Purification and characterization of a Kunitz inhibitor from Poincianella pyramidalis with insecticide activity against the Mediterranean flour moth
Graphical Abstract
Introduction
Peptidase Inhibitors (PIs) are considered an important part of plant defense mechanisms against herbivore and pathogen attack. For nearly 70 years since the first descriptions about PIs [1], [2], a great number of detailed information has been obtained. Thus, the complexity and diversity in this group became increasingly noticeable. Evidence suggests that PIs were a result of an “evolutionary arms race” between pathogens and host plants [3]. Over time, the selective pressure selected organisms which accumulated PIs in tissues with high nutritional value, like seeds and tubers [4], [5], [6]. The sequence analysis of PIs and plant storage proteins revealed a high similarity degree, as observed between sporamin A and miraculin [6], [7]. This fact could be a consequence of numerous protein-folding scaffolds recruitment to PI activity, due to the evolutionary advantage of producing dual-function proteins [3]. Later, the plants also acquired the capability to trigger a systemic response against injuries and herbivory, increasing the production and accumulation of PIs in tissues upon attack [8].
Structural patterns such as molecular weight, polypeptide chains and specificity of inhibitory activity have been used for grouping PIs into families known as Kunitz, Bowman-Birk, Potato I and II, Squash, Cereal and Mustard [9]. However, due to a high number of described molecules a new classification has been adopted. The newest information on PIs is being grouped in the MEROPS database, where data about PIs from plant sources, small-molecules and synthetic inhibitors are deposited [10].
During many years, hundreds of studies investigated the potential of plant PIs in pest control. The advances of molecular biology allowed the expression of PIs in heterologous systems [11] as well as the construction of transgenic plants expressing these proteins [12], [13], [14]. Further, PIs have been tested in the control of diseases related to blood coagulation [15], malaria [9] and cancer [16]. Hence, there is an extreme search for new PI molecules with biotechnological potential.
Poincianella pyramidalis (Fabaceae, Caesalpinioideae) is popularly known as “catingueira” due to the typical biome of the semi-arid northeastern Brazil, known as “caatinga”. Because of its anti-inflammatory and antibacterial activities this species is used in traditional medicine [17]. Earlier analysis revealed a high level of anti-proliferative activity, antioxidant and tannin content [18]. In this work we report the purification, characterization and partial sequencing of PpyTI, a trypsin inhibitor from P. pyramidalis seeds. We evaluated the PpyTI potential to control the insect pest Anagasta kuehniella through bioassays and biochemical analysis.
Section snippets
Materials
We collected the seeds for PpyTI purification in the city of Campo Grande, MS, Brazil (20°29′56.7″S 54°36′49.8″W). Chromatography chemicals and appliances were from GE Healthcare Life Sciences. Electrophoresis reagents and appliances were from Bio-Rad. The bovine trypsin and chymotrypsin, Bovine serum albumin (BSA), Dithiothreitol (DTT), Iodoacetamide (IAA), Nα-Benzoyl-DL-arginine 4-nitroanilide (BAPNA), N-Benzoyl-L-tyrosine p-nitroanilide (BTPNA) and N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide
Purification of P. pyramidalis trypsin inhibitor (PpyTI)
For PpyTI purification we combined two chromatographic steps. First, we chose the size exclusion chromatography on a Sephadex G-75 column. From four separated peaks (Fig. 1A), only the second peak (G-2 peak) showed inhibitory activity against bovine trypsin (Table 1). In order to increase the PpyTI final yield, we lyophilized the pool correspondent to G-2 peak without the dialysis process. Hence, the G-2 peak with salt content was loaded into a Phenyl-Sepharose column. This protocol was adopted
Acknowledgments
We would like to thank CNPq (National Council of Scientific and Technological Development), FINEP (Financier of Studies and Projects) and FUNDECT (Foundation to Support Development of Education, Science and Technology of the State of Mato Grosso do Sul), for supporting this study. We would also like to thank the Mass Spectrometry Laboratory – LNBio for technical support during the mass spectrometry work and Espaço da Escrita – General Coordinator at UNICAMP – for the language services.
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2021, Biomedicine and PharmacotherapyPoincianella pyramidalis (Tul) L.P. Queiroz: A review on traditional uses, phytochemistry and biological-pharmacological activities
2021, Journal of EthnopharmacologyCitation Excerpt :In slides prepared with bone marrow, the extract showed a cytotoxic effect in the highest concentration in the ET of 168 h. No mutagenic effects were observed in bone marrow assays (Chaves et al., 2019). The insecticidal potential of P. pyramidalis seeds was tested by Guimarães et al. (2015). A new inhibitor of the I03 family (Kunitz inhibitor) was purified and characterized from seeds of P. pyramidalis (PpyTI).
GdTI, the first thermostable trypsin inhibitor from Geoffroea decorticans seeds. A novel natural drug with potential application in biomedicine
2020, International Journal of Biological MacromoleculesBiotechnological, biomedical, and agronomical applications of plant protease inhibitors with high stability: A systematic review
2020, Plant ScienceCitation Excerpt :Maize and Sorghum PPIs caused larval mortality ranging from 10 to 77 % when added to the diet of the African cotton leafworm Spodoptera littoralis; with the larval mortality caused by the maize PPI being significantly higher than that of the Sorghum protein, attaining a maximum mortality of 77 % when the larvae were fed a diet containing 0.5 % (w/w) of the maize PPI [190]. In bioassays performed with the Mediterranean meal moth (Anagasta kuehniella), PpyTI, the Kunitz trypsin inhibitor isolated from Poincianella pyramidalis seeds and a member of Group IA, produced a significant decrease in larval weight and survival, as well as an extension of the larval stage [191]. A purified soybean serine-protease inhibitor markedly reduced the mean larval and pupal weight and caused larval and pupal mortality of S. littoralis [188].
A trypsin inhibitor purified from Cassia leiandra seeds has insecticidal activity against Aedes aegypti
2017, Process BiochemistryCitation Excerpt :Generally, Kunitz-type trypsin inhibitors maintain their activity after exposure to high temperatures and large pH variations. ClTI was heat stable (Fig. 6A) like the trypsin inhibitors from Poincianella pyramidalis [57] and Adenanthera pavonina [58] seeds, which maintained their inhibitory activity after incubation within wide temperature range (37–70 and 25–70 °C, respectively) for 30 min. Moreover, a trypsin inhibitor obtained from Pithecellobium dumosum seeds maintained approximately 90% of its activity after exposure to temperatures varying from 37 to 100 °C for 30 min [59].