Genomic and functional characterization of coleopteran insect-specific α-amylase inhibitor gene from Amaranthus species

Abstract

The smallest 32 amino acid α-amylase inhibitor from Amaranthus hypochondriacus (AAI) is reported. The complete gene of pre-protein (AhAI) encoding a 26 amino acid (aa) signal peptide followed by the 43 aa region and the previously identified 32 aa peptide was cloned successfully. Three cysteine residues and one disulfide bond conserved within known α-amylase inhibitors were present in AhAI. Identical genomic and open reading frame was found to be present in close relatives of A. hypochondriacus namely Amaranthus paniculatus, Achyranthes aspera and Celosia argentea. Interestingly, the 3'UTR of AhAI varied in these species. The highest expression of AhAI was observed in A. hypochondriacus inflorescence; however, it was not detected in the seed. We hypothesized that the inhibitor expressed in leaves and inflorescence might be transported to the seeds. Sub-cellular localization studies clearly indicated the involvement of AhAI signal peptide in extracellular secretion. Full length rAhAI showed differential inhibition against α-amylases from human, insects, fungi and bacteria. Particularly, α-amylases from Helicoverpa armigera (Lepidoptera) were not inhibited by AhAI while Tribolium castaneum and Callosobruchus chinensis (Coleoptera) α-amylases were completely inhibited. Molecular docking of AhAI revealed tighter interactions with active site residues of T. castaneum α-amylase compared to C. chinensis α-amylase, which could be the rationale behind the disparity in their IC. Normal growth, development and adult emergence of C. chinensis were hampered after feeding on rAhAI. Altogether, the ability of AhAI to affect the growth of C. chinensis demonstrated its potential as an efficient bio-control agent, especially against stored grain pests.

Proteinaceous α-amylase inhibitors: purification, detection methods, types and mechanisms

“…Engineered/synthetic analogues Abbreviation/ name Microbial [43] Streptomyces [44] Mammalian, [45] bacteria Approximately 74-76 [46] Depsipeptides, [47] β-hairpin Haim, Pain, Tendamistat (HOE 467) Knottin-like [46] Amaranth Insect [17] 32 [15] Analogues [48] and HWTX-chimera AAI γ-Thionin-like [18] Sorghum, [43] cowpea [49] Insect [50] 45-54 [49] SIα1, SIα2, SIα3 CM-proteins [24] Barley, wheat, ragi, rye Mammalian, [39] bacteria, insect 124-160 [24,51] Insertion sitedirected mutants [52] RATI (RBI), 0.19, 0.28 (WMAI-1), 0.53, WRP25, WRP26, BMAI-1 Kunitz-type [53] Barley, wheat, rice, pigeon pea, [54] regia [55] Cereal, insect [56] 176-181 [55] Site-directed mutants in BASI [57] BASI, WASI, RASI Thaumatin-like [58] Maize Insect 173-235 [59] Legume lectin-like [60] Common bean Mammalian, insect, [5] fungi 240-250 [5] α-AI1, α-AI2…”

“…The AAI is the smallest α-amylase protein inhibitor ever found, [15] with only 32 residues and 3 disulfide bonds. The structure of the protein is determined by nuclear magnetic resonance, including a desmin fold, three antiparallel chains, and a characteristic disulfide topology .…”

“…The AAI-TMA complex is characterized by a high degree of complementarity between the interacting surfaces, and the specificity of AAI to insect amylase makes it an excellent candidate for insect resistant genetically modified crops . [15,34] Cereal type Cereal α-amylase inhibitors are composed of 120-160 amino acid residues, forming five disulfide bonds. Among the most studied are exogenous wheat α-amylase inhibitor 0.19 and Eleusine coracana Gaertn bi-function α-amylase/trypsin inhibitor (RBI).…”

Common bean (Phaseolus vulgaris L.) α‐amylase inhibitors as safe nutraceutical strategy against diabetes and obesity: An update review

“…As aforementioned, seven types of natural protein inhibitors of α‐amylases (α‐AIs) have been identified, and six of these, namely the knottin‐like type (Bhide et al, 2017), the γ‐thionin‐like type (Mojica, de Mejia, Granados‐Silvestre, & Menjivar, 2017), the cereal type, the Kunitz type, the thaumatin‐like type, and the lectin‐like type (Richardson, 1991), are extracted from plants. The other type has been isolated from bacteria (Sumitani, Tsujimoto, Kawaguchi, & Arai, 2000).…”

“…The other type has been isolated from bacteria (Sumitani, Tsujimoto, Kawaguchi, & Arai, 2000). The molecular weights of these inhibitors range from 3 to 23 kDa (Bhide et al, 2017). The knottin-like type, the Kunitz-type, and the thaumatin-like type are able to inhibit amylase only in insects (Pereira et al, 1999).…”