Crystallographic studies on human BST-1/CD157 with ADP-ribosyl cyclase and NAD glycohydrolase activities

cADPR is the novel second messenger that elicits calcium release from intracellular calcium stores and works independently of IP(3). In mammals, the ADP-ribosyl cyclase function is found in two membrane proteins, CD38 and BST-1/CD157. These enzymes, exposed extracellularly, bear cADPR hydrolase and NAD glycohydrolase activities. In spite of its functional importance, the structural basis of these enzymatic reactions remains elusive. We determined the crystal structures of the extracellular region of human BST-1 at atomic resolution in the free form and in complexes with five substrate analogues: nicotinamide, NMN, ATPgammaS, ethenoNADP, and ethenoNAD. The three-dimensional structural views of the reaction centre with these ligands revealed the mode of substrate binding and the catalytic mechanism of the multifunctional enzymatic reactions. In each catalytic cleft of the dimeric enzyme, substrates are recognized predominantly through van der Waals interactions with two tryptophan residues, and thereby the N-glycosidic bond of NAD is correctly exposed near a catalytic glutamate residue. Its carboxyl side-chain stabilizes the catalytic intermediate of the S(N)-1 type reaction. This conformation of the catalytic cleft also implies the mechanism of cyclization between the adenine base and the ribose. The three key residues are invariant among the sequences of BST-1, CD38, and Aplysia cyclase, and hence this substrate recognition mode and catalytic scheme appear to be common in the cyclase family.     

The HALTED ROOT gene encoding the 26S proteasome subunit RPT2a is essential for the maintenance of Arabidopsis meristems

In higher plants, post-embryonic development is dependent on the activity of the root and shoot apical meristem (RAM and SAM). The quiescent center (QC) in the RAM and the organizing center (OC) in the SAM are known to be essential for the maintenance of meristematic activity. To understand the mechanism that maintains post-embryonic meristems, we isolated an Arabidopsis mutant, halted root (hlr). In this mutant, the cellular organization was disrupted in post-embryonic meristems both in the root and in the shoot, and their meristematic activity was reduced or became abnormal. We showed that the mutant RAM lost its QC identity after germination, which was specified during embryogenesis, whereas the identity of differentiated tissues was maintained. In the post-embryonic SAM, the expression pattern of a typical OC marker gene, WUSCHEL, was disturbed in the mutant. These observations indicate that the HLR gene is essential to maintain the cellular organization and normal nature of the RAM and SAM. The HLR gene encodes RPT2a, which is a subunit of the 26S proteasome that degrades key proteins in diverse cellular processes. We showed that the HLR gene was expressed both in the RAM and in the SAM, including in the QC and the OC, respectively, and that the activity of proteasomes were reduced in the mutant. We propose that proteasome-dependent programmed proteolysis is required to maintain the meristem integrity both in the shoot and in the root.     

Effects of ghrelin on growth hormone secretion in vivo in ruminants

Ghrelin, a novel endogenous growth hormone (GH) secretagogue, has been shown to exert very potent and specific GH-releasing activity in rats and humans. However, little is known about its GH-releasing activity and endocrine effects in domestic animals. To clarify the effect of ghrelin on GH secretion in vivo in ruminants, plasma GH responses to intra-arterial and intra-hypothalamic injections of rat ghrelin (rGhrelin) were examined in goats and cattle. The intra-arterial injection of 1 microg/kg BW of rGhrelin in ovariectomized goats failed to stimulate GH release, however, a dosage of 3 microg/kg BW significantly increased plasma GH concentrations (P<0.05). GH levels peaked at 15 min after the injection, then decreased to basal concentrations within 1 h after the injection. However, the secretory response to 3 microg/kg BW of rGhrelin was weaker than that of growth hormone-releasing hormone (GHRH) (0.25 microg/kg BW) (P<0.05). An infusion of 10 nmol of ghrelin into the medial basal hypothalamus (arcuate nucleus) significantly stimulated the release of GH in male calves (P<0.05). GH levels began to rise just after the infusions and peaked at 10 min, then decreased to the basal concentrations within 1 h after the injection. The present results show that ghrelin stimulates GH release in ruminants.     

Modulation of erythrocyte membrane mechanical function by protein 4.1 phosphorylation

Erythrocyte membrane mechanical function is regulated by the spectrin-based membrane skeleton composed of alpha- and beta-spectrin, actin, protein 4.1R (4.1R), and adducin. Post-translational modifications of these proteins have been suggested to modulate membrane mechanical function. Indeed, beta-spectrin phosphorylation by casein kinase I has been shown to decrease membrane mechanical stability. However, the effects of the phosphorylation of skeletal proteins by protein kinase C (PKC), a serine/threonine kinase, have not been elucidated. In the present study, we explored the functional consequences of the phosphorylation of 4.1R and adducin by PKC. We identified Ser-312 in 4.1R as the PKC phosphorylation site. Using antibodies raised against phosphopeptides of 4.1R and adducin, we documented significant differences in the time course of phosphorylation of adducin and 4.1R by PKC. Although adducin was phosphorylated rapidly by the activation of membrane-bound atypical PKC by phorbol 12-myristate 13-acetate stimulation, there was a significant delay in the phosphorylation of 4.1R because of delayed recruitment of conventional PKC from cytosol to the membrane. This differential time course in the phosphorylation of 4.1R and adducin in conjunction with membrane mechanical stability measurements enabled us to document that, although phosphorylation of adducin by PKC has little effect on membrane mechanical stability, additional phosphorylation of 4.1R results in a marked decrease in membrane mechanical stability. We further showed that the phosphorylation of 4.1R by PKC results in its decreased ability to form a ternary complex with spectrin and actin as well as dissociation of glycophorin C from the membrane skeleton. These findings have enabled us to define a regulatory role for 4.1R phosphorylation in dynamic regulation of red cell membrane properties.     

Analysis of the interaction between hyaluronan and hyaluronan-binding proteins by capillary affinity electrophoresis: significance of hyaluronan molecular size on binding reaction

We developed a method for the analysis of the interaction between hyaluronan (HA) oligosaccharides and hyaluronan-binding proteins (HABPs) using capillary affinity electrophoresis (CAE). The method is based on high-resolution separation of fluorescent-labeled HA molecules in the presence of hyaluronan-binding proteins at different concentrations by capillary electrophoresis (CE) with laser-induced fluorescent detection. Hyaluronan-binding protein from bovine nasal cartilage interacts strongly with HA decasaccharide or larger oligosaccharides. Effect of the molecular size of HA oligomers clearly showed that longer carbohydrate chains than decasaccharide were required for recognition by HA binding protein. Interestingly, the interaction did not cause retardation of HA oligomers as observed in many binding reactions such as the interaction between pharmaceuticals and serum albumin, but showed disappearance of the oligomer peak. Although we cannot explain the accurate mechanism on the interaction, disappearance is probably due to low equilibrium rate between free and conjugate states. The present technique will be useful to compare the relative binding affinity, and to understand the mechanism on the interaction between hyaluronan and hyaluronan-binding proteins.     

A new fungal lectin recognizing alpha(1-6)-linked fucose in the N-glycan

In this report, we describe a new lectin from the fungus Rhizopus stolonifer that agglutinates rabbit red blood cells. Agglutinating activity was detected in the extract of mycelium-forming spores cultured on agar plates but not in the mycelium-forming no spores from liquid medium. This lectin, which we designated R. stolonifer lectin (RSL), was isolated by affinity chromatography with porcine stomach mucin-Sepharose. SDS-polyacrylamide gel electrophoresis and mass spectral analysis showed that RSL is approximately 4.5 kDa, whereas gel filtration indicated a mass of 28 kDa. This indicates that the lectin is a hexamer of noncovalently associated RSL monomers. RSL activity was very stable, since it was insensitive to heat treatment at 70 degrees C for 10 min. Analysis of RSL binding specificity by both microtiter plate and precipitation assays showed that N-glycans with l-fucose linked to the reducing terminal GlcNAc residues are the most potent inhibitors of RSL binding, whereas N-glycans without alpha(1-6)-linked fucose residues are approximately 100-fold weaker inhibitors of binding. Oligosaccharides with alpha(1-2, -3, and -4) linkages showed no inhibition of binding in these assays. In a mirror resonance biosensor assay, high affinity binding was observed between RSL and the glycopeptide of bovine gamma-globulin, which has N-glycans with alpha(1-6)-linked fucose residues. However, RSL showed only a weak interaction with the glycopeptide of quail ovomucoid, which lacks fucose residues. Finally, capillary affinity electrophoresis studies indicated that RSL binds strongly to N-glycans with alpha(1-6)-linked fucose residues. Together, these results show that RSL recognizes the core structure of N-glycans with alpha(1-6)-linked l-fucose residues. This specificity could make RSL a valuable tool for glycobiological studies.     

Chemokine production by rat macrophages stimulated with streptolysin O from Streptococcus pyogenes

The contribution of streptolysin O (SLO) from Streptococcus pyogenes to neutrophil infiltration in inflammatory lesions was determined by production of cytokine-induced neutrophil chemoattractant (CINC)-1, -2 and -3, and macrophage inflammatory protein (MIP)-1alpha by rat macrophages stimulated with SLO in culture. Active SLO induced the production of CINCs and MIP-1alpha in dose- and time-dependent manners. These inductions were ascertained by chemokine mRNA expression in macrophages. Streptolysin S was without effect. The SLO-cholesterol complex induced the chemokine production in proportion to the residual hemolytic activity of the complex. In addition, the effects of SLO on the chemokine production were confirmed by the injection of active SLO into the preformed air pouch on the back of rats. The infiltration of neutrophils into the pouch fluid (exudate) increased steadily with a lag phase of about 2 hr. The major chemokine found in exudates was MIP-1alpha but not CINCs. In this study, it became clear that active SLO, but not the inactive one, contributed to the production of MIP-1alpha and CINCs in the conditioned medium and in exudates.     

Simultaneous determination of iodate and periodate by capillary zone electrophoresis: application to carbohydrate analysis

Iodate and periodate were rapidly (in 11 min) separated from each other with high column efficiency by capillary zone electrophoresis, using a fused silica tube (50 microns i.d., 80 cm) and 100 mM acetate buffer, pH 4.5, as carrier. On-column uv detection at 222 nm allowed sensitive detection down to the picomole level, and measurement of relative peak area to that of pyromellitic acid (internal standard) enabled reproducible determination of these ions. This method was proved useful for periodate oxidation analysis of various carbohydrates.     

High-performance affinity chromatography of carbohydrate-binding proteins by two-stage separation on a resin carrying a number of oligosaccharides

Oligosaccharides of ovalbumin were released by hydrazinolysis and converted to the glycamine derivatives by reductive amination. The resultant derivatives were immobilized on an epoxy-activated methacrylate polymer. Application of lectins on the column containing the resultant resin, followed by injection of the competing sugars and detection of the eluate using natural fluorescence, allowed differentiation of micro amounts of the lectins, owing to their high specificity. Stepwise elution with various competing sugars also permitted separation of lectins. Application of this method to serum samples enabled detection of various carbohydrate-binding proteins with specific affinity to the injected sugars. This method, based on two-stage separation at the adsorption and elution stages, was highly specific. It was also rapid, reproducible, and sensitive.