Lydia M. Henderson

Localization of Nox2 N-terminus using polyclonal antipeptide antibodies

Nox2/gp91(phox) (where phox is phagocyte oxidase) is the catalytic membrane subunit of the granulocyte NADPH oxidase complex involved in host defence. The current model of membrane topology of Nox2 is based upon the identification of glycosylation sites, of regions that interact with the regulatory cytosolic factors and of the epitopes recognized by antibodies. So far, the localization of the N-terminus of Nox2 was only speculative. In order to clarify this localization, we raised a polyclonal antiserum against the N-terminal sequence M(1)GNWVAVNEGL(11). Purified antibodies recognize the mature protein as a broad band at 91 kDa (glycosylated form) or a band at 55 kDa after deglycosylation. Immunocytochemistry and flow-cytometry analysis show a strong binding of the anti-N-terminal antibodies to differentiated HL60 cells and neutrophils respectively, after permeabilization only. The N-terminus of Nox2 is therefore present in the mature protein and is located to the cytoplasmic side of the plasma membrane.

Inhibition of the neutrophil NADPH oxidase and associated H+ channel by diethyl pyrocarbonate (DEPC), a histidine-modifying agent: evidence for at least two target sites

Diethyl pyrocarbonate (DEPC), a histidine-modifying reagent, has been utilized to demonstrate the importance of histidine residues in the functioning of proteins. In previous studies of the NADPH oxidase, histidine residues have been determined to be important in the ability of gp91(phox) to function as an H(+) pathway and in the binding of haem and FAD. We have investigated the ability of DEPC to inhibit H(+) flux and superoxide generation by human neutrophils. Proton flux through the NADPH oxidase-associated H(+) channel was inhibited by DEPC only if applied simultaneously with an activator of the channel. This suggested that the site modified by DEPC is not accessible in the closed channel. Superoxide generation by the NADPH oxidase was also inhibited by DEPC when applied after or simultaneously with the activator. Translocation of the NADPH oxidase cytosolic components, p67(phox) and p47(phox), to the membrane was unaffected by DEPC. In a cell-free system, DEPC-treated membranes failed to support superoxide generation or the reduction of Iodonitrotetrazolium Violet and showed a loss of the characteristic cytochrome b(558) spectrum. Superoxide generation by DEPC-treated cytosol was inhibited slightly. Therefore it can be concluded that there are two sites within the NADPH oxidase that interact with DEPC, one in the H(+) pathway, only accessible in the activated oxidase, and a second accessible prior to activation of the NADPH oxidase. The latter non-proton pathway DEPC site is located within the membrane components of the NADPH oxidase and is associated with the binding of haem in the enzyme complex.

NADPH oxidase subunit gp91phox: a proton pathway.

SummaryThe generation of superoxide by the NADPH oxidase is an electrogenic process resulting in a rapid depolarisation of the membrane potential of the cell. The efflux of H+ ions through an arachidonate-activatable, Zn2+-inhibitable H+ pathway accompanies the efflux of electrons and provides the necessary charge compensation. Inhibition of H+ flux leads to inhibition of superoxide generation. The protein gp91phox, a transmembrane component of the NADPH oxidase, was demonstrated to be capable of acting as the NADPH oxidase-associated H+ channel in a stable CHO cell line, CHO91. The N-terminal 230 amino acids contain all that is required for the protein to form an H+ channel and specifically histidine 115 is important to the ability of gp91phox to conduct H+ ions. The recording of outward currents from CHO91 cells, in the whole-cell configuration, demonstrated that gp91phox is also capable of functioning as a voltage-gated H+ conductance pathway. The similarity in properties between voltage-elicited outward currents, from both wild type and the mutations, and the arachidonate-activated H+ flux strongly suggests that these H+ pathways are one in the same. Among the recently identified homologues of gp91phox only NOH-1S has so far been demonstrated to also act as an H+ conductance pathway.

The requirement of cytosolic phospholipase A2 for the PMA activation of proton efflux through the N-terminal 230-amino-acid fragment of gp91phox.

The absolute requirement for the 85 kDa cytosolic phospholipase A(2) (cPLA(2)) in the PMA stimulation of proton efflux through the NADPH-oxidase-associated proton channel, has previously been demonstrated using a PLB-985 cell line deficient in cPLA(2) (PLB-D). The flux of protons in Chinese-Hamster ovary (CHO) cells that express the N-terminal 230-amino-acid (NT) fragment of gp91(phox) is activated by arachidonic acid (AA) added externally. To investigate the physiological role of cPLA(2), and the intracellular AA that it releases, in the activation of proton flux through the NT fragment of gp91(phox), this fragment was stably expressed in PLB-985 cells (PLB-985 NT) and in PLB-D cells (PLB-D NT). The expression of the NT fragment of gp91(phox) by itself in PLB-985 did not initiate differentiation and did not alter their ability to undergo differentiation after the addition of DMSO. Addition of PMA induced a proton efflux from undifferentiated PLB-985 NT cells expressing the NT fragment of gp91(phox), which was inhibited by zinc. In contrast, PMA failed to activate proton efflux in undifferentiated PLB-D NT cells, lacking the expression of cPLA(2); however, addition of AA restored the efflux of protons in these cells. These results establish an essential and specific physiological requirement of cPLA(2)-generated AA in the activation of proton flux through the NT fragment of gp91(phox).

Proton conduction through full-length gp91phox requires histidine 115.

Summary. The NADPH oxidase of neutrophils is a transmembrane electron transfer complex, containing a flavin adenine dinucleotide and two hemes, all of which are suggested to be contained within gp91phox, one of four subunits of the enzyme. The transfer of electrons through the NADPH oxidase is associated with an efflux of protons. gp91phox has previously been demonstrated to function as the proton conduction pathway. The mutation of histidines 111, 115, and 119 to leucines and of histidine 115 to leucine within the N-terminal 230-amino-acid fragment of gp91phox has previously been demonstrated to result in the loss of proton conduction through this N-terminal fragment. In this paper we have investigated the role of these histidines in proton conduction by the full-length gp91phox. Stable CHO cell lines were established which expressed full-length gp91phox in which histidines 111, 115, and 119 had been mutated to leucines (CHO91H111/115/119) and in which histidine 115 had been mutated to leucine (CHO91H115L). The expression of gp91phox and its cellular localisation in these cell lines were comparable between wild-type and the mutant gp91phox. The mutation of histidines 111, 115, and 119 to leucines or just histidine 115 to leucine resulted in an almost total loss of both the arachidonate-activated influx and efflux of protons, in comparison with that observed for wild-type gp91phox. Therefore, histidine 115 is required for proton conduction by both full-length gp91phox and the N-terminal 230-amino-acid fragment of gp91phox. Histidine 115 has recently been proposed to act as a coordinating ligand for the outer heme iron of the NADPH oxidase. On the basis of observations for cytochrome c oxidase, we propose a model for this dual role of histidine 115.