Kohei Kazuma

Crystal structure of UDP‐glucose:anthocyanidin 3‐O‐glucosyltransferase from Clitoria ternatea

The anthocyanidin 3-O-glucosyltransferase from Clitoria ternatea (Ct3GT-A) was expressed in Escherichia coli, and the three-dimensional structure of Ct3GT-A was determined using X-ray crystallography. This report describes the architecture of Ct3GT-A, including the structures of the donor- and acceptor-binding sites.

Deficiency of Nicotinamide Mononucleotide Adenylyltransferase 3 (Nmnat3) Causes Hemolytic Anemia by Altering the Glycolytic Flow in Mature Erythrocytes

Background: Nmnat3 is considered a mitochondria-localized NAD synthesis enzyme. However, its physiological function in vivo remains unclear. Results: Loss of Nmnat3 results in drastic depletion of the NAD pool and stalls the glycolytic flow in mature erythrocytes. Conclusion: Nmnat3 deficiency causes splenomegaly and hemolytic anemia in mice. Significance: This report reveals the essential role of Nmnat3 in mature erythrocytes. NAD biosynthesis is of substantial interest because of its important roles in regulating various biological processes. Nicotinamide mononucleotide adenylyltransferase 3 (Nmnat3) is considered a mitochondria-localized NAD synthesis enzyme involved in de novo and salvage pathways. Although the biochemical properties of Nmnat3 are well documented, its physiological function in vivo remains unclear. In this study, we demonstrated that Nmnat3 was localized in the cytoplasm of mature erythrocytes and critically regulated their NAD pool. Deficiency of Nmnat3 in mice caused splenomegaly and hemolytic anemia, which was associated with the findings that Nmnat3-deficient erythrocytes had markedly lower ATP levels and shortened lifespans. However, the NAD level in other tissues were not apparently affected by the deficiency of Nmnat3. LC-MS/MS-based metabolomics revealed that the glycolysis pathway in Nmnat3-deficient erythrocytes was blocked at a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) step because of the shortage of the coenzyme NAD. Stable isotope tracer analysis further demonstrated that deficiency of Nmnat3 resulted in glycolysis stall and a shift to the pentose phosphate pathway. Our findings indicate the critical roles of Nmnat3 in maintenance of the NAD pool in mature erythrocytes and the physiological impacts at its absence in mice.

Structural basis for acceptor-substrate recognition of UDP-glucose: anthocyanidin 3-O-glucosyltransferase from Clitoria ternatea

UDP‐glucose: anthocyanidin 3‐O‐glucosyltransferase (UGT78K6) from Clitoria ternatea catalyzes the transfer of glucose from UDP‐glucose to anthocyanidins such as delphinidin. After the acylation of the 3‐O‐glucosyl residue, the 3′‐ and 5′‐hydroxyl groups of the product are further glucosylated by a glucosyltransferase in the biosynthesis of ternatins, which are anthocyanin pigments. To understand the acceptor‐recognition scheme of UGT78K6, the crystal structure of UGT78K6 and its complex forms with anthocyanidin delphinidin and petunidin, and flavonol kaempferol were determined to resolutions of 1.85 Å, 2.55 Å, 2.70 Å, and 1.75 Å, respectively. The enzyme recognition of unstable anthocyanidin aglycones was initially observed in this structural determination. The anthocyanidin‐ and flavonol‐acceptor binding details are almost identical in each complex structure, although the glucosylation activities against each acceptor were significantly different. The 3‐hydroxyl groups of the acceptor substrates were located at hydrogen‐bonding distances to the Nε2 atom of the His17 catalytic residue, supporting a role for glucosyl transfer to the 3‐hydroxyl groups of anthocyanidins and flavonols. However, the molecular orientations of these three acceptors are different from those of the known flavonoid glycosyltransferases, VvGT1 and UGT78G1. The acceptor substrates in UGT78K6 are reversely bound to its binding site by a 180° rotation about the O1–O3 axis of the flavonoid backbones observed in VvGT1 and UGT78G1; consequently, the 5‐ and 7‐hydroxyl groups are protected from glucosylation. These substrate recognition schemes are useful to understand the unique reaction mechanism of UGT78K6 for the ternatin biosynthesis, and suggest the potential for controlled synthesis of natural pigments.

Bunodosine 391: An Analgesic Acylamino Acid from the Venom of the Sea Anemone Bunodosoma cangicum

A new acylamino acid, bunodosine 391 (BDS 391), was isolated from the venom of the sea anemone Bunodosoma cangicum. The structure was elucidated by spectroscopic analyses (2D NMR, ESIMS/MS) and verified by its synthesis. Intraplantar injection of BDS 391 into the hind paw of a rat induced a potent analgesic effect. This effect was not altered by naloxone (an opioid receptor antagonist), but was completely reversed by methysergide (a serotonin receptor antagonist), indicating that the effect is mediated by activation of serotonin receptors.

Chemical and biological characterization of four new linear cationic α-helical peptides from the venoms of two solitary eumenine wasps

Four novel peptides were isolated from the venoms of the solitary eumenine wasps Eumenes rubrofemoratus and Eumenes fraterculus. Their sequences were determined by MALDI-TOF/TOF (matrix assisted laser desorption/ionization time-of-flight mass spectrometry) analysis, Edman degradation and solid-phase synthesis. Two of them, eumenitin-R (LNLKGLIKKVASLLN) and eumenitin-F (LNLKGLFKKVASLLT), are highly homologous to eumenitin, an antimicrobial peptide from a solitary eumenine wasp, whereas the other two, EMP-ER (FDIMGLIKKVAGAL-NH(2)) and EMP-EF (FDVMGIIKKIAGAL-NH(2)), are similar to eumenine mastoparan-AF (EMP-AF), a mast cell degranulating peptide from a solitary eumenine wasp. These sequences have the characteristic features of linear cationic cytolytic peptides; rich in hydrophobic and basic amino acids with no disulfide bond, and accordingly, they can be predicted to adopt an amphipathic α-helix secondary structure. In fact, the CD (circular dichroism) spectra of these peptides showed significant α-helical conformation content in the presence of TFE (trifluoroethanol), SDS (sodium dodecylsulfate) and asolectin vesicles. In the biological evaluation, all the peptides exhibited a significant broad-spectrum antimicrobial activity, and moderate mast cell degranulation and leishmanicidal activities, but showed virtually no hemolytic activity.

The Extract of Roots of Sophora flavescens Enhances the Recovery of Motor Function by Axonal Growth in Mice with a Spinal Cord Injury

Although axonal extension to reconstruct spinal tracts should be effective for restoring function after spinal cord injury (SCI), chondroitin sulfate proteoglycan (CSPG) levels increase at spinal cord lesion sites, and inhibit axonal regrowth. In this study, we found that the water extract of roots of Sophora flavescens extended the axons of mouse cortical neurons, even on a CSPG-coated surface. Consecutive oral administrations of S. flavescens extract to SCI mice for 31 days increased the density of 5-HT-positive axons at the lesion site and improved the motor function. Further, the active constituents in the S. flavescens extract were identified. The water and alkaloid fractions of the S. flavescens extract each exhibited axonal extension activity in vitro. LC/MS analysis revealed that these fractions mainly contain matrine and/or oxymatrine, which are well-known major compounds in S. flavescens. Matrine and oxymatrine promoted axonal extension on the CSPG-coated surface. This study is the first to demonstrate that S. flavescens extract, matrine, and oxymatrine enhance axonal growth in vitro, even on a CSPG-coated surface, and that S. flavescens extract improves motor function and increases axonal density in SCI mice.

Molecular ion index assisted comprehensive profiling of B-type oligomeric proanthocyanidins in rhubarb by high performance liquid chromatography-tandem mass spectrometry

AbstractProanthocyanidins (PAs) are major anti-diarrhea constituents in rhubarb, one of the frequently used traditional medicines. However, the phytochemical investigation of PAs in rhubarb was hampered by their strenuous purification and identification. In the present study, aiming to clarify the distribution of PAs in different rhubarb species, a molecular ion index was priorly established according to the structural features of B-type PAs, which led to a series of targeted discovery of oligomeric PAs in rhubarb by the HPLC-ESI-MS/MS method. Totally, 66 oligomeric PAs including 27 dimers, 29 trimers, and 10 tetramers were tentatively identified on the basis of their MS/MS spectra from 28 rhubarb samples derived from 5 Rheum species as R. palmatum, R. tanguticum, R. officinale, R. coreanum, and R. laciniatum. It is noteworthy that 6 propelargonidins, 14 prodelphinidins, and 10 procyanidin-tetramers were identified from rhubarb for the first time. The profiling comparison of these oligomeric PAs in different rhubarb samples was achieved by visualizing their abundance in a heat map, which indicated the dominant PAs in rhubarb were procyanidin-dimer and its galloylated derivatives. Graphical AbstractA molecular ion index was priorly established according to the structural features of B-type PAs, which led to a series of targeted discovery of oligomeric PAs in rhubarb by the HPLC-ESI-MS/MS method.

Active Constituents from Drynaria fortunei Rhizomes on the Attenuation of Aβ(25-35)-Induced Axonal Atrophy.

Axonal regeneration might contribute to the restoration of damaged neuronal networks and improvement of memory deficits in a murine Alzheimers disease (AD) model. A search for axonal regenerative drugs was performed to discover novel therapeutic options for AD. In this study, an aqueous extract of Drynaria fortunei rhizomes reversed Aβ25-35-induced axonal atrophy in cultured cortical neurons of mice. Bioassay-guided fractionation of this extract led to the isolation and identification of compounds 1-5. Among them, (2S)-neoeriocitrin (2) and caffeic acid 4-O-glucoside (4) showed significant axonal elongation effects on Aβ25-35-induced atrophy.

Combined Venom Gland Transcriptomic and Venom Peptidomic Analysis of the Predatory Ant Odontomachus monticola

Ants (hymenoptera: Formicidae) have adapted to many different environments and have become some of the most prolific and successful insects. To date, 13,258 ant species have been reported. They have been classified into 333 genera and 17 subfamilies. Except for a few Formicinae, Dolichoderinae, and members of other subfamilies, most ant species have a sting with venom. The venoms are composed of formic acid, alkaloids, hydrocarbons, amines, peptides, and proteins. Unlike the venoms of other animals such as snakes and spiders, ant venoms have seldom been analyzed comprehensively, and their compositions are not yet completely known. In this study, we used both transcriptomic and peptidomic analyses to study the composition of the venom produced by the predatory ant species Odontomachus monticola. The transcriptome analysis yielded 49,639 contigs, of which 92 encoded toxin-like peptides and proteins with 18,106,338 mapped reads. We identified six pilosulin-like peptides by transcriptomic analysis in the venom gland. Further, we found intact pilosulin-like peptide 1 and truncated pilosulin-like peptides 2 and 3 by peptidomic analysis in the venom. Our findings related to ant venom peptides and proteins may lead the way towards development and application of novel pharmaceutical and biopesticidal resources.

Specific expression of the vacuolar iron transporter, TgVit, causes iron accumulation in blue-colored inner bottom segments of various tulip petals.

Several flowers of Tulipa gesneriana exhibit a blue color in the bottom segments of the inner perianth. We have previously reported the inner-bottom tissue-specific iron accumulation and expression of the vacuolar iron transporter, TgVit1, in tulip cv. Murasakizuisho. To clarify whether the TgVit1-dependent iron accumulation and blue-color development in tulip petals are universal, we analyzed anthocyanin, its co-pigment components, iron contents and the expression of TgVit1 mRNA in 13 cultivars which show a blue color in the bottom segments of the inner perianth accompanying yellow- and white-colored inner-bottom petals. All of the blue bottom segments contained the same anthocyanin component, delphinidin 3-rutinoside. The flavonol composition varied with cultivar and tissue part. The major flavonol in the bottom segments of the inner perianth was rutin. The iron content in the upper part was less than that in the bottom segments of the inner perianth. The iron content in the yellow and white petals was higher in the bottom segment of the inner perianth than in the upper tissues. TgVit1 mRNA expression was apparent in all of the bottom tissues of the inner perianth. The result of a reproduction experiment by mixing the constituents suggests that the blue coloration in tulip petals is generally caused by iron complexation to delphinidin 3-rutinoside and that the iron complex is solubilized and stabilized by flavonol glycosides. TgVit1-dependent iron accumulation in the bottom segments of the inner perianth might be controlled by an unknown system that differentiated the upper parts and bottom segments of the inner perianth.