PA1b was first identified by its ability to kill cereal weevils (Sitophilus oryzae, S. zeamais and S. granarius). Several other insect species were tested for their susceptibility to the toxic peptide and it appears that not all insects are susceptible, unrelated to the taxonomy of the species tested. For example, out of three species of aphids, Acyrthosiphon pisum is sensitive, Myzus persicae is not sensitive and Aphis gossypii is only sensitive at high doses). The mosquitoes Culex pipiens [6] and Aedes aegyptii [9] are both highly sensitive which is, of course, of great interest for the potential use of PA1b (on an industrial scale) to control these human and mammalian disease vectors. Some beneficial insects, such as the bee (Apis mellifera) and trichogramma (Trichogramma pretiosum), are insensitive, but both the development and the survival of the lady beetle (Harmonia axyridis) are strongly affected by the toxin [6]. It seems that Coleoptera are generally sensitive (with the exception of Tribolium castaneum) whereas the Lepidoptera are mainly insensitive, with the exception of Ephestia khuniella [3]. Several agronomically important caterpillars (Mamestra brassicae) [6], Spodotera littoralis and Ostrinia nubilalis [10] are not affected by PA1b. However, the cultured cells Sf9, originating from S. frugiperda, displayed high sensitivity to PA1b at low doses [11], indicating that S. frugiperda has all the molecular equipment to trigger the PA1b signal, but that certain insects are able to curtail the toxic effects.

A binding site for PA1b has been found in the weevil gut membranes [12] (see Section 4.1). This receptor is present in all insects, suggesting the existence of a mechanism of insensitivity independent of the receptor. This mechanism of resistance is still unclear, but it is not due to a differential degradation in the insect gut. Indeed, gut extract from the insensitive insect M. brassicae was unable to degrade PA1b [6], as was gut extract from the sensitive weevil [3]. However, Bruchus pisorum (the pea weevil) is a notable exception this being the only insect, among those tested, to feed on legume seeds and also the only one in which the receptor is undetectable in the gut extract. This data suggests that PA1b is the source of insect resistance in Legume seeds and, therefore, adaptation to feeding on Legume seeds necessitates a loss of binding capacity of the toxin [6].

Biological tests performed on mammalian cells [11], bacteria, fungi [6], yeast [13] and nematodes [14] have all shown that PA1b has no effect on these cells or organisms.

However, two biological effects of PA1b have been described outside of the insect world. In plants (specifically a carrot cell culture), it has been found that cell proliferation is enhanced by addition of the soybean homolog of PA1b, leading the authors to suggest that PA1b could act as a new peptidic plant hormone [15] (see Section 4.2 for more details). In mammals, injections of PA1b in mice were found to have a strong hyperglycemic effect, suggesting that PA1b could interfere with glucose metabolism and, in particular, with insulin perception in mammals [16] (see Section 4.3 for more details).

Because of the interesting biological characteristics of PA1b, the molecular mechanism of action and the potential mechanism of resistance existing in insects were amongst the first questions to be addressed. A screening of 90 strains of weevils, belonging to all three species, was used to select four strains, all belonging to the species S. oryzae and fully resistant to the purified toxin, showing that resistance could occur naturally in the absence of selection pressure. Genetic studies have shown that resistance was monogenic and recessive [21], suggesting that the toxicity of PA1b is manifested via a receptor within the insect. With the help of a toxin labeled with iodine 125 (¹²⁵I-PA1b), a saturable, reversible and high affinity (affinity in the nanomolar range) proteinaceous binding site has been characterized in membrane extracts of a susceptible strain of S. oryzae. A similar binding capacity was found in all the sensitive Sitophilus strains, but no specific binding was detectable in the four resistant strains of S. oryzae. This specificity strongly suggests that the characterised binding site is the molecular target of PA1b, and that resistance in S. oryzae implies an absence or modification of this target [12].

PA1b belongs to the knottin family which contains peptides with various biological targets, including enzymes and ionic channels [22]. Moreover, the structural analyses have also revealed similarities between PA1b and the ICK atracotoxin ACTX-Hi:OB4219, isolated from the insectivorous Australian funnel-web spider Hadronyche infensa, which targets an unknown channel [5]. On this basis, electrophysiological studies have been performed using PA1b on Sf9 insect cultured cells. The electrophysiological results clearly show that the toxin depolarizes the membrane of Sf9 cells and a pharmacological study demonstrated that PA1b was able to reproduce the bafilomycin effect, bafilomycin being a fungal inhibitor of V-ATPase [23].

V-ATPase is a proton pump using ATP, first characterized in the vacuolar membranes, but we now know that there are also plasmalemmic forms. V-ATPase is a multimeric protein complex, highly conserved among living species from bacteria to humans. In insects, the protein from Manduca sexta (Lepidoptera) has been closely studied as regards its structure, function and regulation [24,25]. This is an essential protein for insects, especially in the gut, since its action provides the energy required for the absorption of nutrients [25,26]. The insect V-ATPase is composed of 14 subunits organized into two complexes. The membrane complex V₀ has four subunits, including the subunits c and a which form the proton channel, and the V₁ complex is cytosolic and bears the ATPase function [27]. Biochemical tests performed on purified V-ATPase from M. sexta have shown that PA1b inhibits the V₀V₁ enzyme but not the V1 complex alone; the authors concluded that PA1b acts by binding and inhibiting the V₀ complex of V-ATPase in insects [23].

In soybeans, a 43-kDa protein exists which was found to bind insulin and insulin-like growth factor. It was shown that a 4-kDa peptide extract from soybean binds this protein and could compete with insulin for binding. Moreover, the 4-kDa peptide is able to stimulate the phosphorylation activity of the 43-kDa protein. For these reasons, the peptide was named leginsulin and was found to be the soybean isoform of the pea peptide PA1b [28,29]. The expression of leginsulin in cultured carrot cells leads to an enhanced cell proliferation [15]. A glycoprotein homolog to the 43-kDa protein from soybean exists in carrots and binds both insulin and leginsulin [30,31]. Authors then suggested that leginsulin (and, by extension, PA1b) could behave in plants as a hormone-like peptide [15] and that it acts by interacting with the 43-kDa protein, thus generating a cell signal transduction via the phosphorylation activity of the 43-kDa protein. So, a receptor seems to exist in plants (and not only in Legumes, although PA1b has, to date, only been found in Legumes) but it appears to be different from the one present in insects. In fact, the amino acid residues involved in binding were not the same for the interaction with the plant receptor [32] and with the insect receptor [33] (see Section 6).

Recent data from the literature show that subcutaneous injections of aglycin in mice (10 µg/g body weight) causes a transient, but highly significant, increase in blood glucose (+100% in 60 min). In fact, PA1b, aglycin and leginsulin are homologs, and all isoforms of the pea peptide have the same effect on mice. Using surface plasmon resonance biosensor technology, a single aglycin binding protein with an apparent molecular mass of 34-kDa was detected in membrane protein extracts from porcine and mice pancreas. Using affinity chromatography, the protein has been purified and identified, by mass fingerprinting, as a voltage dependant anion-selective channel (VDAC-1), a chloride channel first found in mitochondrial membranes but now known to be present in plasma membranes [16,34]. Thus, the plant peptide could interact with the physiology of mammalian cells (but by injection, not by ingestion as in insects), and particularly with the metabolism of glucose. This effect may be mediated by a receptor different from those found in plants or insects, but a receptor which is, as in insects, an ionic target, the VDAC-1. Linked to the glucose metabolism, electrophysiological studies have been performed on pancreatic β-cells of rat. PA1b may increase the intracellular level of calcium, as does insulin, but using a different mechanism. PA1b seems to act via Ca²⁺ influx through L-type Ca²⁺ channels. As PA1b (leginsulin) could compete with insulin in the plant system, the authors evoke the possibility that PA1b binds to the mammalian insulin receptor [35]. In mammals, the action of PA1b on metabolism seems to be well demonstrated, but the mechanism involved still remains unclear. Whether PA1b acts through the interaction with VDAC-1, with the L-type Ca²⁺ channels or with the insulin receptor is still under investigation. However, in insects, insulin does not compete with PA1b for the receptor, indicating that the receptor in insects (V-ATPase) is different from the one in mammals.

The structure of the pea albumin 1 reference gene (PA1 gene) is depicted in Figure 2. It was first described, in 1986, by Higgins et al. [4]. Two exons, separated by a short intron located inside the coding leader sequence, are present in the gene. The PA1 gene is transcribed as a single mRNA encoding the preproprotein PA1 [molecular weight 13.9 kDa; 130 amino-acids (aa)] which is co-translationally targeted to the endoplasmic reticulum (ER) by cleavage of its signal peptide (2.7 kDa, 26 aa). The proprotein precursor (11.2 kDa, 104 aa) is directed to the vacuole-like, seed-protein storage bodies where it is endoproteolytically cleaved to yield two peptides which, after removal of some carboxyl-terminal amino-acids (propeptides), represent the mature forms of PA1a (6 kDa, 53 aa) (C-terminal fragment) and PA1b (3.8 kDa, 37 aa) (N-terminal fragment) [4].

Although the PA1 gene has been used extensively in genetic engineering, paradoxically little is known about its regulation and, more specifically, upstream cis regulatory elements that could positively or negatively affect the rate of transcription have not yet been identified. However, computational analysis based on the increasing use of growing genomic resources (genome sequencing, EST databases etc.) should soon provide new data on the distribution and tissue-specific expression of PA1 homologous genes, as is already the case for the identification of novel genes including those encoding cysteine-rich peptides [36,37].

Some studies have provided evidence that the PA1 gene is regulated both spatially and temporally. In the pea, regulation of PA1 gene expression during the development of normal, non-deficient seeds appears to be under direct transcriptional control. There is little, or no, transcription of these genes in early development but then transcription progressively increases to reach its highest level midway through development (20 to 22 days after flowering), followed by a sharp decline [4,38]. In the genome of another legume, the narrow-leafed lupin (Lupinus angustifolius), a PA1b-like gene was detected that showed, within the same region, 96% sequence identity with the leginsulin gene from soybeans [39]. The gene was functional at the transcriptional level since the corresponding mRNAs were detected but they were in very low quantities and, almost exclusively, in developing seeds (mainly in cotyledons 40 days after anthesis) [39]. However, unlike in soybeans [29], no evidence was found for the expression of the leginsulin-like gene in the radicle of lupins (two days after germination), suggesting a tight temporal regulation of the gene in developing and germinating seeds [39].

An environmental regulation linked to the plant nutritional status has also been highlighted for PA1 genes. Under conditions of sulfur-deficiency, the levels of the PA1 mRNAs in developing pea seeds were reduced, without major changes in the transcription rates of the corresponding genes [4,38]. Thus, a post-transcriptional mechanism is responsible for these variations. Indeed, it was later confirmed that the PA1 mRNA level was effectively lowered in the leaves of transgenic tobacco grown under sulfur-deficient conditions [40]. To establish which regions of the gene were involved in the process of sulfur sensitivity, the expression level of a reporter gene supplemented with different sets of PA1 gene sequences (e.g., intron) was measured in transgenic tobacco under different conditions of sulfur supply. These experiments have shown that the intron processing was not the post-translational event affected by sulfur nutritional status but instead it was two elements that determined the sulfur responsiveness of the PA1 gene: one was located in the 3’ region between 189 and 323 bp downstream of the PA1 stop codon while the second one, not clearly identified, was within the coding sequence. The mechanism involved in the degradation of the PA1 mRNA during sulfur deficiency has not yet been identified and several models (e.g., mRNA stability) have been proposed [40].

The diversity of PA1b peptides within the same species was initially suggested by the work of Higgins et al. [xref4] which pinpointed four functional genes that were present in the pea genome and expressed in pea cotyledons. To date, nine peptidic isoforms of PA1b have been isolated and biochemically characterized in the garden pea [3,4,29,41,42,43], indicating that these peptides belong to a multigenic family whose members have diverged slightly (Figure 3). Almost all the forms should have retained their insecticidal properties [42] since amino-acid variations are not located inside key positions for the maintenance of toxic activity (see Section 6).

Research and identification of PA1b homologs in Fabaceae seeds were carried out by developing an approach combining three complementary methods: (1) the molecular one consisted of cloning PA1 homologous genomic sequences; (2) the biological method used a PA1b-specific bioassay, defined by a differential toxicity between a susceptible or a resistant strain of the rice weevil, S. oryzae (see Section 3.1), the test being first performed on whole seeds, for a preliminary screening, and then on seeds extracted with different solvants; (3) the biochemical one included the capacity of the extract to compete as a ligand with radiolabeled pea PA1b in its binding to susceptible insect gut membranes, and extracts were also analyzed by HPLC and mass-spectrometry. In this way, new albumin 1 genes, and their associated products, were characterized in the soybean Glycin max and in the bean Phaseolus vulgaris whereas in alfalfa Medicago truncatula no peptide was biochemically detected, despite the presence of high insecticidal activity and of homologous genes [41].

The relevance of the approach described above led to a broader study on 88 additional species scattered amongst the three subfamilies (Caesalpinioideae, Mimosoideae and Papilionoideae) of the Fabaceae [44]. 19 PA1 like genes were characterized from numerous Papilionoideae, but not from Caesalpinioideae or Mimosoideae. The alignment of deduced peptide sequences showed that some amino-acids were particularly well conserved at the structural level; cysteine (6), prolines (5) and some glycines (5), as well as the charged arginine in position 21 (as numbered from the PA1b sequence) ([44]; Figure 3; see also Section 5 in this review). The sequences of these homologues also revealed a two loop peptide sequence highly conserved among Legumes (loop L1, residues 7-15 CSPFE) and a less conserved loop (loop L2, residues 23-31) where the hydrophobic property is conserved (Figure 3)[41,44].These results indicate the importance of these conserved portions (loops L1 and L2) for toxicity.

Thus, to date, in plants the PA1b homologous peptides seem to be strictly legume specific, strongly suggesting that this peptide family is an important line of seed defense against insects from the Fabaceae family.