Purification and characterization of a Kunitz inhibitor from Poincianella pyramidalis with insecticide activity against the Mediterranean flour moth

https://doi.org/10.1016/j.pestbp.2014.12.001Get rights and content

Highlights

  • The partial sequencing revealed homology between PpyTI and Kunitz-type inhibitors.

  • PpyTI possesses a wide broad of thermal and pH stability.

  • The inhibitor showed insecticide activity for Anagasta kuehniella larvae.

  • The PpyTI insecticide effects consist in inhibition of major digestive enzymes.

Abstract

This paper describes the characterization of a trypsin inhibitor from Poincianella pyramidalis seeds (PpyTI). The partial sequencing of PpyTI revealed homology to Kunitz inhibitors, clustered as a member of Family I03 in MEROPS database. PpyTI has a single polypeptide chain of 19,042 Da and presents stability at high temperatures (up to 70 °C) and a wide range of pH. In vitro assays showed that disulfide bridges have an important stabilization role of reactive site in PpyTI, a characteristic shared among several Kunitz inhibitors. Bioassays carried out with the Mediterranean flour moth (Anagasta kuehniella) revealed a significant decrease in both larval weight and survival of PpyTI-fed larvae, besides a larval stage extension. Through biochemical analysis, we demonstrated that the PpyTI insecticide effects were triggered by digestion process commitment, through the inhibition of trypsin and chymotrypsin activities, the major digestive enzymes in this species. The insecticide effects and biochemical characterization of PpyTI encourage further studies using this inhibitor for insect pest control.

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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