The defensive–offensive associations between algae and herbivores determine marine ecology. Brown algae utilize phlorotannin as their chemical defense against the predator Aplysia kurodai , which uses β-glucosidase ( aku BGL) to digest the laminarin in algae into glucose. Moreover, A. kurodai employs Eisenia hydrolysis-enhancing protein (EHEP) as an offense to protect aku BGL activity from phlorotannin inhibition by precipitating phlorotannin. To underpin the molecular mechanism of this digestive–defensive–offensive system, we determined the structures of the apo and tannic acid (TNA, a phlorotannin analog) bound forms of EHEP, as well as the apo aku BGL. EHEP consisted of three peritrophin-A domains arranged in a triangular shape and bound TNA in the center without significant conformational changes. Structural comparison between EHEP and EHEP–TNA led us to find that EHEP can be resolubilized from phlorotannin precipitation at an alkaline pH, which reflects a requirement in the digestive tract. aku BGL contained two GH1 domains, only one of which conserved the active site. Combining docking analysis, we propose the mechanisms by which phlorotannin inhibits aku BGL by occupying the substrate-binding pocket, and EHEP protects aku BGL against this inhibition by binding with phlorotannin to free the aku BGL pocket.
Abstract The defensive-offensive associations between algae and herbivores determine marine ecology. Brown algae utilize phlorotannin as their chemical defense against the predator Aplysia kurodai , which uses β-glucosidase ( aku BGL) to digest the laminarin in algae to glucose. Moreover, A. kurodai employs Eisenia hydrolysis-enhancing protein (EHEP) as an offense to protect aku BGL activity from phlorotannin inhibition by precipitating phlorotannin. To underpin the molecular mechanism of this digestive-defensive-offensive system, we determined the structures of apo and tannic-acid (TNA, a phlorotannin-analog) bound form of EHEP, as well as apo aku BGL. EHEP consisted of three peritrophin-A domains formed in a triangle and bound TNA in the center without significant conformational changes. Structural comparison between EHEP and EHEP–TNA led us to find that EHEP can be resolubilized from phlorotannin-precipitation at an alkaline pH, which reflects a requirement in the digestive tract. aku BGL contained two GH1 domains, only one of which conserved the active site. Combining docking analysis, we propose the mechanisms by which phlorotannin inhibits aku BGL by occupying the substrate-binding pocket, and EHEP protects aku BGL against the inhibition by binding with phlorotannin to free the aku BGL pocket.
The defensive-offensive associations between algae and herbivores determine marine ecology. Brown algae utilize phlorotannin as their chemical defense against the predator Aplysia kurodai , which uses β-glucosidase ( aku BGL) to digest the laminarin in algae to glucose. Moreover, A. kurodai employs Eisenia hydrolysis-enhancing protein (EHEP) as an offense to protect aku BGL activity from phlorotannin inhibition by precipitating phlorotannin. To underpin the molecular mechanism of this digestive-defensive-offensive system, we determined the structures of apo and tannic-acid (TNA, a phlorotannin-analog) bound form of EHEP, as well as aku BGL. EHEP consisted of three peritrophin-A domains formed in a triangle and bound TNA in the center without significant conformational changes. Structural comparison between EHEP and EHEP– TNA led us to find that EHEP can be resolubilized from phlorotannin-precipitation at an alkaline pH, which reflects a requirement in the digestive tract. aku BGL contained two GH1 domains, only one of which conserved the active site. Combining docking analysis, we propose the mechanisms by which phlorotannin inhibits aku BGL by occupying the substrate-binding pocket, and EHEP protects aku BGL against the inhibition by binding with phlorotannin to free the aku BGL pocket.
The defensive-offensive associations between algae and herbivores determine marine ecology. Brown algae utilize phlorotannin as their chemical defense against the predator Aplysia kurodai , which uses β-glucosidase ( aku BGL) to digest the laminarin in algae to glucose. Moreover, A. kurodai employs Eisenia hydrolysis-enhancing protein (EHEP) as an offense to protect aku BGL activity from phlorotannin inhibition by precipitating phlorotannin. To underpin the molecular mechanism of this digestive-defensive-offensive system, we determined the structures of apo and tannic-acid (TNA, a phlorotannin-analog) bound form of EHEP, as well as apo aku BGL. EHEP consisted of three peritrophin-A domains formed in a triangle and bound TNA in the center without significant conformational changes. Structural comparison between EHEP and EHEP–TNA led us to find that EHEP can be resolubilized from phlorotannin-precipitation at an alkaline pH, which reflects a requirement in the digestive tract. aku BGL contained two GH1 domains, only one of which conserved the active site. Combining docking analysis, we propose the mechanisms by which phlorotannin inhibits aku BGL by occupying the substrate-binding pocket, and EHEP protects aku BGL against the inhibition by binding with phlorotannin to free the aku BGL pocket.
Eisenia hydrolysis-enhancing protein (EHEP), which is a novel protein that has been identified in Aplysia kurodai , protects β-glucosidases from phlorotannin inhibition to facilitate the production of glucose from the laminarin abundant in brown algae. Hence, EHEP has attracted attention for its potential applications in producing biofuel from brown algae. In this study, EHEP was purified from the natural digestive fluid of A. kurodai and was crystallized using the sitting-drop vapor-diffusion method. Native and SAD (single-wavelength anomalous diffraction) data sets were successfully collected at resolutions of 1.20 and 2.48 Å using wavelengths of 1.0 and 2.1 Å, respectively, from crystals obtained in initial screening. The crystals belonged to space group P 2 1 2 1 2 1 and contained one EHEP molecule in the asymmetric unit. All 20 S-atom sites in EHEP were located and the phases were determined by the SAD method using the S atoms in the natural protein as anomalous scatterers (native-SAD). After phase improvement, interpretable electron densities were obtained and 58% of the model was automatically built.
Lenvatinib, used for unresectable hepatocellular carcinoma (HCC), causes appetite loss, but the underlying mechanisms, clinical impact, and predictive factors have been unclear. The endocrine factor FGF21 modulates appetite and is involved in cachexia. We evaluated the association between FGF21 level changes during lenvatinib treatment for unresectable HCC and appetite loss. Sixty-three eligible unresectable HCC patients who started lenvatinib treatment between 2018 and 2021 were included. We analyzed FGF21 levels at baseline; 1, 2, and 4 weeks after lenvatinib initiation, and before the onset of appetite loss. Grade ≥ 2 lenvatinib-induced appetite loss led to liver functional reserve deterioration at disease progression and a poor prognosis. Baseline characteristics and serum FGF21 levels were similar between patients with and without appetite loss. However, the serum FGF21 change rate increased significantly at 4 weeks post-lenvatinib initiation in patients with grade ≥ 2 appetite loss, as compared to those without appetite loss. Similar significant increases in the serum FGF21 level change rate were observed prior to grade ≥ 2 appetite loss onset. This suggests that changes in FGF21 levels can be used to predict patients with a greater risk of marked appetite loss and provides insights into the mechanisms underlying lenvatinib-induced appetite loss in patients with HCC.
Recent progress in the techniques of bio-macromolecular crystallography makes crystal structure analysis more powerful and useful for life science. The structure analysis of huge super-molecular (eukaryotic Ribosome, Vault etc.) and membrane proteins related to diseases were successful. Moreover, the structure/fragment drug design using crystal structure analysis method is also becoming reliable. However, crystallization still remains as a major bottleneck for determining bio-macromolecular structures, although many methods have been developed such as crystallization kits, crystallization robot, crystallizing in gel, space, and magnetic field, laser excitation, using antibody, modification of protein surface, and so forth. The current situation of crystallization is still dependent on the accidental method searching for a crystallization reagent and the growth environment since the methodology for obtaining a quality crystal for structure analysis is not established yet. Therefore, further development of more advanced crystallization methods is required to increase the probability of successful crystallization. In principal, probability of successful crystallization could be increased by polymerized molecules with 2 or 3-fold rotation symmetry [1]. We have solved more than 100 structures, and found some fragments which is isolated from core structure, and seem to contribute to form high quality crystal by forming a polymer with 2 or 3-fold axial symmetry. Thus, we developed a novel method by fussing target protein with crystallization tags named 2/3RS-tag. These 2/3RS-tags polymerize target proteins with 2 or 3-fold axial symmetry, and consequently accelerate formation of crystal. We will report and discuss this new method in this presentation.
The defensive-offensive associations between algae and herbivores determine marine ecology. Brown algae utilize phlorotannin as their chemical defense against the predator Aplysia kurodai, which uses β-glucosidase (akuBGL) to digest the laminarin in algae to glucose. Moreover, A. kurodai employs Eisenia hydrolysis-enhancing protein (EHEP) as an offense to protect akuBGL activity from phlorotannin inhibition by precipitating phlorotannin. To underpin the molecular mechanism of this digestive-defensive-offensive system, we determined the structures of apo and tannic-acid (TNA, a phlorotannin-analog) bound form of EHEP, as well as apo akuBGL. EHEP consisted of three peritrophin-A domains formed in a triangle and bound TNA in the center without significant conformational changes. Structural comparison between EHEP and EHEP–TNA led us to find that EHEP can be resolubilized from phlorotannin-precipitation at an alkaline pH, which reflects a requirement in the digestive tract. akuBGL contained two GH1 domains, only one of which conserved the active site. Combining docking analysis, we propose the mechanisms by which phlorotannin inhibits akuBGL by occupying the substrate-binding pocket, and EHEP protects akuBGL against the inhibition by binding with phlorotannin to free the akuBGL pocket.
