![conference banner](/images/conference-banner.jpeg)
Abstract
Background: The O. tesota lectin PF2 is a tetrameric protein with subunits of 33 kDa that recognizes only complex carbohydrates, resistant to proteolytic enzymes and has insecticidal activity against Phaseolus beans pest.
Objective: To explore PF2 lectin features at different protein structural levels and to evaluate the effect of temperature and pH on its functionality and conformational stability. Methods: PF2 lectin was purified by affinity chromatography. Its primary structure was resolved by mass spectrometry and analyzed by bioinformatic tools, including its tertiary structure homology modeling. The effect of temperature and pH on its conformational traits and stability was addressed by dynamic light scattering, circular dichroism, and intrinsic fluorescence. The hemagglutinating activity was evaluated using a suspension of peripheral blood erythrocytes. Results: The proposed PF2 folding comprises a high content of beta sheets. At pH 7 and 25°C, the hydrodynamic diameter (Dh) was found to be 12.3 nm which corresponds to the oligomeric native state of PF2 lectin. Dh increased under the other evaluated pH and temperature conditions, suggesting protein aggregation. At basic pH, PF2 exhibited low conformational stability. The native PF2 (pH 7) retained its full hemagglutinating activity up to 45°C and exhibited one transition state with a melting temperature of 76.8°C. Conclusion: PF2 showed distinctive characteristics found in legume lectins. The pH influences the functionality and conformational stability of the protein. PF2 lectin displayed a relatively narrow thermostability to the loss of secondary structure and hemagglutinating activity.Keywords: Circular dichroism, dynamic light scattering, hemagglutination, homology modeling, intrinsic fluorescence, PF2 lectin, pH, thermostability.
[1]
Lis, H.; Sharon, N. Lectins: Carbohydrate-specific proteins that mediate cellular recognition. Chem. Rev., 1998, 98(2), 637-674.
[http://dx.doi.org/10.1021/cr940413g] [PMID: 11848911]
[http://dx.doi.org/10.1021/cr940413g] [PMID: 11848911]
[2]
Lagarda-Diaz, I.; Guzman-Partida, A.M.; Vazquez-Moreno, L. Legume lectins: proteins with diverse applications. Int. J. Mol. Sci., 2017, 18(6), 1242.
[http://dx.doi.org/10.3390/ijms18061242] [PMID: 28604616]
[http://dx.doi.org/10.3390/ijms18061242] [PMID: 28604616]
[3]
Yau, T.; Dan, X.; Ng, C.C.W.; Ng, T.B. Lectins with potential for anti-cancer therapy. Molecules, 2015, 20(3), 3791-3810.
[http://dx.doi.org/10.3390/molecules20033791] [PMID: 25730388]
[http://dx.doi.org/10.3390/molecules20033791] [PMID: 25730388]
[4]
Sekine, H.; Kenjo, A.; Azumi, K.; Ohi, G.; Takahashi, M.; Kasukawa, R.; Ichikawa, N.; Nakata, M.; Mizuochi, T.; Matsushita, M.; Endo, Y.; Fujita, T. An ancient lectin-dependent complement system in an ascidian: novel lectin isolated from the plasma of the solitary ascidian, Halocynthia roretzi. J. Immunol., 2001, 167(8), 4504-4510.
[http://dx.doi.org/10.4049/jimmunol.167.8.4504] [PMID: 11591777]
[http://dx.doi.org/10.4049/jimmunol.167.8.4504] [PMID: 11591777]
[5]
Macedo, M.L.; Oliveira, C.F.; Oliveira, C.T.; Oliveira, C.T. Insecticidal activity of plant lectins and potential application in crop protection. Molecules, 2015, 20(2), 2014-2033.
[http://dx.doi.org/10.3390/molecules20022014] [PMID: 25633332]
[http://dx.doi.org/10.3390/molecules20022014] [PMID: 25633332]
[6]
Baldwin, R.L. Energetics of protein folding. J. Mol. Biol., 2007, 371(2), 283-301.
[http://dx.doi.org/10.1016/j.jmb.2007.05.078] [PMID: 17582437]
[http://dx.doi.org/10.1016/j.jmb.2007.05.078] [PMID: 17582437]
[7]
Duk, M.; Lisowska, E. Effect of pH on the binding of Vicia graminea lectin to erythrocytes. Dependence on the chemical character of red-cell receptors. Eur. J. Biochem., 1984, 143(1), 73-78.
[http://dx.doi.org/10.1111/j.1432-1033.1984.tb08342.x] [PMID: 6432538]
[http://dx.doi.org/10.1111/j.1432-1033.1984.tb08342.x] [PMID: 6432538]
[8]
Khan, J.M.; Qadeer, A.; Ahmad, E.; Ashraf, R.; Bhushan, B.; Chaturvedi, S.K.; Rabbani, G.; Khan, R.H. Monomeric banana lectin at acidic pH overrules conformational stability of its native dimeric form. PLoS One, 2013, 8(4), e62428.
[http://dx.doi.org/10.1371/journal.pone.0062428] [PMID: 23638080]
[http://dx.doi.org/10.1371/journal.pone.0062428] [PMID: 23638080]
[9]
Carrillo, C.; Cordoba-Diaz, D.; Cordoba-Diaz, M.; Girbés, T.; Jiménez, P. Effects of temperature, pH and sugar binding on the structures of lectins ebulin f and SELfd. Food Chem., 2017, 220, 324-330.
[http://dx.doi.org/10.1016/j.foodchem.2016.10.007] [PMID: 27855907]
[http://dx.doi.org/10.1016/j.foodchem.2016.10.007] [PMID: 27855907]
[10]
Biswas, H.; Chattopadhyaya, R. Thermal, chemical and pH induced unfolding of turmeric root lectin: modes of denaturation. PLoS One, 2014, 9(8), e103579.
[http://dx.doi.org/10.1371/journal.pone.0103579] [PMID: 25140525]
[http://dx.doi.org/10.1371/journal.pone.0103579] [PMID: 25140525]
[11]
Agrawal, S.B.; Ghosh, D.; Gaikwad, S.M. Investigation of structural and saccharide binding transitions of Bauhinia purpurea and Wisteria floribunda lectins. Arch. Biochem. Biophys., 2019, 662, 134-142.
[http://dx.doi.org/10.1016/j.abb.2018.12.003] [PMID: 30529570]
[http://dx.doi.org/10.1016/j.abb.2018.12.003] [PMID: 30529570]
[12]
Gautam, A.K.; Srivastava, N.; Nagar, D.P.; Bhagyawant, S.S. Biochemical and functional properties of a lectin purified from the seeds of Cicer arietinum L. 3 Biotech, 2018, 8(6), 272.
[13]
Zhao, J.; He, S.; Tang, M.; Sun, X.; Zhang, Z.; Ye, Y.; Cao, X.; Sun, H. Low-pH induced structural changes, allergenicity and in vitro digestibility of lectin from black turtle bean (Phaseolus vulgaris L.). Food Chem., 2019, 283, 183-190.
[http://dx.doi.org/10.1016/j.foodchem.2018.12.134] [PMID: 30722859]
[http://dx.doi.org/10.1016/j.foodchem.2018.12.134] [PMID: 30722859]
[14]
Vazquez-Moreno, L.; Ortega-Nieblas, M.; Burgueño, M.R.; Clamont, G.R. Purification of complex carbohydrate specific lectins from Olneya tesota seeds using tandem affinity chromatography. Int. J. Biochromatography, 2000, 5, 83-90.
[15]
Urbano-Hernandez, G. Identificacion de Estructuras Glicosıdicas Reconocidas Por La Lectina PF2. Fetuına y Tejidos Linfoides; Centro de Investigacio’n en Alimentacion y Desarrollo A.C. Hermosillo: Sonora, México, 2007.
[16]
Madeira, F.; Park, Y.M.; Lee, J.; Buso, N.; Gur, T.; Madhusoodanan, N.; Basutkar, P.; Tivey, A.R.N.; Potter, S.C.; Finn, R.D.; Lopez, R. The EMBL-EBI search and sequence analysis tools APIs in 2019. Nucleic Acids Res., 2019, 47(W1), W636-W641.
[http://dx.doi.org/10.1093/nar/gkz268] [PMID: 30976793]
[http://dx.doi.org/10.1093/nar/gkz268] [PMID: 30976793]
[17]
Hulo, N.; Bairoch, A.; Bulliard, V.; Cerutti, L.; Cuche, B.A.; de Castro, E.; Lachaize, C.; Langendijk-Genevaux, P.S.; Sigrist, C.J.A. The 20 years of PROSITE. Nucleic Acids Res., 2008, 36(Database issue), D245-D249.
[PMID: 18003654]
[PMID: 18003654]
[18]
Kumar, S.; Stecher, G.; Li, M.; Knyaz, C.; Tamura, K. MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Mol. Biol. Evol., 2018, 35(6), 1547-1549.
[http://dx.doi.org/10.1093/molbev/msy096] [PMID: 29722887]
[http://dx.doi.org/10.1093/molbev/msy096] [PMID: 29722887]
[19]
Kelley, L.A.; Mezulis, S.; Yates, C.M.; Wass, M.N.; Sternberg, M.J.E. The Phyre2 web portal for protein modeling, prediction and analysis. Nat. Protoc., 2015, 10(6), 845-858.
[http://dx.doi.org/10.1038/nprot.2015.053] [PMID: 25950237]
[http://dx.doi.org/10.1038/nprot.2015.053] [PMID: 25950237]
[20]
Sippl, M.J. Recognition of errors in three-dimensional structures of proteins. Proteins, 1993, 17(4), 355-362.
[http://dx.doi.org/10.1002/prot.340170404] [PMID: 8108378]
[http://dx.doi.org/10.1002/prot.340170404] [PMID: 8108378]
[21]
Wiederstein, M.; Sippl, M.J. ProSA-web: interactive web service for the recognition of errors in three-dimensional structures of proteins. Nucleic Acids Res., 2007, 35, W407-410.
[22]
Micsonai, A.; Wien, F.; Kernya, L.; Lee, Y-H.; Goto, Y.; Réfrégiers, M.; Kardos, J. Accurate secondary structure prediction and fold recognition for circular dichroism spectroscopy. Proc. Natl. Acad. Sci. USA, 2015, 112(24), E3095-E3103.
[http://dx.doi.org/10.1073/pnas.1500851112] [PMID: 26038575]
[http://dx.doi.org/10.1073/pnas.1500851112] [PMID: 26038575]
[23]
Liener, I.E. Toxic factors in edible legumes and their elimination. Am. J. Clin. Nutr., 1962, 1, 281-298.
[http://dx.doi.org/10.1093/ajcn/11.4.281] [PMID: 13930568]
[http://dx.doi.org/10.1093/ajcn/11.4.281] [PMID: 13930568]
[24]
Lagarda-Diaz, I.; Guzman-Partida, A.M.; Urbano-Hernandez, G.; Ortega-Nieblas, M.M.; Robles-Burgueño, M.R.; Winzerling, J.; Vazquez-Moreno, L. Insecticidal action of PF2 lectin from Olneya tesota (Palo Fierro) against Zabrotes subfasciatus larvae and midgut glycoconjugate binding. J. Agric. Food Chem., 2009, 57(2), 689-694.
[http://dx.doi.org/10.1021/jf802557m] [PMID: 19102651]
[http://dx.doi.org/10.1021/jf802557m] [PMID: 19102651]
[25]
Nagae, M.; Soga, K.; Morita-Matsumoto, K.; Hanashima, S.; Ikeda, A.; Yamamoto, K.; Yamaguchi, Y. Phytohemagglutinin from Phaseolus vulgaris (PHA-E) displays a novel glycan recognition mode using a common legume lectin fold. Glycobiology, 2014, 24(4), 368-378.
[http://dx.doi.org/10.1093/glycob/cwu004] [PMID: 24436051]
[http://dx.doi.org/10.1093/glycob/cwu004] [PMID: 24436051]
[26]
Ramos, M.V.; Grangeiro, T.B.; Cavada, B.S.; Shepherd, I.; Lopes, R.O. de M.; Sampaio, A.H. Carbohydrate/Glycan-binding specificity of legume lectins in respect to their proposed biological functions. Braz. Arch. Biol. Technol., 2000, 43(4), 349-359.
[http://dx.doi.org/10.1590/S1516-89132000000400001]
[http://dx.doi.org/10.1590/S1516-89132000000400001]
[27]
Schwarz, F.; Aebi, M. Mechanisms and principles of N-linked protein glycosylation. Curr. Opin. Struct. Biol., 2011, 21(5), 576-582.
[http://dx.doi.org/10.1016/j.sbi.2011.08.005] [PMID: 21978957]
[http://dx.doi.org/10.1016/j.sbi.2011.08.005] [PMID: 21978957]
[28]
Khan, F.; Ahmad, A.; Khan, M.I. Chemical, thermal and pH-induced equilibrium unfolding studies of Fusarium solani lectin. IUBMB Life, 2007, 59(1), 34-43.
[http://dx.doi.org/10.1080/15216540601178075] [PMID: 17365178]
[http://dx.doi.org/10.1080/15216540601178075] [PMID: 17365178]
[29]
He, S.; Simpson, B.K.; Ngadi, M.O.; Xue, S.J.; Shi, J.; Ma, Y. pH stability study of lectin from black turtle bean (Phaseolus vulgaris) as influenced by guanidinium-HCl and thermal treatment. Protein Pept. Lett., 2015, 22(1), 45-51.
[http://dx.doi.org/10.2174/0929866521666140909155556] [PMID: 25213796]
[http://dx.doi.org/10.2174/0929866521666140909155556] [PMID: 25213796]
[30]
Sturm, A.; Chrispeels, M.J. The high mannose oligosaccharide of phytohemagglutinin is attached to asparagine 12 and the modified oligosaccharide to asparagine 60. Plant Physiol., 1986, 81(1), 320-322.
[http://dx.doi.org/10.1104/pp.81.1.320] [PMID: 16664800]
[http://dx.doi.org/10.1104/pp.81.1.320] [PMID: 16664800]
[31]
Bischof, J.C.; He, X. Thermal stability of proteins. Ann. N. Y. Acad. Sci., 2005, 1066, 12-33.
[http://dx.doi.org/10.1196/annals.1363.003] [PMID: 16533916]
[http://dx.doi.org/10.1196/annals.1363.003] [PMID: 16533916]
[32]
Gaikwad, S.M.; Islam Khan, M. pH-dependent aggregation of oligomeric Artocarpus hirsuta lectin on thermal denaturation. Biochem. Biophys. Res. Commun., 2003, 311(2), 254-257.
[http://dx.doi.org/10.1016/j.bbrc.2003.09.206] [PMID: 14592406]
[http://dx.doi.org/10.1016/j.bbrc.2003.09.206] [PMID: 14592406]
[33]
Leavitt, R.D.; Felsted, R.L.; Bachur, N.R. Biological and biochemical properties of Phaseolus vulgaris isolectins. J. Biol. Chem., 1977, 252(9), 2961-2966.
[PMID: 853039]
[PMID: 853039]
[34]
Castillo-Villanueva, A.; Caballero-Ortega, H.; Abdullaev-Jafarova, F.; Garfias, Y.; del Carmen Jiménez-Martínez, M.; Bouquelet, S.; Martínez, G.; Mendoza-Hernández, G.; Zenteno, E. Lectin from Phaseolus acutifolius var. escumite: chemical characterization, sugar specificity, and effect on human T-lymphocytes. J. Agric. Food Chem., 2007, 55(14), 5781-5787.
[http://dx.doi.org/10.1021/jf063644k] [PMID: 17567024]
[http://dx.doi.org/10.1021/jf063644k] [PMID: 17567024]
[35]
Toyama, J.; Tanaka, H.; Horie, A.; Uchiyama, T.; Akashi, R. Purification and characterization of Anti-H lectin from the seed of Momordica Charantia and the inter-specific differences of hemagglutinating activity in cucurbitaceae. Asian J. Plant Sci., 2008, 7(7), 647-653.
[http://dx.doi.org/10.3923/ajps.2008.647.653]
[http://dx.doi.org/10.3923/ajps.2008.647.653]
[36]
Jawade, A.A.; Pingle, S.K.; Tumane, R.G.; Sharma, A.S.; Ramteke, A.S.; Jain, R.K. Isolation and characterization of lectin from the leaves of Euphorbia tithymaloides (L.). Trop. Plant Res., 2016, 3(3), 634-641.
[http://dx.doi.org/10.22271/tpr.2016.v3.i3.083]
[http://dx.doi.org/10.22271/tpr.2016.v3.i3.083]
[37]
Sitohy, M.; Doheim, M.; Badr, H. Isolation and characterization of a lectin with antifungal activity from Egyptian Pisum sativum seeds. Food Chem., 2007, 104(3), 971-979.
[http://dx.doi.org/10.1016/j.foodchem.2007.01.026]
[http://dx.doi.org/10.1016/j.foodchem.2007.01.026]