Hartmut Kayser

Two-dimensional peptide mapping by reversed-phase column chromatography, applied to the sequence determination of cytochrome c from the wild type and a mutant of the butterfly, pieris brassicae

Abstract Two-dimensional peptide mapping has been very effective in the characterization of protein digests, particularly for the detection of small structural differences between homologous proteins. The classical thin-layer strategy, which exploits differences in charge and hydrophobicity, has been realized as a method based on reversed-phase high-performance liquid chromatography. An initial fractionation at pH 7.2 with 100 m M potassium phosphate, followed by chromatography with 0.1% trifluoroacetic acid, has been applied to chymotryptic digests of cytochromes c . The use of UV-transparent and (in the final stage) volatile solvents allows detection and rapid recovery of nanomole amounts of peptides suitable for sequence determination. As an example of the application of this method we report the comparison of two variants of cytochrome c from the butterfly, Pieris brassicae , one being the wild type and the other a spontaneous mutant isolated from a laboratory colony. The single residue difference was easily detected and identified.

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-β-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.

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

Abstract 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 ∼10,000-fold purification with final yields of up to 25% of electrophoretically pure PBGS with a specific activity of ∼160 µmol PBG h-1 mg-1 at 37°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.

Conversion of [14C]β-Carotene to its 2-hydroxy and 3-hydroxy metabolites by two moth species

Abstract 1. 1. [ 14 C]β-Carotene was fed to last instar larvae of two Notodontid moth species, Cerura vinula L. and Phalera bucephala L. 2. 2. Label was specifically incorporated to 3.8% into β,β-caroten-2-ol by C. vinula and to 65% into β,β-caroten-3-ol (cryptoxanthin) by Ph. bucephala . This is the first direct demonstration of carotenoid metabolism in insects. 3. 3. In Ph. bucephala pupae no sexual difference in the relative amounts of cryptoxanthin could be stated. 4. 4. The capability to transform dietary carotenoids in Lepidoptera is discussed.

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.

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.

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.