A Domain of p47phox That Interacts with Human Neutrophil Flavocytochrome b558

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. is formed. In this functional capacity, Cyt b has been established as an essential component of the respiratory burst oxidase, although little published experimental data describe its topology in the membrane. Determination of structural and functional aspects of epitopes bound by specific antibodies can provide information about the protein against which the antibody is directed (11). Antipeptide and antisubunit polyclonal antibodies against regions of Cyt b have been used to locate the corresponding epitopes (12, 13) and information derived from identification of epitope mimetics has led to the description of anti-Cyt b "antibody imprints" (14). We continue to elucidate the structure of Cyt b domains recognized by monoclonal antibodies to better define its transmembrane topology and gain insight into its functional organization.Reported epitope mapping data for monoclonal antibodies (mAbs) specific for Cyt b indicate that they bind cytosolic aspects of the protein (15-19). However, it has been reported that mAb 7D5 (19) binds an extracellular Cyt b epitope on intact neutrophils derived from normal but not CGD patients that lack Cyt b (20). Thus, 7D5 has proven useful in the determination of Cyt b up-regulation as an indicator of neutrophil activation and granule exocytosis (21) and for the identification of individuals deficient in Cyt b (20,22,23). However, neither the subunit location nor chemical nature of the 7D5 epitope has been elucidated.In our current studies, we have used phage display and immunological analyses to identify the 7D5 epitope on Cyt b. Although we confirmed the inability of 7D5 to recognize Cyt b on immunoblots, we found that 7D5 immunoprecipitated detergent-solubilized Cyt b heterodimer containing its fully processed 91kDa form of gp91 phox and its 65-kDa precursor. Additionally, it precipitated the deglycosylated gp91 phox core protein, 2 suggesting that neither the mature nor high man-* This work was performed during the tenure of the following American Heart Association awards: postdoctoral fellowships 9704584S

Section: -K-n -P/r -(X)-v-r-g-qmentioning

confidence: 89%

Phage Display Epitope Mapping of Human Neutrophil Flavocytochromeb 558

Burritt

DeLeo²,

McDonald

et al. 2001

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“…Two structural differences between p47 phox and p41 suggest that an auto-inhibited conformation involving intramolecular SH3 domain contacts is not assumed by p41: (i) the PXXP motif present in the PX domain of several proteins is absent in p41, and (ii) most of the phosphorylated serine residues in the carboxyl-terminal portion, as well as flanking sequences within the polybasic, auto-inhibitory domain of p47 phox (residues 299 -340) are absent. Interestingly, sequence homologous to p47 phox residues 318 -329 (RLSQDAYRRNRSV), shown to be involved in interactions with p67 phox and the flavocytochrome b (21,22), is retained in the carboxyl-terminal domain of human p41 (residues 293-304: LLSGTGFRGGDD). These observations suggest that p41 adopts an "open" conformation under resting conditions and that regulation of p41 is achieved by other mechanisms.…”

Section: Homologues Of P47 Phox and P67 Phox Support Nad(p)h Oxidase mentioning

confidence: 99%

Proteins Homologous to p47 and p67 Support Superoxide Production by NAD(P)H Oxidase 1 in Colon Epithelial Cells

Geiszt

Lekstrom

Witta

et al. 2003

257

246

Superoxide production by phagocytes involves activation of a multi-component NADPH oxidase. Recently, several homologues of the catalytic component of the phagocyte oxidase, gp91 phox , were identified in various tissues. Here we describe two proteins, p41 and p51, with significant homology to two cytosolic components of the phagocytic oxidase, p47 phox and p67 phox . Like p47 phox , p41 contains an amino-terminal Phox homology domain, two SH3 domains, and a conserved carboxylterminal, proline-rich motif. Similarly, p51 is homologous to p67 phox , containing four amino-terminal tetratrico-peptide repeats, a conserved "activation domain" motif, a PB1 domain, and a carboxyl-terminal SH3 domain. The highest levels of p41 transcript are detected in the colon and in other gastrointestinal tissues that express Nox1, the predominant gp91 phox homologue in these tissues. In contrast, the p51 transcript showed a more widespread expression pattern, suggesting that it may support other tissue-specific oxidases. Mouse colon in situ hybridization detected both transcripts in the epithelial cells of colon crypts. Heterologous co-expression of p41 and p51 significantly enhances the superoxide-generating activity of Nox1-expressing cells; thus, p41 and p51 appear to be novel regulators of Nox1. These proteins also support the activity of gp91 phox , albeit at much lower levels than the cytosolic phox counterparts. Our results suggest colon epithelial cells contain a multi-component NAD(P)H oxidase with a molecular architecture similar to the phagocytic oxidase.

“…p47 phox contains a total of seven tryptophan residues. Interestingly, five of the tryptophans are located in the region of p47 phox containing both Src homology 3 (SH3) domains (28,29) and the cationic flavocytochrome b/p67 phox binding domain (30,31), all of which appear to be masked in the resting cell. In addition, Sumimoto et al (32) recently reported that mutation of a tryptophan (residue 193) to arginine in the first SH3 domain of p47 phox resulted in a nonfunctional p47 phox , possibly by altering the charge distribution in this key functional domain of p47…”

mentioning

confidence: 99%

Analysis of Activation-induced Conformational Changes in p47 Using Tryptophan Fluorescence Spectroscopy

Swain

Helgerson

Davis

et al. 1997

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100

Activation of the neutrophil NADPH oxidase requires translocation of cytosolic proteins p47phox , p67 phox , and Rac to the plasma membrane or phagosomal membrane, where they assemble with membrane-bound flavocytochrome b. During this process, it appears that p47 phox undergoes conformational changes, resulting in the exposure of binding sites involved in assembly and activation of the oxidase. In the present study, we have directly evaluated activation-induced conformational changes in p47 phox using tryptophan fluorescence and circular dichroism spectroscopy. Treatment of p47 phox with amphiphilic agents known to activate the NADPH oxidase (SDS and arachidonic acid) caused a dose-dependent quenching in the intrinsic tryptophan fluorescence of p47 phox , whereas treatment with a number of other amphiphilic agents that failed to activate the oxidase had no effect on p47 phox fluorescence. In addition, the concentration range of activating agents required to induce changes in fluorescence correlated with the concentration range of these agents that induced maximal NADPH oxidase activity in a cell-free assay system. We next determined if activation by phosphorylation caused the same type of conformational changes in p47 phox . Protein kinase C phosphorylation of p47 phox in vitro resulted in comparable quenching of fluorescence, which also correlated directly with NADPH oxidase activity. Finally, the circular dichroism (CD) spectrum of p47 phox was significantly changed by the addition of SDS, whereas treatment with a non-activating detergent had no effect on the CD spectrum. These results support the conclusion that activation by amphiphilic agents results in changes in the secondary structure of p47 phox . Thus, our studies provide direct evidence linking conformational changes in p47 phox to the NADPH oxidase activation/assembly process and also further support the hypothesis that amphiphile-mediated activation of the NADPH oxidase induces changes in p47 phox that are similar to those mediated by phosphorylation in vivo.