Membrane proteinase 3 and its interactions within microdomains of neutrophil membranes

Ram Fridlich, Alina David, Irit Aviram*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


Proteinase3 (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 FcγRIIIb. 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, FcγRIIIb, the β2 integrin CD11b/CD18 as well as the membrane and cytosolic subunits of the NADPH oxidase, p22 phox and p47phox/p67phox. Treatment of neutrophils with phosphatidylinositol-specific phospholipase C (PI-PLC) reduced amounts of PR3 and FcγRIIIb in HMW complexes isolated from the treated cells, supporting our previous suggestion that FcγRIIIb acts as a membrane adaptor for PR3. FcγRIIIb 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.

Original languageEnglish
Pages (from-to)117-125
Number of pages9
JournalJournal of Cellular Biochemistry
Issue number1
StatePublished - 1 Sep 2006


  • Membrane complexes
  • Neutrophils
  • Proteinase 3
  • Rafts


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