Protein cross-linking by peroxidase: Possible mechanism for sclerotization of insect cuticle

Incubation of test proteins with horseradish peroxidase in the presence of hydrogen peroxide and a catechol resulted in polymerization and precipitation of test proteins. SDS-PAGE readily revealed the generation of dimers, trimers, and higher oligomers in the reaction mixture. With the exception of 3,4-dihydroxyphenylalanine, dopamine, and norepinephrine, most other catechols tested participated in protein polymerization. The inability of these three catechols to accomplish polymerization is attributed to their high rate of intramolecular cyclization, which results in melanin formation. Radioactive studies with [³H]N-acetyldopamine clearly reveal both intermolecular and intramolecular cross-linking of test proteins by peroxidase. Based on these studies a possible mechanism for sclerotization and the biological significance of peroxidase in cuticle is discussed.     

The isolation of ketocatechols from insect cuticle and their possible rôle in sclerotization

Several catechol-derivatives have been isolated from acid hydrolysates of insect hard cuticle, and the two major compounds were identified as a hydroxy-ketocatechol, 2-hydroxy-3′,4′-dihydroxyacetophenone, and the corresponding aldehyde, 3,4-dihydroxyphenylglyoxal. It is shown that the glyoxal is formed from the former compound during hydrolysis.     

Identification of the namH gene, encoding the hydroxylase responsible for the N-glycolylation of the mycobacterial peptidoglycan

The peptidoglycan of most bacteria consists of a repeating disaccharide unit of beta-1,4-linked N-acetylmuramic acid and N-acetylglucosamine. However, the muramic acid moieties of the mycobacterial peptidoglycan are N-glycolylated, not N-acetylated. This is a rare modification seen only in the peptidoglycan of mycobacteria and five other closely related genera of bacteria. The N-glycolylation of sialic acids is a unique carbohydrate modification that has been studied extensively in eukaryotes. However, the significance of the N-glycolylation of bacterial peptidoglycan is unknown. The goal of this project was to identify the gene encoding the hydroxylase responsible for the N-glycolylation of the mycobacterial peptidoglycan. We developed a novel assay for the mycobacterial UDP-N-acetylmuramic acid hydroxylation reaction and demonstrated that Mycobacterium smegmatis has an enzyme activity that can convert UDP-N-acetylmuramic acid to UDP-N-glycolylmuramic acid. We identified the gene namH encoding the mycobacterial UDP-N-acetylmuramic acid hydroxylase by computer data base searching and motif comparisons with the eukaryotic enzymes responsible for the N-glycolyation of sialic acids. The namH gene is not essential for in vitro growth as we were successful in deleting the gene in M. smegmatis. The M. smegmatis mutant is devoid of UDP-N-acetylmuramic acid hydroxylase activity and synthesizes only N-acetylated muropeptide precursors. Furthermore, the mutant exhibits increased susceptibility to beta-lactam antibiotics and lysozyme. Our studies suggest that the N-glycolylation of mycobacterial peptidoglycan may play a role in lysozyme resistance or may contribute to the structural stability of the cell wall architecture.     

3-O-Hydrosulphato-4-hydroxyphenethylamine (dopamine 3-O-sulphate), a metabolite involved in the sclerotization of insect cuticle

Dopamine 3-O-sulphate (3-O-hydrosulphato-4-hydroxyphenethylamine) was isolated from newly ecdysed cockroaches, Periplaneta americana (L.), and its structure established by chemical and physical techniques and by synthesis. Relatively high concentrations (about 1mumol/g wet. wt.) of dopamine 3-O-sulphate exist in the newly ecdysed insect, and these concentrations decrease sharply as sclerotization of the cuticle proceeds. At least 40% of the radioactivity of (14)C-labelled dopamine 3-O-sulphate injected into newly ecdysed nymphs was recovered in the sclerotized cuticle 7-12 days after the injection. However, less than 1% of the radioactivity of injected dopamine 3-O-[(35)S]sulphate was recovered, and this value was not appreciably different from that for the incorporation of Na(2) (35)SO(4). Apparently, little or none of the sulphate moiety of dopamine 3-O-sulphate is incorporated directly into the cuticle as the intact sulphate ester. These observations are discussed in relation to current concepts of cuticular sclerotization in insects.     

From bioassays to Drosophila genetics: strategies for characterizing an essential insect neurohormone, bursicon

We describe the molecular analysis and cellular expression of the insect peptide neurohormone, bursicon. Bursicon triggers the sclerotization of the soft insect cuticle after ecdysis. Using protein elution analyses from SDS gels, we determined the molecular weight of bursicon from different insects to be approximately 30 kDa. Four partial peptide sequences of Periplaneta americana bursicon were obtained from purified nerve cord homogenates separated on two-dimensional gels. Antibodies produced against one of the sequences identified the cellular location of bursicon in different insects and showed that bursicon is co-produced with crustacean cardioactive peptide (CCAP) in the same neurons in all insects tested so far. Additionally, using the partial peptide sequences, we successfully searched the Drosophila genome project for the gene encoding bursicon. With Drosophila as a tool, we can now verify the function of the sequence using transgenic flies. Sequence comparisons also allowed us to verify that bursicon is conserved, corroborating the older data from bioassays and immunohistochemical analyses. The sequence of bursicon will enable further analysis of its function, release, and evolution.     

Identification of a novel insect neuropeptide,CNMa and its receptor

To identify ligands for orphan GPCRs, we searched novel neuropeptide genes in the Drosophila melanogaster genome. Here, we describe CNMa, a novel cyclic neuropeptide that is a highly potent and selective agonist for the orphan GPCR, CG33696 (CNMaR). Phylogenetic analysis revealed that arthropod species have two paralogous CNMaRs, but many taxa retain only one. Drosophila CNMa potently activates CNMaR-2 from Apis mellifera, suggesting both receptors are functional. Although CNMa is conserved in most arthropods, Lepidoptera lack the CNMa gene. However, they retain the CNMaR gene. Bombyx CNMaR showed low sensitivity to Drosophila CNMa, hinting toward the existence of additional CNMaR ligand(s).     

Identification of the yeast gene encoding the tRNA m1G methyltransferase responsible for modification at position 9

Methylation of tRNA at the N-1 position of guanosine to form m(1)G occurs widely in nature. It occurs at position 37 in tRNAs from all three kingdoms, and the methyltransferase that catalyzes this reaction is known from previous work of others to be critically important for cell growth in Escherichia coli and the yeast Saccharomyces cerevisiae. m(1)G is also widely found at position 9 in eukaryotic tRNAs, but the corresponding methyltransferase was unknown. We have used a biochemical genomics approach with a collection of purified yeast GST-ORF fusion proteins to show that m(1)G(9) formation of yeast tRNA(Gly) is associated with ORF YOL093w, named TRM10. Extracts lacking Trm10p have undetectable levels of m(1)G(9) methyltransferase activity but retain normal m(1)G(37) methyltransferase activity. Yeast Trm10p purified from E. coli quantitatively modifies the G(9) position of tRNA(Gly) in an S-adenosylmethionine-dependent fashion. Trm10p is responsible in vivo for most if not all m(1)G(9) modification of tRNAs, based on two results: tRNA(Gly) purified from a trm10-Delta/trm10-Delta strain is lacking detectable m(1)G; and a primer extension block occurring at m(1)G(9) is removed in trm10-Delta/trm10-Delta-derived tRNAs for all 9 m(1)G(9)-containing species that were testable by this method. There is no obvious growth defect of trm10-Delta/trm10-Delta strains. Trm10p bears no detectable resemblance to the yeast m(1)G(37) methyltransferase, Trm5p, or its orthologs. Trm10p homologs are found widely in eukaryotes and many archaea, with multiple homologs in several metazoans, including at least three in humans.     

Identification, cloning, and expression of the Scytalidium acidophilum XYL1 gene encoding for an acidophilic xylanase

We cloned XYL1, a Scytalidium acidophilum gene encoding for an acidophilic family 11 xylanase. The XYL1p protein was expressed in Pichia pastoris using the pPICZalphaA expression plasmid. The secreted protein was purified by TAXI affinity column chromatography. The purified XYL1p showed an optimum activity at pH 3.2 and 56 degrees C. The Michaelis-Menten constants were determined.     

Comparison between the sclerotization of adult and larval cuticle in Schistocerca gregaria

Femur cuticle from fifth instar larvae of the desert locust, Schistocerca gregaria, has been characterized with respect to composition, rate of deposition, and rate of sclerotization. The results are compared with those from adult cuticle of the same species. The protein compositions of the two types of cuticle are very similar, but the rates of deposition of both protein and chitin are different. The main difference is, however, that sclerotization is restricted to the first day after ecdysis in larval cuticle, whereas in adult cuticle sclerotization continues for at least a couple of weeks. The result is that the endocuticle remains untanned in the larvae, whereas in the adults the whole cuticle becomes tanned.     

Identification of the gene encoding hydroxyacid-oxoacid transhydrogenase, an enzyme that metabolizes 4-hydroxybutyrate

To identify the sequence of hydroxyacid-oxoacid transhydrogenase (HOT), responsible for the oxidation of 4-hydroxybutyrate in mammalian tissues, we have purified this enzyme from rat liver and obtained partial sequences of proteins coeluting with the enzymatic activity in the last purification step. One of the identified proteins was 'iron-dependent alcohol dehydrogenase', an enzyme encoded by a gene present on human chromosome 8q 13.1 and distantly related to bacterial 4-hydroxybutyrate dehydrogenases. The identification of this protein as HOT was confirmed by showing that overexpression of the mouse homologue in HEK cells resulted in the appearance of an enzyme catalyzing the alpha-ketoglutarate-dependent oxidation of 4-hydroxybutyrate to succinate semialdehyde.     

Observation of an anisotropic texture inside the wax layer of insect cuticle

The outermost part of insect cuticles is very often covered with wax, which prevents desiccation and serves for chemical communication in many species. Earlier studies on cuticular waxes have mainly focused on their chemical composition revealing complex mixtures of lipids. In the absence of information on their physical organization, cuticular waxes have been considered isotropic. Here we report the presence of parallel stripes in the wax layer of the carapace of the scarab beetle, Chrysina gloriosa, with a textural periodicity of ca. 28 nm, as revealed by electron microscopy of transverse sections. Observations at oblique incidence argue for a layered organization of the wax, which might be related to a layer-by-layer deposition of excreted wax. Our findings may lay the foundation for further studies on the internal structure of cuticular waxes for other insects.     

Purification, partial sequencing and characterization of an insect membrane dipeptidyl aminopeptidase that degrades the insect neuropeptide proctolin.

Two proctolin-binding proteins solubilized from 1600 cockroach hindgut membranes were purified 1000-fold using five chromatography steps. Twenty-five micrograms of protein were recovered from the final size-exclusion chromatography as a single peak eluting at 74 kDa, whereas two major bands at 80 and 76 kDa were identified after silver staining of electrophoresis gels. The fragments, sequenced by tandem mass spectrometry and the Edman method, revealed a high homology with rat liver dipeptidyl aminopeptidase (DPP) III and a significant homology between the cockroach-purified proteins. From analysis of the Drosophila genome sequence database, it was possible to identify a putative DPP sharing high homology with the sequences obtained from the cockroach purified proteins and with the rat DPP III. Anti-(rat liver DPP III) Ig reacted specifically with both cockroach-purified proteins in Western blot analysis. The purified proteins removed the N-terminal dipeptide from the insect myotropic neuropeptide proctolin (Arg-Tyr-Leu-Pro-Thr) with a Km value of 3.8 +/- 1.1 microM. The specific DPP III inhibitor tynorphin prevented the degradation of proctolin by the purified insect DPP (IC50 = 0.68 microM). These results provide strong evidence that the cockroach-purified proteins represent an insect membrane DPP, presumably present in Drosophila, and that it is closely related to vertebrate DPP III.     

Sugar responsible and tissue specific expression of a gene encoding AtCIPK14, an Arabidopsis CBL-interacting protein kinase

Expression of AtCIPK14 (an Arabidopsis CBL-interacting protein kinase 14) was induced by metabolic sugars. Two A/T-rich sequences similar to elements involved in sugar-inducible expression of other genes were found within the -183 bp 5' region of the AtCIPK14 promoter that was responsible for the sugar induction. Histochemical analysis using a reporter gene indicated vascular-specific expression of AtCIPK14.     

Gene structure and expression of the gene from Beauveria bassiana encoding bassiasin I, an insect cuticle-degrading serine protease

Genomic and cDNA encoding Beauveria bassiana bassiasin I, a potential cuticle-degrading serine protease, were isolated and analysed. Bassiasin I gene is comprised of 1137 bp (379 aa) and 3 introns which are 69, 62 and 68 bp long. The comparison of a deduced amino acid sequence with Metarhizium anisopliae Pr1, B. bassiana Pr1, and proteinase K showed high homology. When the cDNA including the intact signal peptide was expressed in E. coli, a clear proteolytic-degraded zone on LB-skimmed milk plates was observed.     

Gene expression and morphogenesis during the deposition of Drosophila wing cuticle

The exoskeleton of insects and other arthropods is a very versatile material that is characterized by a complex multilayer structure. In Sobala and Adler (2016) we analyzed the process of wing cuticle deposition by RNAseq and electron microscopy. In this extra view we discuss the unique aspects of the envelope the first and most outermost layer and the gene expression program seen at the end of cuticle deposition. We discussed the role of undulae in the deposition of cuticle and how the hydrophobicity of wing cuticle arises.     

Sclerotin and lipid in the waterproofing of the insect cuticle

In all the cuticles studied waterproofing is effected by extracuticular material, a mixture of sclerotin precursors and lipids, exuded from the tubular filaments of the pore canals. In Rhodnius larval abdomen it is a layer of thickness similar to the outer epicuticle, believed to be composed of 'sclerotin' and wax, in Schistocerca larval sternal cuticle and in Carausius sternal cuticle it is similar. In Tenebrio adult sternal cuticle of the abdomen, in both the extracuticular exudation and the contents of the distal endings of the tubular filaments, the wax component is obscured by hard 'sclerotin'. In Manduca larva a very thin layer of 'sclerotin' and wax is covered by an irregular wax layer, average 0.75 micron, twice the thickness of the inner epicuticle. In Periplaneta and Blattella the abdominal cuticle is covered by a soft waxy layer, often about 1 micron thick, which is mixed with argentaffin material. Below this is a very thin waterproof layer of wax and 'sclerotin' continuous with the contents of the tubular filaments, which is readily removed by adsorptive dusts. In Apis adult abdominal terga free wax plus sclerotin precursors form a thin layer which is known to be removed by adsorptive dusts. In Calliphora larva there is a very thin layer of the usual mixed wax and sclerotin and below this a thick (0.5 micron) layer, lipid staining and strongly osmiophil, likewise extracuticular and exuded from the epicuticular channels. This material (which is often called 'outer epicuticle') has the same staining and resistance properties as the true outer epicuticle on which it rests. In the abdomen of Calliphora adult the waterproofing wax-sclerotin mixture forms a thin layer over the entire cuticle including the surface of the microtrichia. There is also a thin detachable layer of free wax on the surface.