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Günter Schäfer - One of the best experts on this subject based on the ideXlab platform.
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Cytochrome B558 566 from the archaeon sulfolobus acidocaldarius has a unique asn linked highly branched hexasaccharide chain containing 6 sulfoquinovose
FEBS Journal, 2000Co-Authors: Ulrich Zähringer, Hermann Moll, Thomas Hettmann, Yuriy A. Knirel, Günter SchäferAbstract:Cytochrome B558/566 from the archaeon Sulfolobus acidocaldarius (DSM 639) has been described as a novel highly glycosylated membrane-bound b-type hemoprotein [Hettmann, T., Schmidt, C. L., Anemuller, S., Zahringer, U., Moll, H., Petersen, A. & Schafer, G. (1998) J. Biol. Chem.273, 12032–12040]. The purified Cytochrome B558/566 was characterized by MALDI MS as a 64-kDa (glyco)protein expressing 17% glycosylation. Detailed chemical studies showed that it was exclusively O-mannosylated with monosaccharides and N-glycosylated with at least seven hexasaccharide units having the same unique structure. The hexasaccharide was released by cleavage with peptide:N-glycosidase (PNGase) F and found to consist of two residues each of Man and GlcNAc and one residue each of Glc and 6-deoxy-6-sulfoglucose (6-sulfoquinovose). The last sugar has been known as a component of glycolipids of plants and some prokaryotes, but has not been hitherto found in bacterial glycoproteins. Digestion with trypsin/pronase gave a mixture of glycopeptides with the same Asn-linked hexasaccharide chain, from which an N-glycosylated Tyr-Asn dipeptide was purified by gel chromatography and anion-exchange HPLC. Studies of the degradation products using methylation analysis, ESI MS, MALDI MS, and 1H and 13C NMR spectroscopy, including 1H,13C HMQC and NOESY experiments, established the structure of the unique Asn-linked hexasaccharide chain of Cytochrome B558/566.
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Cytochrome B558/566 from the archaeon Sulfolobus acidocaldarius has a unique Asn-linked highly branched hexasaccharide chain containing 6-sulfoquinovose.
European Journal of Biochemistry, 2000Co-Authors: Ulrich Zähringer, Hermann Moll, Thomas Hettmann, Yuriy A. Knirel, Günter SchäferAbstract:Cytochrome B558/566 from the archaeon Sulfolobus acidocaldarius (DSM 639) has been described as a novel highly glycosylated membrane-bound b-type hemoprotein [Hettmann, T., Schmidt, C. L., Anemuller, S., Zahringer, U., Moll, H., Petersen, A. & Schafer, G. (1998) J. Biol. Chem.273, 12032–12040]. The purified Cytochrome B558/566 was characterized by MALDI MS as a 64-kDa (glyco)protein expressing 17% glycosylation. Detailed chemical studies showed that it was exclusively O-mannosylated with monosaccharides and N-glycosylated with at least seven hexasaccharide units having the same unique structure. The hexasaccharide was released by cleavage with peptide:N-glycosidase (PNGase) F and found to consist of two residues each of Man and GlcNAc and one residue each of Glc and 6-deoxy-6-sulfoglucose (6-sulfoquinovose). The last sugar has been known as a component of glycolipids of plants and some prokaryotes, but has not been hitherto found in bacterial glycoproteins. Digestion with trypsin/pronase gave a mixture of glycopeptides with the same Asn-linked hexasaccharide chain, from which an N-glycosylated Tyr-Asn dipeptide was purified by gel chromatography and anion-exchange HPLC. Studies of the degradation products using methylation analysis, ESI MS, MALDI MS, and 1H and 13C NMR spectroscopy, including 1H,13C HMQC and NOESY experiments, established the structure of the unique Asn-linked hexasaccharide chain of Cytochrome B558/566.
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Cytochrome B558/566 from the archaeon Sulfolobus acidocaldarius. A novel highly glycosylated, membrane-bound b-type hemoprotein.
The Journal of biological chemistry, 1998Co-Authors: Thomas Hettmann, Ulrich Zähringer, Hermann Moll, Christian L. Schmidt, Stefan Anemüller, Arnd Petersen, Günter SchäferAbstract:In this study we re-examined the inducible Cytochrome B558/566 from the archaeon Sulfolobus acidocaldarius (DSM 639), formerly thought to be a component of a terminal oxidase (Becker, M., and Schafer, G. (1991) FEBS Lett. 291, 331-335). An improved purification method increased the yield of the protein and allowed more detailed investigations. Its molecular mass and heme content have been found to be 64,210 Da and 1 mol of heme/mol of protein, respectively. It is only detectable in cells grown at low oxygen tensions. The composition of the growth medium also exerts significant influence on the Cytochrome B558/566 content of S. acidocaldarius membranes. The Cytochrome exhibits an extremely high redox potential of +400 mV and shows no CO reactivity; a ligation other than a His/His-coordination of axial ligands appears likely. It turned out to be highly glycosylated (more than 20% of its molecular mass are sugar residues) and is probably exposed to the outer surface of the plasma membrane. The sugar moiety consists of several O-glycosidically linked mannoses and at least one N-glycosidically linked hexasaccharide comprising two glucoses, two mannoses, and two N-acetyl-glucosamines. The gene of the Cytochrome (cbsA) has been sequenced, revealing an interesting predicted secondary structure with two putative alpha-helical membrane anchors flanking the majority of a mainly beta-pleated sheet structure containing unusually high amounts of serine and threonine. A second gene (cbsB) was found to be cotranscribed. The latter displays extreme hydrophobicity and is thought to form a functional unit with Cytochrome B558/566 in vivo, although it did not copurify with the latter. Sequence comparisons show no similarity to any entry in data banks indicating that this Cytochrome is indeed a novel kind of b-type hemoprotein. A Cytochrome c analogous function in the pseudoperiplasmic space of S. acidocaldarius is discussed.
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Cytochrome B558 566 from the archaeon sulfolobus acidocaldarius a novel highly glycosylated membrane bound b type hemoprotein
Journal of Biological Chemistry, 1998Co-Authors: Thomas Hettmann, Ulrich Zähringer, Hermann Moll, Christian Schmidt, Stefan Anemüller, Arnd Petersen, Günter SchäferAbstract:In this study we re-examined the inducible Cytochrome B558/566 from the archaeon Sulfolobus acidocaldarius (DSM 639), formerly thought to be a component of a terminal oxidase (Becker, M., and Schafer, G. (1991) FEBS Lett. 291, 331-335). An improved purification method increased the yield of the protein and allowed more detailed investigations. Its molecular mass and heme content have been found to be 64,210 Da and 1 mol of heme/mol of protein, respectively. It is only detectable in cells grown at low oxygen tensions. The composition of the growth medium also exerts significant influence on the Cytochrome B558/566 content of S. acidocaldarius membranes. The Cytochrome exhibits an extremely high redox potential of +400 mV and shows no CO reactivity; a ligation other than a His/His-coordination of axial ligands appears likely. It turned out to be highly glycosylated (more than 20% of its molecular mass are sugar residues) and is probably exposed to the outer surface of the plasma membrane. The sugar moiety consists of several O-glycosidically linked mannoses and at least one N-glycosidically linked hexasaccharide comprising two glucoses, two mannoses, and two N-acetyl-glucosamines. The gene of the Cytochrome (cbsA) has been sequenced, revealing an interesting predicted secondary structure with two putative alpha-helical membrane anchors flanking the majority of a mainly beta-pleated sheet structure containing unusually high amounts of serine and threonine. A second gene (cbsB) was found to be cotranscribed. The latter displays extreme hydrophobicity and is thought to form a functional unit with Cytochrome B558/566 in vivo, although it did not copurify with the latter. Sequence comparisons show no similarity to any entry in data banks indicating that this Cytochrome is indeed a novel kind of b-type hemoprotein. A Cytochrome c analogous function in the pseudoperiplasmic space of S. acidocaldarius is discussed.
Françoise Morel - One of the best experts on this subject based on the ideXlab platform.
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Molecular interface of S100A8 with Cytochrome B558 and NADPH oxidase activation.
PloS one, 2012Co-Authors: Sylvie Berthier, Marie-hélène Paclet, Minh Vu Chuong Nguyen, Athan Baillet, Marc-andré Hograindleur, Benoît Polack, Françoise MorelAbstract:S100A8 and S100A9 are two calcium binding Myeloid Related Proteins, and important mediators of inflammatory diseases. They were recently introduced as partners for phagocyte NADPH oxidase regulation. However, the precise mechanism of their interaction remains elusive. We had for aim (i) to evaluate the impact of S100 proteins on NADPH oxidase activity; (ii) to characterize molecular interaction of either S100A8, S100A9, or S100A8/S100A9 heterocomplex with Cytochrome B558; and (iii) to determine the S100A8 consensus site involved in Cytochrome B558/S100 interface. Recombinant full length or S100A9-A8 truncated chimera proteins and ExoS-S100 fusion proteins were expressed in E. coli and in P. aeruginosa respectively. Our results showed that S100A8 is the functional partner for NADPH oxidase activation contrary to S100A9, however, the loading with calcium and a combination with phosphorylated S100A9 are essential in vivo. Endogenous S100A9 and S100A8 colocalize in differentiated and PMA stimulated PLB985 cells, with Nox2/gp91phox and p22phox. Recombinant S100A8, loaded with calcium and fused with the first 129 or 54 N-terminal amino acid residues of the P. aeruginosa ExoS toxin, induced a similar oxidase activation in vitro, to the one observed with S100A8 in the presence of S100A9 in vivo. This suggests that S100A8 is the essential component of the S100A9/S100A8 heterocomplex for oxidase activation. In this context, recombinant full-length rS100A9-A8 and rS100A9-A8 truncated 90 chimera proteins as opposed to rS100A9-A8 truncated 86 and rS100A9-A8 truncated 57 chimeras, activate the NADPH oxidase function of purified Cytochrome B558 suggesting that the C-terminal region of S100A8 is directly involved in the molecular interface with the hemoprotein. The data point to four strategic 87HEES90 amino acid residues of the S100A8 C-terminal sequence that are involved directly in the molecular interaction with Cytochrome B558 and then in the phagocyte NADPH oxidase activation.
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New p22-phox monoclonal antibodies: identification of a conformational probe for Cytochrome b 558.
Journal of innate immunity, 2009Co-Authors: Yannick Campion, Françoise Morel, Sylvie Berthier, Algirdas J. Jesaitis, Minh Vu Chuong Nguyen, Athan Baillet, Alexei Grichine, Yvan Herenger, Marie-hélène PacletAbstract:The phagocyte NADPH oxidase, belonging to the NADPH oxidase family (Nox), is dedicated to the production of bactericidal reactive oxygen species. The enzyme catalytic center is the Cytochrome B558, formed by 2 subunits, Nox2 (gp91-phox) and p22-phox. Cytochrome B558 activation results from a conformational change induced by cytosolic regulatory proteins (p67-phox, p47-phox, p40-phox and Rac). The catalytic subunit is Nox2, while p22-phox is essential for both Nox2 maturation and the membrane anchorage of regulatory proteins. Moreover, it has been shown to be necessary for novel Nox activity. In order to characterize both p22-phox topology and Cytochrome B558 conformational change, 6 monoclonal antibodies were produced against purified Cytochrome B558. Phage display epitope mapping combined with a truncation analysis of recombinant p22-phox allowed the identification of epitope regions. Some of these antibodies almost completely inhibited in vitro reconstituted NADPH oxidase activity. Data analysis identified antibodies that recognized epitopes involved in either Nox2 maturation or Nox2 activation. Moreover, flow cytometry analysis and confocal microscopy performed on stimulated neutrophils showed that the monoclonal antibody 12E6 bound preferentially active Cytochrome B558. These monoclonal antibodies provided novel and unique probes to investigate maturation, activation and activity, not only of Nox2 but also of novel Nox.
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Regulation of phagocyte NADPH oxidase activity: identification of two Cytochrome B558 activation states.
FASEB Journal, 2007Co-Authors: Marie-hélène Paclet, Sylvie Berthier, Lauriane Kuhn, Jérôme Garin, Françoise MorelAbstract:Activation of the phagocyte NADPH oxidase (phox) requires the association of cytosolic proteins (p67-phox, p47-phox, p40-phox, and Rac1/2) with the membrane Cytochrome B558, leading to a hemoprotein conformation change. To clarify this mechanism, the phagocyte NADPH oxidase complex was isolated through Cytochrome B558 purification after three chromatographic steps. The purified neutrophil complex was constitutively active in the absence of an amphiphile agent with a maximum turnover (125 mol O2(-) x s(-1) x mol heme b(-1)), indicating that Cytochrome B558 has been activated by cytosolic proteins and is in an "open conformation," able to transfer a maximum rate of electrons. In contrast, the phox complex prepared with B lymphocyte cytosol shows a lower constitutive turnover (approximately 50 mol O2(-) x s(-1) x mol heme b(-1)). Analysis of phox complex components by Western blot and mass spectrometry showed the presence of cytosolic factors (especially p67-phox) and structural proteins (moesin, ezrin). To investigate the difference in activity of phox complexes, we evaluated the effect of MRP8 and MRP14, specifically expressed in neutrophils, on the activity of the B lymphocyte complex. MRPs induce the switch between the partially and the fully "open" Cytochrome B558 conformation. Moreover, their effect was independent of p67-phox. Data point out two potential Cytochrome B558 activation states.
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[Molecular aspects of chronic granulomatous disease. "the NADPH oxidase complex"].
2007Co-Authors: Françoise MorelAbstract:Chronic granulomatous disease (CGD) is a hereditary illness generally occurring in childhood, in the form of recurrent severe infections. The main pathogens are staphylococci and aspergilli. It results from a failure of professional phagocytes, and particularly neutrophils, to produce superoxide ions O2- and their derivatives, which protect cells from bacterial, invasion through an oxidative and toxic defence mechanism. At an infection site. contact between the neutrophils and microorganisms or an inflammatory mediator triggers a respiratory burst, which results in the activation of the NADPH oxidase enzyme complex. NADPH depletes surrounding oxygen to yield O2-. In its active form. NADPH oxidase is an assembly of two components, namely the membrane Cytochrome B558 (consisting o two subunits, gp91-phox and p22-phox) and soluble protein factors present in the resting neutrophil cytoplasm. Transfer of these cytosolic factors and their anchorage to Cytochrome B558 determines the activity of NADPH oxidase. The respiratory burst lasts no more than a few minutes, but the precise mechanisms underlying its termination are not well known. In chronic granulomatous disease, neutrophils have lost their bactericidal capacity The most frequent form is hereditary and X-linked; in this case, the affected gene is CYBB, which encodes gp91-phox, the catalytic subunit of Cytochrome B558. In autosomal and recessive forms of CGD the mutations affect the genes encoding p22-phox, p67-phox or p47-phox. We have unraveled the assembly mechanisms of the NADPH oxidase complex and have demonstrated that the cytosolic factor p67-phox is the determining element: it triggers both the assembly and the activation of NI4DPH oxidase. Binding of p67-phox to Cytochrome B558 induces a gradual conformational change of Cytochrome B558, which then becomes capable of transferring electrons produced in the cytoplasm from NADPH to oxygen, reducing the latter to O2-. The isolation of NADPH oxidase in its active and assembled form has allowed us to identify the activation partners of the oxidase complex. We also demonstrated that calcium-binding myeloid-related proteins (MRP). that are abundant in neutrophil cytoplasm, play a fundamental role in this activation. CGD patient management is essentially based on long-term high-dose prophylactic antibiotic administration. Gene therapy is promising but some distance away from practical application. We are currently investigating a new therapeutic concept that consists of transferring Cytochrome B558 protein directly into deficient cells (initially the PLB 985 X cell line), encapsulated in proteoliposomes, which are hydrophobic.
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Leu505 of Nox2 is crucial for optimal p67phox-dependent activation of the flavoCytochrome B558 during phagocytic NADPH oxidase assembly.
Journal of Leukocyte Biology, 2007Co-Authors: Franck Fieschi, Françoise Morel, Didier Grunwald, Marie-hélène Paclet, Yannick Campion, Philippe Gaudin, Marie José StasiaAbstract:The role of Leu505 of Nox2 on the NADPH oxidase activation process was investigated. An X-CGD PLB-985 cell line expressing the Leu505Arg Nox2 mutant was obtained, exactly mimicking the phenotype of a previously published X91+-CGD case. In a reconstituted cell-free system (CFS), NADPH oxidase and iodonitrotetrazolium (INT) reductase activities were partially maintained concomitantly with a partial cytosolic factors translocation to the plasma membrane. This suggests that assembly and electron transfer from NADPH occurred partially in the Leu505Arg Nox2 mutant. Moreover, in a simplified CFS using purified mutant Cytochrome B558 and recombinant p67phox, p47phox, and Rac1proteins, we found that the Km for NADPH and for NADH was about three times higher than those of purified WT Cytochrome B558, indicating that the Leu505Arg mutation induces a slight decrease of the affinity for NADPH and NADH. In addition, oxidase activity can be extended by increasing the amount of p67phox in the simplified CFS assay. However, the maximal reconstituted oxidase activity using WT purified Cytochrome B558 could not be reached using mutant Cytochrome B558. In a three-dimensional model of the C-terminal tail of Nox2, Leu505 appears to have a strategic position just at the entry of the NADPH binding site and at the end of the alpha-helical loop (residues 484-504), a potential cytosolic factor binding region. The Leu505Arg mutation seems to affect the oxidase complex activation process through alteration of cytosolic factors binding and more particularly the p67phox interaction with Cytochrome B558, thus affecting NADPH access to its binding site.
Ulrich Zähringer - One of the best experts on this subject based on the ideXlab platform.
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Cytochrome B558 566 from the archaeon sulfolobus acidocaldarius has a unique asn linked highly branched hexasaccharide chain containing 6 sulfoquinovose
FEBS Journal, 2000Co-Authors: Ulrich Zähringer, Hermann Moll, Thomas Hettmann, Yuriy A. Knirel, Günter SchäferAbstract:Cytochrome B558/566 from the archaeon Sulfolobus acidocaldarius (DSM 639) has been described as a novel highly glycosylated membrane-bound b-type hemoprotein [Hettmann, T., Schmidt, C. L., Anemuller, S., Zahringer, U., Moll, H., Petersen, A. & Schafer, G. (1998) J. Biol. Chem.273, 12032–12040]. The purified Cytochrome B558/566 was characterized by MALDI MS as a 64-kDa (glyco)protein expressing 17% glycosylation. Detailed chemical studies showed that it was exclusively O-mannosylated with monosaccharides and N-glycosylated with at least seven hexasaccharide units having the same unique structure. The hexasaccharide was released by cleavage with peptide:N-glycosidase (PNGase) F and found to consist of two residues each of Man and GlcNAc and one residue each of Glc and 6-deoxy-6-sulfoglucose (6-sulfoquinovose). The last sugar has been known as a component of glycolipids of plants and some prokaryotes, but has not been hitherto found in bacterial glycoproteins. Digestion with trypsin/pronase gave a mixture of glycopeptides with the same Asn-linked hexasaccharide chain, from which an N-glycosylated Tyr-Asn dipeptide was purified by gel chromatography and anion-exchange HPLC. Studies of the degradation products using methylation analysis, ESI MS, MALDI MS, and 1H and 13C NMR spectroscopy, including 1H,13C HMQC and NOESY experiments, established the structure of the unique Asn-linked hexasaccharide chain of Cytochrome B558/566.
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Cytochrome B558/566 from the archaeon Sulfolobus acidocaldarius has a unique Asn-linked highly branched hexasaccharide chain containing 6-sulfoquinovose.
European Journal of Biochemistry, 2000Co-Authors: Ulrich Zähringer, Hermann Moll, Thomas Hettmann, Yuriy A. Knirel, Günter SchäferAbstract:Cytochrome B558/566 from the archaeon Sulfolobus acidocaldarius (DSM 639) has been described as a novel highly glycosylated membrane-bound b-type hemoprotein [Hettmann, T., Schmidt, C. L., Anemuller, S., Zahringer, U., Moll, H., Petersen, A. & Schafer, G. (1998) J. Biol. Chem.273, 12032–12040]. The purified Cytochrome B558/566 was characterized by MALDI MS as a 64-kDa (glyco)protein expressing 17% glycosylation. Detailed chemical studies showed that it was exclusively O-mannosylated with monosaccharides and N-glycosylated with at least seven hexasaccharide units having the same unique structure. The hexasaccharide was released by cleavage with peptide:N-glycosidase (PNGase) F and found to consist of two residues each of Man and GlcNAc and one residue each of Glc and 6-deoxy-6-sulfoglucose (6-sulfoquinovose). The last sugar has been known as a component of glycolipids of plants and some prokaryotes, but has not been hitherto found in bacterial glycoproteins. Digestion with trypsin/pronase gave a mixture of glycopeptides with the same Asn-linked hexasaccharide chain, from which an N-glycosylated Tyr-Asn dipeptide was purified by gel chromatography and anion-exchange HPLC. Studies of the degradation products using methylation analysis, ESI MS, MALDI MS, and 1H and 13C NMR spectroscopy, including 1H,13C HMQC and NOESY experiments, established the structure of the unique Asn-linked hexasaccharide chain of Cytochrome B558/566.
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Cytochrome B558/566 from the archaeon Sulfolobus acidocaldarius. A novel highly glycosylated, membrane-bound b-type hemoprotein.
The Journal of biological chemistry, 1998Co-Authors: Thomas Hettmann, Ulrich Zähringer, Hermann Moll, Christian L. Schmidt, Stefan Anemüller, Arnd Petersen, Günter SchäferAbstract:In this study we re-examined the inducible Cytochrome B558/566 from the archaeon Sulfolobus acidocaldarius (DSM 639), formerly thought to be a component of a terminal oxidase (Becker, M., and Schafer, G. (1991) FEBS Lett. 291, 331-335). An improved purification method increased the yield of the protein and allowed more detailed investigations. Its molecular mass and heme content have been found to be 64,210 Da and 1 mol of heme/mol of protein, respectively. It is only detectable in cells grown at low oxygen tensions. The composition of the growth medium also exerts significant influence on the Cytochrome B558/566 content of S. acidocaldarius membranes. The Cytochrome exhibits an extremely high redox potential of +400 mV and shows no CO reactivity; a ligation other than a His/His-coordination of axial ligands appears likely. It turned out to be highly glycosylated (more than 20% of its molecular mass are sugar residues) and is probably exposed to the outer surface of the plasma membrane. The sugar moiety consists of several O-glycosidically linked mannoses and at least one N-glycosidically linked hexasaccharide comprising two glucoses, two mannoses, and two N-acetyl-glucosamines. The gene of the Cytochrome (cbsA) has been sequenced, revealing an interesting predicted secondary structure with two putative alpha-helical membrane anchors flanking the majority of a mainly beta-pleated sheet structure containing unusually high amounts of serine and threonine. A second gene (cbsB) was found to be cotranscribed. The latter displays extreme hydrophobicity and is thought to form a functional unit with Cytochrome B558/566 in vivo, although it did not copurify with the latter. Sequence comparisons show no similarity to any entry in data banks indicating that this Cytochrome is indeed a novel kind of b-type hemoprotein. A Cytochrome c analogous function in the pseudoperiplasmic space of S. acidocaldarius is discussed.
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Cytochrome B558 566 from the archaeon sulfolobus acidocaldarius a novel highly glycosylated membrane bound b type hemoprotein
Journal of Biological Chemistry, 1998Co-Authors: Thomas Hettmann, Ulrich Zähringer, Hermann Moll, Christian Schmidt, Stefan Anemüller, Arnd Petersen, Günter SchäferAbstract:In this study we re-examined the inducible Cytochrome B558/566 from the archaeon Sulfolobus acidocaldarius (DSM 639), formerly thought to be a component of a terminal oxidase (Becker, M., and Schafer, G. (1991) FEBS Lett. 291, 331-335). An improved purification method increased the yield of the protein and allowed more detailed investigations. Its molecular mass and heme content have been found to be 64,210 Da and 1 mol of heme/mol of protein, respectively. It is only detectable in cells grown at low oxygen tensions. The composition of the growth medium also exerts significant influence on the Cytochrome B558/566 content of S. acidocaldarius membranes. The Cytochrome exhibits an extremely high redox potential of +400 mV and shows no CO reactivity; a ligation other than a His/His-coordination of axial ligands appears likely. It turned out to be highly glycosylated (more than 20% of its molecular mass are sugar residues) and is probably exposed to the outer surface of the plasma membrane. The sugar moiety consists of several O-glycosidically linked mannoses and at least one N-glycosidically linked hexasaccharide comprising two glucoses, two mannoses, and two N-acetyl-glucosamines. The gene of the Cytochrome (cbsA) has been sequenced, revealing an interesting predicted secondary structure with two putative alpha-helical membrane anchors flanking the majority of a mainly beta-pleated sheet structure containing unusually high amounts of serine and threonine. A second gene (cbsB) was found to be cotranscribed. The latter displays extreme hydrophobicity and is thought to form a functional unit with Cytochrome B558/566 in vivo, although it did not copurify with the latter. Sequence comparisons show no similarity to any entry in data banks indicating that this Cytochrome is indeed a novel kind of b-type hemoprotein. A Cytochrome c analogous function in the pseudoperiplasmic space of S. acidocaldarius is discussed.
Marie José Stasia - One of the best experts on this subject based on the ideXlab platform.
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CYBA encoding p22phox, the Cytochrome B558 alpha polypeptide: gene structure, expression, role and physiopathology
Gene, 2016Co-Authors: Marie José StasiaAbstract:P22(phox) is a ubiquitous protein encoded by the CYBA gene located on the long arm of chromosome 16 at position 24, containing six exons and spanning 8.5 kb. P22(phox) is a critical component of the superoxide-generating nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs). It is associated with NOX2 to form Cytochrome B558 expressed mainly in phagocytes and responsible for the killing of microorganisms when bacterial and fungal infections occur. CYBA mutations lead to one of the autosomal recessive forms of chronic granulomatous disease (AR22(0)CGD) clinically characterized by recurrent and severe infections in early childhood. However, p22(phox) is also the partner of NOX1, NOX3 and NOX4, but not NOX5, which are analogs of NOX2, the first identified member of the NOX family. P22(phox)-NOX complexes have emerged as one of the most relevant sources of reactive oxygen species (ROS) in tissues and cells, and are associated with several diseases such as cardiovascular and cerebrovascular diseases. The p22(phox)-deficient mouse strain nmf333 has made it possible to highlight the role of p22(phox) in the control of inner ear balance in association with NOX3. However, the relevance of p22(phox) for NOX3 function remains uncertain because AR22(0)CGD patients do not suffer from vestibular dysfunction. Finally, a large number of genetic variations of CYBA have been reported, among them the C242T polymorphism, which has been extensively studied in association with coronary artery and heart diseases, but conflicting results continue to be reported.
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Identification of NOX2 regions for normal biosynthesis of Cytochrome B558 in phagocytes highlighting essential residues for p22phox binding.
Biochemical Journal, 2014Co-Authors: Sylvain Beaumel, Didier Grunwald, Franck Fieschi, Marie José StasiaAbstract:Cytochrome B558, the redox core of the NADPH oxidase (NOX) complex in phagocytes, is composed of NOX2 and p22phox, the synthesis of which is intimately connected but not fully understood. We reproduced 10 rare X-minus chronic granulomatous disease (CGD) mutations of highly conserved residues in NOX1-NOX4, in X0-CGD PLB-985 cells in order to analyse their impact on the synthesis of Cytochrome B558. According to the impact of these mutations on the level of expression of NADPH oxidase 2 (NOX2) and its activity, mutants were categorized into group A (W18C, E309K, K315del and I325F), characterized by a linear relationship between NOX2 expression and NOX activity, and group B (H338Y, P339H, G389A and F656-F570del), showing an absence of NOX activity associated with variable levels of NOX2 expression. These last residues belong to the FAD-binding pocket of NOX2, suggesting that this functional domain also plays a role in the structural integrity of NOX2. Finally, we observed an abnormal accumulation of p65 (65-kDa monomer), the NOX2 precursor and p65-p22phox dissociation in the W18C, E309K, I325F and G389A mutants, pointing out a possible role of the first transmembrane domain (Trp18), and the region between the membrane and the dehydrogenase domain of NOX2 (Glu309, Ile325 and Gly389), in the binding with p22phox.
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characterization of superoxide overproduction by the d loopnox4 nox2 Cytochrome B558 in phagocytes differential sensitivity to calcium and phosphorylation events
Biochimica et Biophysica Acta, 2011Co-Authors: Laure Carrichon, Sylvain Beaumel, Algirdas J. Jesaitis, Antoine Picciocchi, Franck Debeurme, F. Defendi, Marie-claire Dagher, Marie José StasiaAbstract:Abstract NADPH oxidase is a crucial element of phagocytes involved in microbicidal mechanisms. It becomes active when membrane-bound Cytochrome B558, the redox core, is assembled with cytosolic p47phox, p67phox, p40phox, and rac proteins to produce superoxide, the precursor for generation of toxic reactive oxygen species. In a previous study, we demonstrated that the potential second intracellular loop of Nox2 was essential to maintaining NADPH oxidase activity by controlling electron transfer from FAD to O2. Moreover, replacement of this loop by the Nox4-D-loop (D-loopNox4-Nox2) in PLB-985 cells induced superoxide overproduction. In the present investigation, we demonstrated that both soluble and particulate stimuli were able to induce this superoxide overproduction. Superoxide overproduction was also observed after phosphatidic acid activation in a purified cell-free-system assay. The highest oxidase activity was obtained after ionomycin and fMLF stimulation. In addition, enhanced sensitivity to Ca2+ influx was shown by thapsigargin, EDTA, or BTP2 treatment before fMLF activation. Mutated Cytochrome B558 was less dependent on phosphorylation triggered by ERK1/2 during fMLF or PMA stimulation and by PI3K during OpZ stimulation. The superoxide overproduction of the D-loopNox4-Nox2 mutant may come from a change of responsiveness to intracellular Ca2+ level and to phosphorylation events during oxidase activation. Finally the D-loopNox4-Nox2-PLB-985 cells were more effective against an attenuated strain of Pseudomonas aeruginosa compared to WT-Nox2 cells. The killing mechanism was biphasic, an early step of ROS production that was directly bactericidal, and a second oxidase-independent step related to the amount of ROS produced in the first step.
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Characterization of superoxide overproduction by the D-LoopNox4-Nox2 Cytochrome B558 in phagocytes—Differential sensitivity to calcium and phosphorylation events
Biochimica et biophysica acta, 2010Co-Authors: Laure Carrichon, Sylvain Beaumel, Algirdas J. Jesaitis, Antoine Picciocchi, Franck Debeurme, F. Defendi, Marie-claire Dagher, Marie José StasiaAbstract:Abstract NADPH oxidase is a crucial element of phagocytes involved in microbicidal mechanisms. It becomes active when membrane-bound Cytochrome B558, the redox core, is assembled with cytosolic p47phox, p67phox, p40phox, and rac proteins to produce superoxide, the precursor for generation of toxic reactive oxygen species. In a previous study, we demonstrated that the potential second intracellular loop of Nox2 was essential to maintaining NADPH oxidase activity by controlling electron transfer from FAD to O2. Moreover, replacement of this loop by the Nox4-D-loop (D-loopNox4-Nox2) in PLB-985 cells induced superoxide overproduction. In the present investigation, we demonstrated that both soluble and particulate stimuli were able to induce this superoxide overproduction. Superoxide overproduction was also observed after phosphatidic acid activation in a purified cell-free-system assay. The highest oxidase activity was obtained after ionomycin and fMLF stimulation. In addition, enhanced sensitivity to Ca2+ influx was shown by thapsigargin, EDTA, or BTP2 treatment before fMLF activation. Mutated Cytochrome B558 was less dependent on phosphorylation triggered by ERK1/2 during fMLF or PMA stimulation and by PI3K during OpZ stimulation. The superoxide overproduction of the D-loopNox4-Nox2 mutant may come from a change of responsiveness to intracellular Ca2+ level and to phosphorylation events during oxidase activation. Finally the D-loopNox4-Nox2-PLB-985 cells were more effective against an attenuated strain of Pseudomonas aeruginosa compared to WT-Nox2 cells. The killing mechanism was biphasic, an early step of ROS production that was directly bactericidal, and a second oxidase-independent step related to the amount of ROS produced in the first step.
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Leu505 of Nox2 is crucial for optimal p67phox-dependent activation of the flavoCytochrome B558 during phagocytic NADPH oxidase assembly.
Journal of Leukocyte Biology, 2007Co-Authors: Franck Fieschi, Françoise Morel, Didier Grunwald, Marie-hélène Paclet, Yannick Campion, Philippe Gaudin, Marie José StasiaAbstract:The role of Leu505 of Nox2 on the NADPH oxidase activation process was investigated. An X-CGD PLB-985 cell line expressing the Leu505Arg Nox2 mutant was obtained, exactly mimicking the phenotype of a previously published X91+-CGD case. In a reconstituted cell-free system (CFS), NADPH oxidase and iodonitrotetrazolium (INT) reductase activities were partially maintained concomitantly with a partial cytosolic factors translocation to the plasma membrane. This suggests that assembly and electron transfer from NADPH occurred partially in the Leu505Arg Nox2 mutant. Moreover, in a simplified CFS using purified mutant Cytochrome B558 and recombinant p67phox, p47phox, and Rac1proteins, we found that the Km for NADPH and for NADH was about three times higher than those of purified WT Cytochrome B558, indicating that the Leu505Arg mutation induces a slight decrease of the affinity for NADPH and NADH. In addition, oxidase activity can be extended by increasing the amount of p67phox in the simplified CFS assay. However, the maximal reconstituted oxidase activity using WT purified Cytochrome B558 could not be reached using mutant Cytochrome B558. In a three-dimensional model of the C-terminal tail of Nox2, Leu505 appears to have a strategic position just at the entry of the NADPH binding site and at the end of the alpha-helical loop (residues 484-504), a potential cytosolic factor binding region. The Leu505Arg mutation seems to affect the oxidase complex activation process through alteration of cytosolic factors binding and more particularly the p67phox interaction with Cytochrome B558, thus affecting NADPH access to its binding site.
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A novel mutation at a probable heme-binding ligand in neutrophil Cytochrome B558 in atypical X-linked chronic granulomatous disease.
Human genetics, 1998Co-Authors: Masahiko Tsuda, Mizuho Kaneda, Takeshi Sakiyama, Ichiro Inana, Misao Owada, Chika Kiryu, Takuo Shiraishi, Katsuko KakinumaAbstract:A membrane-bound Cytochrome B558, a heterodimer consisting of gp91-phox and p22-phox, is a critical component of the superoxide (O2-)-generating reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in phagocytes. Chronic granulomatous disease (CGD) is characterized by recurrent bacterial infection caused by a defect of the oxidase. Both subunits are absent from phagocytes in typical X-linked recessive CGD patients who are primarily defective in gp91-phox. We report here an atypical case of X-linked CGD in which neutrophils showed a complete absence of O2–-forming NADPH oxidase activity, but a small amount (about 10% of control) of both subunits was detected by immunoblot analysis. Spectrophotometric studies of the neutrophils with a recently developed sensitive method gave no evidence for the heme spectrum in the Cytochrome B558 of this CGD. Reverse transcription/polymerase chain reaction and sequence analysis revealed a C to T transition replacing histidine at amino acid position 101 (His101) by tyrosine in gp91-phox. These results provide evidence that His101 of gp91-phox is the one of the heme-binding ligands of Cytochrome B558.
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Spectrophotometric determination of neutrophil Cytochrome B558 of chronic granulomatous disease
Acta paediatrica Japonica : Overseas edition, 1998Co-Authors: Chika Kiryu, Mizuho Kaneda, Masahiko Tsuda, Takeshi Sakiyama, Ichiro Inana, Takuo Shiraishi, Toru Fujinaga, Akira Nishida, Katsuko KakinumaAbstract:BACKGROUND Chronic granulomatous disease (CGD) is an inherited disease characterized clinically by severe recurrent bacterial infections from infancy. This disease is a disorder of the formation of superoxide (O2-) by the neutrophil NADPH oxidase system, mostly due to defects in Cytochrome B558 (cyt B558), which is one of the oxidase components. Diagnosis of CGD has been performed by the assay of the O2- forming activity, immunological determination of defects in the oxidase components, and or spectrophotometry of cyt B558. However, spectrophotometric analysis of the b-type heme is difficult with small amounts of blood from infant CGD patients, as the limited amounts of neutrophils are contaminated with a relatively high ratio of hemoglobin (Hb) that interferes with the heme spectrum of cyt B558. This report presents an accurate method for the spectrophotometric analysis of cyt B558 in a small amount of CGD neutrophils that were treated with CO gas in a safe procedure instead of the previously reported CO-bubbling method. METHODS AND RESULTS The difference of the reduced minus oxidized cyt B558 spectrum was measured under no interference from oxy Hb at the alpha and beta bands and differentiated as d[delta A]/d lambda (lambda = wavelength) to obtain further evidence for the defects of the cyt B558 heme spectrum. The interference from CO-insensitive met Hb was eliminated by subtracting the absorption peak at the Soret (gamma) band of the contaminating met Hb, which was estimated from the CO-treated and untreated spectra of the same, hemolyzed sample. CONCLUSIONS This spectrophotometric method is feasible for the determination of abnormality and heme content of cyt B558 with a small amount of CGD neutrophils in 10-20 mL of blood even in the presence of contaminating Hb.
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Spectroscopic identification of the heme axial ligation of Cytochrome B558 in the NADPH oxidase of porcine neutrophils
FEBS letters, 1995Co-Authors: Hirotada Fujii, Toshiaki Miki, Katsuko Kakinuma, Michael G. Finnegan, Brian R. Crouse, Michael K. JohnsonAbstract:Abstract The combination of electron paramagnetic resonance (EPR), near-infrared magnetic circular dichroism (NIR-MCD) and resonance Raman (RR) spectroscopies at cryogenic temperatures has been used to identify the axial heme ligation of the low spin Cytochrome B558 component of NADPH oxidase from porcine blood neutrophils. The EPR and NIR-MCD results indicate the presence of two distinct forms in frozen solution; one with a low field g-value at 3.23 and porphyrin(π)-to-Fe(III) charge transfer maximum at 1660 nm and the other a low field g-value at 3.00 and porphyrin(π)-to-Fe(III) charge transfer maximum at 1510 nm. On the basis of these properties and the RR studies, both are attributed to forms of Cytochrome B558 with bis-histidine axial ligation. The origin of the observed heterogeneity, the location and identity of the specific histidines involved in ligating the heme, and the role of the heme prosthetic group in O2− production are discussed in light of these results.
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Electron spin resonance studies on neutrophil Cytochrome B558. Evidence that low-spin heme iron is essential for O2-. generating activity.
The Journal of biological chemistry, 1995Co-Authors: Hirotada Fujii, Toshiaki Miki, Michael K. Johnson, Michael G. Finnegan, Lucia S. Yoshida, Katsuko KakinumaAbstract:Abstract Cytochrome B558 purified from pig neutrophils was studied to characterize the spin state of the heme iron in relation to its O generating activity. ESR spectra of Cytochrome B558 either from resting or stimulated neutrophils showed a low-spin hemoprotein with g1,2,3 of 3.2, 2.1, and 1.3 (estimated). At physiological pH, the oxidized Cytochrome B558 is in a purely low-spin state. On lowering or raising pH from 7, the spin state changes to high-spin. The ESR spectrum of high-spin Cytochrome B558 was identical to that of methemoglobin, suggesting that the axial-ligand type in both hemoproteins may be the same, i.e. histidine is the fifth ligand. The ratio of the low-spin to high-spin heme in Cytochrome B558 was evaluated by magnetic circular dichroism spectroscopy. The pH of Cytochrome B558 was varied to form different ratios of the low-spin to high-spin states of the heme, and its O generating activity was examined in cell-free systems. O forming activity decreased concomitant with loss of the low-spin heme, which provides direct evidence that the low-spin state of Cytochrome B558 is essential to generate O and the heme retains the low-spin state through the redox cycle.
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MODULATION OF THE HEME ENVIRONMENT OF NEUTROPHIL Cytochrome B558 TO A Cytochrome P450-LIKE STRUCTURE BY PYRIDINE
The Journal of biological chemistry, 1995Co-Authors: Hirotada Fujii, Toshiaki Miki, Takashi Yonetani, Katsuko KakinumaAbstract:The effect of pyridine on the heme environment of Cytochrome B558 was studied using ESR and optical absorption spectroscopy in relation to the O2(-)-generating activity in the NADPH oxidase system of stimulated pig neutrophils. As the concentration of pyridine increased, the absorption maxima of the alpha- and gamma-bands of Cytochrome B558 shifted which correlated with a concomitant decrease in O2(-)-generating activity. In addition, the g = 3.2 signal of Cytochrome B558 decreased with the concomitant appearance of a new ESR spectrum that strikingly resembled that of Cytochrome P450. The results suggest that pyridine induces a structural modification in the heme environment of Cytochrome B558 by shifting the 5th heme ligand (histidine) to a nearby thiolate group without direct binding of pyridine to the heme. The existence of a reactive thiolate near the heme iron was confirmed by pretreatment of blocked Cytochrome B558 with p-chloromercuribenzoate, which completely inhibited the formation of the Cytochrome P450-like ESR spectrum. The results provide further evidence that a low-spin heme iron of Cytochrome B558 with a g-value of 3.2 is essential to the O2(-)-forming reaction of the NADPH oxidase system. From sequence alignments of Cytochrome P450 with those of large and small subunits of Cytochrome B558, the heme in Cytochrome B558 appears to be specifically associated with the large subunit.
