Xu, P; (2006) Proteomic analysis of the neutrophil cytoskeleton, defining the function of grancalcin. Doctoral thesis , UCL (University College London).

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Abstract

The cytoskeleton plays an essential role in neutrophil adhesion, chemotaxis, and phagocytosis, however, the composition and regulation of function of the neutrophil cytoskeleton are still poorly understood. To elucidate some of these questions, we analysed the proteome of the cytosolic, membrane and phagosome skeleton. Using proteomic approach, we identified 138 proteins associated with the neutrophil cytoskeleton, among them 72 proteins in the cytosol, 39 in the membrane and 26 associated with the phagosome skeleton. Some of the newly reported proteins associating with the cytoskeleton encompass n- acetylglucosamine kinase, phosphoglycerate mutase 1, prohibitin, ficolin-1 (M-ficolin, ficolin A), phosphogluconate dehydrogenase, glucosidase, transketolase, major vault protein, valosin-containing protein, aldehyde dehydrogenase and LCRP8. Furthermore, we report for the first time that filamin, grancalcin, L-plastin and IQGAP1 are associated with the phagosome skeleton in neutrophils. Our mass spectrometric analysis also suggested the previously unknown post-translational modification of grancalcin, a protein of unknown function. By using a high-speed co-sedimentation assay, we found that grancalcin directly binds to F-actin with a dissociation constant of 2.5x1 OM and a stoichiometric ratio of 1.1:1. Furthermore, the identification of F-actin as a novel binding partner of grancalcin, indicates a novel mechanism for the association of this protein with cytoskeleton that is potentially relevant for actin dynamics. This study constitutes the broadest investigation to date of the neutrophil cytoskeletal proteome and identifies a series of proteins not previously reported to be associated with this subcellular compartment. The work based on grancalcin deficient mice suggested it has defects in adhesion and spreading on fibronectin-coated surface and this could be due to the reduced expression of surface integrins ct4pi and ctsPi in the grancalcin-deficient mice. Taken together, our results suggested that grancalcin could be a protein linking the integrins and cytoskeleton.

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