Proteomic Analysis of Human Neutrophil Granules

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

Human Neutrophils Secrete Bioactive Paucimannosidic Proteins from Azurophilic Granules into Pathogen-Infected Sputum

“…Human ␤-mannosidase may be responsible for yielding amannosylated di-(M0) or tri-(M0F) saccharides. The detection of ␣-and ␤-mannosidases in isolated azurophilic granules (34,38) and the promyelocyte-specific expression of genes coding for the corresponding mannosidases support their association with protein paucimannosylation. By overlaying our observations on the existing map of the mammalian N-glycosylation machinery (3), we propose a new spatio-temporal restricted biosynthetic route enabling paucimannosylation of human neutrophil proteins (Fig.…”

“…4H). M3(F) truncation to M2(F), M1(F), and M0(F) may be facilitated by human ␣-and ␤-mannosidases previously identified in the azurophilic granules of human neutrophils (34,38); promyelocytic stage-specific expression of the corresponding mannosidase genes, i.e. MAN2A2/MAN2B2 (coding for ␣-mannosidases, which hydrolyze terminal ␣1,3/6-linked mannosides) and MANBA (coding for ␤-mannosidase, which hydrolyzes terminal ␤-linked mannosides), was indeed observed.…”

Impaired Host Defense in Mice Lacking ONZIN

“…Proteomics of neutrophils and cell lines after ingestion of bioparticles is implicating an ever-increasing number of proteins in this complex process (54,55). Our studies show that ONZIN is associated with granules in activated neutrophils.…”

Subproteome analysis of the neutrophil cytoskeleton

“…The early time point, when granule content is not yet delivered to phagosomes, is also the most plausible explanation for the fact that our detergent-insoluble phagosome preparation did not contain any of the typical granular proteins [24]. Phagosome proteome in macrophages has been studied more extensively than in neutrophils [6,25].…”