Bioinformatics for comprehensive finding and analysis of glycosyltransferases

Bioinformatics is a very powerful tool in the field of glycoproteomics as well as genomics and proteomics. As a part of the Glycogene Project (GG project), we have developed a novel bioinformatics system for the comprehensive identification and in silico cloning of human glycogenes. Using our system, a total of 105 candidate human glycogenes were identified and then engineered for heterologous expression. Of these candidates, 38 recombinant proteins were successfully identified for their enzyme activity and substrate specificity. We also classified 47 out of 60 carbohydrate-active enzyme glycosyltransferase families into 4 superfamilies using the profile Hidden Markov Model method. On the basis of our classification and the relationship between glycosylation pathways and superfamilies, we propose the evolution of glycosyltransferases.     

Lidocaine depresses splenocyte immune functions following trauma-hemorrhage in mice

Traumatic and/or surgical injury as well as hemorrhage induces profound suppression of cellular immunity. Although local anesthetics have been shown to impair immune responses, it remains unclear whether lidocaine affects lymphocyte functions following trauma-hemorrhage (T-H). We hypothesized that lidocaine will potentiate the suppression of lymphocyte functions after T-H. To test this, we randomly assigned male C3H/HeN (6–8 wk) mice to sham operation or T-H. T-H was induced by midline laparotomy and 90 min of hemorrhagic shock (blood pressure 35 mmHg), followed by fluid resuscitation (4x shed blood volume in the form of Ringer lactate). Two hours later, the mice were killed and splenocytes and bone marrow cells were isolated. The effects of lidocaine on concanavalin A-stimulated splenocyte proliferation and cytokine production in both sham-operated and T-H mice were assessed. The effects of lidocaine on LPS-stimulated bone marrow cell proliferation and cytokine production were also assessed. The results indicate that T-H suppresses cell proliferation, Th1 cytokine production, and MAPK activation in splenocytes. In contrast, cell proliferation, cytokine production, and MAPK activation in bone marrow cells were significantly higher 2 h after T-H compared with shams. Lidocaine depressed immune responses in splenocytes; however, it had no effect in bone marrow cells in either sham or T-H mice. The enhanced immunosuppressive effects of lidocaine could contribute to the host's enhanced susceptibility to infection following T-H. shock; bone marrow cells     

Spontaneous absolute asymmetric synthesis in the presence of achiral silica gel in conjunction with asymmetric autocatalysis

An enantiomerically enriched pyrimidyl alkanol with either S or R configurations was obtained stochastically from the reaction between pyrimidine-5-carbaldehyde and diisopropylzinc in the presence of achiral silica gel in conjunction with asymmetric autocatalysis with amplification of chirality.     

How does stochasticity in colonization accelerate the speed of invasion in a cellular automaton model?

We investigate the speed of invasion waves for a single species generated by stochastic short- and/or long-distance colonizations in a time-continuous cellular automaton (CA) model on a two-dimensional homogenous landscape. By simulating the CA models, we demonstrate that stochasticity can dramatically increase the speed of invasion compared to the corresponding deterministic CA model or the corresponding one-dimensional stochastic CA model. To explain this phenomenon, we first develop a mathematical model for the invasion involving only short-distance colonization (i.e., colonization only occurs from the eight adjacent cells), and present several approximation methods for solving the model. Our analyses show that the increased wave speed in the stochastic model is due to irregularity in the shape of the wavefront. Further extension of this model to include long-distance colonization demonstrates that stochasticity influences speeds to even greater extents in this case. Using dimension analysis, we deduced a semi-empirical formula for the speed as a function of three parameters intrinsic to short- and long-distance colonization, which agrees well with simulation results. Based on these results, we discuss how important stochasticity in colonization and spatial dimensionality are in the acceleration of invasion speed.     

Relationship between the dough quality and content of specific glutenin proteins in wheat mill streams, and its application to making flour suitable for instant Chinese noodles

The content of specific proteins such as high-molecular-weight glutenin subunits HMW-GS 5+10 and low-molecular-weight glutenin subunits LMW-GS KS2 in wheat mill streams of extra-strong Kachikei 33 wheat was quantified by SDS-PAGE and 2D-PAGE. The mill streams showed varied quantities of HMW-GS 5+10 (0.077 to 2.007 mg/g of mill stream), LMW-GS KS2 (0.018 to 0.586 mg/g of mill stream) and total protein (9.42% to 18.98%). The contents of these specific proteins in the mill streams were significantly correlated with the SDS sedimentation volume and the mixing properties, which are respective indices of specific loaf volume and dough strength. The contents of these specific glutenin proteins in the mill streams were therefore found to be significantly important for improving the dough quality suitable for bread and Chinese noodles. Accordingly, we present here the application of this information to the development of an effective method for producing mill streams with high quality and yield that are suitable for instant Chinese noodles.     

Development of a colorimetric assay for determining the amount of H2O2 generated in tobacco cells in response to elicitors and its application to study of the structure-activity relationship of flagellin-derived peptides

Plants exhibit various defense responses after recognizing elicitor molecules derived from pathogenic microorganisms and host plants. In this study, we developed an improved colorimetric assay for quantifying the generation of H2O2 in plant cells, one of the defense responses, to evaluate elicitor activity quantitatively. H2O2 is detected using a dye, N-(carboxymethylaminocarbonyl)-4,4'-bis(dimethylamino)-diphenylamine sodium salt (DA-64), which can be measured by conventional spectrometers in a highly sensitive and quantitative manner. Using this method, we successfully measured the elicitor activity of flagellin-derived peptides in cultured tobacco cells, and identified several structural features of the peptides important for the elicitor activity. The results suggest that the structural factors required for expression of the elicitor activity differ slightly among plant species. The efficient and sensitive assay developed in this study should be useful not only for studying structure-activity relationships, but also for the screening of novel compounds that can induce defense responses.     

Production of (R)-3-amino-3-phenylpropionic acid and (S)-3-amino-3-phenylpropionic acid from (R,S)-N-acetyl-3-amino-3-phenylpropionic acid using microorganisms having enantiomer-specific amidohydrolyzing activity

(R)-3-Amino-3-phenylpropionic acid ((R)-beta-Phe) and (S)-3-amino-3-phenylpropionic acid ((S)-beta-Phe) are key compounds on account of their use as intermediates in synthesizing pharmaceuticals. Enantiomerically pure non-natural amino acids are generally prepared by enzymatic resolution of the racemic N-acetyl form, but despite the intense efforts this method could not be used for preparing enantiomerically pure beta-Phe, because the effective enzyme had not been found. Therefore, screening for microorganisms capable of amidohydrolyzing (R,S)-N-acetyl-3-amino-3-phenylpropionic acid ((R,S)-N-Ac-beta-Phe) in an enantiomer-specific manner was performed. A microorganism having (R)-enantiomer-specific amidohydrolyzing activity and another having both (R)-enantiomer- and (S)-enantiomer-specific amidohydrolyzing activities were obtained from soil samples. Using 16S rDNA analysis, the former organism was identified as Variovorax sp., and the latter as Burkholderia sp. Using these organisms, enantiomerically pure (R)-beta-Phe (>99.5% ee) and (S)-beta-Phe (>99.5% ee) with a high molar conversion yield (67%-96%) were obtained from the racemic substrate.     

Crystal structure of GlcAT-S, a human glucuronyltransferase, involved in the biosynthesis of the HNK-1 carbohydrate epitope

The HNK-1 carbohydrate epitope is found in various neural cell adhesion molecules. Two glucuronyltransferases (GlcAT-P and GlcAT-S) are involved in the biosynthesis of HNK-1 carbohydrate. Our previous study on the crystal structure of GlcAT-P revealed the reaction and substrate recognition mechanisms of this enzyme. Comparative analyses of the enzymatic activities of GlcAT-S and GlcAT-P showed that there are notable differences in the acceptor substrate specificities of these enzymes. To elucidate differences between their specificities, we now solved the crystal structure of GlcAT-S. Residues interacting with UDP molecule, which is a part of the donor substrate, are highly conserved between GlcAT-P and GlcAT-S. On the other hand, there are some differences between these proteins in the manner they recognize their respective acceptor substrates. Phe245, one of the most important GlcAT-P residues for the recognition of acceptors, is a tryptophan in GlcAT-S. In addition, Val320, which is located on the C-terminal long loop of the neighboring molecule in the dimer and critical in the recognition of the acceptor sugar molecule by the GlcAT-P dimer, is an alanine in GlcAT-S. These differences play key roles in establishing the distinct specificity for the acceptor substrate by GlcAT-S, which is further supported by site-directed mutagenesis of GlcAT-S and a computer-aided model building of GlcAT-S/substrate complexes.     

Netrin 1 regulates ventral tangential migration of guidepost neurons in the lateral olfactory tract

In the developing nervous system, functional neural networks are constructed with intricate coordination of neuronal migrations and axonal projections. We have previously reported a ventral tangential migration of a special type of cortical neurons, lot cells, in the mouse embryo. These neurons originate from the ventricular zone of the entire neocortex, tangentially migrate in the surface layer of the neocortex into the ventral direction, align in the future pathway of the lateral olfactory tract (LOT) and eventually guide the projection of LOT axons. In this study, we developed an organotypic culture system to investigate the regulation of this cell migration in the developing telencephalon. Our data show that the neocortex contains the signals that direct lot cells ventrally, that the ganglionic eminence excludes lot cells by repelling the migration and that lot cells are attracted to netrin 1, an axon guidance factor. Furthermore, we demonstrate that mutations in the genes encoding netrin 1 and its functional receptor Dcc lead to inappropriate distribution of lot cells and subsequent partial disruption of LOT projection. These results suggest that netrin 1 regulates the migration of lot cells and LOT projections, possibly by ensuring the correct distribution of these guidepost neurons.     

Molecular cloning and characterization of a novel human beta1,3-glucosyltransferase, which is localized at the endoplasmic reticulum and glucosylates O-linked fucosylglycan on thrombospondin type 1 repeat domain

Protein O-linked fucosylation is an unusual glycosylation associated with many important biological functions such as Notch signaling. Two fucosylation pathways synthesizing O-fucosylglycans have been reported on cystein-knotted proteins, that is, on epidermal growth factor-like (EGF-like) domains and on thrombospondin Type 1 repeat (TSR) domains. We report here the molecular cloning and characterization of a novel beta1,3-glucosyltransferase (beta3Glc-T) that synthesizes a Glcbeta1,3Fucalpha- structure on the TSR domain. We found a novel glycosyltransferase gene with beta1,3-glycosyltransferase (beta3GT) motifs in databases. The recombinant enzyme expressed in human embryonic kidney 293T (HEK293T) cells exhibited glucosyltransferase activity toward fucose-alpha-para-nitrophenyl (Fucalpha-pNp). Thin-layer chromatography (TLC) analysis revealed that the product of the recombinant enzyme migrated to the same position as did the product of endogenous beta3Glc-T of Chinese hamster ovary (CHO) cells. The two products could be digested by beta-glucosidase from almond and by exo-1,3-beta-glucanase from Trichoderma sp. These results strongly suggested that the product has the structure of Glcbeta1-3Fuc. Therefore, we named this novel enzyme beta3Glc-T. Immunostaining revealed that FLAG-tagged beta3Glc-T is an enzyme residing in the endoplasmic reticulum (ER) via retention signal, "REEL," which is a KDEL-like sequence, at the C-terminus. The TSR domain expressed in Escherichia coli was first fucosylated by the recombinant protein O-fucosyltransferase 2 (POFUT2), after which it became an acceptor substrate for the recombinant beta3Glc-T, which could apparently transfer Glc to the fucosylated TSR domain. Our results suggest that a novel glycosyltransferase, beta3Glc-T, contributes to the elongation of O-fucosylglycan and that this occurs specifically on TSR domains.