title: Membrane proteinase 3 and its interactions within microdomains of neutrophil membranes.
abstract: Proteinase 3 (PR3) is a serine protease of neutrophil granules released to the medium or into the phagocytic vesicle upon neutrophil stimulation. A fraction of the enzyme is thought to associate with the cell membrane yielding membrane PR3 (mPR3). In autoimmune disorders characterized by the presence of antineutrophil cytoplasmic antibodies (ANCA), the reaction of the latter with their target antigen mPR3 activates the cell inflicting injuries on the surrounding tissues. In a previous communication we provided evidence for the presence of mPR3 in lipid rafts obtained by lysis of neutrophils in Triton X-100 and for the mediation of PR3 binding to the membrane by a glycosylphosphatidylinositol (GPI)-anchored neutrophil protein, possibly FcgammaRIIIb. In the current study we employed the mild detergent Brij 58 to isolate high molecular weight (HMW) protein complexes in the void volume of a Sepharose 4B gel filtration minicolumn. HMW complexes of unstimulated neutrophils comprised PR3, FcgammaRIIIb, the beta2 integrin CD11b/CD18 as well as the membrane and cytosolic subunits of the NADPH oxidase, p22phox and p47phox/p67phox. Treatment of neutrophils with phosphatidylinositol-specific phospholipase C (PI-PLC) reduced amounts of PR3 and FcgammaRIIIb in HMW complexes isolated from the treated cells, supporting our previous suggestion that FcgammaRIIIb acts as a membrane adaptor for PR3. FcgammaRIIIb of HMW fractions co-immunoprecipitated with PR3, indicating their presence in the same protein complex. Since HMW fractions contained also the majority of biotinylated proteins obtained by the reaction of neutrophils with a membrane impermeable biotinylating agent Sulfo-NHS-biotin, it was concluded that HMW proteins were derived from cell membranes. Lipid rafts isolated from Brij 58-lysed neutrophils were similar in their protein composition to the HMW complexes but not identical.
authors.affiliation: The Department of Biochemistry, Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv 69978, Israel.
authors.forename: Ram
authors.initials: R
authors.lastname: Fridlich
authors.forename: Alina
authors.initials: A
authors.lastname: David
authors.forename: Irit
authors.initials: I
authors.lastname: Aviram
mesh_terms: D015703:Antigens, CD / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002592:Cetomacrogol / Q000737:chemistry; D002847:Chromatography, Agarose; D003902:Detergents / Q000737:chemistry; D058851:GPI-Linked Proteins; D006801:Humans; D021962:Membrane Microdomains / Q000378:metabolism*; D046912:Multiprotein Complexes; D053146:Myeloblastin; D019255:NADPH Oxidases / Q000378:metabolism; D009504:Neutrophils / Q000378:metabolism*; D043265:Phosphatidylinositol Diacylglycerol-Lyase / Q000378:metabolism; D054801:Phosphoinositide Phospholipase C; D010750:Phosphoproteins / Q000378:metabolism; D017452:Receptors, IgG / Q000378:metabolism; D012697:Serine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D015703:Antigens, CD; D003902:Detergents; C428200:FCGR3B protein, human; D058851:GPI-Linked Proteins; D046912:Multiprotein Complexes; D010750:Phosphoproteins; D017452:Receptors, IgG; C079789:neutrophil cytosol factor 67K; D002592:Cetomacrogol; D019255:NADPH Oxidases; C086986:CYBA protein, human; C060505:neutrophil cytosolic factor 1; D054801:Phosphoinositide Phospholipase C; D012697:Serine Endopeptidases; D053146:Myeloblastin; D043265:Phosphatidylinositol Diacylglycerol-Lyase