Farnesyl biliverdins IXα are novel ligands of biliproteins from moths of the Noctuoidea superfamily: A chemosystematic view of the Lepidoptera.

Bilins, derived from biliverdin IXα, are known from animals, plants and microorganisms, where they play vital roles as light-absorbing pigments. Bilins occur also in many insects. Recently, we discovered in insects a novel structural type of bilins with a farnesyl substituent at pyrrole ring A of biliverdin IXα. The first of these unusual bilins with a molecular mass of 852 (C48H60O10N4) was identified in Cerura vinula, subsequently in Spodoptera littoralis; both species are members of the Noctuoidea superfamily of moths. From an evolutionary point of view, it was of interest to examine other species and families of this monophyletic clade. Here, we show that other moths species in this clade (three Notodontidae species, one Erebidae species, and one Noctuidae species) have farnesylated biliverdins IXα that are present as a mixture of three bilins, differing by the number of oxygen atoms (O8-10). These bilins are associated with typical hemolymph storage proteins, which were identified by mass spectroscopic sequencing of tryptic peptides as arylphorins (a class of 500-kDa hexamerins) in the Notodontidae and Erebidae families, and as 350-kDa very high-density lipoproteins in the Noctuidae family. Circular dichroism spectroscopy revealed that the bilins adopt opposite conformations in complex with the two different classes of proteins. At present, farnesylated biliverdins and IXα-isomers of bilins in general are known only from species of the Noctuoidea clade; the sister clades of Bombycoidea and Papilionoidea synthesise the IXγ-isomer of biliverdin and derivatives thereof.

cDNA sequences of two arylphorin subunits of an insect biliprotein: phylogenetic differences and gene duplications during evolution of hexamerins-implications for hexamer formation.

Arylphorins represent a conserved class of hexameric ∼500 kDa insect hemolymph glycoproteins, rich in aromatic amino acids, which are produced in large quantities at the larval stage as reserves for metamorphosis and egg development. The recently isolated arylphorin from the moth Cerura vinula is unique in being complexed to a novel farnesylated bilin. Protein sequencing suggested the presence of two different ∼85 kDa subunits. Here, we report the complete coding sequences of two cDNAs encoding two arylphorins subunits with 67% identity and calculated physicochemical characteristics in agreement with the isolated holoprotein. Our phylogenetic analyses of the hexamerins revealed monophyletic origins not only for each of the arylphorins and methionine-rich proteins (H-type and M-type), the two major classes of hexamerins, but also for the minor groups of arylphorin-like and riboflavin-binding hexamerins. We named the latter proteins X-type (mixed type) hexamerins because they share sequence features with both major groups, and they show unique deletions and insertions at conserved sites located on the protein surface. We present a phylogenetic tree of lepidopteran hexamerins, which is in agreement with actual systematics. Overall, duplications of hexamerin genes occurred independently in several lepidopteran lineages. We also analyzed the hexamerin sequences for key parameters, which characterize each type of hexamerins. Based on the crystal structure of the homomeric arylphorin from Antheraea pernyi, we present a model for the heteromeric Cerura protein focusing on the role of N-glycan structures in stabilizing the hexamer structure.

Binding of imidacloprid, thiamethoxam and N-desmethylthiamethoxam to nicotinic receptors of Myzus persicae: pharmacological profiling using neonicotinoids, natural agonists and antagonists.

BACKGROUND: The increasing structural diversity of the neonicotinoid class of insecticides presently used in crop protection calls for a more detailed analysis of their mode of action at their cellular targets, the nicotinic acetylcholine receptors. RESULTS: Comparative radioligand binding studies using membranes of Myzus persicae (Sulzer) and representatives of the chloropyridyl subclass (imidacloprid), the chlorothiazolyl subclass (thiamethoxam), the tetrahydrofuranyl subclass (dinotefuran), as well as the novel sulfoximine type (sulfoxaflor), which is not a neonicotinoid, reveal significant differences in the number of binding sites, the displacing potencies and the mode of binding interference. Furthermore, the mode of interaction of [3 H]thiamethoxam and the nicotinic antagonists methyllycaconitine and dihydro-ß-erythroidine is unique, with Hill values of >1, clearly different to the values of around unity for [3 H]imidacloprid and [3 H]N-desmethylthiamethoxam. The interaction of [3 H]N-desmethylthiamethoxam with the agonist (-)nicotine is also characterised by a Hill value of >1. CONCLUSIONS: There is no single conserved site or mode of binding of neonicotinoids and related nicotinic ligands to their target receptor, but a variety of binding pockets depending on the combination of receptor subunits, the receptor subtype, its functional state, as well as the structural flexibility of both the binding pockets and the ligands. © 2016 Society of Chemical Industry.

Very high-density lipoprotein and vitellin as carriers of novel biliverdins IXα with a farnesyl side-chain presumably derived from heme A in Spodoptera littoralis.

Bilins in complex with specific proteins play key roles in many forms of life. Biliproteins have also been isolated from insects; however, structural details are rare and possible functions largely unknown. Recently, we identified a high-molecular weight biliprotein from a moth, Cerura vinula, as an arylphorin-type hexameric storage protein linked to a novel farnesyl biliverdin IXα; its unusual structure suggests formation by cleavage of mitochondrial heme A. In the present study of another moth, Spodoptera littoralis, we isolated two different biliproteins. These proteins were identified as a very high-density lipoprotein (VHDL) and as vitellin, respectively, by mass spectrometric sequencing. Both proteins are associated with three different farnesyl biliverdins IXα: the one bilin isolated from C. vinula and two new structurally closely related bilins, supposed to be intermediates of heme A degradation. The different bilin composition of the two biliproteins suggests that the presumed oxidations at the farnesyl side-chain take place mainly during egg development. The egg bilins are supposedly transferred from hemolymph VHDL to vitellin in the female. Both biliproteins show strong induced circular dichroism activity compatible with a predominance of the M-conformation of the bilins. This conformation is opposite to that of the arylphorin-type biliprotein from C. vinula. Electron microscopy of the VHDL-type biliprotein from S. littoralis provided a preliminary view of its structure as a homodimer and confirmed the biochemically determined molecular mass of ∼350 kDa. Further, images of S. littoralis hexamerins revealed a 2 × 3 construction identical to that known from the hexamerin from C. vinula.

Structure of a novel farnesylated bilin from an insect--formation by α-cleavage of heme A of mitochondrial cytochrome c oxidases?

Biliproteins are present in almost all forms of life, and many of them play vital roles in photobiology. The bilin ligand of a recently characterized 500-kDa biliprotein from an insect has been isolated and its structure elucidated with chemical and spectroscopic techniques (UV-visible, IR, MS, NMR, and CD). This blue pigment, named CV-bilin, represents a unique high molecular mass derivative of biliverdin IXα, with an unusual 10E-configuration and a molecular mass of 852 Da, corresponding to C48H60N4O10. The high mass of this open-chain tetrapyrrole results from the presence of an epoxi-dihydroxyethylfarnesyl substituent at C-18 and a hydroxymethyl substituent at C-13. This substitution pattern exactly reflects that of heme A of mitochondrial cytochrome c oxidases with a hydroxyethylfarnesyl chain and a formyl group at corresponding positions of the cyclic tetrapyrrole. As no other natural product is known to show these structural features (heme O, the precursor of heme A, has a methyl group at C-13), this bilin is presumed to be derived from heme A by cleavage of the α-methine bridge and oxidative modifications at C-13 and the hydroxyethylfarnesyl chain. Possibly, a bilin structurally related to this insect bilin is also produced in other organisms as a result of mitochondrial turnover or degradation. As CV-bilin in complex with a specific protein is accumulated at the end of larval life, stored in the pupa, and finally transferred to the oocytes, a possible role of the free or protein-bound pigment in egg or embryonic development is discussed.

C-26 vs. C-27 hydroxylation of insect steroid hormones: regioselectivity of a microsomal cytochrome P450 from a hormone-resistant cell line.

Hydroxylation of steroids at one of the side chain terminal methyl groups, commonly linked to C-26, represents an important regulatory step established in many phyla. Discrimination between the two sites, C-26 and C-27, requires knowing the stereochemistry of the products. 26-Hydroxylation of the insect steroid hormone 20-hydroxyecdysone by a microsomal cytochrome P450 was previously found to be responsible for hormonal resistance in a Chironomus cell line mainly producing the (25S)-epimer of 20,26-dihydroxyecdysone. Here, we studied the 25-desoxy analog of 20-hydroxyecdysone, ponasterone A, to elucidate the stereochemistry of the expected 26-hydroxy product, inokosterone, which occurs as C-25 epimers in nature. We identified the predominant metabolite as the C-25 R epimer of inokosterone on comparison by RP-HPLC with the (25R)- and (25S)-epimers the stereochemistry of which was confirmed by X-ray crystallography. (25R)-inokosterone was further oxidized to the 26-aldehyde identified by mass spectroscopy, borohydride reduction and metabolic transformation to 26-carboxylic acid. The (25S)-epimers of inokosterone and its aldehyde were minor products. With 20-hydroxyecdysone as substrate, we newly identified the (25R)-epimer of 20,26-dihydroxyecdysone as a minor product. In conclusion, the present stereochemical studies revealed high regioselectivity of the Chironomus enzyme to hydroxylate both steroids at the same methyl group, denoted C-27.

Isolation, characterisation and molecular imaging of a high-molecular-weight insect biliprotein, a member of the hexameric arylphorin protein family.

The abundant blue hemolymph protein of the last instar larvae of the moth Cerura vinula was purified and characterized by protein-analytical, spectroscopic and electron microscopic methods. Amino acid sequences obtained from a large number of cleavage peptides revealed a high level of similarity of the blue protein with arylphorins from a number of other moth species. In particular, there is a high abundance of the aromatic amino acids tyrosine and phenylalanine amounting to about 19% of total amino acids and a low content of methionine (0.8%) in the Cerura protein. The mass of the native protein complex was studied by size-exclusion chromatography, analytical ultracentrifugation, dynamic light scattering and scanning transmission electron microscopy and found to be around 500 kDa. Denaturating gel electrophoresis and mass spectrometry suggested the presence of two proteins with masses of about 85 kDa. The native Cerura protein is, therefore, a hexameric complex of two different subunits of similar size, as is known for arylphorins. The protein was further characterized as a weakly acidic (pI approximately 5.5) glycoprotein containing mannose, glucose and N-acetylglucosamine in an approximate ratio of 10:1:1. The structure proposed for the most abundant oligosaccharide of the Cerura arylphorin was the same as already identified in arylphorins from other moths. The intense blue colour of the Cerura protein is due to non-covalent association with a bilin of novel structure at an estimated protein subunit-to-ligand ratio of 3:1. Transmission electron microscopy of the biliprotein showed single particles of cylindrical shape measuring about 13 nm in diameter and 9 nm in height. A small fraction of particles of the same diameter but half the height was likely a trimeric arylphorin dissociation intermediate. Preliminary three-dimensional reconstruction based on averaged transmission electron microscopy projections of the individual particles revealed a double-trimeric structure for the hexameric Cerura biliprotein complex, suggesting it to be a dimer of trimers.

Porphobilinogen synthase from the butterfly, Pieris brassicae: purification and comparative characterization.

Porphobilinogen represents a key building block of tetrapyrroles serving as functional ligands of many vitally important proteins. Here we report the first purification of porphobilinogen synthase (PBGS) from whole insects by sequentially employing two modes of native electrophoresis on polyacrylamide gels subsequent to more conventional procedures. Using adults of Pieris brassicae L. (Lepidoptera: Pieridae) we achieved approximately 10,000-fold purification with final yields of up to 25% of electrophoretically pure PBGS with a specific activity of approximately 160 micromol PBG h(-1) mg(-1) at 37 degrees C and an affinity of 0.36 mM to its substrate 5-aminolevulinic acid. Enzyme activity was inhibited by the substrate mimics, levulinic acid and succinylacetone, and by chelating agents. PBGS behaved as a relatively heat-stable octameric complex of 292.3 kDa composed of 36.5 kDa subunits. Most general features of this insect PBGS were comparable to those published for other animal PBGS enzymes, while remarkable differences were found to the reported recombinant Drosophila enzyme. Moreover, rabbit antiserum directed against purified Pieris PBGS revealed significant immunological differences among insect PBGS enzymes from a wide range of orders contrasting to the overall evolutionary conserved features of this enzyme.

Stable free radicals in insect cuticles: electron spin resonance spectroscopy reveals differences between melanization and sclerotization.

Insect cuticles (exuviae; cast skins) were examined for the first time by ESR spectroscopy for the presence of stable free radicals, as found in melanins. All cuticles, except those from a locust albino strain, irrespective of the presence of melanin, provided single-line signals of varied g-values and linewidths. The ESR signals of melanins, isolated or in cuticles, were characterized by g-values <2.004 and small linewidths in the range of 4-6G, while sclerotized cuticles, lacking melanin, showed g-values >2.004 and broad linewidths of 5-11 G. The melanin spectra were comparable to those reported for eumelanins with indol-based monomers. Minor signals ascribed to pheomelanins were found in several probes. The 'sclerotin' spectra were broader and displayed unresolved hyperfine structure in some cases. As for melanins, the location and environment of the radicals in cuticles giving rise to the two types of ESR spectra could not be assigned. Changes in the radical environment due to insecticide or solvent treatment can be detected by ESR spectroscopy.

Butterfly wings, a new site of porphyrin synthesis and cleavage: studies on the expression of the lipocalin bilin-binding protein in Pieris brassicae.

The bilin-binding protein (BBP), a member of the lipocalin protein superfamily, is synthesized mainly in last instar larvae and in late pupae and newly emerged adults of Pieris brassicae, as previously reported. Here we present results from Northern blot analysis of the BBP gene transcript and from in vitro studies of holo-BBP biosynthesis with isolated wings using [14C]5-aminolevulinic acid as a precursor to the bilin ligand, [14C]-amino acids to label the apo-protein and inhibitors for both processes. Our combined data clearly demonstrate that BBP, which accumulates around pupa-adult transformation, is produced as holoprotein in the developing wings, while the BBP gene transcript is no longer detected in the rest of the body. Forewings and hind wings behave markedly different as the latter represent the major site of BBP synthesis, in agreement with the unequal distribution of BBP in the wings. The presence of an active pathway of porphyrin synthesis and cleavage in insect wings, shown here for the first time, and the role of the biliprotein during wing development remains an enigma so far. As part of this work sequences of fragments of the genes for actin and glyceraldehyde-3-phosphate dehydrogenase were obtained and examined as reference house-keeping genes in the expression studies.

The insecticide pymetrozine selectively affects chordotonal mechanoreceptors.

Pymetrozine is a neuroactive insecticide but its site of action in the nervous system is unknown. Based on previous studies of symptoms in the locust, the feedback loop controlling the femur-tibia joint of the middle leg was chosen to examine possible targets of the insecticide. The femoral chordotonal organ, which monitors joint position and movement, turned out to be the primary site of pymetrozine action, while interneurons, motoneurons and central motor control circuitry in general did not noticeably respond to the insecticide. The chordotonal organs associated with the wing hinge stretch receptor and the tegula were influenced by pymetrozine in the same way as the femoral chordotonal organ, indicating that the insecticide affects chordotonal sensillae in general. Pymetrozine at concentrations down to 10(-8) mol l(-1) resulted in the loss of stimulus-related responses and either elicited (temporary) tonic discharges or eliminated spike activity altogether. Remarkably, pymetrozine affected the chordotonal organs in an all-or-none fashion, in agreement with previous independent studies. Other examined sense organs did not respond to pymetrozine, namely campaniform sensillae on the tibia and the subcosta vein, hair sensillae of the tegula (type I sensillae), and the wing hinge stretch receptor (type II sensillae).

Developmental profiles of 5-aminolevulinate, porphobilinogen and porphobilinogen synthase activity in Pieris brassicae related to the synthesis of the bilin-binding protein.

The bilin-binding protein (BBP) occurs as a major soluble protein in haemolymph, fat body, epidermis and wings of Pieris brassicae. It is a member of the lipocalin protein superfamily with yet unknown function. Here, we studied the developmental regulation of tetrapyrrole biosynthesis that provides the bilin ligand as the predominating end product. The levels of the precursors 5-aminolevulinate (ALA) and porphobilinogen (PBG) varied during larval-pupal transition in accordance with the activity of the related enzyme porphobilinogen synthase (PBGS). During adult development, both precursors were low while PBGS activity increased parallel to the formation of BBP, as shown in previous work. A competitive inhibitor of PBGS was partially purified from the meconium and characterised as a heat-stabile acidic compound. Label from [14C]ALA, injected into developing pupae of different age, was found to 80% in the hind wings and to 20% in the forewings after adult eclosion, reflecting the unequal distribution of BBP between the pairs of wings. This contrasted to the activity of PBGS that was equally active in forewings and hind wings. Together with the variation of enzyme activity during wing development our results led us propose that the (hind) wings may play a role in the synthesis of the tetrapyrrole ligand of BBP.

The serotonergic system is involved in feeding inhibition by pymetrozine. Comparative studies on a locust (Locusta migratoria) and an aphid (Myzus persicae).

Pymetrozine inhibits feeding in aphids immediately after application without producing visible neurotoxic effects, as previously reported. In the present work, Locusta migratoria, though not a plant-sucking insect, was found to respond to pymetrozine by displaying unique symptoms, which were lifting and stretching of the hindlegs, in addition to inhibition of feeding. In locust, pymetrozine enhanced spontaneous spike discharge of the metathoracic and suboesophageal ganglia in situ at nanomolar concentrations. Similarly, pymetrozine increased the spontaneous rhythmic contractions of the isolated foregut with maximal effects also in the nanomolar range. The actions of pymetrozine were counteracted by biogenic amine receptor antagonists and mimicked by serotonin, not by dopamine and octopamine. Moreover, pymetrozine and serotonin strongly potentiated the effects of each other. Pymetrozine was inactive at all neurotransmitter receptors present on isolated locust neuronal somata, and at all other examined neuronal sites. In Myzus persicae, electrical penetration graph experiments revealed that serotonin, like pymetrozine, inhibited stylet penetration, and strongly enhanced the action of pymetrozine, comparable to the locust. Amine receptor antagonists were not specifically active in the aphid. We conclude from the present results that pymetrozine acts via a novel mechanism that is linked to the signalling pathway of serotonin.

Comparative analysis of neonicotinoid binding to insect membranes: I. A structure-activity study of the mode of [3H]imidacloprid displacement in Myzus persicae and Aphis craccivora.

Neonicotinoids bind selectively to insect nicotinic acetylcholine receptors with nanomolar affinity to act as potent insecticides. While the members of the neonicotinoid class have many structural features in common, it is not known whether they also share the same mode of binding to the target receptor. Previous competition studies with [3H]imidacloprid, the first commercialised neonicotinoid, indicated that thiamethoxam, representing a novel structural sub-class, may bind in a different way from that of other neonicotinoids. In the present work we analysed the mode of [3H]imidacloprid displacement by established neonicotinoids and newly synthesized analogues in the aphids Myzus persicae Sulzer and Aphis craccivora Koch. We found two classes of neonicotinoids with distinct modes of interference with [3H]imidacloprid, described as direct competitive inhibition and non-competitive inhibition, respectively. Competitive neonicotinoids were acetamiprid, nitenpyram, thiacloprid, clothianidin and nithiazine, whereas thiamethoxam and the N-methyl analogues of imidacloprid and clothianidin showed non-competitive inhibition. The chloropyridine or chlorothiazole heterocycles, the polar pharmacophore parts, such as nitroimino, cyanoimino and nitromethylene, and the cyclic or acyclic structure of the pharmacophore were not relevant for the mode of inhibition. Consensus structural features of the neonicotinoids were defined for the two mechanisms of interaction with [3H]imidacloprid binding. Furthermore, two sub-classes of non-competitive inhibitors can be discriminated on the basis of their Hill coefficients for imidacloprid displacement. We conclude from the present data that the direct competitors share the binding site with imidacloprid, whereas non-competitive compounds, like thiamethoxam, bind to a different site or in a different mode.

Comparative analysis of neonicotinoid binding to insect membranes: II. An unusual high affinity site for [3H]thiamethoxam in Myzus persicae and Aphis craccivora.

Neonicotinoids represent a class of insect-selective ligands of nicotinic acetylcholine receptors. Imidacloprid, the first commercially used neonicotinoid insecticide, has been studied on neuronal preparations from many insects to date. Here we report first intrinsic binding data of thiamethoxam, using membranes from Myzus persicae Sulzer and Aphis craccivora Koch. In both aphids, specific binding of [3H]thiamethoxam was sensitive to temperature, while the absolute level of non-specific binding was not affected. In M persicae, binding capacity (Bmax) for [3H]thiamethoxam was ca 450 fmol mg(-1) of protein at 22 degrees C and ca 700 fmol mg(-1) of protein at 2 degrees C. The negative effect of increased temperature was reversible and hence not due to some destructive process. The affinity for [3H]thiamethoxam was less affected by temperature: Kd was ca 11 nM at 2 degrees C and ca 15 nM at 22 degrees C. The membranes also lost binding sites for [3H]thiamethoxam during prolonged storage at room temperature, and upon freezing and thawing. In A craccivora, [3H]thiamethoxam was bound with a capacity of ca 1000 fmol mg(-1) protein and an affinity of ca 90 nM, as measured at 2 degrees C. Overall, the in vitro temperature sensitivity of [3H]thiamethoxam binding was in obvious contrast to the behaviour of [3H]imidacloprid studied in parallel. Moreover, the binding of [3H]thiamethoxam was inhibited by imidacloprid in a non-competitive mode, as shown with M persicae. In our view, these differences demonstrate that thiamethoxam and imidacloprid, which represent different structural sub-classes of neonicotinoids, do not share the same binding site or mode. This holds also for other neonicotinoids, as we report in a companion article.

Diastereomeric ecdysteroids with a cyclic hemiacetal in the side chain produced by cytochrome P450 in hormonally resistant insect cells.

A microsomal cytochrome P450 from a cell line of the insect Chironomus tentans has been shown to hydroxylate the steroid hormone 20-hydroxyecdysone at C(26) to yield 20,26-dihydroxyecdysone, P1, which is further metabolized to P2 and P3. Based on (1)H NMR studies, acetonide formation and quantum chemical calculations, P2 and P3 represent novel slowly interconvertible geometrical isomers, occurring at a 3:1 ratio, presumably arising from hemiacetal formation between the 26-aldehyde group and the 22R-hydroxyl group to build a tetrahydropyran ring in the side chain. The stereochemistry at C(26) was S in P2 (trans-diol) and R in P3 (cis-diol), respectively. Both metabolites showed S configuration at C(25). With Chironomus cells, P2/P3 was inactive as both a hormonal agonist and antagonist, whereas 20,26-dihydroxyecdysone (P1) showed weak agonist activity. Thus, cytochrome P450-mediated inactivation of 20-hydroxyecdysone may be responsible for the hormonal insensitivity observed in some subclones of this cell line.