Journal of Biological Chemistry
Volume 296, January–June 2021, 100144
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Research Article
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Hyper-truncated Asn355- and Asn391-glycans modulate the activity of neutrophil granule myeloperoxidase

https://doi.org/10.1074/jbc.RA120.016342Get rights and content
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Myeloperoxidase (MPO) plays essential roles in neutrophil-mediated immunity via the generation of reactive oxidation products. Complex carbohydrates decorate MPO at discrete sites, but their functional relevance remains elusive. To this end, we have characterised the structure–biosynthesis–activity relationship of neutrophil MPO (nMPO). Mass spectrometry demonstrated that nMPO carries both characteristic under-processed and hyper-truncated glycans. Occlusion of the Asn355/Asn391-glycosylation sites and the Asn323-/Asn483-glycans, located in the MPO dimerisation zone, was found to affect the local glycan processing, thereby providing a molecular basis of the site-specific nMPO glycosylation. Native mass spectrometry, mass photometry and glycopeptide profiling revealed significant molecular complexity of diprotomeric nMPO arising from heterogeneous glycosylation, oxidation, chlorination and polypeptide truncation variants and a previously unreported low-abundance monoprotomer. Longitudinal profiling of maturing, mature, granule-separated and pathogen-stimulated neutrophils demonstrated that nMPO is dynamically expressed during granulopoiesis, unevenly distributed across granules and degranulated upon activation. We also show that proMPO-to-MPO maturation occurs during early/mid-stage granulopoiesis. While similar global MPO glycosylation was observed across conditions, the conserved Asn355-/Asn391-sites displayed elevated glycan hyper-truncation, which correlated with higher enzyme activities of MPO in distinct granule populations. Enzymatic trimming of the Asn355-/Asn391-glycans recapitulated the activity gain and showed that nMPO carrying hyper-truncated glycans at these positions exhibits increased thermal stability, polypeptide accessibility and ceruloplasmin-mediated inhibition potential relative to native nMPO. Finally, molecular modelling revealed that hyper-truncated Asn355-glycans positioned in the MPO-ceruloplasmin interface are critical for uninterrupted inhibition. Here, through an innovative and comprehensive approach, we report novel functional roles of MPO glycans, providing new insight into neutrophil-mediated immunity.

Keywords

myeloperoxidase
N-glycosylation
neutrophil
granule
biosynthesis
activity
inhibition
ceruloplasmin
granulopoiesis
degranulation

Abbreviations

AUC
area-under-the-curve
Az granule
azurophilic granule
Az-MPO
azurophilic granule-resident MPO
BN
band neutrophil
CD
circular dichroism
CytB/I
cytochalasin B and ionomycin
Dg-MPO
degranulated MPO
EIC
extracted ion chromatogram
Endo H
endoglycosidase H
ER
endoplasmic reticulum
Fuc (F)
α-L-fucose
Ge granule
gelatinase granule
Ge-MPO
gelatinase granule-resident MPO
GlcNAc
N-acetyl-β-D-glucosamine
H2O2
hydrogen peroxide
HOCl
hypochlorous acid
KRG buffer
Krebs-Ringer buffer with glucose
LC-MS/MS
liquid chromatography tandem mass spectrometry
LDH
lactate dehydrogenase
LFQ
label-free quantitation
Man
α/β-D-mannose
MD
molecular dynamics
MM
metamyelocyte
MOI
multiplicity-of-infection
MPO
myeloperoxidase
nMPO
neutrophil-derived myeloperoxidase (unfractionated)
PDB
Protein Data Bank
PGC
porous graphitised carbon
PM
promyelocyte
PMN
polymorphonuclear cell (neutrophil)
PMN-nMPO
myeloperoxidase from derived from resting (circulating) neutrophils
RMSD
root mean squared deviation
RMSF
root mean squared fluctuation
SD
standard deviation
Se/Pl
secretory vesicle and plasma membrane fraction
Se/Pl-MPO
secretory vesicle/plasma membrane-resident MPO
SN
maturing neutrophil with segmented nuclei
Sp granule
specific granule
Sp-MPO
specific granule-resident MPO
TMB
3,3ʹ,5,5ʹ-tetramethylbenzidine

Cited by (0)

Dr Harry C. Tjondro recently completed his PhD in Molecular Sciences from Macquarie University, Sydney, Australia. His research focuses on performing deep structural characterization of key neutrophil glycoproteins including myeloperoxidase using glycomics, glycopeptide, and glycoprotein-based mass spectrometry to decode how the unusual neutrophil glycosylation impacts neutrophil function in health and relevant diseases such as cystic fibrosis and sepsis. He can be found at Analytical Glycoimmunology Group, @HarryTjondro (Twitter).

Julian Ugonotti is a first-year PhD candidate, Department of Molecular Sciences, Macquarie University, Sydney, Australia. His research aims to utilize glycomics, glycoproteomics, and functional assays to investigate the immune-modulatory roles of protein paucimannosylation on key neutrophil glycoproteins, such as myeloperoxidase, and more broadly throughout the human innate immune system. He can be found at Analytical Glycoimmunology Group, @JUgonotti (Twitter).

Dr Rebeca Kawahara is an Early Career Fellow—Cancer Institute NSW, Department of Molecular Sciences, Macquarie University, Sydney, Australia. Her current focus is to understand how protein N-glycosylation modulates the tumor microenvironment and impacts the immune- and cancer-related anti- and proinflammatory processes in colorectal cancer. She can be found at Analytical Glycoimmunology Group and http://publicationslist.org/rebeca.kawahara.

Sayantani Chatterjee is a final-year PhD candidate, Department of Molecular Sciences, Macquarie University, Sydney, Australia. Her research focuses on performing structural characterization of mannose-containing glycoproteins from lysosomes and granular neutrophils in healthy and diseased states using glycomics and glycoproteomics to decode the extracellular roles of this modification in inflammation and cancer. She can be found at Analytical Glycoimmunology Group, @csayantani9 (Twitter).

This article contains supporting information.

These authors contributed equally to this work.