Isolation, purification and characterization of an N-acetyl-D-lactosamine binding mitogenic and anti-proliferative lectin from tubers of a cobra lily Arisaema utile Schott


Lectins are the carbohydrate-binding proteins of non-immune origin which have been the subject of intense investigation over the last few decades owing to the variety of interesting biological properties. Most of the lectins which have been purified and characterized from plants have been obtained from dicotyledons. In the present study a lectin was purified from tubers of a monocot plant Arisaema utile (AUL) Schott by affinity chromatography on asialofetuin-linked amino activated silica beads. AUL gave a single band in SDS-PAGE at pH 8.3 corresponding to subunit Mr 13.5 kDa. The native molecular mass of AUL was 54 kDa suggesting a homotetrameric structure. AUL gave multiple bands in isoelectric focusing and in native PAGE at pH 8.3. AUL was inhibited by N-acetyl-D-lactosamine (Lac NAc), a disaccharide and asialofetuin, a complex desialylated serum glycoprotein. When treated with denaturing agents, the lectin was stable in the presence of urea (3 M), thiourea (4 M) and guanidine HCl (4 M). AUL was a glycoprotein with a carbohydrate content of 1.2%. Complete loss of activity was observed upon modification of tryptophan residues of the lectin. The activity was reduced to 25% after modification of tyrosine. Chemical modification of arginine, histidine, serine and cysteine residues of AUL did not affect its activity. Using Far UV CD spectra the estimated secondary structure was 37% α-helix, 25% β-sheet and 38% random contributions. The lectin showed potent mitogenic response towards human lymphocytes. In vitro anti-proliferative assay using 11 human cancer cell lines resulted in 50% inhibition of six cell lines viz. SW-620, HCT-15, SK-N-SH, IMR-32, Colo-205 and HT-29 at 38, 42, 43, 49, 50 and 89 µg/ml, respectively.

Share and Cite:

Dhuna, V. , Dhuna, K. , Singh, J. , Saxena, A. , Agrawal, S. and Kamboj, S. (2010) Isolation, purification and characterization of an N-acetyl-D-lactosamine binding mitogenic and anti-proliferative lectin from tubers of a cobra lily Arisaema utile Schott. Advances in Bioscience and Biotechnology, 1, 79-90. doi: 10.4236/abb.2010.12012.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Goldstein, I.J., Hughes, R.C., Monsigny, M., Osawa, T. and Sharon, N. (1980) What should be called a lectin? Nature, 285(5760), 66.
[2] Sharon, N. and Lis, H. (1989) Lectins as cell recognition molecules. Science, 246(4927), 227-234.
[3] Sharon, N. and Lis, H. (2003) Lectins. 2nd Edition, Kluwer Academic Publishers, Dordrecht.
[4] Nowell, P.C. (1960) Phytohemagglutinin: An initiator of mitosis in cultures of normal human leucocytes. Cancer research, 20, 462-464.
[5] Harris, H. and Robson, E.B. (1963) Precipitin reactions between extracts of seeds of Canavalia ensiformis (jack bean) and normal and pathological serum proteins. Vox Sanguinis, 8(3), 348-355.
[6] Aub, J.C., Sanford, B.H. and Wang, L.H. (1965) Reactions of normal and leukemic cell surfaces to a wheat germ agglutinin. Proceedings of the National Academy of Sciences, USA, 54, 400-402.
[7] Brittinger, G. and Konig, E. (1969) Lymphocyte stimulation by pokeweed mitogen (pwm). Klin. Wochenschr, 47, 1307-1313.
[8] Krickeberg, H., Mauff, G., Mertens, T., Plum, G. and Heitmann, K. (1990) Lymphocyte proliferation in aidsrelated complex/walter-reed 5 patients: response to herpes simplex virus and tuberculin antigen and mitogen during intravenous immunoglobulin treatment,” Vox Sanguinis, 59, 38-43.
[9] Gabius, H.J. (1987) Endogenous lectins in tumors and the immune system. Cancer investigations, 5, 39-46.
[10] Zarkovic, N., Vukovic, T., Loncaric, I., Miletic, M., Zarkovic, K. and Borovic, S. (2001) An overview on anticancer activities of the Viscum album extract isorel. Cancer Biotherapy and Radiopharmaceuticals, 16, 5562.
[11] Singh, J., Singh, J. and Kamboj, S.S. (2004) A novel mitogenic and antiproliferative lectin from a wild cobra lily. Arisaema flavum. Biochemical and Biophysical Research Communications, 318, 1057-1065.
[12] Kaur, A., Kamboj, S.S., Singh, J., Saxena, A.K. and Dhuna, V. (2005) Isolation of a novel N-acetyl-D-lactosamine specific lectin from Alocasia cucullata. Biotechnology Letters, 27, 1815-1820.
[13] Bantel, H., Engels, I.H., Voelter, W., Schulze-osthoff, K. and Wesselborg, S. (1999) Mistletoe lectin activates caspase-8/flice independently of death receptor signaling and enhances anticancer drug-induced apoptosis. Cancer Research, 59, 2083-2090.
[14] Park, R., Kim, M.S., So, H.S., Jung, B.H., Moon, S.R. and Chung, S.Y. (2000) Activation of cJun N-terminal kinase 1 (JNK1) in mistletoe lectin II-induced apoptosis of human myeloleukemic U937 cells. Biochemical Pharmacology, 60, 1685-1691.
[15] Ohba, H., Bakalova, R., Moriwaki, S. and Nakamura, O. (2002) Fractionation of normal and leukemic t-cells by lectin affinity column chromatography. Cancer letters, 184, 207-214.
[16] Shangary, S., Singh, J., Kamboj, S.S., Kamboj, K.K. and R. S. Sandhu (1995) Purification and properties of four monocot lectins from the family araceae. Phytochemistry, 40, 449-455.
[17] Dhuna, V., Singh, J., Kamboj, S.S., Singh, J., Shanmugavel and Saxena, A.K. (2005) Purification and characterization of a lectin from Arisaema tortuosum schott having in-vitro anticancer activity against human cancer cell lines. Journal of Biochemistry and Molecular Biology, 38, 526-532.
[18] Lowry, O.H., Rosebrough, N.J., Farr, A.R. and Randall, R.J. (1951) Protein measurements with folin-phenol reagent. The Journal of Biological Chemistry, 193, 265275.
[19] Spiro, R.G. (1966) Analysis of sugars found in glycoproteins. Methods in Enzymology, 8, 3-26.
[20] Reisfeld, R.A., Lewis, O.J. and Williams, D.E. (1962) Disc electrophoresis of basic proteins and peptides on polyacrylamide gels. Nature, 145, 281-283.
[21] Davis, B.J. (1964) Disc electrophoresis: Methods and applications to human serum proteins. Annals of the New York Academy of Sciences, 121, 404-427.
[22] Bryan, J.K. (1977) Molecular weights of proteins multimers from polyacrylamide gel electrophoresis. Analytical Biochemistry, 78, 513-519.
[23] Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 277, 680-685.
[24] Robertson, E.F., Dannelly, H.K., Malloy, P.J. and Reeve, H.C. (1987) Rapid isoelectric focusing in a vertical polyacrylamide minigel system. Analytical Biochemistry, 167, 290-294.
[25] Andrews, P. (1964) Estimation of the molecular weights of proteins by sephadex gel-filtration. Biochemical Journal, 91, 222-233.
[26] Paulova, M., Entlicher, G., Ticha, M., Kostir, J.V. and Kocourek, J. (1971) Studies of phytohemagglutinins. Vii. Effect of Mn2+ and Ca2+ on hemagglutinin of phytohemagglutinin of Pisum sativum l. Biochimica et Biophysica Acta, 237, 513-518.
[27] Spande, T.F. and Witkop, B. (1967) Determination of tryptophan content of protein with n-bromosuccinimide. Methods in Enzymology, 11, 498-532.
[28] Horton, H.R. and Koshland, Jr., D.E. (1972) Modification of proteins with active benzyl halides. Methods in Enzymology, 25, 468-477.
[29] Riordan, J.F., Wacker, W.E.C. and Vallee, B.L. (1965) N-acteyl imidazole: A reagent for determination of free tyrosyl residues of proteins. Biochemistry, 4, 1758-1765.
[30] Wang, H., Ye, X.Y. and Ng, T.B. (2001) Purification of chrysancorin, a novel antifungal protein with mitogenic activity from garland chrysanthemum seeds. Biological Chemistry, 382, 947-951.
[31] Mosmann, T. (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods, 65, 55-63.
[32] Boyum, A. (1968) Separation of leukocytes from blood and bone marrow. Scandinavian Journal of Clinical and Laboratory Investigation, 21, 77-89.
[33] Monks, A., Scudiero, D., Skehan, P., Shoemaker, R., Paul, K. and Vistica, D. (1991) Feasibility of a high-flux anticancer drug screen using a diverse panel of cultured human tumor cell lines. Journal of the National Cancer Institute, 83, 757-766.
[34] van Damme, E.J., Goossens, K., Smeets, K., van Leuven, F., Verhaert, P. and Peumans, W.J. (1995) The major tuber storage protein of araceae species is a lectin. Characterization and molecular cloning of the lectin from Arum maculatum. Plant Physiology, 107, 11471158.
[35] Bains, J.S., Dhuna, V., Singh, J., Kamboj, S.S., Nijjar, K. K. and Agrewala, J.N. (2005) Novel lectins from rhizomes of two acorus species with mitogenic activity and inhibitory potential towards murine cancer cell lines. International Immunopharmacology, 5, 1470-1478.
[36] van Damme, E.J.M., Goldstein, I.J. and Peumans, W.J. (1991) A comparative study of mannose-binding lectins from amaryllidaceae and alliaceae. Photochemistry, 30, 509-514.
[37] Ito, N., Imai, S., Haga, S., Nagaike, C., Morimura, Y. and Hatake, K. (1996) Localization of binding sites of Ulex europaeus, Helix pomatia and Griffonia simplicifolia i-b4 lectins and analysis of their backbone structures by several glycosidases and poly-n-acetyllactosamine-specific lectins in human breast carcinoma. Histochemistry and Cell Biology, 106, 331-339.
[38] Green, E.D., Adelt, G., Baenziger, J.U., Wilson, S. and van Halbeek, H. (1988) The asparagine-linked oligosaccharides on bovine fetuin. Structural analysis of n-glycanase-released oligosaccharides by 500-megahertz 1h nmr spectroscopy. The Journal of Biological Chemistry, 263, 18253-18268.
[39] van Damme, E.J.M., Allen, A.K. and Peumans, W.J. (1988) Related mannose-specific lectins from different species of the family amaryllidaceae. Plant Physiology, 73, 52-57.
[40] Chandra, N.R., Prabu, M.M., Suguna, K. and Vijayan, M. (2001) Structural similarity and functional diversity in proteins containing the legume lectin fold. Protein Engineering, 11, 857-866.
[41] Pang, Y., Shen, G.A., Liao, Z.H., Yao, J.H., Fei, J., Sun, X.F. and Tang, X. (2003) Molecular cloning and characterization of a novel lectin gene from Zephyranthes candida. DNA Sequence, 14, 163-167.
[42] Hayes, C.E. and Goldstein, I.J. (1974) An alphad-galactosyl-binding lectin from bandeiraea simplicifolia seeds. Isolation by affinity chromatography and characterization. The Journal of Biological Chemistry, 249, 1904-1914.
[43] van Damme, E.J., Smeets, K., Torrekens, S., van Leuven, F., Goldstein, I.J. and Peumans, W.J. (1992) The closely related homomeric and heterodimeric mannosebinding lectins from garlic are encoded by one-domain and two-domain lectin genes, respectively. European Jou nal of Biochemistry, 206, 413-420.
[44] Devyani, N. and Mala, R. (1998) Structural and functional role of tryptophan in xylanase from an extremophilic bacillus: Assessment of the active site. Biochemical and Biophysical Research Communications, 249, 207-212.
[45] Zand, R., Agrawal, B.B.L. and Goldstein, I.J. (1971) pH-dependent conformational changes of concanavalin A. Proceedings of the National Academy of Sciences, USA, 68, 2173-2376.
[46] Kaur, M., Singh, K., Rup, P.J., Saxena, A.K., Khan, R.H., Ashraf, M.T., Kamboj, S.S. and Singh, J. (2006) A tuber lectin from Arisaema Helleborifolium schott with antiinsect activity against melon fruit fly Bactrocera Cucurbitae (coquillett) and anti-cancer effect on human cancer cell lines. Archives of Biochemistry and Biophysics, 445, 156-165.
[47] Desai, N.N., Allen, A.K. and Neuberger, A. (1983) The properties of potato (Solanum tuberosum) lectin after deglycosylation by triflouromethanesulphonic acid. Biochemical Journal, 211, 273-276,
[48] Konozy, E.H.E., Mulay, R., Faca, V., Aard, R.J., Greene, L.J., Roque-barriera, M.C., Sabharwal, S. and Bhide, S.V. (2002) Purification, some properties of a d-galactosebinding leaf lectin from Erythrina indica and further characterization of seed lectin. Biochimie, 84, 1035-1043.
[49] Konozy, E.H.E., Bernardes, E.S., Rosa, C., Faca, V. Greene, L.J. and Ward, R.J. (2003) Isolation, purification, and physicochemical characterization of a d-galactosebinding lectin from seeds of Erythrina speciosa. Archives of Biochemistry and Biophysics, 410, 222-229.
[50] Sultan, N.A.M., Kenoth, R. and Swamy, M.J. (2004) Purification, physicochemical characterization, saccharide specificity, and chemical modification of a gal/galnac specific lectin from the seeds of Trichosanthes dioica,” Archives of Biochemistry and Biophysics, 432, 212-221.
[51] Yu, L.G., Fernig, D.G., White, M.R., Spiller, D.G., Appleton, P. and Evans, R.C. (1999) Edible mushroom (Agaricus bisporus) lectin, which reversibly inhibits epithelial cell proliferation, blocks nuclear localization sequence-dependent nuclear protein import. The Journal of Biological Chemistry, 274, 4890-4899.
[52] Wang, H.X., Ng, T.B., Liu, W.K., Ooi, V.E. and Chang, S.T. (1995) Isolation and characterization of two distinct lectins with antiproliferative activity from the cultured mycelium of the edible mushroom Tricholoma mongolicum. International Journal of Peptide and Protein Research, 46, 508-513.
[53] Ngai, P.H. and Ng, T.B. (2004) A mushroom (Ganoderma capense) lectin with spectacular thermostability, potent mitogenic activity on splenocytes, and antiproliferative activity toward tumor cells. Biochemical and Biophysical Research Communications, 314, 988-993.

Copyright © 2023 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.