Abstract
Stimulated exocytosis of intracellular granules plays a critical role in conversion of inactive, circulating neutrophils to fully activated cells capable of chemotaxis, phagocytosis, and bacterial killing. The functional changes induced by exocytosis of each of the granule subsets, gelatinase (tertiary) granules, specific (secondary) granules, and azurophil (primary) granules, are poorly defined. To improve the understanding of the role of exocytosis of these granule subsets, a proteomic analysis of the azurophil, specific, and gelatinase granules from human neutrophils was performed. Two different methods for granule protein identification were applied. First, two-dimensional (2D) gel electrophoresis followed by MALDI-TOF MS analysis of peptides obtained by in-gel trypsin digestion of proteins was performed. Second, peptides from tryptic digests of granule membrane proteins were separated by two-dimensional microcapillary chromatography using strong cation exchange and reverse phase microcapillary high pressure liquid chromatography and analyzed with electrospray ionization tandem mass spectrometry (2D HLPC ESI-MS/MS). Our analysis identified 286 proteins on the three granule subsets, 87 of which were identified by MALDI MS and 247 were identified by 2D HPLC ESI-MS/MS. The increased sensitivity of 2D HPLC ESI-MS/MS, however, resulted in identification of over 500 proteins from subcellular organelles contaminating isolated granules. Defining the proteome of neutrophil granule subsets provides a basis for understanding the role of exocytosis in neutrophil biology. Additionally, the described methods may be applied to mobilizable compartments of other secretory cells.
Highlights
Stimulated exocytosis of intracellular granules plays a critical role in conversion of inactive, circulating neutrophils to fully activated cells capable of chemotaxis, phagocytosis, and bacterial killing
Granule subsets were analyzed by Western blotting for CD66b and by ELISA for gelatinase and myeloperoxidase (MPO), markers for gelatinase and azurophil granules, respectively (Fig. 1)
The Western blot showed that CD66b was present in the specific granule fraction but essentially absent in gelatinase granule and azurophil granule fractions
Summary
Stimulated exocytosis of intracellular granules plays a critical role in conversion of inactive, circulating neutrophils to fully activated cells capable of chemotaxis, phagocytosis, and bacterial killing. The functional changes induced by exocytosis of each of the granule subsets, gelatinase (tertiary) granules, specific (secondary) granules, and azurophil (primary) granules, are poorly defined. To improve the understanding of the role of exocytosis of these granule subsets, a proteomic analysis of the azurophil, specific, and gelatinase granules from human neutrophils was performed. Stimulated exocytosis of membrane-bound compartments plays a critical role in converting inactive, circulating neutrophils to fully activated cells capable of directed migration, phagocytosis, and killing of microbes [1]. Specific, and gelatinase granules contain various enzymes and host defense proteins in their luminal spaces, whereas their membranes contain receptors, signaling proteins, adhesion molecules, and enzymes [2, 3]. The controlled exocytosis of neutrophil granules allows sequential acquisition of functional responses and targeted delivery of toxic granule proteins, thereby reducing damage to normal host tissue
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