Anionic lipid-induced conformational changes in human phagocyte flavocytochrome b precede assembly and activation of the NADPH oxidase complex.

Phagocyte NADPH oxidases generate superoxide at high rates in defense against infectious agents, a process regulated by second messenger anionic lipids using incompletely understood mechanisms. We reconstituted the catalytic core of the human neutrophil NADPH oxidase, flavocytochrome b (Cyt b) in 99% phosphatidylcholine vesicles in order to correlate anionic lipid-dependent conformational changes in membrane-bound Cyt b and oxidase activity. The anionic lipid 10:0 phosphatidic acid (10:0 PA) specifically induced conformational changes in Cyt b as measured by a combination of fluorescence resonance energy transfer methods and size exclusion chromatography. The fluorescence lifetime of a complex between Cyt b and Cascade Blue-derivatized anti-p22(phox) antibody (CCB-CS9), increased after exposure to 10:PA by ∼50% of the change observed when the complex is dissociated, indicating a structural rearrangement of p22(phox) and/or the Cyt b heme prosthetic groups. Half of the quenching relaxation occurred at 10:0 PA concentrations permissive to less than 10% full NADPH oxidase activity, but saturated near the saturation in activity in a matched cell-free oxidase assay. We conclude that anionic lipids modulate the conformation of Cyt b in the membrane and suggest they may serve to modulate the structure of Cyt b as a control mechanism for the NADPH oxidase.

Single-step immunoaffinity purification and functional reconstitution of human phagocyte flavocytochrome b.

Human neutrophil flavocytochrome b (Cyt b) is a heterodimeric, integral membrane protein that generates high levels of superoxide in the multisubunit NADPH oxidase complex. Since Cyt b is currently isolated in limited quantities, improved methods for purification from low levels of starting membranes (from both neutrophils and other expressing cell types) are important for the analysis of structure and catalytic mechanism. In the present study, the epitope-mapped monoclonal antibody CS9 was coupled to Sepharose beads and used as an affinity matrix for single-step immunoaffinity purification of Cyt b. Following solubilization of both human neutrophil and PLB-985 membrane fractions in the nonionic detergent octylglucoside, Cyt b was absorbed on the CS9-Sepharose affinity matrix and purified protein was eluted under non-denaturing conditions with an epitope-mimicking peptide. The high efficiency of this isolation procedure allowed Cyt b to be reproducibly purified from readily obtainable levels of starting membrane fractions (9x10(8) cell equivalents of neutrophil membranes and 2x10(9) cell equivalents of PLB-985 membranes). Since Cyt b could be affinity-purified in the detergent octylglucoside, high-level functional reconstitution was carried out directly on elution fractions by simple addition of solubilized phospholipid and subsequent dialysis for detergent removal. To our knowledge, this study describes the most efficient method for generating purified, functionally-reconstituted Cyt b and should facilitate analyses that require a highly-defined NADPH oxidase system.

Characterization of surface structure and p47phox SH3 domain-mediated conformational changes for human neutrophil flavocytochrome b.

The heterodimeric, integral membrane protein flavocytochrome b (Cyt b) is the catalytic core of the phagocyte NADPH oxidase and generates superoxide which plays a critical role in host defense. To better define the activation of superoxide production by this multisubunit enzyme complex, Cyt b-specific monoclonal antibodies (mAbs) and the p47phox SH3 domains (p47SH3AB) were used in the present study as probes to map surface structure and conformational dynamics in human neutrophil Cyt b. In pull-down and co-immunoprecipitation studies with detergent-solubilized Cyt b, the oxidase-inhibitory mAb CS9 was shown to share an overlapping binding site with p47SH3AB on the C-terminal region of the p22phox subunit. Similar studies demonstrated a surprising lack of overlap between the mAb 44.1 and CS9/p47SH3AB binding sites, and they indicated that the oxidase-inhibitory mAb NL7 binds a region physically separated from the p22phox C-terminal domain. Resonance energy transfer and size exclusion chromatography confirmed the above results for functionally reconstituted Cyt b and provided evidence that binding of both mAb CS9 and p47SH3AB altered the conformation of Cyt b. Further support that binding of the p47phox SH3 domains modulates the structure of Cyt b was obtained using a cell-free assay system where p47SH3AB enhanced superoxide production in the presence of a p67phox (1-212)-Rac1(Q61L) fusion protein. Taken together, this study further characterizes the structure of human neutrophil Cyt b in both detergent micelles and reconstituted membrane bilayers, and it provides evidence that the cytosolic regulatory subunit p47phox modulates the conformation of Cyt b (in addition to serving as an adapter protein) during oxidase activation.

Analysis of human phagocyte flavocytochrome b(558) by mass spectrometry.

The catalytic core of the phagocyte NADPH oxidase is a heterodimeric integral membrane protein (flavocytochrome b (Cyt b)) that generates superoxide and initiates a cascade of reactive oxygen species critical for the host inflammatory response. In order to facilitate structural characterization, the present study reports the first direct analysis of human phagocyte Cyt b by matrix-assisted laser desorption/ionization and nanoelectrospray mass spectrometry. Mass analysis of in-gel tryptic digest samples provided 73% total sequence coverage of the gp91(phox) subunit, including three of the six proposed transmembrane domains. Similar analysis of the p22(phox) subunit provided 72% total sequence coverage, including assignment of the hydrophobic N-terminal region and residues that are polymorphic in the human population. To initiate mass analysis of Cyt b post-translational modifications, the isolated gp91(phox) subunit was subject to sequential in-gel digestion with Flavobacterium meningosepticum peptide N-glycosidase F and trypsin, with matrix-assisted laser desorption/ionization and liquid chromatography-mass spectrometry/mass spectrometry used to demonstrate that Asn-132, -149, and -240 are genuinely modified by N-linked glycans in human neutrophils. Since the PLB-985 cell line represents an important model system for analysis of the NADPH oxidase, methods were developed for the purification of Cyt b from PLB-985 membrane fractions in order to confirm the appropriate modification of N-linked glycosylation sites on the recombinant gp91(phox) subunit. This study reports extensive sequence coverage of the integral membrane protein Cyt b by mass spectrometry and provides analytical methods that will be useful for evaluating posttranslational modifications involved in the regulation of superoxide production.