Mapping of functional domains in the p22(phox) subunit of flavocytochrome b(559) participating in the assembly of the NADPH oxidase complex by "peptide walking".
The superoxide-generating NADPH oxidase complex of phagocytes consists of a membranal heterodimeric flavocytochrome (cytochrome b(559)), composed of gp91(phox) and p22(phox) subunits, and four cytosolic proteins, p47(phox), p67(phox), p40(phox), and the small GTPase Rac (1 or 2). All redox stations involved in electron transport from NADPH to oxygen are located in gp91(phox). NADPH oxidase activation is the consequence of assembly of cytochrome b(559) with cytosolic proteins, a process reproducible in a cell-free system, consisting of phagocyte membranes, and recombinant cytosolic components, activated by an anionic amphiphile. p22(phox) is believed to act as a linker between the cytosolic components and gp91(phox). We applied "peptide walking" to mapping of domains in p22(phox) participating in NADPH oxidase assembly. Ninety one synthetic overlapping pentadecapeptides, spanning the p22(phox) sequence, were tested for the ability to inhibit NADPH oxidase activation in the cell-free system and to bind individual cytosolic NADPH oxidase components. We conclude the following. 1) The p22(phox) subunit of cytochrome b(559) serves as an anchor for both p47(phox) and p67(phox). 2) p47(phox) binds not only to the proline-rich region, located at residues 151-160 in the cytosolic C terminus of p22(phox), but also to a domain (residues 51-63) located on a loop exposed to the cytosol. 3) p67(phox) shares with p47(phox) the ability to bind to the proline-rich region (residues 151-160) and also binds to two additional domains, in the cytosolic loop (residues 81-91) and at the start of the cytosolic tail (residues 111-115). 4) The binding affinity of p67(phox) for p22(phox) peptides is lower than that of p47(phox). 5) Binding of both p47(phox) and p67(phox) to proline-rich p22(phox) peptides occurs in the absence of an anionic amphiphile. A revised membrane topology model of p22(phox) is proposed, the core of which is the presence of a functionally important cytosolic loop (residues 51-91).
Dahan
Iris
I
Julius Friedrich Cohnheim-Minerva Center for Phagocyte Research and the Ela Kodesz Institute of Host Defense against Infectious Diseases, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel.
Issaeva
Irina
I
Gorzalczany
Yara
Y
Sigal
Natalia
N
Hirshberg
Miriam
M
Pick
Edgar
E
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002474:Cell-Free System; D003573:Cytochrome b Group / Q000737:chemistry*; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006168:Guinea Pigs; D020128:Inhibitory Concentration 50; D007477:Ions; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D009252:NADPH Dehydrogenase / Q000737:chemistry*; D019255:NADPH Oxidases / Q000737:chemistry* / Q000378:metabolism; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry; D010587:Phagocytosis; D010750:Phosphoproteins / Q000737:chemistry*; D045332:Photosystem II Protein Complex; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
D003573:Cytochrome b Group; D007477:Ions; D026901:Membrane Transport Proteins; D019151:Peptide Library; D010455:Peptides; D010750:Phosphoproteins; D045332:Photosystem II Protein Complex; C079789:neutrophil cytosol factor 67K; C019313:cytochrome b559; D019255:NADPH Oxidases; C086986:CYBA protein, human; C060505:neutrophil cytosolic factor 1; D009252:NADPH Dehydrogenase