Glycoproteome remodelling and granule-specificN-glycosylation accompany neutrophil granulopoiesis

Abstract

Neutrophils store microbicidal glycoproteins in cytosolic granules to fight intruding pathogens, but their granule distribution and formation mechanism(s) during granulopoiesis remain unmapped. Herein, we perform comprehensive spatiotemporal N-glycoproteome profiling of isolated granule populations from blood-derived neutrophils and during their maturation from bone marrow-derived progenitors using glycomics-assisted glycoproteomics. Interestingly, the granules of resting neutrophils exhibited distinctive glycophenotypes including, most strikingly, peculiar highly truncated N-glycosylation in the azurophilic granules. Excitingly, proteomics and transcriptomics data from discrete myeloid progenitor stages revealed that profound glycoproteome remodelling underpins the promyelocytic-to-metamyelocyte transition and that remodelling is driven primarily by changes in protein expression and less by the glycosylation machinery. Notable exceptions were the oligosaccharyltransferase subunits responsible for initiation of N-glycoprotein biosynthesis that were strongly expressed in early myeloid progenitors correlating with high glycosylation efficiencies of the azurophilic granule proteins. Our study provides spatiotemporal insights into the complex neutrophil N-glycoproteome featuring an intriguing granule-specific N-glycosylation formed by dynamic remodelling during myeloid progenitor-to-neutrophil maturation.

Profound N-glycan remodelling accompanies MHC-II immunopeptide presentation

“…To this end, we firstly mined the available PGC-LC-MS/MS-based glycomics data of the cellular N-glycome of MDA-MB-231 and THP-1 from which 29 and 41 N-glycan structures spanning 22 and 29 monosaccharide compositions, respectively, were confidently identified (Supplementary Tables S1, S2). Hyper-truncated chitobiose core N-glycans (GlcNAc 1-2 Fuc 0-1 ) play recognised roles in innate immunity and cancer (32,33) and have been reported on MHC-II immunopeptides (9) yet are ineffectively profiled by PGC-LC-MS/MS-based glycomics; thus, these short N-glycans were manually added to the glycan search space.…”

Tumor- and immune-derivedN-acetyl-β-D-hexosaminidase drive colorectal cancer and stratify patient risk

“…While protein paucimannosylation is a constitutively expressed and tissue-wide N-glycan modification in plants and lower vertebrates and until recently considered absent in mammals 34,35 , our developing knowledge of paucimannose in the context of human glycobiology points to a tissue-restricted and context-dependent expression of this non-canonical class of N-glycoproteins in humans 23 with reports mainly related to immunity 26,28,36 and cancer [37][38][39][40][41] including several in CRC 14,[42][43][44] . These recent observations of non-canonical paucimannosylation in cancer biospecimens including those reported in this study were critically enabled by technology improvements in the emerging field of systems glycobiology including the advent of powerful quantitative glycomics and glycoproteomics 45 .…”

“…Building on our recent reports that circulating innate immune cells (e.g. neutrophils, monocytes) constitutively express and store paucimannosidic proteins that may be released upon activation [26][27][28] and the established knowledge that innate immune cells abundantly infiltrate the CRC TME 29,30 , these observations led us to hypothesize that the strong paucimannosidic signatures in the tumor tissues may, in part, originate from infiltrating immune cells and, in part, arise from aberrations in the glycosylation machinery of the growing epithelial cancer cells (Figure 2D).…”

Glycoproteome remodeling and organelle-specific N -glycosylation accompany neutrophil granulopoiesis

“…All LC-MS/MS data and metadata have been deposited in public repositories. Specifically, glyco/proteomics LC-MS/MS data were deposited to PRIDE ( PXD039387 and PXD021131 ) ( 72 , 73 ). Glycomics LC-MS/MS raw data were deposited to GlycoPOST ( GPST000315 ) ( 74 ).…”

“…Specifically, glyco/proteomics LC-MS/MS data were deposited to PRIDE ( PXD039387 and PXD021131 ) ( 72 , 73 ). Glycomics LC-MS/MS raw data were deposited to GlycoPOST ( GPST000315 ) ( 74 ). Previously published data were used for this work (PRIDE: PXD013785 and https://blueprint.haem.cam.ac.uk/neutrodiff ) ( 75 , 76 ).…”