The NADPH oxidase responsible for generatio of superoxide anion and related microbicidal oxidants by phagocytes Is assembled from at least five distinct proteins. TWo are cytosolic components (p47-phox and p67-phox) that contain Src homology 3 (SH3) domine and associate with a transmembrane cytochrome bss5 upon activation. We show here that the SH3 do of p47-phox bind to proine-rich sequences in p47-phx Itself and the p22-phox subunit of cytochrome b5ss. Binding of the p47-phox SH3 domains to p22-phox was abolished by a mutation in one proline-rich sequence (Pro'S' -* Gin) noted in a distinct form of chronic granulomatous disease and was inhibited by a short prolinerich synthetic peptide corresonding to resIdues 149-162 of p22-phox. Expression of mutated p22-phox did not restore oxidase activity to p22-phox-defclent B cells and did not enable p22-phox-dependent translocation of p47-phox to membranes in phorbol ester-stimulated cells. We also show that the cytosolic oxidase components associate with one another through the C-terminal SH3 domain of p67-phox and a proline-rich C-terminal sequence in p47-phox. These SH3 target sites conform to consensus features deduced from SH3 binding sites in other systems. We propose a model in which the oxidase complex assembles through a mechanism involving SH3 domains of both cytosolic proteins and cognate proline-rich targets in other oxidase components.Neutrophils respond to a number of stimuli with a burst of oxygen consumption and production of superoxide. This "respiratory burst" is attributed to NADPH oxidase, an enzyme whose importance is evident in patients with chronic granulomatous disease (CGD) (1), who suffer from increased susceptibility to bacterial and fungal infections due to deficient production ofmicrobicidal oxidants by phagocytes. The NADPH oxidase is composed of five essential components, four that are affected in different genetic types of CGD (2-6) and a fifth component, the Ras-related small GTPase p21, that bestows guanine nucleotide sensitivity to the enzyme complex (7-10). The functional center of the oxidase is flavocytochrome b558 (11, 12), composed of two subunits (gp9l-phox and p22-phox) that accept electrons from cytosolic NADPH and donate them to molecular oxygen. Two cytosolic factors, p47-and p67-phox, translocate to the membrane and associate with the cytochrome upon activation (13,14), although their precise functions remain unclear. Both factors contain two Src homology 3 (SH3) domains (3,4) shown to be critical for assembly of these proteins into the active, membrane-bound oxidase complex (15, 16).SH3 domains are conserved 60-aa sequences found in a variety of intracellular signaling and cytoskeletal proteins in organisms ranging from yeast to man (17,18 (19)(20)(21)(22)(23)(24).In light of recent observations showing that SH3 domains of both p67-and p47-phox are essential for oxidase assembly (15, 16), we examined the roles of these domains in the interactions of several oxidase components. In this report we describe specific SH...

Eosinophil cationic protein (ECP) is one of two RNase A-superfamily ribonucleases found in secretory granules of human eosinophilic leukocytes. Although the physiologic function of eosinophils [and thus of the two eosinophil ribonucleases, ECP and eosinophil-derived neurotoxin (EDN)] remains controversial, we have recently shown that isolated human eosinophils promote ribonuclease-dependent toxicity toward extracellular virions of the single-stranded RNA virus, respiratory syncytial virus, group B (RSV-B). We have also shown that recombinant human EDN (rhEDN) can act alone as a ribonuclease-dependent antiviral agent. In this work, we provide a biochemical characterization of recombinant human ECP (rhECP) prepared in baculovirus, and demonstrate that rhECP also promotes ribonuclease-dependent antiviral activity. The rhECP described here is N-glycosylated, as is native ECP, and has approximately 100-fold more ribonuclease activity than non-glycosylated rhECP prepared in bacteria. The enzymatic activity of rhECP was sensitive to inhibition by placental ribonuclease inhibitor (RI). Although rhECP was not as effective as rhEDN at reducing viral infectivity (500 nM rhECP reduced infectivity of RSV-B approximately 6 fold; 500 nM rhEDN, >50 fold), the antiviral activity appears to be unique to the eosinophil ribonucleases; no reduction in infectivity was promoted by bovine RNase A, by the amphibian ribonuclease, onconase, nor by the closely-related human ribonuclease, RNase k6. Interestingly, combinations of rhEDN and rhECP did not result in either a synergistic or even an additive antiviral effect. Taken together, these results suggest that that the interaction between the eosinophil ribonucleases and the extracellular virions of RSV-B may be specific and saturable.

Src homology 3 (SH3) domains mediate specific protein‐protein interactions crucial for signal transduction and protein subcellular localization. Upon phagocyte stimulation, two SH3 domain‐containing cytosolic components of the NADPH oxidase, p47phox and p67phox, are recruited to the membrane where they interact with flavocytochrome b558 to form an activated microbicidal oxidase. Deletion analysis of p47phox and p67phox in transfected K562 cells demonstrated multiple SH3‐mediated interactions between p47phox and the transmembrane flavocytochrome b558 and also between the cytosolic components themselves. The core region of p47phox (residues 151–284), spanning both SH3 domains, was required for flavocytochrome‐dependent translocation and oxidase activity in whole cells. Furthermore, translocation of p67phox occurred through interactions of its N‐terminal domain (residues 1–246) with p47phox SH3 domains. Both of these interactions were promoted by PMA activation of cells and were influenced by the presence of other domains in both cytosolic factors. Deletion analysis also revealed a third SH3 domain‐mediated interaction involving the C‐termini of both cytosolic factors, which also promoted p67phox membrane translocation. These data provide evidence for a central role for p47phox in regulation of oxidase assembly through several SH3 domain interactions.