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

Nox4 B-loop Creates an Interface between the Transmembrane and Dehydrogenase Domains*

By targeting redox-sensitive amino acids in signaling proteins, the NADPH oxidase (Nox) family of enzymes link reactive oxygen species to physiological processes. We previously analyzed the sequences of 107 Nox enzymes and identified conserved regions that are predicted to have important functions in Nox structure or activation. One such region is the cytosolic B-loop, which in Nox1–4 contains a conserved polybasic region. Previous studies of Nox2 showed that certain basic residues in the B-loop are important for activity and translocation of p47phox/p67phox, suggesting this region participates in subunit assembly. However, conservation of this region in Nox4, which does not require p47phox/p67phox, suggested an additional role for the B-loop in Nox function. Here, we show by mutation of Nox4 B-loop residues that this region is important for Nox4 activity. Fluorescence polarization detected binding between Nox4 B-loop peptide and dehydrogenase domain (Kd = 58 ± 12 nm). This interaction was weakened with Nox4 R96E B-loop corresponding to a mutation that also markedly decreases the activity of holo-Nox4. Truncations of the dehydrogenase domain localize the B-loop-binding site to the N-terminal half of the NADPH-binding subdomain. Similarly, the Nox2 B-loop bound to the Nox2 dehydrogenase domain, and both the Nox2 and Nox4 interactions were dependent on the polybasic region of the B-loop. These data indicate that the B-loop is critical for Nox4 function; we propose that the B-loop, by binding to the dehydrogenase domain, provides the interface between the transmembrane and dehydrogenase domains of Nox enzymes

Critical roles for p22(phox) in the structural maturation and subcellular targeting of Nox3

Otoconia are small biominerals in the inner ear that are indispensable for the normal perception of gravity and motion. Normal otoconia biogenesis requires Nox3, a Nox (NADPH oxidase) highly expressed in the vestibular system. In HEK-293 cells (human embryonic kidney cells) transfected with the Nox regulatory subunits NoxO1 (Nox organizer 1) and NoxA1 (Nox activator 1), functional murine Nox3 was expressed in the plasma membrane and exhibited a haem spectrum identical with that of Nox2, the electron transferase of the phagocyte Nox. In vitro Nox3 cDNA expressed an ∼50 kDa primary translation product that underwent N-linked glycosylation in the presence of canine microsomes. RNAi (RNA interference)-mediated reduction of endogenous p22(phox), a subunit essential for stabilization of Nox2 in phagocytes, decreased Nox3 activity in reconstituted HEK-293 cells. p22(phox) co-precipitated not only with Nox3 and NoxO1 from transfectants expressing all three proteins, but also with NoxO1 in the absence of Nox3, indicating that p22(phox) physically associated with both Nox3 and with NoxO1. The plasma membrane localization of Nox3 but not of NoxO1 required p22(phox). Moreover, the glycosylation and maturation of Nox3 required p22(phox) expression, suggesting that p22(phox) was required for the proper biosynthesis and function of Nox3. Taken together, these studies demonstrate critical roles for p22(phox) at several distinct points in the maturation and assembly of a functionally competent Nox3 in the plasma membrane