Phagocytosis is a part of immune response. IgG opsonized particles of greater than 1 um are recognized by Fcy receptors on the surface of professional phagocytes such as macrophages and neutrphils. IgGs are part of the immune system and is a cognate ligand of the Fc receptor. Live Cell Affinity Receptor chromatography (LARC) was used to capture an activated Fcy receptor supramolecular complex from the surface of live human neutrophils, by allowing IgG opsonized microbeads to bind to the cell surface.
The cells were burst in PBS, collected and digested along side with controls. Isolated FcyR complex was analysed by LC-MS/MS. Fc and control experiment lists of SEQUEST correlated proteins were screened for a total cumulative score of at least 2400 and a minimum of three different peptides. This served as the basis of protein involvement in the FcyR mediated phagocytosis, which were then searched with iHOP for their interaction partners. Gathered interactions were then exported and Cytoscape, Osprey and String algorithms were used to generate network of interacting proteins. PAKs2-4 and PAK6 were detected with LARC. PAK2 and PAK4 were predicted by algorithms to have a central role in particle uptake. From Western Blotting, endogenous PAKs2-4 and PAK6 were detected in murine macrophages. Immunofluorescent staining was then used to verify the presence of these proteins in the forming phagosome and showed localization of PAKs to the phagosome. The same effect was observed with transfection of GFP constructs of PAKs. Upon transfection with dominant negative PAKs reduction in phagocytosis was observed.