Expression, purification, crystallization and X-ray diffraction analysis of ChiL, a chitinase from Chitiniphilus shinanonensis

Acta Crystallographica Section F. F7(12):1516-1520 (2015)journal articl

Solution Structure of IseA, an Inhibitor Protein of DL-Endopeptidases from Bacillus subtilis, Reveals a Novel Fold with a Characteristic Inhibitory Loop

In Bacillus subtilis, LytE, LytF, CwlS, and CwlO are vegetative autolysins, DL-endopeptidases in the NlpC/P60 family, and play essential roles in cell growth and separation. IseA (YoeB) is a proteinaceous inhibitor against the DL-endopeptidases, peptidoglycan hydrolases. Overexpression of IseA caused significantly long chained cell morphology, because IseA inhibits the cell separation DL-endopeptidases post-translationally. Here, we report the first three-dimensional structure of IseA, determined by NMR spectroscopy. The structure includes a single domain consisting of three alpha-helices, one 3(10)-helix, and eight beta-strands, which is a novel fold like a "hacksaw." Noteworthy is a dynamic loop between beta 4 and the 3(10)-helix, which resembles a "blade." The electrostatic potential distribution shows that most of the surface is positively charged, but the region around the loop is negatively charged. In contrast, the LytF active-site cleft is expected to be positively charged. NMR chemical shift perturbation of IseA interacting with LytF indicated that potential interaction sites are located around the loop. Furthermore, the IseA mutants D100K/D102K and G99P/G101P at the loop showed dramatic loss of inhibition activity against LytF, compared with wild-type IseA, indicating that the beta 4-3(10) loop plays an important role in inhibition. Moreover, we built a complex structure model of IseA-LytF by docking simulation, suggesting that the beta 4-3(10) loop of IseA gets stuck deep in the cleft of LytF, and the active site is occluded. These results suggest a novel inhibition mechanism of the hacksaw-like structure, which is different from known inhibitor proteins, through interactions around the characteristic loop regions with the active-site cleft of enzymes.ArticleJOURNAL OF BIOLOGICAL CHEMISTRY. 287(53):44736-44748 (2012)journal articl

Flight density of aquatic insect fauna over river water surface in the middle reaches of the Shinano River, mainly among Caddisflies (Trichoptera).

We focused on the relative number of flying adult caddisflies on the river surface captured by sticky board traps with the aim of elucidating differences in the distribution pattern of caddisfly larvae along the slope in the middle reaches of the Shinano River. The individual number of adult caddisflies caught increased from April and decreased from October. Even in the same middle reaches of a river, there was a large difference geographically in the species captured. Hydroptila sp. was caught mainly downstream of the Taishyobashi Bridge, Psychomyia acutipennis (Ulmer 1908) in the vicinity of the Taishyobashi Bridge, and Stenopsyche marmorata Navás 1920 upstream of the Awasabashi Bridge. It is known that the slope of the Shinano River bed suddenly becomes less and the flow rate slower in the area from the Taishyobashi Bridge to the Awasabashi Bridge, and it was shown that the species composition and number of aquatic insects caught changes with the change in the slope of the river bed.ArticleZoosymposia.10:203-213(2016)journal articl

Molecular cloning, gene expression analysis, and recombinant protein expression of novel silk proteins from larvae of a retreat-maker caddisfly, Stenopsyche marmorata

Retreat-maker larvae of Stenopsyche marmorata, one of the major caddisfly species in Japan, produce silk threads and adhesives to build food capture nets and protective nests in water. Research on these underwater adhesive silk proteins potentially leads to the development of new functional biofiber materials. Recently, we identified four major S. marmorata silk proteins (Smsps), Smsp-1, Smsp-2, Smsp-3, and Smsp-4 from silk glands of S. marmorata larvae. In this study, we cloned full-length cDNAs of Smsp-2, Smsp-3, and Smsp-4 from the cDNA library of the S. marmorata silk glands to reveal the primary sequences of Smsps. Homology search results of the deduced amino acid sequences indicate that Smsp-2 and Smsp-4 are novel proteins. The Smsp-2 sequence [167 amino acids (aa)] has an array of GYD-rich repeat motifs and two (SX)(4)E motifs. The Smsp-4 sequence (132 aa) contains a number of GW-rich repeat motifs and three (SX)(4)E motifs. The Smsp-3 sequence (248 aa) exhibits high homology with fibroin light chain of other caddisflies. Gene expression analysis of Smsps by real-time PCR suggested that the gene expression of Smsp-1 and Smsp-3 was relatively stable throughout the year, whereas that of Smsp-2 and Smsp-4 varied seasonally. Furthermore, Smsps recombinant protein expression was successfully performed in Escherichia coli. The study provides new molecular insights into caddisfly aquatic silk and its potential for future applications. (C) 2015 Elsevier Inc. All rights reserved.ArticleBIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS. 464(3):814-819 (2015)journal articl

Characterization of silk gland ribosomes from a bivoltine caddisfly, Stenopsyche marmorata: translational suppression of a silk protein in cold conditions

Larval Stenopsyche marmorata constructs food capture nets and fixed retreats underwater using self-produced proteinaceous silk fibers. In the Chikuma River (Nagano Prefecture, Japan) S. marmorata has a bivoltine life cycle; overwintering larvae grow slowly with reduced net spinning activity in winter. We recently reported constant transcript abundance of S. marmorata silk protein 1 (Smsp-1), a core S. marmorata silk fiber component, in all seasons, implying translational suppression in the silk gland during winter. Herein, we prepared and characterized silk gland ribosomes from seasonally collected S. marmorata larvae. Ribosomes from silk glands immediately frozen in liquid nitrogen (LN2) after dissection exhibited comparable translation elongation activity in spring, summer, and autumn. Conversely, silk glands obtained in winter did not contain active ribosomes and Smsp-1. Ribosomes from silk glands immersed in ice-cold physiological saline solution for approximately 4 h were translationally inactive, despite summer collection and Smsp-1 expression. The ribosomal inactivation occurs because of defects in the formation of 80S ribosomes, presumably due to splitting of 60S subunits containing 28S rRNA with central hidden break, in response to cold stress. These results suggest a novel-type ribosome-regulated translation control mechanism.ArticleBiochemical and Biophysical Research Communications.469(2):210-215(2016)journal articl