Hyper-truncated Asn355- and Asn391-glycans modulate the activity of neutrophil granule myeloperoxidase

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 remain 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.

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Hyper-Truncated Glycans Augment the Activity of Neutrophil Granule Myeloperoxidase

10.1101/2020.07.24.219956 ◽

2020 ◽

Author(s):

Harry C. Tjondro ◽

Julian Ugonotti ◽

Rebeca Kawahara ◽

Sayantani Chatterjee ◽

Ian Loke ◽

...

Keyword(s):

Mass Spectrometry ◽

Native Mass Spectrometry ◽

Oxidation Products ◽

Multifaceted Approach ◽

Functional Roles ◽

Glycosylation Sites ◽

Functional Relevance ◽

Relationship Of ◽

First Time ◽

Insight Into

AbstractMyeloperoxidase (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 remain 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 monomer. 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, structural 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.SignificanceMyeloperoxidase (MPO) is an important microbicidal glycoprotein critical for fighting pathogens. We report, for the first time, the intriguingly complex relationship between glycobiology and MPO immune function by demonstrating that uncommon and strategically positioned hyper-truncated glycans both elevate the activity and the inhibition potential of this pathogen-combating enzyme. We have used a multifaceted approach employing integrated biomolecular analytics to generate new insights into the sugar code of MPO. The findings described in this study improve our understanding of key innate immune processes and may guide future glycoengineering efforts aiming to generate therapeutically relevant recombinant MPO products with tuneable activity and inhibition potential tailored to biomedical applications involving persisting and severe pathogen infections.

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State-of-the-Art Native Mass Spectrometry and Ion Mobility Methods to Monitor Homogeneous Site-Specific Antibody-Drug Conjugates Synthesis

Pharmaceuticals ◽

10.3390/ph14060498 ◽

2021 ◽

Vol 14 (6) ◽

pp. 498

Author(s):

Evolène Deslignière ◽

Anthony Ehkirch ◽

Bastiaan L. Duivelshof ◽

Hanna Toftevall ◽

Jonathan Sjögren ◽

...

Keyword(s):

Mass Spectrometry ◽

Gas Phase ◽

Ion Mobility ◽

State Of The Art ◽

Native Mass Spectrometry ◽

Site Specific ◽

Drug Conjugates ◽

Conjugation Process ◽

Antibody Drug

Antibody-drug conjugates (ADCs) are biotherapeutics consisting of a tumor-targeting monoclonal antibody (mAb) linked covalently to a cytotoxic drug. Early generation ADCs were predominantly obtained through non-selective conjugation methods based on lysine and cysteine residues, resulting in heterogeneous populations with varying drug-to-antibody ratios (DAR). Site-specific conjugation is one of the current challenges in ADC development, allowing for controlled conjugation and production of homogeneous ADCs. We report here the characterization of a site-specific DAR2 ADC generated with the GlyCLICK three-step process, which involves glycan-based enzymatic remodeling and click chemistry, using state-of-the-art native mass spectrometry (nMS) methods. The conjugation process was monitored with size exclusion chromatography coupled to nMS (SEC-nMS), which offered a straightforward identification and quantification of all reaction products, providing a direct snapshot of the ADC homogeneity. Benefits of SEC-nMS were further demonstrated for forced degradation studies, for which fragments generated upon thermal stress were clearly identified, with no deconjugation of the drug linker observed for the T-GlyGLICK-DM1 ADC. Lastly, innovative ion mobility-based collision-induced unfolding (CIU) approaches were used to assess the gas-phase behavior of compounds along the conjugation process, highlighting an increased resistance of the mAb against gas-phase unfolding upon drug conjugation. Altogether, these state-of-the-art nMS methods represent innovative approaches to investigate drug loading and distribution of last generation ADCs, their evolution during the bioconjugation process and their impact on gas-phase stabilities. We envision nMS and CIU methods to improve the conformational characterization of next generation-empowered mAb-derived products such as engineered nanobodies, bispecific ADCs or immunocytokines.

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Expanding the structural analysis capabilities on an Orbitrap-based mass spectrometer for large macromolecular complexes

The Analyst ◽

10.1039/c7an01629h ◽

2018 ◽

Vol 143 (1) ◽

pp. 100-105 ◽

Cited By ~ 43

Author(s):

Kyle L. Fort ◽

Michiel van de Waterbeemd ◽

Dmitriy Boll ◽

Maria Reinhardt-Szyba ◽

Mikhail E. Belov ◽

...

Keyword(s):

Mass Spectrometry ◽

Structural Analysis ◽

Mass Spectrometer ◽

Biological Systems ◽

Native Mass Spectrometry ◽

Macromolecular Complexes ◽

Insight Into

Native mass spectrometry can provide insight into the structure of macromolecular biological systems.

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Native Mass Spectrometry and Surface Induced Dissociation Provide Insight into the Post-Translational Modifications of Tetrameric AQP0 Isolated from Bovine Eye Lens

Analytical Chemistry ◽

10.1021/acs.analchem.1c04322 ◽

2022 ◽

Author(s):

Sophie R. Harvey ◽

Carla O’Neale ◽

Kevin L. Schey ◽

Vicki H. Wysocki

Keyword(s):

Mass Spectrometry ◽

Native Mass Spectrometry ◽

Eye Lens ◽

Post Translational Modifications ◽

Surface Induced Dissociation ◽

Insight Into

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Mechanistic Insight into the Assembly of the HerA-NurA Helicase-Nuclease DNA End Resection Complex using Native Mass Spectrometry

Biophysical Journal ◽

10.1016/j.bpj.2017.11.2433 ◽

2018 ◽

Vol 114 (3) ◽

pp. 440a

Author(s):

Zainab Ahdash ◽

Andy M. Lau ◽

Robert Thomas Byrne ◽

Katja Lammens ◽

Paula J. Booth ◽

...

Keyword(s):

Mass Spectrometry ◽

Native Mass Spectrometry ◽

Mechanistic Insight ◽

Dna End Resection ◽

End Resection ◽

Insight Into

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A Novel Insight into the Ozone–Skin Lipid Oxidation Products Observed by Secondary Electrospray Ionization High-Resolution Mass Spectrometry

Environmental Science & Technology ◽

10.1021/acs.est.0c05100 ◽

2020 ◽

Vol 54 (21) ◽

pp. 13478-13487

Author(s):

Jiafa Zeng ◽

Majda Mekic ◽

Xin Xu ◽

Gwendal Loisel ◽

Zhen Zhou ◽

...

Keyword(s):

Mass Spectrometry ◽

High Resolution ◽

Electrospray Ionization ◽

High Resolution Mass Spectrometry ◽

Oxidation Products ◽

Skin Lipid ◽

Lipid Oxidation Products ◽

Secondary Electrospray Ionization ◽

Insight Into ◽

Resolution Mass

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Native mass spectrometry analyses of chaperonin complex TRiC/CCT reveal subunit N-terminal processing and re-association patterns

Scientific Reports ◽

10.1038/s41598-021-91086-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Miranda P. Collier ◽

Karen Betancourt Moreira ◽

Kathy H. Li ◽

Yu-Chan Chen ◽

Daniel Itzhak ◽

...

Keyword(s):

Mass Spectrometry ◽

Protein Folding ◽

Insect Cells ◽

Native Mass Spectrometry ◽

Cellular Protein ◽

Small Population ◽

Relative Stabilities ◽

Assembly Mechanism ◽

And Function ◽

Insight Into

AbstractThe eukaryotic chaperonin TRiC/CCT is a large ATP-dependent complex essential for cellular protein folding. Its subunit arrangement into two stacked eight-membered hetero-oligomeric rings is conserved from yeast to man. A recent breakthrough enables production of functional human TRiC (hTRiC) from insect cells. Here, we apply a suite of mass spectrometry techniques to characterize recombinant hTRiC. We find all subunits CCT1-8 are N-terminally processed by combinations of methionine excision and acetylation observed in native human TRiC. Dissociation by organic solvents yields primarily monomeric subunits with a small population of CCT dimers. Notably, some dimers feature non-canonical inter-subunit contacts absent in the initial hTRiC. This indicates individual CCT monomers can promiscuously re-assemble into dimers, and lack the information to assume the specific interface pairings in the holocomplex. CCT5 is consistently the most stable subunit and engages in the greatest number of non-canonical dimer pairings. These findings confirm physiologically relevant post-translational processing and function of recombinant hTRiC and offer quantitative insight into the relative stabilities of TRiC subunits and interfaces, a key step toward reconstructing its assembly mechanism. Our results also highlight the importance of assigning contacts identified by native mass spectrometry after solution dissociation as canonical or non-canonical when investigating multimeric assemblies.

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HAP40 orchestrates huntingtin structure for differential interaction with polyglutamine expanded exon 1

10.1101/2021.04.02.438217 ◽

2021 ◽

Author(s):

Rachel J. Harding ◽

Justin C. Deme ◽

Johannes F. Hevler ◽

Sem Tamara ◽

Alexander Lemak ◽

...

Keyword(s):

Mass Spectrometry ◽

Huntington's Disease ◽

Huntington’S Disease ◽

Domain Architecture ◽

Native Mass Spectrometry ◽

Polyglutamine Expansion ◽

X Ray Scattering ◽

Exon 1 ◽

Functional Relevance

AbstractHuntington’s disease results from expansion of a glutamine-coding CAG tract in the huntingtin (HTT) gene, producing an aberrantly functioning form of HTT. Both wildtype and disease-state HTT form a hetero-dimer with HAP40 of unknown functional relevance. We demonstrate in vivo that HTT and HAP40 cellular abundance are coupled. Integrating data from a 2.6 Å cryo-electron microscopy structure, cross-linking mass spectrometry, small-angle X-ray scattering, and modeling, we provide a near-atomic-level view of HTT, its molecular interaction surfaces and compacted domain architecture, orchestrated by HAP40. Native mass-spectrometry reveals a remarkably stable hetero-dimer, potentially explaining the cellular inter-dependence of HTT and HAP40. The polyglutamine tract containing N-terminal exon 1 region of HTT is dynamic, but shows greater conformational variety in the mutant than wildtype exon 1. By providing novel insight into the structural consequences of HTT polyglutamine expansion, our data provide a foundation for future functional and drug discovery studies targeting Huntington’s disease.

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