α-Glucosidase from a strain of deep-sea Geobacillus: a potential enzyme for the biosynthesis of complex carbohydrates

An α-glucosidase from Geobacillus sp. strain HTA-462, one of the deepest sea bacteria isolated from the sediment of the Mariana Trench, was purified to homogeneity and estimated to be a 65-kDa protein by SDS-PAGE. At low ion strength, the enzyme exists in the homodimeric form (130 kDa). It is a thermo- and alkaline-stable enzyme with a half-life of 13.4 h and a maximum hydrolytic activity at 60°C and pH 9.0 in 15 mM glycine–NaOH buffer. The enzyme exclusively hydrolyzed α-1,4-glycosidic linkages of oligosaccharides in an exo-type manner. The enzyme had an overwhelming transglycosylation activity and glycosylated various non-sugar molecules when maltose was used as a sugar donor. It converted maltose to isomaltose. The gene encoding the enzyme was cloned and sequenced. The recombinant enzyme could be extracellularly overproduced by Bacillus subtilis harboring its gene and preserved the primary properties of the native enzyme. Site-directed mutagenesis experiments showed that Asp98 is essential for the enzyme activity in addition to Asp199, Asp326, and Glu256.     

GSK-3beta regulates phosphorylation of CRMP-2 and neuronal polarity

Neurons are highly polarized and comprised of two structurally and functionally distinct parts, an axon and dendrites. We previously showed that collapsin response mediator protein-2 (CRMP-2) is critical for specifying axon/dendrite fate, possibly by promoting neurite elongation via microtubule assembly. Here, we showed that glycogen synthase kinase-3beta (GSK-3beta) phosphorylated CRMP-2 at Thr-514 and inactivated it. The expression of the nonphosphorylated form of CRMP-2 or inhibition of GSK-3beta induced the formation of multiple axon-like neurites in hippocampal neurons. The expression of constitutively active GSK-3beta impaired neuronal polarization, whereas the nonphosphorylated form of CRMP-2 counteracted the inhibitory effects of GSK-3beta, indicating that GSK-3beta regulates neuronal polarity through the phosphorylation of CRMP-2. Treatment of hippocampal neurons with neurotrophin-3 (NT-3) induced inactivation of GSK-3beta and dephosphorylation of CRMP-2. Knockdown of CRMP-2 inhibited NT-3-induced axon outgrowth. These results suggest that NT-3 decreases phosphorylated CRMP-2 and increases nonphosphorylated active CRMP-2, thereby promoting axon outgrowth.     

Phosphorylation by Rho kinase regulates CRMP-2 activity in growth cones

Collapsin response mediator protein 2 (CRMP-2) enhances the advance of growth cones by regulating microtubule assembly and Numb-mediated endocytosis. We previously showed that Rho kinase phosphorylates CRMP-2 during growth cone collapse; however, the roles of phosphorylated CRMP-2 in growth cone collapse remain to be clarified. Here, we report that CRMP-2 phosphorylation by Rho kinase cancels the binding activity to the tubulin dimer, microtubules, or Numb. CRMP-2 binds to actin, but its binding is not affected by phosphorylation. Electron microscopy revealed that CRMP-2 localizes on microtubules, clathrin-coated pits, and actin filaments in dorsal root ganglion neuron growth cones, while phosphorylated CRMP-2 localizes only on actin filaments. The phosphomimic mutant of CRMP-2 has a weakened ability to enhance neurite elongation. Furthermore, ephrin-A5 induces phosphorylation of CRMP-2 via Rho kinase during growth cone collapse. Taken together, these results suggest that Rho kinase phosphorylates CRMP-2, and inactivates the ability of CRMP-2 to promote microtubule assembly and Numb-mediated endocytosis, during growth cone collapse.     

Nuclear translocation of the heterotrimeric CCAAT binding factor of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> is dependent on two redundant localising signals in a single subunit

The CCAAT-binding complex in the Aspergillus species, also known as the Hap complex, consists of at least three subunits, namely HapB, HapC and HapE. Each Hap subunit contains an evolutionary conserved core domain. Recently, we have found that the HapC and HapE subunits do not carry a nuclear localisation signal. Furthermore, when in complex with HapB, they are transported into the nucleus via a ‘piggy back mechanism’ in A. nidulans. To extend our findings to other filamentous fungi, we examined the nuclear localisation of the A. oryzae Hap subunits by analysing several GFP fusion proteins with these Hap subunits in the hap deletion strains of A. nidulans. The nuclear translocation of the A. oryzae complex was found to be dependent on two redundant localising signals in HapB.     

Dietary unripe apple polyphenol inhibits the development of food allergies in murine models

The incidence of type I allergic disorders has been increasing worldwide, particularly, the hypersensitivity to food. We first showed that apple condensed tannin (ACT) intake would inhibit the development of the oral sensitization and that the inhibition could correlate with the rise in the population of TCR(gamma)delta-T cells in the intestinal intraepithelial lymphocytes (IEL) using W/W(V) mice and B10A mice which were ovalbumin (OVA)-orally sensitized. Serum OVA-specific immunoglobulin E and immunoglobulin G1 titers in the OVA-orally sensitized W/W(V) and B10A mice ad libitium fed ACT were extremely inhibited compared to those of the control. The ACT intakes of OVA-sensitized W/W(V) and B10A mice inhibited the immediate reduction of the body temperature or the rise in serum histamine induced by active systemic anaphylaxis. The proportions of the TCR(gamma)delta-T cells in the IEL of the OVA-orally sensitized W/W(V) and B10A mice ad libitium fed ACT were significantly greater than that in the controls. Furthermore, ACT feeding by itself could induce the rise in the percentage of the TCR(gamma)delta-T cells among the IEL of the W/W(V) and B10A mice. This suggests that the ACT intake may prevent the development of food allergies and this effect could be correlated with the rise in the percentage of TCR(gamma)delta-T cells among the IEL.     

Synthesis of digalactosyl diacylglycerols and their structure-inhibitory activity on human lanosterol synthase

Digalactosyl and monogalactocyl diacylglycerols (DGDG and MGDG), which were identified as anti-hyperlipemia active components in Colocasia esculenta (Taro), were synthesized. The inhibitory activity of DGDG, MGDG and related compounds on human lanosterol synthase was evaluated as anti-hyperlipemic activity. DGDG with two myristoyl groups at both sn-1 and sn-2 positions and with an oleoyl group at the sn-1 position showed the most potent activity.     

Recombinant porcine zona pellucida glycoproteins expressed in Sf9 cells bind to bovine sperm but not to porcine sperm

The zona pellucida, which surrounds the mammalian oocyte, consists of the ZPA, ZPB, and ZPC glycoproteins and plays roles in species-selective sperm-egg interactions via its carbohydrate moieties. In the pig, this activity is conferred by tri- and tetraantennary complex type chains; in cattle, it is conferred by a chain of 5 mannose residues. In this study, porcine zona glycoproteins were expressed as secreted forms, using the baculovirus-Sf9 insect cell system. The sperm binding activities of the recombinant proteins were examined in three different assays. The assays clearly demonstrated that recombinant ZPB bound bovine sperm weakly but did not bind porcine sperm; when recombinant ZPC was also present, bovine sperm binding activity was greatly increased, but porcine sperm still was not bound. The major sugar chains of ZPB were pauci and high mannose type chains that were similar in structure to the major neutral N-linked chain of the bovine zona. In fact, the nonreducing terminal alpha-mannose residues were necessary for the sperm binding activity. These results show that the carbohydrate moieties of zona glycoproteins, but not the polypeptide moieties, play an essential role in species-selective recognition of porcine and bovine sperm. Moreover, Asn to Asp mutations at either of two of the N-glycosylation sites of ZPB, residue 203 or 220, significantly reduced the sperm binding activity of the ZPB/ZPC mixture, whereas a similar mutation at the third N-glycosylation site, Asn-333, had no effect on binding. These results suggest that the N-glycans located in the N-terminal half of the ZP domain of porcine ZPB are involved in sperm-zona binding.