Muhammad IMRAN, Ahmed AKREM, Qamar SAEED, Sohaib MEHMOOD, Arslan ALI, Aisha MUNAWAR, Binish KHALIQ, Farzeen ANWAR, Friedrich BUCK, Saber HUSSAIN, Ahsan SAEED, Muhammad YASIN ASHRAF

Model prediction of a Kunitz-type trypsin inhibitor protein from seeds of Acacia nilotica L. with strong antimicrobial and insecticidal activity

A Kunitz-type trypsin inhibitor protein has been purified and characterized from seeds of Acacia nilotica L. LC-MS/MS analysis of Acacia nilotica trypsin inhibitor (AnTI) provided the N-terminal fragment of 11 amino acids which yielded 100% identity with already reported Kunitz-type trypsin inhibitor protein of Acacia confusa (AcTI) in UniProtKB database search. SDS-PAGE showed a single band of ~21 kDa under nonreduced condition and appearance of a daughter band (17 kDa) in the presence of β-mercaptoethanol indicating the presence of interchain disulfide linkage typical for Kunitz-type trypsin inhibitors. AnTI was purified from seed extract by using a combination of anion exchange and gel filtration chromatography. Since AnTI showed maximum homology with AcTI, a molecular structure of AcTI was predicted which showed highly β-sheeted molecular conformation similar to crystallographic structure of Enterolobium contortisiliquum trypsin inhibitor (EcTI). AnTI (20 μg) produces significant population inhibition against different human pathogenic bacteria along strong antifungal activity (50 μg). Entomotoxin potential of AnTI was evaluated against two stored grain insect pests Tribolium castaneum (Herbst) (Tenebrionidae: Coleoptera) and Sitophilus oryzae (Linnaeus) (Curculionidae: Coleoptera). Statistically significant mortality of T. castaneum adults was observed at 1.5 mg after 15 days in comparison to control. Additionally, number of total eggs, larvae, pupae, adults, and their male/female ratio were also severely reduced in comparison to control. Similarly, two generation progeny of S. oryzae was studied after mixing AnTI with rice kernels. Mean percent mortality of adult population was significantly higher after 9 days of exposure in comparison to control group. AnTI significantly reduced the F1 generation while little mortality was observed for F2 generation. Exploration of such potent molecules is the prerequisite of our time regarding the anticipation of postantibiotic era and the development of insect resistance against chemical pesticides.

PDF

___

Ahmad M, Zaman F, Sharif T, Ch MZ (2008). Antidiabetic and hypolipidemic effects of aqueous methanolic extract of Acacia nilotica pods in alloxan-induced diabetic rabbits. Scandinavian Journal of Laboratory Animal Sciences 35 (1): 29-34. doi: 10.23675/sjlas.v35i1.135
Ahmed AK, Johnson KA (2000). Horticultural development of Australian native edible plants. Australian Journal of Botany 48 (4): 417-426.doi: 10.1071/BT99042
Alasbahi R, Melzig M (2008). Screening of some Yemeni medicinal plants for inhibitory activity against peptidases. Die PharmazieAn International Journal of Pharmaceutical Sciences 63 (1): 86-88. doi: 10.1691/ph.2008.7633
Aliyu B (2006). Some ethno-medicinal plants of the Savannah Regions of West Africa description and phytochemicals. Triumph Publishing Company 1: 135-152.
Amsterdam D (1996). Susceptibility testing of antimicrobials in liquid media. Antibiotics in Laboratory Medicine.
Armstrong WB, Taylor TH, Kennedy AR, Melrose RJ, Messadi DV et al. (2013). Bowman birk inhibitor concentrate and oral leukoplakia: a randomized phase IIb trial. Cancer Prevention Research 6 (5): 410-418. doi: 10.1158/1940-6207
Aviles-Gaxiola S, Chuck-Hernández C, Serna Saldívar SO (2018). Inactivation methods of trypsin inhibitor in legumes: a review. Journal of Food Science 83 (1): 17-29. doi: 10.1111/1750- 3841.13985
Batista IF, Oliva MLV, Araujo MS, Sampaio MU, Richardson M et al. (1996). Primary structure of a Kunitz-type trypsin inhibitor from Enterolobium contortisiliquum seeds. Phytochemistry 41 (4): 1017-1022. doi: 10.1016/0031-9422(95)00710-5
Bendre AD, Ramasamy S, Suresh C (2018). Analysis of Kunitz inhibitors from plants for comprehensive structural and functional insights. International Journal of Biological Macromolecules 113: 933-943. doi: 10.1016/j. ijbiomac.2018.02.148
Benkert P, Biasini M, Schwede T (2010). Toward the estimation of the absolute quality of individual protein structure models. Bioinformatics 27 (3): 343-350. doi: 10.1093/bioinformatics/ btq662
Biasini M, Bienert S, Waterhouse A, Arnold K, Studer G et al. (2014). SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Research 42 (1): 252-258. doi: 10.1093/nar/gku340
Boyle VJ, Fancher ME, Ross RW (1973). Rapid, modified KirbyBauer susceptibility test with single, high-concentration antimicrobial disks. Antimicrobial Agents and Chemotherapy 3 (3): 418-424. doi: 10.1128/AAC.3.3.418
Cardona C, Posso CE, Kornegay J, Valor J, Serrano M (1989). Antibiosis effects of wild dry bean accessions on the Mexican bean weevil and the bean weevil (Coleoptera: Bruchidae). Journal of Economic Entomology 82 (1): 310-315. doi: 10.1093/ jee/82.1.310
Cruz AC, Massena FS, Migliolo L, Macedo LL, Monteiro NK et al. (2013). Bioinsecticidal activity of a novel Kunitz trypsin inhibitor from Catanduva (Piptadenia moniliformis) seeds. Plant Physiology and Biochemistry 70: 61-68. doi: 10.1016/j. plaphy.2013.04.023
da Silva Bezerra C, de Oliveira CFR, Machado OLT, de Mello GSV, da Rocha Pitta MG et al. (2016). Exploiting the biological roles of the trypsin inhibitor from Inga vera seeds: a multifunctional Kunitz inhibitor. Process Biochemistry 51 (6): 792-803. doi: 10.1016/j.procbio.2016.03.008
Dabhade AR, Mokashe NU, Patil UK (2016). Purification, characterization, and antimicrobial activity of nontoxic trypsin inhibitor from Albizia amara Boiv. Process Biochemistry 51 (5): 659-674. doi: 10.1016/j.procbio.2016.02.015
De Leo F, Gallerani R (2002). The mustard trypsin inhibitor 2 affects the fertility of Spodoptera littoralis larvae fed on transgenic plants. Insect Biochemistry and Molecular Biology 32 (5): 489- 496. doi: 10.1016/S0965-1748(01)00126-6
Desjardins P, Conklin D (2010). NanoDrop microvolume quantitation of nucleic acids. Journal of Visualized Experiments (45): e2565. doi: 10.3791/2565
Dias LP, Oliveira JT, Rocha-Bezerra LC, Sousa DO, Costa HP et al. (2017). A trypsin inhibitor purified from Cassia leiandra seeds has insecticidal activity against Aedes aegypti. Process Biochemistry 57: 228-238. doi: 10.1016/j.procbio.2017.03.015
Dokka MK, Davuluri SP (2014). Antimicrobial activity of a trypsin inhibitor from the seeds of Abelmoschus moschatus L. International Journal of Current Microbiology and Applied Sciences 3 (5): 184-199
Duncan AJ, In G (1991). Toxic Substances in Crop Plants. Cambridge, UK: The Royal Society of Chemistry
Eriksson AE, Matthews BW, Cousens LS (1993). Refinement of the structure of human basic fibroblast growth factor at 1.6 Å resolution and analysis of presumed heparin binding sites by selenate substitution. Protein Science 2 (8): 1274-1284. doi: 10.1002/pro.5560020810
Furstenberg-Hagg J, Zagrobelny M, Bak S (2013). Plant defense against insect herbivores. International Journal of Molecular Sciences 14 (5): 10242-10297. doi: 10.3390/ijms140510242
Guimarães LC, de Oliveira CFR, Marangoni S, de Oliveira DGL, Macedo MLR (2015). Purification and characterization of a Kunitz inhibitor from Poincianella pyramidalis with insecticide activity against the Mediterranean flour moth. Pesticide Biochemistry and Physiology 118:1-9. doi: 10.1016/j. pestbp.2014.12.001
Heibges A, Glaczinski H, Ballvora A, Salamini F, Gebhardt C (2003). Structural diversity and organization of three gene families for Kunitz-type enzyme inhibitors from potato tubers (Solanum tuberosum L.). Molecular Genetics and Genomics 269 (4): 526- 534. doi: 10.1007/s00438-003-0860-0
Heibges A, Salamini F, Gebhardt C (2003). Functional comparison of homologous members of three groups of Kunitz-type enzyme inhibitors from potato tubers (Solanum tuberosum L.). Molecular Genetics and Genomics 269 (4): 535-541. doi: 10.1007/s00438-003-0861-z
Hewick RM, Hunkapiller MW, Hood LE, Dreyer WJ (1981). A gasliquid solid phase peptide and protein sequenator. Journal of Biological Chemistry 256 (15): 7990-7997
Jain E, Bairoch A, Duvaud S, Phan I, Redaschi N et al. (2009). Infrastructure for the life sciences: design and implementation of the UniProt website. BMC Bioinformatics 10 (1): 136. doi: 10.1186/1471-2105-10-136
Jamal F, Pandey PK, Singh D, Khan M (2013). Serine protease inhibitors in plants: nature’s arsenal crafted for insect predators. Phytochemistry Reviews 12 (1): 1-34. doi: 10.1007/s11101- 012-9231-y
Kim J-Y, Park S-C, Hwang I, Cheong H, Nah J-W et al. (2009). Protease inhibitors from plants with antimicrobial activity. International Journal of Molecular Sciences 10 (6): 2860-2872. doi: 10.3390/ijms10062860
Kim J-Y, Park S-C, Kim M-H, Lim H-T, Park Y et al. (2005). Antimicrobial activity studies on a trypsin–chymotrypsin protease inhibitor obtained from potato. Biochemical and Biophysical Research Communications 330 (3): 921-927. doi: 10.1016/j.bbrc.2005.03.057
Konala G, Seva L, Davuluri SP (2012). Antimicrobial activity of prickly chaff (Achyranthes aspera) seed trypsin inhibitor. International Journal of Pharmaceutical Sciences and Research 3 (9): 3241. doi: 10.1.1.299.9896&rep
Kortt AA, Jermyn MA (1981). Acacia proteinase inhibitors: purification and properties of the trypsin inhibitors from Acacia elata seed. European Journal of Biochemistry 115 (3): 551-558. doi: 10.1111/j.1432-1033.1981.tb06238.x
Laemmli UK (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685
Laskowski R, MacArthur M, Thornton J (2006). PROCHECK: validation of protein-structure coordinates. International Tables for Crystallography F: 722-725
Lopes J, Valadares N, Moraes D, Rosa JC, Araújo H et al. (2009). Physico-chemical and antifungal properties of protease inhibitors from Acacia plumosa. Phytochemistry 70 (7): 871- 879. doi: 10.1016/j.phytochem.2009.04.009
Macedo MLR, Ribeiro SF, Taveira GB, Gomes VM, de Barros KM et al. (2016). Antimicrobial activity of ILTI, a Kunitz‐type trypsin inhibitor from Inga laurina (SW.) Willd. Current Microbiology 72 (25): 538-544. doi: 10.1007/s00284-015-0970-z
Major IT, Constabel CP (2008). Functional analysis of the Kunitz trypsin inhibitor family in poplar reveals biochemical diversity and multiplicity in defense against herbivores. Plant Physiology 146 (3): 888-903. doi: 10.1104/pp.107.106229
Maslin B, McDonald M (2006). AcaciaSearch: evaluation of Acacia as a woody crop option for southern Australia. Acacia Utilisation and Management–Adding Value 26:86
Pandey PK, Singh D, Singh R, Sinha MK, Singh S et al. (2016). Cassia fistula seed’s trypsin inhibitor (s) as antibiosis agent in Helicoverpa armigera pest management. Biocatalysis and Agricultural Biotechnology 6:202-208. doi: 10.1016/j. bcab.2016.04.005
Park Y, Choi BH, Kwak J-S, Kang C-W, Lim H-T et al. (2005). Kunitztype serine protease inhibitor from potato (Solanum tuberosum L. cv. Jopung). Journal of Agricultural and Food Chemistry 53 (16): 6491-6496. doi: 10.1021/jf0505123
Rao K, Suresh C (2007). Bowman–Birk protease inhibitor from the seeds of Vigna unguiculata forms a highly stable dimeric structure. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics 1774 (10): 1264-1273. doi: 10.1016/j. bbapap.2007.07.009
Revina T, Speranskaya A, Kladnitskaya G, Shevelev A, Valueva T (2004). Subtilisin protein inhibitor from potato tubers. Biochemistry (Moscow) 69 (10): 1092-1098. doi: 10.1023/B:B IRY.0000046882.99647.21
Silva RG, Vasconcelos IM, Acrísio Filho J, Carvalho AF, Souza TM et al. (2015). Castor bean cake contains a trypsin inhibitor that displays antifungal activity against Colletotrichum gloeosporioides and inhibits the midgut proteases of the dengue mosquito larvae. Industrial Crops and Products 70:48-55. doi: 10.1016/j.indcrop.2015.02.058
Sumikawa JT, Nakahata AM, Fritz H, Mentele R, Sampaio MU et al. (2006). A Kunitz-type glycosylated elastase inhibitor with one disulfide bridge. Planta Medica 72 (5): 393-397. doi: 10.1055/s2005-916237
Tripathi VR, Sahasrabuddhe AA, Kumar S, Garg SK (2014). Purification and characterization of a trypsin inhibitor from Senna tora active against midgut protease of podborer. Process Biochemistry 49 (2): 347-355. doi: 10.1016/j. procbio.2013.11.007
Yamchi A, Ben C, Rossignol M, Zareie SR, Mirlohi A et al. (2018). Proteomics analysis of Medicago truncatula response to infection by the phytopathogenic bacterium Ralstonia solanacearum points to jasmonate and salicylate defence pathways. Cellular Microbiology 20 (4): e12796. doi: 10.1111/ cmi.12796
Yang X, Li J, Wang X, Fang W, Bidochka MJ et al. (2006). Psc-AFP, an antifungal protein with trypsin inhibitor activity from Psoralea corylifolia seeds. Peptides 27 (7): 1726-1731. doi: 10.1016/j. peptides.2006.01.020
Zhou D, Lobo YA, Batista IF, Marques-Porto R, Gustchina A et al. (2013). Crystal structures of a plant trypsin inhibitor from Enterolobium contortisiliquum (EcTI) and of its complex with bovine trypsin. PloS One 8 (4): e62252. doi: 10.1371/journal. pone.0062252