Proteome profiling of human neutrophil granule subsets, secretory vesicles, and cell membrane: correlation with transcriptome profiling of neutrophil precursors

Abstract

Neutrophils are indispensable in the innate immune defense against invading microorganisms. Neutrophils contain SVs and several subsets of granules that are essential for their function. Proteins present in neutrophil SVs and granules are synthesized during terminal granulopoiesis in the bone marrow. The heterogeneity of granules, as determined by marker proteins characteristic of each granule subset, is thought to result from differences in the biosynthetic windows of major classes of granule proteins, a process referred to as targeting by timing. Qualitative proteomic analysis of neutrophil granules, SVs, and plasma membrane has been performed before. Here, we performed subcellular fractionation on freshly isolated human neutrophils by nitrogen cavitation and density centrifugation on a four-layer Percoll gradient. Granule subsets were pooled and subjected to SDS-PAGE, and gel pieces were in-gel-digested with trypsin. The resulting peptides were analyzed using LTQ Orbitrap XL tandem MS. A total of 1292 unique proteins were identified and grouped, according to the neutrophil fraction, in which they displayed maximal expression. In addition to various known neutrophil proteins, several uncharacterized proteins were found, as well as proteins not described previously in neutrophils. To study the correlation between mRNA expression in neutrophil precursors and the localization of their cognate proteins, the distribution of 126 identified proteins was compared with their mRNA expression profiles. The neutrophil subcellular proteome profiles presented here may be used as a database in combination with the mRNA array database to predict and test the presence and localization of proteins in neutrophil granules and membranes.

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

“…abundance of MPO, neutrophil elastase (NE), eosinophil peroxidase, lactoferrin, catalase, and aminopeptidase N (see supplemental Table 2 for supporting proteome data). The neutrophil-specific proteome (as predicted from a relatively unique transcriptional profile of human neutrophils among other immune cell types (36)) was significantly represented in the CF sputum proteome (63% of 672 human proteins) (34) (Fig. 2A), which was supported by morphology-based identification by microscopy of sputum neutrophils (data not shown).…”

“…Utilizing sequon-based (NX(T/S), X P) prediction of N-glycosylation and published granule proteome libraries of human neutrophils (34), the putative N-glycoproteins of CF sputum were shown to localize to all four main subcellular granular compartments of the human neutrophil, i.e. azurophil, specific, gelatinase/ficolin granules, and secretory vesicles.…”

“…Further evidence supporting strong neutrophilic molecular signatures in sputum was obtained by the near identical N-glycomes of CF sputum proteins and blood-derived 2 ); A, NeuAc; H, Hex; N, GlcNAc; F, fucose. C, the granular distribution of predicted N-glycoproteins identified in CF sputum using neutrophilic granule libraries (34) shows that the putative glycoproteins (when not considering their type of N-glycosylation) localize to all four main compartments of the human neutrophil, but preferentially to the azurophilic granules (red) (see supplemental Tables 1 and 2 for more). Graphics modified and used with permission from Blausen Medical (Blausen Gallery 2014).…”

“…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.…”

“…Subcellular Localization of Neutrophilic Proteins-The subcellular localizations were established using granule-specific proteome libraries of human neutrophils (34) to classify identified sputum proteins and by immunocytochemistry of DMSO-differentiated, fixed (4% (v/v) formaldehyde), and membrane-permeabilized (0.5% (v/v) Triton X-100 in PBS (aq), 20 min, 25°C) HL-60 cells plated on 0.02% (v/v) polylysinecoated coverslips using the following antibodies: mouse antihuman myeloperoxidase (MPO) IgG (1:2000), rabbit anti-human Hex A IgG (1:500) (Santa Cruz Biotechnology), the paucimannose-recognizing Mannitou IgM (undiluted concentration) (12), goat anti-mouse IgG Alexa Fluor 546 (1:400), and goat anti-rabbit IgG Alexa Fluor 488 (1:400). Nuclei were DAPI-stained.…”