Structural basis of the catalytic reaction mechanism of novel 1,2-alpha-L-fucosidase from Bifidobacterium bifidum

1,2-alpha-L-fucosidase (AfcA), which hydrolyzes the glycosidic linkage of Fucalpha1-2Gal via an inverting mechanism, was recently isolated from Bifidobacterium bifidum and classified as the first member of the novel glycoside hydrolase family 95. To better understand the molecular mechanism of this enzyme, we determined the x-ray crystal structures of the AfcA catalytic (Fuc) domain in unliganded and complexed forms with deoxyfuconojirimycin (inhibitor), 2'-fucosyllactose (substrate), and L-fucose and lactose (products) at 1.12-2.10 A resolution. The AfcA Fuc domain is composed of four regions, an N-terminal beta region, a helical linker, an (alpha/alpha)6 helical barrel domain, and a C-terminal beta region, and this arrangement is similar to bacterial phosphorylases. In the complex structures, the ligands were buried in the central cavity of the helical barrel domain. Structural analyses in combination with mutational experiments revealed that the highly conserved Glu566 probably acts as a general acid catalyst. However, no carboxylic acid residue is found at the appropriate position for a general base catalyst. Instead, a water molecule stabilized by Asn423 in the substrate-bound complex is suitably located to perform a nucleophilic attack on the C1 atom of L-fucose moiety in 2'-fucosyllactose, and its location is nearly identical near the O1 atom of beta-L-fucose in the products-bound complex. Based on these data, we propose and discuss a novel catalytic reaction mechanism of AfcA.     

Imaging the interaction of αv integrin-GFP in osteosarcoma cells with RFP-expressing host stromal cells and tumor-scaffold collagen in the primary and metastatic tumor microenvironment

Human osteosarcoma 143B cells were previously stably transfected with an α_v integrin green flourescent protein (GFP) vector. 143B cells expressing α_v integrin-GFP were transplanted orthotopically in the tibia of transgenic nude mice ubiquitously expressing red fluorescent protein (RFP). The primary tumors acquired RFP-expressing stroma and were passaged orthotopically in the tibia in noncolored nude mice, which maintained the RFP stroma. The interaction of α_v integrin-GFP expression in 143B cells with RFP-expressing host stromal cells was observed by confocal microscopy using the Olympus FV1000. Collagen fibers were imaged simultaneously in reflectance mode. The RFP-expressing stroma included cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs) which persisted even 3 weeks after passage to nontransgenic nude mice. CAFs expressing RFP were aligned between collagen fibers and cancer cells expressing α_v integrin-GFP. Six weeks after transplantation, pulmonary metastases expressing α_v integrin-GFP could be identified. TAMs expressing RFP accompanied metastasized osteosarcoma cells expressing α_v integrin-GFP in the lung. The current study demonstrates the importance of α_v integrin interaction with stromal elements in osteosarcoma.     

Effects of Tween 80 and sodium fluoride on extracellular glucosyltransferase production and membrane lipids of Streptococcus mutans

1. Both Tween 80 and sodium fluoride significantly enhanced total extracellular glucosyltransferase activities of Streptococcus mutans. 2. Water-insoluble and water-soluble glucan formation were uniformly increased by Tween 80, whereas fluoride stimulated only water-soluble glucan formation. 3. Elevated glucan formation was due to an increase in enzymes secreted from bacterial cells. 4. Fatty acid composition and phospholipid content in bacterial membrane were changed by Tween 80, although sodium fluoride scarcely showed these changes. 5. Comparative results suggest that modulation of membrane lipids participates in mutansucrase production but not in dextransucrase production of S. mutans.     

Arginine-specific ADP-ribosyltransferase on the surface of gizzard smooth muscle cells and the involvement of phosphatidylinositol 3-kinase in maintaining the activity of this transferase

An arginine-specific ADP-ribosyltransferase activity was detected in chicken gizzard smooth muscle, and the specific activity is highest in the membrane fraction. This transferase is released from the membrane fraction by phosphatidylinositol-specific phospholipase C (PI-PLC), suggesting that it is a glycosylphosphatidylinositol (GPI)-anchored protein. When primary cultured gizzard smooth muscle cells (SMCs) were incubated with [adenylate-(32)P]NAD, several proteins were labeled. The labeling was inhibited by preincubation of the cells with PI-PLC, or by the addition of L-arginine to the reaction, and was sensitive to hydroxylamine treatment. The activity of the transferase was maintained in differentiated SMCs cultured with insulin, but was dramatically decreased concomitantly with cell dedifferentiation induced by serum or a specific PI3-kinase inhibitor, LY294002. These results indicate that the GPI-anchored arginine-specific ADP-ribosyltransferase is expressed on the surface of differentiated SMCs and can modify several cell surface proteins. Our results also suggest that PI3-kinase is involved in the regulation of transferase activity during differentiation.     

A cryptic melibiose transporter gene possessing a frameshift from Citrobacter freundii

Wild-type Citrobacter freundii cannot grow on melibiose as a sole source of carbon. The melibiose transporter gene melB was cloned from a C. freundii mutant M4 that could utilize melibiose as a sole carbon source. Although the cloned melB gene is closely similar to the melB genes of other bacteria, it is cryptic because of a frameshift mutation. Site-directed mutagenesis was used to construct a functional melB gene by deleting one nucleotide, resulting in the production of an active melibiose transporter. The active MelB transporter could utilize Na(+) and H(+) as coupling cations to melibiose transport. The amino acid sequence of the C. freundii MelB was found to be most similar to those of Salmonella typhimurium and Escherichia coli MelB. These facts are consistent with the phylogenetic relationship of bacteria and the cation coupling properties of the melibiose transporters.     

Enhancement of human cord blood CD34+ cell-derived NK cell cytotoxicity by dendritic cells

NK cells and dendritic cells (DCs) are both important in the innate host defense. However, the role of DCs in NK cell-mediated cytotoxicity is unclear. In this study, we designed two culture systems in which human cord blood CD34(+) cells from the same donor were induced to generate NK cells and DCs, respectively. Coculture of the NK cells with DCs resulted in significant enhancement of NK cell cytotoxicity and IFN-gamma production. However, NK cell cytotoxicity and IFN-gamma production were not increased when NK cells and DCs were grown together separated by a transwell membrane. Functional studies demonstrated that 1) concanamycin A, a selective inhibitor of perforin/granzyme B-based cytolysis, blocked DC-stimulated NK cytotoxicity against K562 cells; and 2) neutralizing mAb against Fas ligand (FasL) significantly reduced DC-stimulated NK cytotoxicity against Fas-positive Jurkat cells. In addition, a marked increase of FasL mRNA and FasL protein expression was observed in DC-stimulated NK cells. The addition of neutralizing mAb against IL-18 and IL-12 significantly suppressed DC-stimulated NK cell cytotoxicity. Neutralizing IFN-gamma Ab almost completely inhibited NK cell cytotoxicity against Jurkat cells. These observations suggest that DCs enhance NK cell cytotoxicity by up-regulating both perforin/granzyme B- and FasL/Fas-based pathways. Direct interaction between DCs and NK cells is necessary for DC-mediated enhancement of NK cell cytotoxicity. Furthermore, DC-derived IL-18 and IL-12 were involved in the up-regulation of NK cell cytotoxicity, and endogenous IFN-gamma production plays an important role in Fas-mediated cytotoxicity.     

Endothelium-dependent relaxation by cilostazol, a phosphodiesteras III inhibitor, on rat thoracic aorta

The relaxation effect of cilostazol, a phosphodiesterase III inhibitor, on the thoracic aorta was investigated. Cilostazol induced the relaxation of the thoracic aorta precontracted by phenylephrine in a concentration-dependent manner. The concentration-dependent relaxation was shifted to the right in the endothelium denuded aorta compared with that of intact endothelium, suggesting that this relaxation was partly dependent on endothelium. Cilostazol-induced relaxation of thoracic aorta tone was reversed by treatment with N(G)-nitro L-arginine (L-NNA), a competitive inhibitor of nitric oxide (NO) synthase. Cilostazol also significantly increased the NO level in the porcine thoracic aorta. In rats treated with cilostazol, the urinary excretion of nitrites, a stable metabolite of NO, and basal production of NO of the aortic ring were significantly greater than in those without treatment. These findings indicate that cilostazol-induced vasodilation of the rat thoracic aorta was dependent on the endothelium, which released NO from aortic endothelial cells.     

Purification and further characterization of enteropeptidase from porcine duodenum

Enteropeptidase [EC 3.4.21.9] is a membrane-bound serine endopeptidase present in the duodenum that converts trypsinogen to trypsin. We previously cloned the cDNA of the porcine enzyme and deduced its entire amino acid sequence [M. Matsushima et al. (1994) J. Biol. Chem. 269, 19976-19982]. In the present study, we purified the porcine enzyme approximately 2,200-fold in a 12% yield from a duodenal mucosal extract to apparent homogeneity by an improved procedure comprising four steps of chromatography including benzamidine-Sepharose affinity chromatography. Lectin blotting analysis suggested that the enzyme is glycosylated mainly with N-linked carbohydrate chains of the tri- and/or tetraantennary complex type. The H and L chains of the enzyme were separated into two major bands upon SDS-PAGE under reducing conditions, suggesting that the enzyme mainly comprises two isoforms, a higher molecular weight form and a lower molecular weight form. The enzyme was also separated by lectin affinity chromatography into two major fractions, named isoforms I and II, which corresponded to the higher and lower molecular weight forms, respectively. These two isoforms appeared to be different only in the carbohydrate moiety, having essentially the same enzymatic properties. The enzyme was optimally active at pH 8.0 toward Gly-Asp-Asp-Asp-Asp-Lys-beta-naphthylamide, and was inhibited strongly by various serine proteinase inhibitors. Furthermore, it was also strongly inhibited by E-64 [L-trans-epoxysuccinyl-leucylamide-(4-guanido)-butane], a cysteine proteinase inhibitor. Substrate specificity studies involving various synthetic peptides indicated that acidic residues at the P2, P3, and/or P4 positions are especially favorable for maximal activity, but are not absolutely necessary, at least in the cases of peptide substrates.     

Uncompetitive inhibition of superoxide generation by a synthetic peptide corresponding to a predicted NADPH binding site in gp91-phox, a component of the phagocyte respiratory oxidase

The large subunit of cytochrome b558, gp91-phox, is believed to play a key role in superoxide generation in neutrophils by accepting electrons from NADPH and donating them to molecular oxygen. We found that a peptide corresponding to a predicted NADPH binding site in gp91-phox inhibited superoxide generation in a cell-free system consisting of neutrophil membrane and cytosol. Minimum essential sequence for the inhibition was KSVWYK, which corresponded to residues 420-425 (IC50 = 30 microM). Unlike other peptides known to inhibit the reaction, this peptide was effective even when added to the system after activation or to activated membrane from stimulated neutrophils. Furthermore, the peptide inhibited superoxide generation in a membrane system activated without cytosol. Kinetic analysis revealed that the peptide inhibited the reaction uncompetitively. These results suggest that the peptide combines with the activated cytochrome b558-NADPH complex and thereby inhibits electron transfer from NADPH to molecular oxygen.