Structures of the N-linked carbohydrate of ascorbic acid oxidase from zucchini

N-glycan moiety of ascorbic acid oxidase from zucchini (Cucurbita pepo) has been described to be a core-pentasaccharide with a xylose [D'Andrea et al. (1988) Glycoconjugate J 5:151-7]. Ascorbic acid oxidase is sometimes used to characterize antibodies directed against carbohydrate determinants on plant glycoproteins. To prevent misinterpretations of immunological data, the structure of the N-glycan of ascorbic acid oxidase has been reinvestigated. The oligosaccharides were released by almond N-glycosidase and analysed as their pyridylamino derivatives by 2D-HPLC and exoglycosidase digestions. The main structure resembled the typical complex plant N-glycan consisting of a core-pentasaccharide decorated with xylose and 3-linked fucose. The other abundant species lacked the fucose residue. Small amounts of these glycans carried a GlcNAc residue on the 6-arm. Therefore, ascorbic acid oxidase will not only react with antibodies directed against the xylosylated region but also with those binding to N-glycans with 3-linked fucose.     

Porcine α-1,3-galactosyltransferase: full length cDNA cloning, genomic organization, and analysis of splicing variants

Full length cDNA and genomic DNA of porcine -1,3-galactosyltransferase were isolated, and their structures were analysed. The coding region was encoded by six exons as in the mouse, and the length of each exon was conserved between the two species. The porcine exons were designated Exon 4–9, since in the mouse coding exons started from Exon 4. Introns tended to be longer in the porcine gene; the distance from Exon 4 to the 3-end of Exon 9 was 24 kb, while this region was 18 kb in the mouse gene. The cDNA structure was extended from the previous data to the 3-end and to the 5 side of the cDNA. In addition to a cDNA clone with all coding exons, clones lacking parts of these exons were isolated and their structures were determined. One variant lacked Exon 5; the second, Exons 5 and 6; and the third, Exons 5, 6 and 7. The last variant was not found in the mouse, and cDNA transfection of this variant yielded scarcely any enzymatic activity using asialo 1-acid glycoprotein as a substrate, and decreased activity using N-acetyllactosamine as a substrate.     

Effect of Na+ and K+ Ions on the Initial Crystallization Process of Lysozyme in the Presence of D2O and H2O

In the initial stage of the crystallization of egg-white lysozyme, monomeric lysozyme aggregated rapidly to form a nucleus in the presence of high salt concentrations. In the present studies, we examined the initial aggregation process of lysozyme (initial crystallization process of lysozyme) in D₂O/H₂O with sodium ions or potassium ions, and investigated the relationship between the surface hydrophobicity and the aggregation rate of lysozyme. The effect of sodium ions or potassium ions on the initial aggregation process of lysozyme in D₂O was clearly different from H₂O. The initial aggregation rate of lysozyme in H₂O was slower than in D₂O. In the case of H₂O, the initial aggregation rate was about the same in both ions. But in the case of D₂O, the initial aggregation rate was affected by the ion species and the value was lower in potassium ions than in sodium ions. These results suggest that the interaction between lysozyme molecules is stronger in D₂O than in H₂O. Furthermore, sodium ions have a stronger effect on the interaction than potassium ions in the case of D₂O. There was a good correlation among the initial aggregation rate, surface hydrophobicity, and -potential of lysozyme. The hydrophobic interaction may be an important active force in the initial aggregation process of lysozyme.     

Cleavage Specificities of Individual Members of Kallikrein Family of Proteins on Synthetic Peptide Containing the Bradykinin Sequence

We have analyzed the effect on bond specificity of various isolated members of the mouse kallikrein family of proteins on a synthetic peptide containing the bradykinin sequence. The cleavage pattern shows the selected specificity of these proteases toward the synthetic peptide. The Phe–His bond (positions 11–12) in the synthetic peptide was favorably cleaved by most of the members in this family, including gamma nerve growth factor. On the other hand, the Lys–Arg bond (position 3–4) was found to be susceptible only to -NGF. The combination of these cleavages could result in the degradation of bradykinin in vivo.     

Characterization of β-D-xyloside-initiated glycosaminoglycan synthesized by human skin fibroblasts in the presence of tunicamycin

Human skin fibroblasts were incubated with a fluorogenic xyloside, 4-methylumbelliferyl--D-xyloside (Xyl-MU), in the presence or absence of tunicamycin. The xyloside-initiated glycosaminoglycans (GAG-MUs) were isolated from the culture medium, and their structures characterized. When the cells were incubated with Xyl-MU in the presence of 0.2 g ml–1 tunicamycin, the synthesis of GAG-MU was increased about three fold, compared with the control value in the absence of tunicamycin (cells exposed to Xyl-MU alone). The structures of GAG-MUs synthesized in the presence or absence of tunicamycin were compared by HPLC analysis using gel-filtration and ion-exchange columns, enzymatic digestion, and unsaturated disaccharide composition analysis. The data indicated that cells incubated with tunicamycin produced more undersulfated and shorter GAG-MUs than cells without tynicamycin. These results suggest that tunicamycin inhibits the elongation and sulfation of glycosaminoglycan (GAG) chains and that, as a result, GAG-MUs with shorter chains and undersulfated residues, but possessing a large number of GAG chains, are synthesized in the presence of tunicamycin.     

N-glycan structures of a recombinant mouse soluble Fcγ receptor II

N-glycans of a recombinant mouse soluble Fc receptor II (sFcRII) expressed in baby hamster kidney cells were released from glycopeptides by digestion with glycoamidase A (from sweet almond), and the reducing ends of the oligosaccharides were reductively aminated with 2-aminopyridine. The derivatized N-glycans were separated and structurally identified by a three-dimensional high-performance liquid chromatography (HPLC) mapping technique on three kinds of HPLC columns [Takahashi, et al. (1995) Anal. Biochem. 226: 139–46]. Eighteen different major N-glycan structures were identified, of which six were neutral (45%), five mono-sialyl (49%), one di-sialyl (4.6%), five tri-sialyl (1.1%), and one tetra-sialyl (0.3%). All N-glycan structures determined were complex type with fucosylation at the N-acetylglucosamine residue of the reducing end, and N-acetylneuraminic acid, when present, was -(2,3)-linked. The existence of a unique structure containing both N-acetylgalactosamine and -(2,3)-N-acetylneuraminic acid residues at the reducing ends, as below, was confirmed by MALDI-TOF mass spectrometry.     

Disruption of the ribosomal P complex leads to stress-induced autophagy

The human ribosomal P complex, which consists of the acidic ribosomal P proteins RPLP0, RPLP1, and RPLP2 (RPLP proteins), recruits translational factors, facilitating protein synthesis. Recently, we showed that overexpression of RPLP1 immortalizes primary cells and contributes to transformation. Moreover, RPLP proteins are overexpressed in human cancer, with the highest incidence in breast carcinomas. It is thought that disruption of the P complex would directly affect protein synthesis, causing cell growth arrest and eventually apoptosis. Here, we report a distinct mechanism by which cancer cells undergo cell cycle arrest and induced autophagy when RPLP proteins are downregulated. We found that absence of RPLP0, RPLP1, or RPLP2 resulted in reactive oxygen species (ROS) accumulation and MAPK1/ERK2 signaling pathway activation. Moreover, ROS generation led to endoplasmic reticulum (ER) stress that involved the EIF2AK3/PERK-EIF2S1/eIF2α-EIF2S2-EIF2S3-ATF4/ATF-4- and ATF6/ATF-6-dependent arms of the unfolded protein response (UPR). RPLP protein-deficient cells treated with autophagy inhibitors experienced apoptotic cell death as an alternative to autophagy. Strikingly, antioxidant treatment prevented UPR activation and autophagy while restoring the proliferative capacity of these cells. Our results indicate that ROS are a critical signal generated by disruption of the P complex that causes a cellular response that follows a sequential order: first ROS, then ER stress/UPR activation, and finally autophagy. Importantly, inhibition of the first step alone is able to restore the proliferative capacity of the cells, preventing UPR activation and autophagy. Overall, our results support a role for autophagy as a survival mechanism in response to stress due to RPLP protein deficiency.     

The C-terminal extension of bacterial flavodoxin-reductases: Involvement in the hydride transfer mechanism from the coenzyme

To study the role of the mobile C-terminal extension present in bacterial class of plant type NADP(H):ferredoxin reductases during catalysis, we generated a series of mutants of the Rhodobacter capsulatus enzyme (RcFPR). Deletion of the six C-terminal amino acids beyond alanine 266 was combined with the replacement A266Y, emulating the structure present in plastidic versions of this flavoenzyme. Analysis of absorbance and fluorescence spectra suggests that deletion does not modify the general geometry of FAD itself, but increases exposure of the flavin to the solvent, prevents a productive geometry of FAD:NADP(H) complex and decreases the protein thermal stability. Although the replacement A266Y partially coats the isoalloxazine from solvent and slightly restores protein stability, this single change does not allow formation of active charge-transfer complexes commonly present in the wild-type FPR, probably due to restraints of C-terminus pliability. A proton exchange process is deduced from ITC measurements during coenzyme binding. All studied RcFPR variants display higher affinity for NADP⁺ than wild-type, evidencing the contribution of the C-terminus in tempering a non-productive strong (rigid) interaction with the coenzyme. The decreased catalytic rate parameters confirm that the hydride transfer from NADPH to the flavin ring is considerably hampered in the mutants. Although the involvement of the C-terminal extension from bacterial FPRs in stabilizing overall folding and bent-FAD geometry has been stated, the most relevant contributions to catalysis are modulation of coenzyme entrance and affinity, promotion of the optimal geometry of an active complex and supply of a proton acceptor acting during coenzyme binding.     

Knockdown of miR-210 decreases hypoxic glioma stem cells stemness and radioresistance

Glioma contains abundant hypoxic regions which provide niches to promote the maintenance and expansion of glioma stem cells (GSCs), which are resistant to conventional therapies and responsible for recurrence. Given the fact that miR-210 plays a vital role in cellular adaption to hypoxia and in stem cell survival and stemness maintenance, strategies correcting the aberrantly expressed miR-210 might open up a new therapeutic avenue to hypoxia GSCs. In the present study, to explore the possibility of miR-210 as an effective therapeutic target to hypoxic GSCs, we employed a lentiviral-mediated anti-sense miR-210 gene transfer technique to knockdown miR-210 expression and analyze phenotypic changes in hypoxic U87s and SHG44s cells. We found that hypoxia led to an increased HIF-2α mRNA expression and miR-210 expression in GSCs. Knockdown of miR-210 decreased neurosphere formation capacity, stem cell marker expression and cell viability, and induced differentiation and G0/G1 arrest in hypoxic GSCs by partially rescued Myc antagonist (MNT) protein expression. Knockdown of MNT could reverse the gene expression changes and the growth inhibition resulting from knockdown of miR-210 in hypoxic GSCs. Moreover, knockdown of miR-210 led to increased apoptotic rate and Caspase-3/7 activity and decreased invasive capacity, reactive oxygen species (ROS) and lactate production and radioresistance in hypoxic GSCs. These findings suggest that miR-210 might be a potential therapeutic target to eliminate GSCs located in hypoxic niches.