Bioinsecticide PA1b alleviates lipopolysaccharide-induced inflammation and impairment of bovine mammary epithelial cells.

Pea Albumin 1, subunit b (PA1b) is a 37 amino acid peptide. It was extracted from pea seeds and showed significant insecticidal activity against certain insects, such as the mosquitoes Culex pipiens and Aedes aegyptii, cereal weevils (genus Sitophilus), and certain species of aphids. Considering that pea seeds are regularly consumed by humans and mammals, PA1b is assumed to be a promising bioinsecticide with no allergenicity or toxicity to hosts. To clarify this aspect, PA1b was applied to bovine mammary epithelial cells challenged with lipopolysaccharide (LPS). The results revealed that LPS induced inflammatory cytokine tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL6) and monocyte chemoattractant protein 1 (MCP-1) secretion, while PA1b depressed these cytokines release via inhibiting NF-κB signaling activation. In addition, PA1b protected mammary epithelial cells from impairment caused by LPS, because it reduced cell membrane permeability and subsequently reconstructed mammary epithelial cell viability. Moreover, it inhibited cell apoptosis accompanied with alleviated oxidative stress. Furthermore, PA1b prevented opening of mitochondrial permeability transition pores, in turn up-regulated mitochondrial membrane potential and ATP production. Therefore, PA1b improved mitochondrial function, which contributed to re-construction of mammary epithelial cell viability. In conclusion, PA1b alleviates LPS-induced inflammation of bovine mammary epithelial cells via inhibiting NF-κB signaling activation and protects bovine mammary epithelial cells by improving mitochondrial function. PA1b is a good therapeutic survival factor for mammary epithelial cells.

Heterologous production of the insecticidal pea seed albumin PA1 protein by Pichia pastoris and protein engineering to potentiate aphicidal activity via fusion to snowdrop lectin Galanthus nivalis agglutinin; GNA).

BACKGROUND: New bioinsecticides with novel modes of action are urgently needed to minimise the environmental and safety hazards associated with the use of synthetic chemical pesticides and to combat growing levels of pesticide resistance. The pea seed albumin PA1b knottin peptide is the only known proteinaceous inhibitor of insect vacuolar adenosine triphosphatase (V-ATPase) rotary proton pumps. Oral toxicity towards insect pests and an absence of activity towards mammals makes Pa1b an attractive candidate for development as a bioinsecticide. The purpose of this study was to investigate if Pichia pastoris could be used to express a functional PA1b peptide and if it's insecticidal activity could be enhanced via engineering to produce a fusion protein comprising the pea albumin protein fused to the mannose-specific snowdrop lectin (Galanthus nivalis agglutinin; GNA). RESULTS: We report the production of a recombinant full-length pea albumin protein (designated PAF) and a fusion protein (PAF/GNA) comprised of PAF fused to the N-terminus of GNA in the yeast Pichia pastoris. PAF was orally toxic to pea (Acyrthosiphon pisum) and peach potato (Myzus persicae) aphids with respective, Day 5 LC50 values of 54 µM and 105 µM derived from dose-response assays. PAF/GNA was significantly more orally toxic as compared to PAF, with LC50 values tenfold (5 µM) and 3.3-fold (32 µM) lower for pea and peach potato aphids, respectively. By contrast, no phenotypic effects were observed for worker bumble bees (Bombus terristrus) fed PAF, GNA or PAF/GNA in acute toxicity assays. Confocal microscopy of pea aphid guts after pulse-chase feeding fluorescently labelled proteins provides evidence that enhanced efficacy of the fusion protein is attributable to localisation and retention of PAF/GNA to the gut epithelium. In contact assays the fusion protein was also found to be significantly more toxic towards A. pisum as compared to PAF, GNA or a combination of the two proteins. CONCLUSIONS: Our results suggest that GNA mediated binding to V-type ATPase pumps acts to potentiate the oral and contact aphicidal activity of PAF. This work highlights potential for the future commercial development of plant protein-based bioinsecticides that offer enhanced target specificity as compared to chemical pesticides, and compatibility with integrated pest management strategies.

PA1b inhibitor binding to subunits c and e of the vacuolar ATPase reveals its insecticidal mechanism.

The vacuolar ATPase (V-ATPase) is a 1MDa transmembrane proton pump that operates via a rotary mechanism fuelled by ATP. Essential for eukaryotic cell homeostasis, it plays central roles in bone remodeling and tumor invasiveness, making it a key therapeutic target. Its importance in arthropod physiology also makes it a promising pesticide target. The major challenge in designing lead compounds against the V-ATPase is its ubiquitous nature, such that any therapeutic must be capable of targeting particular isoforms. Here, we have characterized the binding site on the V-ATPase of pea albumin 1b (PA1b), a small cystine knot protein that shows exquisitely selective inhibition of insect V-ATPases. Electron microscopy shows that PA1b binding occurs across a range of equivalent sites on the c ring of the membrane domain. In the presence of Mg·ATP, PA1b localizes to a single site, distant from subunit a, which is predicted to be the interface for other inhibitors. Photoaffinity labeling studies show radiolabeling of subunits c and e. In addition, weevil resistance to PA1b is correlated with bafilomycin resistance, caused by mutation of subunit c. The data indicate a binding site to which both subunits c and e contribute and inhibition that involves locking the c ring rotor to a static subunit e and not subunit a. This has implications for understanding the V-ATPase mechanism and that of inhibitors with therapeutic or pesticidal potential. It also provides the first evidence for the position of subunit e within the complex.

Basic 7S globulin in plants.

Soybean seed basic 7S globulin (Bg7S)-like proteins are found in many plant species. Bg7S was originally thought to be a major seed storage protein but was later found to be multifunctional, with stress response, antibacterial activity, hormone receptor-like activity. Moreover, functional differences between Bg7S proteins from legumes and other plants have been revealed. In non-leguminous plants, Bg7S molecules inhibit the invasion of pathogenic microorganisms. However, although leguminous plants have a peptide called leg-insulin that can bind to Bg7S, non-leguminous plants do not have leginsulin. Bg7S in leguminous plants and other plants may have evolved in functionally different directions. Several homologs of Bg7S in plants are reported, but there is no homolog of this protein in peas, suggesting that the pea evolution might have followed a different route when compared to other leguminous plants. Although the functions of Bg7S are well documented in plants, recent studies suggest that this protein is also important in controlling blood glucose level, blood pressure and plasma cholesterol level, and cancer cell antiproliferative actions.

Host range of the potential biopesticide Pea Albumin 1b (PA1b) is limited to insects.

The Pea Albumin 1 subunit b (PA1b) peptide is an entomotoxin extracted from legume seeds with lethal activity towards several insect pests. Its toxic activity occurs after the perception of PA1b by a plasmalemmic proton pump (V-ATPase) in the insects. Assays revealed that PA1b showed no activity towards mammalian cells displaying high V-ATPase activity. Similarly, PA1b displayed no binding activity and no biological activity towards other non-insect organisms. We demonstrate here that binding to labelled PA1b was found in all the insect families tested, regardless of the sensitivity or insensitivity of the individual species. The coleopteran Bruchidae, which are mainly legume seed pests, were found to be fully resistant. A number of insect species were seen to be insensitive to the toxin although they exhibited binding activity for the labelled PA1b. The fruit fly, Drosophila melanogaster (Diptera), was generally insensitive when maintained on an agar diet, but the fly appeared to be sensitive to PA1b in bioassays using a different diet. In conclusion, the PA1b toxin provides legumes with a major source of resistance to insects, and insects feeding on legume seeds need to overcome this plant resistance by disrupting the PA1b - V-ATPase interaction.

Pea Albumin 1 subunit b (PA1b), a promising bioinsecticide of plant origin.

PA1b (Pea Albumin 1, subunit b) is a peptide extract from pea seeds showing significant insecticidal activity against certain insects, such as cereal weevils (genus Sitophilus), the mosquitoes Culex pipiens and Aedes aegyptii, and certain species of aphids. PA1b has great potential for use on an industrial scale and for use in organic farming: it is extracted from a common plant; it is a peptide (and therefore suitable for transgenic applications); it can withstand many steps of extraction and purification without losing its activity; and it is present in a seed regularly consumed by humans and mammals without any known toxicity or allergenicity. The potential of this peptide to limit pest damage has stimulated research concerning its host range, its mechanism of action, its three-dimensional structure, the natural diversity of PA1b and its structure-function relationships.