containing appropriate protease inhibitors and sodium chloride. Different 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. Ammonium sulfate precipitation The crude extract was subjected to ammonium sulfate [(NH4)2SO4] precipitation 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 min. The supernatant was retained while the precipitate (residue) was redissolved in 2 ml of PBS. 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 precipitation were separated on a 15% acrylamide gel. The protein sample was treated with 6x SDS buffer and boiled for 5 minutes at 100℃. Proteins were cooled and loaded into the wells and electrophoresed at 80 V for 4 hours. After electrophoresis was complete, gels were stained with Commassie Brilliant Blue R-250. A standard molecular weight protein ladder ranging from 14.3 to 97.4 kDa was also processed and electrophoresed under similar conditions. Protein estimation Protein content at various stages, including crude extracts, was estimated according to the protocol described by Lowry et al. (LOWRY et al. 1951). ).ResultsAmong the different weed plant seeds, only Calotropis gigantea seeds showed the highest hemagglutination activity(Titer-16), determined by a two-fold serial dilution technique using rabbit erythrocytes (Table 1). Besides Calotropis gigantean, Lantana camara seeds also showed hemagglutination activity but with a lower titer (04). Since the maximum hemagglutination activity was observed in the Calotropis gigantean plant, further studies were carried out using this plant for lectin isolation, hapten inhibition test, etc. Calotropis gigantean lectin (CGL) recognized all blood group erythrocytes equally. Since the lectin agglutinated rabbit erythrocytes, the following human erythrocytes of blood groups A, B and O were used for the test and found that CGL did not discriminate between erythrocytes of blood groups A, B and O. However, the lectin bound with varying intensity and recognized erythrocytes of blood group “O” with the maximum titer (64) while erythrocytes of blood group “B” with the lowest titer (08). These results are shown in Figure 1. For further studies, blood group O erythrocytes were used due to the easy availability of red blood cells. The CGL lectin is strongly inhibited by mucin glycoproteins. To determine the carbohydrate specificity of the lectin, various monosaccharides, disaccharides and glycoproteins were used. to perform a 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 the 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 could be another reason why this lectin is non-blood group specific in nature. 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 shown in Figure 3, the lectin exhibited constant stability of its activity between 40 °C and 60 °C. Although the titer decreased in the 40°C-60°C treatments, the same activity remained for several days. This may be due to the inactivation of proteases present in the extract. Additionally, lectin activity was also stable for at least 7 days when stored at room temperature. Maximum hemagglutination activity of CGL was found in 30–60% of ammonium sulfate saturation. Then, ammonium sulfate precipitation of the crude extract was performed to fractionate the proteins. . The results of the ammonium sulfate precipitation are shown in Figure 4. The results indicate that the lectin concentration increased in 30 to 60% of the ammonium sulfate precipitated fraction, as evidenced by the increase in hemagglutination activity (title 64). It is clear from Figure 4 that some of the contaminated proteins can be removed by this step. The 0-30% fraction showed some hemagglutination activity with titer 08. This may be due to the residual presence of lectin in this fraction. Although a good amount of protein was precipitated in a fraction of 60–80%, it did not show any hemagglutination activity. An SDS-PAGE analysis of partially purified lectin. SDS-polyacrylamide gel electrophoresis of the crude and sulfate-precipitated fractions..

Essay / Isolation of Non-Blood Group Specific Lectin from Calotropis Gigantean Seeds -PAGEProtein estimationResultsCGL lectin is strongly inhibited by mucin glycoproteins. clusionAltered expression of cell surface glycans can act as markers for various diseases, including cancer and AIDS. Identification of these altered glycans can be easily accomplished using glycan-binding proteins, particularly antibodies and lectins. Therefore, it is still important to identify and isolate novel lectins with varied carbohydrate specificity that can be used as diagnostic markers for different diseases. The present study describes the isolation and carbohydrate specificity of the lectin from Calotropis gigantea seeds. Calotropis gigantea lectin (CGL) showed blood group non-specificity and is strongly inhibited by mucin glycoprotein glycans. Ammonium sulfate precipitation of the crude extract of Calotropis gigantean results in concentration of hemagglutination activity at 30-60% saturation. The lectin retained its activity when exposed to a temperature of up to 50°C for 1 h. As Calotropis gigantean is commonly used as a medicinal plant, the lectin of 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 should not be banned”?Get the 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 carbohydrate ligands (Zeng et al. 2012). Understanding the qualitative and quantitative expression of these glycans, which tend to change depending on the state of the cell, provides useful information indicating whether the cell is normal or diseased as well as their mechanisms. Among the different molecules that recognize carbohydrates qualitatively and quantitatively are lectins (Sharon and Lis 2004). Lectins are carbohydrate-binding proteins of non-immune origin that recognize glycans specifically expressed on the surface of cells or free in solutions. This glycan recognition property of lectins has been exploited in different fields of life sciences (Sharon and Lis 2004). Certain lectins bind specifically to tumor-associated carbohydrates and therefore have the potential to serve as biomarkers to differentiate between normal and cancerous states of mammalian cells. Many of these specific glycans are considered disease markers and are both diagnostic and therapeutic targets (Brockhausen I. 2006). Lectins from plant sources were the first proteins of this class to be studied, and to date, most lectins studied thus far come primarily from plant sources. Since the discovery of the first castor lectin by Stillmark in 1888, numerous lectins from almost all plant parts have been reported (Goldstein and Poretz 1986). Although many plant lectins have been studied for their structural details, the physiological role of these proteins is still poorly understood. Recently, much speculation has been attributed to plant lectins “as storage proteins”, “as defense molecules», in symbiosis. A number of lectins have been isolated from plant storage tissues (seed or vegetative storage tissues) where they represent a very large proportion of the total protein content of the tissues (Van Damme et al. 1995). Some plant lectins have been implicated in the plant defense mechanism (Mirelman et al. 1975). On the other hand, certain 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), ongoing research work has been undertaken to detect the presence of lectin activity in weed plants and to isolate them from the same source. The study describes the isolation and partial purification of Calotropis gigantea lectin and its carbohydrate specificity. Material and methods Calotropis gigantea seeds were collected during the month of March from the botanical garden of 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-tetramethylethylenediamine (TEMED) and Commassie brilliant blue were from either 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 of analytical grade unless specified by company names. Methods Lectin extraction from Calotropis gigantea seeds To extract lectin, Calotropis gigantea seeds were collected, washed with distilled water and dried. Then, the seeds were homogenized (5 g in 25 ml) using a mortar and pestle at room temperature with phosphate-buffered saline (pH 7.2; 100 mM), containing 200 mM of EDTA and 200 mM PMSF (phenylmethylsulfonyl fluoride). The extraction procedure was carried out overnight at 4°C. The extract was filtered through muslin cloth and clarified by centrifugation at 8,000 rpm for 15 min at 4°C. 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 of different blood groups (A, B and O) was collected in 1 ml of 4% sodium citrate solution. Erythrocytes were separated by centrifugation at 1,500 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. The total volume is measured and a final concentration of 0.025% trypsin was added and incubated at 37ºC for 1 h. Excess trypsin was removed by repeated washes in saline and finally adjusted to an OD of 3.5 at 660 nm and used for the hemagglutination test and inhibition tests. Hemagglutination (HA) test To perform the hemagglutination test, U-bottom 96-well microtiter plates were used. Initially, 50 μl of saline was added to all wells of a respective row. Then, to the first well of each row, 50 µl of test solution was added and a serial dilution of 2 was performed until the 11th well. From the 11th well, 50 ul were discarded. 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 onesaline and erythrocytes were included as negative controls. The plates were incubated at room temperature for 1 h and visualized. Plates were photographed and geometric mean titers (GMT) were calculated. The highest dilution of the extract causing visible agglutination was arbitrarily considered the "titer" and the minimum concentration of protein required for agglutination was considered MCA which is equivalent to "one unit of hemagglutinating activity ( 1 HAU). The specific hemagglutination activity was expressed as activity in 1 mg of (unit mg -1) protein. Hapten Inhibition Assay Inhibition assays were performed by incubating the lectin sample in serially diluted sugar/glycoproteins prior to the addition of erythrocytes in 25 µl of test solution. The lowest concentration of the sugar/glycoprotein, which inhibits agglutination, was taken as the hapten inhibitor titer. In the 10th well, saline is added in place of the sugar/glycoprotein solutions, while in the 11th well, saline is added in place of the lectin. These wells served as positive and negative controls, respectively, for the inhibition studies. 12th well serving as a regular control which had only received 50 μl of saline solution and erythrocyte suspension. 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, inhibition of lectin activity was visualized and photographed as previously described and the minimum inhibitory concentration (MIC), defined as “the lowest concentration of the sugar/glycoprotein, which inhibited agglutination” was observed. was determined for each sugar/glycoprotein. In order to know the optimal pH for lectin activity, lectin was extracted in different buffers with varied pH. For extraction, the same procedure was followed as described above containing appropriate protease inhibitors and sodium chloride. Different 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. Ammonium sulfate precipitation The crude extract was subjected to ammonium sulfate [(NH4)2SO4] precipitation 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 min. The supernatant was retained while the precipitate (residue) was redissolved in 2 ml of PBS. 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 precipitation were separated on a 15% acrylamide gel. The protein sample was treated with 6x SDS buffer and boiled for 5 minutes at 100℃. Proteins were cooled and loaded into the wells and electrophoresed at 80 V for 4 hours. After electrophoresis was complete, gels were stained with Commassie Brilliant Blue R-250. A standard molecular weight protein ladder ranging from 14.3 to 97.4 kDa was also processed and electrophoresed under similar conditions. Protein estimation Protein content at various stages, including crude extracts, was estimated according to the protocol described by Lowry et al. (LOWRY et al. 1951). ).ResultsAmong the different weed plant seeds, only Calotropis gigantea seeds showed the highest hemagglutination activity(Titer-16), determined by a two-fold serial dilution technique using rabbit erythrocytes (Table 1). Besides Calotropis gigantean, Lantana camara seeds also showed hemagglutination activity but with a lower titer (04). Since the maximum hemagglutination activity was observed in the Calotropis gigantean plant, further studies were carried out using this plant for lectin isolation, hapten inhibition test, etc. Calotropis gigantean lectin (CGL) recognized all blood group erythrocytes equally. Since the lectin agglutinated rabbit erythrocytes, the following human erythrocytes of blood groups A, B and O were used for the test and found that CGL did not discriminate between erythrocytes of blood groups A, B and O. However, the lectin bound with varying intensity and recognized erythrocytes of blood group “O” with the maximum titer (64) while erythrocytes of blood group “B” with the lowest titer (08). These results are shown in Figure 1. For further studies, blood group O erythrocytes were used due to the easy availability of red blood cells. The CGL lectin is strongly inhibited by mucin glycoproteins. To determine the carbohydrate specificity of the lectin, various monosaccharides, disaccharides and glycoproteins were used. to perform a 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 the 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 could be another reason why this lectin is non-blood group specific in nature. 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 shown in Figure 3, the lectin exhibited constant stability of its activity between 40 °C and 60 °C. Although the titer decreased in the 40°C-60°C treatments, the same activity remained for several days. This may be due to the inactivation of proteases present in the extract. Additionally, lectin activity was also stable for at least 7 days when stored at room temperature. Maximum hemagglutination activity of CGL was found in 30–60% of ammonium sulfate saturation. Then, ammonium sulfate precipitation of the crude extract was performed to fractionate the proteins. . The results of the ammonium sulfate precipitation are shown in Figure 4. The results indicate that the lectin concentration increased in 30 to 60% of the ammonium sulfate precipitated fraction, as evidenced by the increase in hemagglutination activity (title 64). It is clear from Figure 4 that some of the contaminated proteins can be removed by this step. The 0-30% fraction showed some hemagglutination activity with titer 08. This may be due to the residual presence of lectin in this fraction. Although a good amount of protein was precipitated in a fraction of 60–80%, it did not show any hemagglutination activity. An SDS-PAGE analysis of partially purified lectin. SDS-polyacrylamide gel electrophoresis of the crude and sulfate-precipitated fractions..

Navigation

« Prev 1 2 3 4 5 Next »

Get In Touch