Dataset Information

Spatiotemporal glycoproteome remodelling in neutrophils

ABSTRACT: Neutrophils are short-lived yet vital innate immune cells equipped with bioactive glycoproteins packed in cytosolic granules that can readily be mobilised to elicit an effective and timely response against invading pathogens. Since the dynamic glycosylation processes underpinning the dramatic metamorphosis associated with myeloid progenitor-to-neutrophil differentiation remain unmapped, we here perform a comprehensive spatiotemporal profiling of the complex N-glycoproteome of isolated granule populations from blood-derived neutrophils and during maturation of bone marrow-derived progenitors using glycomics-assisted glycoproteomics. Firstly, glycomics indicated that the granule populations of mature (resting) neutrophils exhibit distinctive glycophenotypes including, most strikingly, unusual paucimannosidic- and monoantennary complex-type N-glycans in azurophilic granules. The granule-specific N-glycosylation features were recapitulated by glycoproteomics, which also uncovered extensive site- and protein-specific N-glycosylation decorating 4,772 N-glycopeptides from 352 N-glycoproteins across the neutrophil granules. Excitingly, comprehensive mining of proteomics and transcriptomics data collected from discrete myeloid progenitor stages for glycopeptide and glyco-enzyme expression revealed that dramatic glycoproteome remodelling underpins the promyelocytic-to-metamyelocyte transition and that remodelling is driven predominantly by proteome expression changes rather than modulation of the glycosylation machinery. Notable exceptions were the oligosaccharyltransferase subunits responsible for initiation of N-glycoprotein biosynthesis that were strongly reduced with myeloid differentiation leading to reduced N-glycosylation efficiency in late-stage neutrophil maturation and in corresponding granules. Our study provides new spatiotemporal insights into the dynamic neutrophil N-glycoproteome, which exhibits intriguingly complex site-, protein- and granule-specific N-glycosylation features and which undergoes strong remodelling during myeloid progenitor-to-neutrophil metamorphosis.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Neutrophil, Blood

SUBMITTER: Rebeca Kawahara

LAB HEAD: Morten Thaysen-Andersen

PROVIDER: PXD039387 | Pride | 2023-09-18

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20171221_Fusion_BenP_Morten_EnrichedV2_VA.msf	Msf
	20171221_Fusion_BenP_Morten_EnrichedV2_VA.raw	Raw
	20171221_Fusion_BenP_Morten_EnrichedV2_VB.msf	Msf
	20171221_Fusion_BenP_Morten_EnrichedV2_VB.raw	Raw
	20171221_Fusion_BenP_Morten_EnrichedV2_VC.msf	Msf

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Publications

Glycoproteome remodeling and organelle-specific <i>N</i>-glycosylation accompany neutrophil granulopoiesis.

Kawahara Rebeca R Ugonotti Julian J Chatterjee Sayantani S Tjondro Harry C HC Loke Ian I Parker Benjamin L BL Venkatakrishnan Vignesh V Dieckmann Regis R Sumer-Bayraktar Zeynep Z Karlsson-Bengtsson Anna A Bylund Johan J Thaysen-Andersen Morten M

Proceedings of the National Academy of Sciences of the United States of America 20230828 36

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 comprehensively profile the neutrophil <i>N</i>-glycoproteome with spatiotemporal resolution by analyzing four key types of intracellular organelles isolated from blood-derived neutrophils and during their maturation from bone marrow-derived progenitors using a glycomics-guided glycoproteomics ...[more]

PMID: 37639587

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Project description:Myeloperoxidase (MPO), an important diprotomeric glycoprotein in neutrophil-mediated immunity, produces microbicidal hypohalous acids, but the underpinning glycobiology remains elusive. Deep characterisation of neutrophil-derived MPO (nMPO) using advanced mass spectrometry demonstrated that under-processed oligomannosidic- and hyper-truncated paucimannosidic- and N-acetyl-β-D-glucosamine (GlcNAc) core-type asparagine-linked glycans decorate the protein. Occlusion of Asn355 and Asn391 and sterical hindrance of Asn323- and Asn483-glycans located in the MPO dimerisation zone were found to shape the local glycan processing thereby providing a molecular basis of the site-specific nMPO glycosylation. Native mass spectrometry, mass photometry, and glycopeptide profiling revealed extreme molecular complexity of diprotomeric nMPO arising from heterogeneous glycosylation, oxidation, chlorination and polypeptide truncation variants, and a lower-abundance monomer. Longitudinal profiling of maturing, mature, granule-separated, and pathogen-activated neutrophils demonstrated that MPO is dynamically expressed during granulopoiesis, unevenly distributed across granules and rapidly degranulated, but surprisingly carries uniform glycosylation across conditions. Complete proMPO-to-MPO maturation evidently occur during early/mid-stage granulopoiesis. The conserved Asn355- and Asn391-sequons displayed elevated GlcNAc signatures and higher oxidation and chlorination activity of the secretory vesicle/plasma membrane-resident MPO relative to MPO from other granules. Endoglycosidase H-treated nMPO displaying Asn355-/Asn391-GlcNAcylation recapitulated the activity gain and showed increased thermal stability and polypeptide accessibility relative to untreated nMPO as measured by activity assays, circular dichroism and molecular dynamics. Endoglycosidase H-treated nMPO also demonstrated elevated ceruloplasmin-mediated inhibition relative to nMPO. Modelling revealed that hyper-truncated Asn355-glycans positioned in the MPO:ceruloplasmin interface are critical for uninterrupted inhibition. We report on novel bimodal roles of the peculiar MPO glycosylation providing new insight into neutrophil glycobiology.

Project description:Background Plasma proteins can be modified by addition of sugar residues by non-enzymatic glycation as well as glycosylation. While glycation is a hallmark of hyperglycemia, glycosylation is a complex, enzyme-catalyzed post-translational modification by heterogeneous sugar chains and is present on a majority of plasma proteins. N-linked glycosylation occurs on asparagine residues occurring predominantly on a canonical N-glycosylation motif (Asn-X-Ser/Thr) although non-canonical N-glycosylation motifs Asn-X-Cys/Val have also been reported to be glycosylated less frequently. Albumin is the most abundant protein in human plasma whose glycation has been implicated in end-organ damage in advanced diabetes mellitus, and as such, has both diagnostic and therapeutic implications. However, albumin has long been regarded as a non-glycosylated protein owing to the absence of canonical motifs. Albumin contains two non-canonical N-glycosylation motifs, of which one was recently reported to be glycosylated with two possible attached glycans. Methods To investigate N-linked glycosylation of abundant serum proteins in greater detail, we passed healthy donor samples through a multiple affinity removal spin (MARS14) column followed by trypsin digestion of bound proteins and subsequent glycopeptide enrichment by size-exclusion chromatography (SEC) or mixed-mode anion-exchange (MAX), in parallel. Global analysis of intact glycopeptides was performed by LC-MS/MS to evaluate potential glycosylation at canonical as well as non-canonical sites. PNGase F treatment was carried out to confirm N-linked glycosylation at non-canonical sites Asn-X-Cys/Val. Glycopeptides on albumin at non-canonical sites were further characterized by MS3 fragmentation from immunoprecipitated albumin to confirm the attached glycans. To assess if the observed glycosylation was a general phenomenon, targeted analysis using parallel reaction monitoring (PRM) was carried out on an independent set of twenty human serum samples. Finally, to test whether albumin glycosylation is unique to human albumin or is a general phenomenon, bovine and rabbit serum albumins were subjected to MAX chromatography and LC-MS/MS analysis. Results We report that human albumin is modified by N-linked glycosylation at two sites, Asn68(-Glu-Val) and Asn123-(Glu-Cys), both of which occur in non-canonical N-glycosylation motifs. We detected N-glycopeptides at both sites bearing four complex mono- and bi-antennary N-linked glycans, with Hex5HexNAc4NeuAc2 and Hex5HexNAc4NeuAc1 being the most abundant glycans. These findings were validated by analyzing deglycosylated peptides and MS3-based fragmentation of glycopeptides. Glycosylation at both sites was confirmed by targeted MS in twenty donor samples. Finally, we report that the Asn residue on bovine and rabbit serum albumins that corresponds to the highly conserved Asn123 in human albumin is also N-glycosylated with complex sialylated glycans similar to those observed in human albumin. Conclusions Albumin is a conserved glycoprotein with multiple N-linked glycosylation sites. This glycosylation of albumin has potential functional implications and applications in medicine.