Lepidopteran wing scales contain abundant cross-linked film-forming histidine-rich cuticular proteins

Scales are symbolic characteristic of Lepidoptera; however, nothing is known about the contribution of cuticular proteins (CPs) to the complex patterning of lepidopteran scales. This is because scales are resistant to solubilization, thus hindering molecular studies. Here we succeeded in dissolving developing wing scales from Bombyx mori, allowing analysis of their protein composition. We identified a distinctive class of histidine rich (His-rich) CPs (6%–45%) from developing lepidopteran scales by LC-MS/MS. Functional studies using RNAi revealed CPs with different histidine content play distinct and critical roles in constructing the microstructure of the scale surface. Moreover, we successfully synthesized films in vitro by crosslinking a 45% His-rich CP (BmorCPR152) with laccase2 using N-acetyl- dopamine or N-β-alanyl-dopamine as the substrate. This molecular study of scales provides fundamental information about how such a fine microstructure is constructed and insights into the potential application of CPs as new biomaterials

Functional Evolution of Duplicated Odorant-Binding Protein Genes, Obp57d and Obp57e, in Drosophila

Odorant-binding proteins (OBPs) are extracellular proteins found in insect chemosensilla, where they participate in the sensing of odors, tastes, and pheromones. Although a large number of OBP genes have been identified in insect genomes, their molecular functions and biological roles have been clarified in limited cases. Two OBP genes, Obp57d and Obp57e, were involved in the evolution of host-plant preference in Drosophila sechellia. Comparative analyses of the Obp57d/e genomic sequences from 27 closely related species suggested that the two genes arose by tandem gene duplication and functionally diverged from each other. In this study, the functional evolution of Obp57d and Obp57e was examined by in vitro binding assays using recombinant proteins synthesized in a bacterial system. Compared to the ancestral Dpse\OBP57de, Dmel\OBP57d was more specialized to tridecanoic acid while Dmel\OBP57e was generalized regarding their binding affinity, suggesting that the two OBP genes underwent subfunctionalization and neofunctionalization. A behavioral analysis using knockout flies supported that the biological role is different between OBP57d and OBP57e in vivo. Site-directed mutagenesis of the evolutionarily conserved amino acids revealed that these residues play an important role in protein folding. These findings provide a clue to understanding how the repertoire of OBP genes is maintained in a genome under natural selection

Endothelin Type B Receptor–Induced Sustained Ca2+ Influx Involves Gq/11/Phospholipase C–Independent, p38 Mitogen-Activated Protein Kinase–Dependent Activation of Na+/H+ Exchanger

The mechanism for sustained Ca2+ influx activated by G protein-coupled receptors was examined. In Chinese hamster ovary cells expressing recombinant human endothelin type B receptor (ETBR) and endogenous P2Y receptor (P2Y-R), endothelin-1 elicited a sustained Ca2+ influx depending on Gq/11 protein, phospholipase C (PLC), Na+/H+ exchanger (NHE) and p38 mitogen-activated protein kinase (p38MAPK), whereas P2Y-R-induced sustained Ca2+ influx was negligible. Functional study showed that NHE activation by ETBR was mediated via p38MAPK but not Gq/11/PLC, while that by P2Y-R involves only Gq/11/PLC/p38MAPK. These results suggest that Gq/11/PLC-independent NHE activation via p38MAPK plays an important role in ETBR-mediated sustained Ca2+ influx

p-values of the pairwise Wilcoxon rank sum test with adjustment by Holm's method for multiple comparisons for the results of the oviposition site selection assay.

<p>p-values of the pairwise Wilcoxon rank sum test with adjustment by Holm's method for multiple comparisons for the results of the oviposition site selection assay.</p

Site-directed mutagenesis of the evolutionarily conserved amino acids.

<p>(A) Aligned sequence Logo representation of OBP57d and OBP57e. Among the 16 evolutionarily conserved sites (highlighted by yellow for the OBP signature cysteins and green for the others) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029710#pone.0029710-Matuo1" target="_blank">[19]</a>, 11 were selected for site-directed mutagenesis in Dmel\OBP57d (indicated by black circles; numbers indicate the position in Dmel\OBP57d). (B) Expression of the 11 mutated OBPs in the insoluble cytoplasmic fraction was confirmed by SDS-PAGE.</p

Behavioral response to tridecanoic acid in the OBP mutants.

<p>Behavioral responses of the <i>D. melanogaster</i> knockout flies for <i>Obp57d</i> and <i>Obp57e</i> to tridecanoic acid were examined by the oviposition site selection assay. Preference index (PI) = (<i>N_acid</i>−<i>N_cont</i>)/(<i>N_acid</i>+<i>N_cont</i>), where <i>N_acid</i> and <i>N_cont</i> are the number of eggs laid on tridecanoic acid-containing and control media, respectively. A total of 48 individuals were examined in the six independent replicates. (A) <i>w¹¹¹⁸</i>, (B) <i>Obp57d^KO</i>, (C) <i>Obp57e^KO</i>, and (D) <i>Obp57d+e^KO</i>.</p

Comparisons of the binding specificity among Dmel\OBP57d, Dmel\OBP57e, and Dpse\OBP57de.

<p>Binding affinity to various ligands was compared among the three OBPs using the quenching value (Q, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029710#pone-0029710-g002" target="_blank">Figure 2</a>). A higher 1/Q value means higher affinity to the ligand. Bars represent the means of three independent replicates, and error bars indicate standard error.</p

Binding affinity of the recombinant OBPs to various compounds.

<p>The binding affinity to C6, C8–15 fatty acids and C13 compounds with different functional groups was examined by the <i>in vitro</i> binding assay using intrinsic fluorescence. (A, B) Dmel\OBP57d, (C, D) Dmel\OBP57e, and (E, F) Dpse\OBP57de. The ligand solution was added to a 1 µM OBP solution. The Y axis indicates the relative fluorescence intensity at 340 nm. Mean values of three independent replicates are shown. C13Alc, C13Ald, and C13ME indicate 1-tridecanol, 1-tridecanal, and methyl tridecanoate, respectively.</p