BBA - General Subjects (v.1780, #6)

Glycosylation is the major modification of proteins, and alters their structures, functions and localizations. Glycosylation of secretory and surface proteins takes place in the endoplasmic reticulum and Golgi apparatus in eukaryotic cells and is classified into four modification pathways, namely N- and O-linked glycosylations, glycosylphosphatidylinositol (GPI)-anchor and C-mannosylation. These modifications are accomplished by sequential addition of single monosaccharides (O-linked glycosylation and C-mannosylation) or en bloc transfer of lipid-linked oligosaccharides (N-linked glycosylation and GPI) onto the proteins. The glycosyltransferases involved in these glycosylations are categorized into two classes based on the type of sugar donor, namely nucleotide-sugars and dolichol-phosphate-sugars, in which the sugar moiety is mannose or glucose. The sugar transfer from dolichol-phosphate-sugars occurs exclusively on the luminal side of the endoplasmic reticulum and is utilized in all four glycosylation pathways. In this review, we focus on the biosynthesis of dolichol-phosphate-mannose, and particularly on the mammalian enzyme complex involved in the reaction.
Keywords: Dolichol-phosphate mannose; DPM1; Biosynthesis; Glycosylation;

A trifunctional enzyme with glutathione S-transferase, glutathione peroxidase and superoxide dismutase activity by Fei Yan; Wen-kui Yang; Xin-yang Li; Ting-ting Lin; Yan-ni Lun; Feng Lin; Shao-wu Lv; Gang-lin Yan; Jun-qiu Liu; Jia-cong Shen; Ying Mu; Gui-min Luo (869-872).
Superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione S-transferase (GST) and glutathione reductase (GR) play crucial roles in balancing the production and decomposition of reactive oxygen species (ROS) in living organisms. These enzymes act cooperatively and synergistically to scavenge ROS, as not one of them can singlehandedly clear all forms of ROS. In order to imitate the synergy of the enzymes, we designed and generated a recombinant protein, which comprises of a Schistosoma japonicum GST (SjGST) and a bifunctional 35-mer peptide with SOD and GPX activities. The engineered protein demonstrated SOD, GPX and GST activities simultaneously. This trifunctional enzyme with SOD, GPX and GST activities is expected to be the best ROS scavenger.
Keywords: Reactive oxygen species; Trifunctional enzyme; Glutathione S-transferase; Superoxide dismutase; Glutathione peroxidase; Enzyme mimic; Fusion protein; Metal incorporation;

Acetyl-Coenzyme A acyltransferase 2 attenuates the apoptotic effects of BNIP3 in two human cell lines by Wei Cao; Nansong Liu; Shuai Tang; Lei Bao; Li Shen; Hanying Yuan; Xin Zhao; Hong Lu (873-880).
BNIP3 is a unique pro-apoptotic protein which belongs to the BH3-only subset of the Bcl-2 family and localizes on mitochondrial membrane. Despite the inherent difficulty of identifying binding partners for membrane proteins, several binding partners for BNIP3 have been identified. In this study, a modified split-ubiquitin membrane yeast two-hybrid system was constructed and used to identify acetyl-Coenzyme A acyltransferase 2 (ACAA2) as a new BNIP3 binding partner. The interaction between BNIP3 and ACAA2 was confirmed by pull-down and co-immunoprecipitation assays. ACAA2 was also found to co-localize with BNIP3 in mitochondria. Furthermore, the apoptosis induced by over-expressed BNIP3 via transfection or hypoxia treatment was abolished by ACAA2 in human hepatocellular carcinoma HepG2 cells and osteosarcoma U-2 OS cells. These results strongly suggest that ACAA2 be a functional BNIP3 binding partner and provide a possible linkage between fatty acid metabolism and apoptosis of cells.
Keywords: BNIP3; ACAA2; Split-ubiquitin membrane yeast two-hybrid system; Apoptosis; Mitochondrial membrane potential;

Papain polymerizes l-glutamic acid diethyl ester (Glu-di-OEt) regioselectively, resulting in the formation of poly (γ-ethyl α-l-glutamic acid) with various degrees of polymerization of less than 13. Reaction temperatures below 20 °C were appropriate for the reaction in terms of suppression of non-enzymatic degradation of Glu-di-OEt and an increase in the peptide yield, while the reaction was preceded by a pronounced induction period. Mass spectrometric analyses of the reaction conducted at 0 °C revealed that the accumulation of the initial dimerization product, l-glutamyl-l-glutamic acid triethyl ester (Glu-Glu-tri-OEt), was limited during the induction period, and that a sequential polymer derived from a further elongation of the dimer was the tetramer, but not the trimer. Kinetic analyses of acyl transfer reactions with Glu-di-OEt and Glu-Glu-tri-OEt as acyl acceptors and Nα-benzoyl-l-arginine ethyl ester as an acyl donor affirmed that Glu-Glu-tri-OEt bound more strongly than Glu-di-OEt both to the S- and S′-subsites of papain. Therefore, what occurred during the initial stage of the polymerization was interpreted as follows: the rate of the papain-catalyzed dimerization of Glu-di-OEt was extremely slow, once Glu-Glu-tri-OEt was initially synthesized it exclusively bound to the active site of papain, and then papain utilized the dimer in polymerization effectively rather than the monomer.
Keywords: Papain; Protease-catalyzed peptide synthesis; Oligoglutamate; Glutamic acid; Induction period;

To further elucidate the impact of fermentative stress on Saccharomyces cerevisiae wine strains, we have here evaluated markers of oxidative stress, oxidative damage and antioxidant response in four oenological strains of S. cerevisiae, relating these to membrane integrity, ethanol production and cell viability during fermentation in high-sugar-containing medium. The cells were sampled at different fermentation stages and analysed by flow cytometry to evaluate membrane integrity and accumulation of reactive oxygen species (ROS). At the same time, catalase and superoxide dismutase activities, trehalose accumulation, and protein carbonylation and degradation were measured. The results indicate that the stress conditions occurring during hypoxic fermentation in high-sugar-containing medium result in the production of ROS and trigger an antioxidant response. This involves superoxide dismutase and trehalose for the protection of cell structures from oxidative damage, and protein catabolism for the removal of damaged proteins. Cell viability, membrane integrity and ethanol production depend on the extent of oxidative damage to cellular components. This is, in turn, related to the ‘fitness’ of each strain, which depends on the contribution of individual cells to ROS accumulation and scavenging. These findings highlight that the differences in individual cell resistances to ROS contribute to the persistence of wine strains during growth under unfavourable culture conditions, and they provide further insights into our understanding of yeast behaviour during industrial fermentation.
Keywords: Fermentative stress; Wine yeast; ROS; Membrane permeability; Protein catabolism; Trehalose; Viability;

Monocarboxylate transporter (MCT)-1 is up-regulated by PPARα by Bettina König; Alexander Koch; Karen Giggel; Batsuch Dordschbal; Klaus Eder; Gabriele I. Stangl (899-904).
Peroxisome proliferator-activated receptor (PPAR)-α mediates an adaptive response to fasting by up-regulation of genes involved in fatty acid oxidation and ketone body synthesis. Ketone bodies are transferred in and out of cells by monocarboxylate transporter (MCT)-1. In this study we observed for the first time that activation of PPARα in rats by clofibrate treatment or fasting increased hepatic mRNA concentration of MCT1. In Fao rat hepatoma cells, incubation with the PPARα agonist WY 14,643 increased mRNA concentration of MCT1 whereas the PPARγ agonist troglitazone did not. To elucidate whether up-regulation of MCT1 is indeed mediated by PPARα we treated wild-type and PPARα-null mice with WY 14,643. In wild-type mice, treatment with WY 14,643 increased mRNA concentrations of MCT1 in liver, kidney and small intestine whereas no up-regulation was observed in PPARα-null mice.
Keywords: Monocarboxylate transporter 1; Peroxisome proliferator-activated receptor (PPAR)-α; PPARα-null mice;

Functional role of N-glycosylation from ADAM10 in processing, localization and activity of the enzyme by Cristina Escrevente; Vanessa A. Morais; Sascha Keller; Cláudio M. Soares; Peter Altevogt; Júlia Costa (905-913).
A disintegrin and metalloprotease 10 (ADAM10) is a type I transmembrane glycoprotein with four potential N-glycosylation sites (N267, N278, N439 and N551), that cleaves several plasma membrane proteins. In this work, ADAM10 was found to contain high-mannose and complex-type glycans. Individual N-glycosylation site mutants S269A, T280A, S441A, T553A were constructed, and results indicated that all sites were occupied. T280A was found to accumulate in the endoplasmic reticulum as the non-processed precursor of the enzyme. Furthermore, it exhibited only residual levels of metalloprotease activity in vivo towards the L1 cell adhesion molecule, as well as in vitro, using a ProTNF-alpha peptide as substrate. S441A showed increased ADAM10 susceptibility to proteolysis. Mutation of N267, N439 and N551 did not completely abolish enzyme activity, however, reduced levels were found. ADAM10 is sorted into secretory vesicles, the exosomes. Here, a fraction of ADAM10 from exosomes was found to contain more processed N-linked glycans than the cellular enzyme. In conclusion, N-glycosylation is crucial for ADAM10 processing and resistance to proteolysis, and results suggest that it is required for full-enzyme activity.
Keywords: ADAM10; N-linked glycosylation; L1 shedding; Ovarian carcinoma; Exosomes;

NeoR6 inhibits HIV-1-CXCR4 interaction without affecting CXCL12 chemotaxis activity by Aviva Lapidot; Amnon Peled; Alexander Berchanski; Boaz Pal; Orit Kollet; Tsvee Lapidot; Gadi Borkow (914-920).
Aminoglycoside-arginine conjugates (AACs) are multi-target HIV-1 inhibitors. The most potent AAC is neomycin hexa-arginine conjugate, NeoR6. We here demonstrate that NeoR6 interacts with CXCR4 without affecting CXCL12–CXCR4 ordinary chemotaxis activity or loss of CXCR4 cell surface expression. Importantly, NeoR6 alone does not affect cell migration, indicating that NeoR6 interacts with CXCR4 at a distinct site that is important for HIV-1 entry and mAb 12G5 binding, but not to CXCL12 binding or signaling sites. This is further supported by our modeling studies, showing that NeoR6 and CXCL12 bind to two distinct sites on CXCR4, in contrast with other CXCR4 inhibitors, e.g. T140 and AMD3100. This complementary utilization of chemical, biology, and computation analysis provides a powerful approach for designing anti-HIV-1 drugs without interfering with the natural function of CXCL12/CXCR4 binding.
Keywords: Aminoglycoside-arginine conjugate; HIV-1 inhibitor; CXCL12–CXCR4 chemotaxis; Computer-aided molecular modeling and docking;

mAtNOS1 regulates mitochondrial functions and apoptosis of human neuroblastoma cells by Mordhwaj S. Parihar; Arti Parihar; Zhonghai Chen; Rafal Nazarewicz; Pedram Ghafourifar (921-926).
mAtNOS1 is a novel gene recently reported in mammalian cells with functions that are not fully understood. The present study generated human neuroblastoma SHSY cells over- and underexpressing mAtNOS1 and shows that mAtNOS1 is involved in regulating mitochondrial nitric oxide, mitochondrial transmembrane potential, protein tyrosine nitration, cytochrome c release, and apoptosis of those cells.
Keywords: SHSY; Mitochondria; Nitric oxide; Transmembrane potential; Apoptosis;

Kinetics of hydrogen peroxide elimination by astrocytes and C6 glioma cells by Nobuo Makino; Takeshi Mise; Jun-ichi Sagara (927-936).
Oxidative stress is implicated in a variety of disorders including neurodegenerative diseases, and H2O2 is important in the generation of reactive oxygen and oxidative stress. In this study, we have examined the rate of extracellular H2O2 elimination and relevant enzyme activities in cultured astrocytes and C6 glioma cells and have analyzed the results based on a mathematical model. As compared with other types of cultured cells, astrocytes showed higher activity of glutathione peroxidase (GPx) but lower activities for GSH recycling. C6 cells showed relatively low GPx activity, and treatment of C6 cells with dibutyryl-cAMP, which induces astrocytic differentiation, increased catalase activity and H2O2 permeation rate but exerted little effect on other enzyme activities. A mathematical model [N. Makino, K. Sasaki, N. Hashida, Y. Sakakura, A metabolic model describing the H2O2 elimination by mammalian cells including H2O2 permeation through cytoplasmic and peroxisomal membranes: comparison with experimental data, Biochim. Biophys. Acta 1673 (2004) 149–159.], which includes relevant enzymes and H2O2 permeation through membranes, was found to be fitted well to the H2O2 concentration dependences of removal reaction with the permeation rate constants as variable parameters. As compared with PC12 cells as a culture model for neuron, H2O2 removal activity of astrocytes was considerably higher at physiological H2O2 concentrations. The details of the mathematical model are presented in Appendix.
Keywords: Antioxidant enzyme; Catalase; Glutathione peroxidase; Hydrogen peroxide; Membrane permeation; Modeling and simulation;