A C-type lectin isolated from the skin of Japanese bullhead shark (Heterodontus japonicus) binds a remarkably broad range of sugars and induces blood coagulation
The aim of this study was to determine the physiological role of skin lectins of the Japanese bullhead shark (Heterodontus japonicus). A skin extract was subjected to affinity chromatography using seven different sugars as ligands. Molecular mass and N-terminal amino acid sequence analyses indicated elution of the same protein by each of the seven respective cognate ligands from sugar affinity columns. The predicted amino acid sequence encoded by the cDNA of this protein [designated as H. japonicus C-type-lectin (HjCL)] identified it as a novel fish subgroup VII C-type lectin evolutionarily related to snake venom lectins. ...
Source: Journal of Biochemistry - April 28, 2015 Category: Biochemistry Authors: Tsutsui, S., Dotsuta, Y., Ono, A., Suzuki, M., Tateno, H., Hirabayashi, J., Nakamura, O. Tags: Regular Papers Source Type: research
Identification of diphtheria toxin R domain mutants with enhanced inhibitory activity against HB-EGF
Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a ligand of EGF receptor, is involved in the growth and malignant progression of cancers. Cross-reacting material 197, CRM197, a non-toxic mutant of diphtheria toxin (DT), specifically binds to the EGF-like domain of HB-EGF and inhibits its mitogenic activity, thus CRM197 is currently under evaluation in clinical trials for cancer therapy. To develop more potent DT mutants than CRM197, we screened various mutant proteins of R domain of DT, the binding site for HB-EGF. A variety of R-domain mutant proteins fused with maltose-binding protein were produced a...
Source: Journal of Biochemistry - April 28, 2015 Category: Biochemistry Authors: Suzuki, K., Mizushima, H., Abe, H., Iwamoto, R., Nakamura, H., Mekada, E. Tags: Regular Papers Source Type: research
Bacillus subtilis degSU operon is regulated by the ClpXP-Spx regulated proteolysis system
The DegS-DegU two-component regulatory system regulates many cellular events in Bacillus subtilis. Genes for DegSU constitutes an operon directed by the P1 promoter and downstream degU is autoregulated via the P3 promoter activated by phosphorylated DegU. In the Gram-positive bacteria, Spx plays a major role in the protection system against oxidative stresses as a transcriptional regulator. Spx is a substrate of the ATP-dependent ClpXP protease. It regulates diamide-stress regulon in addition to many genes with unknown functions. We have found that null mutations for clpX and clpP, which encode the subunits for the proteas...
Source: Journal of Biochemistry - April 28, 2015 Category: Biochemistry Authors: Shiwa, Y., Yoshikawa, H., Tanaka, T., Ogura, M. Tags: Regular Papers Source Type: research
Mutation analysis and molecular modeling for the investigation of ligand-binding modes of GPR84
GPR84 is a G protein-coupled receptor for medium-chain fatty acids. Capric acid and 3,3'-diindolylmethane are specific agonists for GPR84. We built a homology model of a GPR84-capric acid complex to investigate the ligand-binding mode using the crystal structure of human active-state β2-adrenergic receptor. We performed site-directed mutagenesis to subject ligand-binding sites to our model using GPR84-Giα fusion proteins and a [35S]GTPS-binding assay. We compared the activity of the wild type and mutated forms of GPR84 by [35S]GTPS binding to capric acid and diindolylmethane. The mutations L100D `Ballesteros&nda...
Source: Journal of Biochemistry - April 28, 2015 Category: Biochemistry Authors: Nikaido, Y., Koyama, Y., Yoshikawa, Y., Furuya, T., Takeda, S. Tags: Regular Papers Source Type: research
Phosphatidylethanolamine plasmalogen enhances the inhibiting effect of phosphatidylethanolamine on {gamma}-secretase activity
Plasmalogens (Pls) are widely distributed in the biological membrane of animals and certain anaerobic bacteria, but their functions in the cell membrane are still poorly understood. Decrease of phosphatidylethanolamine plasmalogen (PEPls) in the brain tissue of patients with Alzheimer's disease prompted us to investigate the effect of the membrane phosphorus lipid composition on the activity of -secretase that produces amyloid-beta protein (Aβ). To clarify the effect of phospholipids, including PEPls, on Aβ production, -secretase activity was measured in an in vitro assay using yeast microsomes and reconstituted ...
Source: Journal of Biochemistry - April 28, 2015 Category: Biochemistry Authors: Onodera, T., Futai, E., Kan, E., Abe, N., Uchida, T., Kamio, Y., Kaneko, J. Tags: Regular Papers Source Type: research
Ral GTPases: crucial mediators of exocytosis and tumourigenesis
The Ral guanosine triphosphatases (GTPases), RalA and RalB, are members of the Ras superfamily of small GTPases. Research on Ral GTPases and their functions over the past 25 years has revealed the essential involvement of these GTPases in unique and diverse cellular processes including exocyst-mediated exocytosis and related cellular activities. Moreover, it is increasingly appreciated that the aberrant activation of Ral GTPases is one of the major causes of human tumourigenesis induced by oncogenic Ras. Recent evidence suggests that Ral signalling pathways may be potential therapeutic targets for the treatment of human ca...
Source: Journal of Biochemistry - April 28, 2015 Category: Biochemistry Authors: Shirakawa, R., Horiuchi, H. Tags: JB Reviews Source Type: research
Hepatocyte growth factor and Met in drug discovery
Activation of the hepatocyte growth factor (HGF)–Met pathway evokes dynamic biological responses that support the morphogenesis, regeneration and survival of cells and tissues. A characterization of conditional Met knockout mice indicates that the HGF–Met pathway plays important roles in the regeneration, protection and homeostasis of cells such as hepatocytes, renal tubular cells and neurons. Preclinical studies in disease models have indicated that recombinant HGF protein and expression plasmid for HGF are biological drug candidates for the treatment of patients with diseases or injuries that involve impaired...
Source: Journal of Biochemistry - April 28, 2015 Category: Biochemistry Authors: Sakai, K., Aoki, S., Matsumoto, K. Tags: JB Reviews Source Type: research
Quantitative analysis of the interaction between L-methionine derivative and oligonucleotides
This study explores the use of l-methionine derivative as a potential affinity ligand for nucleic acids purification. The l-methionine derivative is synthesized by activation of the carboxylic acid group with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide follow by immobilization on amine sensor surface, previously activated and treated with ethylenediamine. Their affinity towards oligonucleotides has been determined by surface plasmon resonance biosensor. The highest affinity is found for cytosine and thymine, followed by adenine, whereas the lowest affinity is found for guanine. For hetero-oligonucle...
Source: Journal of Biochemistry - March 27, 2015 Category: Biochemistry Authors: Mota, E., Sousa, F., Queiroz, J. A., Cruz, C. Tags: Regular Papers Source Type: research
Isolation and functional analysis of yeast ubiquitin ligase Rsp5 variants that alleviate the toxicity of human {alpha}-synuclein
The essential ubiquitin ligase Rsp5 is a key enzyme involved in the degradation of abnormal or unfavourable proteins in the yeast Saccharomyces cerevisiae. Overexpression of human α-synuclein (α-syn), a small lipid-binding protein implicated in several neurodegenerative diseases, in S. cerevisiae leads to growth inhibition due to many intracellular defects, including accumulation of reactive oxygen species (ROS). Here, to understand the mechanism of Rsp5-mediated detoxification of α-syn, we isolated novel Rsp5 variants (T255A, D295G, P343S and N427D), which conferred α-syn tolerance to yeast cells. ...
Source: Journal of Biochemistry - March 27, 2015 Category: Biochemistry Authors: Wijayanti, I., Watanabe, D., Oshiro, S., Takagi, H. Tags: Regular Papers Source Type: research
Protocadherin-9 involvement in retinal development in Xenopus laevis
Biological roles of most protocadherins (Pcdhs) are a largely unsolved problem. Therefore, we cloned cDNA for Xenopus laevis protocadherin-9 and characterized its properties to elucidate the role. The deduced amino acid sequence was highly homologous to those of mammalian protocadherin-9 s. X. laevis protocadherin-9 expressed from the cDNA in L cells showed basic properties similar to those of mammalian Pcdhs. Expression of X. laevis protocadherin-9 was first detected in stage-31 embryos and increased as the development proceeded. In the later stage embryos and the adults, the retina strongly expressed protocadherin-9, whi...
Source: Journal of Biochemistry - March 27, 2015 Category: Biochemistry Authors: Izuta, Y., Taira, T., Asayama, A., Machigashira, M., Kinoshita, T., Fujiwara, M., Suzuki, S. T. Tags: Regular Papers Source Type: research
The side chain of a glycosylated asparagine residue is important for the stability of isopullulanase
N-glycosylation has been shown to be important for the stability of some glycoproteins. Isopullulanase (IPU), a polysaccharide-hydrolyzing enzyme, is a highly N-glycosylated protein, and IPU deglycosylation results in a decrease in thermostability. To investigate the function of N-glycan in IPU, we focused on an N-glycosylated residue located in the vicinity of the active site, Asn448. The thermostabilities of three IPU variants, Y440A, N448A and S450A, were 0.5–8.4°C lower than the wild-type enzyme. The crystal structure of endoglycosidase H (Endo H)-treated N448A variant was determined. There are four IPU molec...
Source: Journal of Biochemistry - March 27, 2015 Category: Biochemistry Authors: Miyazaki, T., Yashiro, H., Nishikawa, A., Tonozuka, T. Tags: Regular Papers Source Type: research
The peroxidase activity of bleomycin-Fe3+ is associated with damage to biological components
In this study, bleomycin-Fe3+ steadily oxidized tetramethylbenzidine (TMB) in the presence of peroxides. However, the ability of bleomycin-Fe3+ to function as a peroxidase was extremely low compared with that of other peroxidases. A characteristic property of bleomycin-Fe3+ different from that observed for other peroxidases is its ability to oxidize TMB at the similar rate at both a pH 5 and 8 in the presence of lipid hydroperoxide (LOOH). In the present experiments, hydroxyl radicals (HO•) were generated only when bleomycin-Fe3+ was incubated with H2O2 at a pH of 5. No generation of HO• was observed during the i...
Source: Journal of Biochemistry - March 27, 2015 Category: Biochemistry Authors: Miura, T. Tags: Regular Papers Source Type: research
In situ visualization of a glycoform of transferrin: localization of {alpha}2,6-sialylated transferrin in the liver
We previously found that a lectin, Sambucus sieboldiana agglutinin (SSA), bound to α2,6-sialylated glycan epitopes on transferrin and inhibited anti-transferrin antibody binding to the antigen in ELISA (SSA inhibition). Here we report that SSA inhibition is applicable to immunohistochemistry, localizing α2,6-sialylated transferrin in the liver. Immunohistochemistry using anti-transferrin polyclonal antibody revealed that transferrin was detected in hepatocytes near interlobular veins. Addition of SSA lectin markedly attenuated the staining. Sialidase treatment of a liver section abolished SSA binding and concom...
Source: Journal of Biochemistry - March 27, 2015 Category: Biochemistry Authors: Matsumoto, Y., Saito, T., Hoshi, K., Ito, H., Kariya, Y., Nagae, M., Yamaguchi, Y., Hagiwara, Y., Kinoshita, N., Wada, I., Saito, K., Honda, T., Hashimoto, Y. Tags: Regular Papers Source Type: research
Heterologous expression of L-lysine {alpha}-oxidase from Scomber japonicus in Pichia pastoris and functional characterization of the recombinant enzyme
In this study, a synthetic gene coding for Scomber japonicus l-lysine α-oxidase, originally termed AIP (for apoptosis-inducing protein), was expressed in Pichia pastoris, and the recombinant enzyme (rAIP) was purified and characterized. rAIP exhibited essentially the same substrate specificity as the native enzyme, catalyzing the oxidative deamination of l-lysine as an exclusive substrate. rAIP was N-glycosylated and remained active over a wide range of pH, with an optimal pH of 7.5. The enzyme was stable in the pH range from 4.5 to 10.0 and was thermally stable up to 60°C. A molecular modelling of rAIP and a com...
Source: Journal of Biochemistry - March 27, 2015 Category: Biochemistry Authors: Tani, Y., Omatsu, K., Saito, S., Miyake, R., Kawabata, H., Ueda, M., Mihara, H. Tags: Regular Papers Source Type: research
Engineering of a 3'-sulpho-Gal{beta}1-4GlcNAc-specific probe by a single amino acid substitution of a fungal galectin
Among sulphated glycans, little is known about 3'-sulphation because of the lack of useful probes. In the course of molecular engineering of a fungal galectin from Agrocybe cylindracea, we found that a single substitution of Glu86 with alanine resulted in acquisition of specific binding for the 3'-sulpho-Galβ1-4GlcNAc structure. Extensive glyco-technological analysis revealed that this property was obtained in a ‘loss-of-function’ manner. Though this mutant (E86A) had low total affinity, it showed substantial binding to a naturally occurring N-glycan, of which the terminal galactose is 3-sulphated. Moreove...
Source: Journal of Biochemistry - March 27, 2015 Category: Biochemistry Authors: Hu, D., Huang, H., Tateno, H., Nakakita, S.-i., Sato, T., Narimatsu, H., Yao, X., Hirabayashi, J. Tags: Rapid Communication Source Type: research
