scholarly journals Hyper-Truncated Glycans Augment the Activity of Neutrophil Granule Myeloperoxidase

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.

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

2020 ◽  
pp. jbc.RA120.016342
Author(s):  
Harry C Tjondro ◽  
Julian Ugonotti ◽  
Rebeca Kawahara ◽  
Sayantani Chatterjee ◽  
Ian Loke ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Native Mass Spectrometry ◽  
Oxidation Products ◽  
Site Specific ◽  
Functional Roles ◽  
Glycosylation Sites ◽  
Molecular Complexity ◽  
Functional Relevance ◽  
Relationship Of ◽  
Insight Into

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.

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

The Analyst ◽  
2018 ◽  
Vol 143 (1) ◽  
pp. 100-105 ◽  
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.

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

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

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

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.

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

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.

scholarly journals Native Mass Spectrometry Gives Insight into the Allosteric Binding Mechanism of M2 Pyruvate Kinase to Fructose-1,6-Bisphosphate

Biochemistry ◽  
2018 ◽  
Vol 57 (11) ◽  
pp. 1685-1689 ◽  
Author(s):  
Agni F. M. Gavriilidou ◽  
Finn P. Holding ◽  
Daniel Mayer ◽  
Joseph E. Coyle ◽  
Dmitry B. Veprintsev ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Pyruvate Kinase ◽  
Native Mass Spectrometry ◽  
Binding Mechanism ◽  
Fructose 1,6 Bisphosphate ◽  
Insight Into

scholarly journals An Update of the Virion Proteome of Kaposi Sarcoma-Associated Herpesvirus

Viruses ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 1382
Author(s):  
Ramina Nabiee ◽  
Basir Syed ◽  
Jesus Ramirez Castano ◽  
Rukhsana Lalani ◽  
Jennifer E. Totonchy
Keyword(s):  
Mass Spectrometry ◽  
Kaposi Sarcoma ◽  
Protein Composition ◽  
Viral Proteins ◽  
Novel Proteins ◽  
Flight Mass Spectrometry ◽  
Associated Proteins ◽  
Cellular Components ◽  
First Time ◽  
Insight Into

The virion proteins of Kaposi sarcoma-associated herpesvirus (KSHV) were initially characterized in 2005 in two separate studies that combined the detection of 24 viral proteins and a few cellular components via LC-MS/MS or MALDI-TOF. Despite considerable advances in the sensitivity and specificity of mass spectrometry instrumentation in recent years, leading to significantly higher yields in detections, the KSHV virion proteome has not been revisited. In this study, we have re-examined the protein composition of purified KSHV virions via ultra-high resolution Qq time-of-flight mass spectrometry (UHR-QqTOF). Our results confirm the detection of all previously reported virion proteins, in addition to 17 other viral proteins, some of which have been characterized as virion-associated using other methods, and 10 novel proteins identified as virion-associated for the first time in this study. These results add KSHV ORF9, ORF23, ORF35, ORF48, ORF58, ORF72/vCyclin, K3, K9/vIRF1, K10/vIRF4, and K10.5/vIRF3 to the list of KSHV proteins that can be incorporated into virions. The addition of these proteins to the KSHV virion proteome provides novel and important insight into early events in KSHV infection mediated by virion-associated proteins. Data are available via ProteomeXchange with identifier PXD022626.

scholarly journals A New Native Mass Spectrometry Platform Identifies Inhibitors of the HSP90 – HOP Protein-Protein Interaction

2020 ◽  
Author(s):  
Clinton Veale ◽  
Mateos-Jimenez, Maria ◽  
Michaelone Vaaltyn ◽  
Ronel Müller ◽  
Matodzi Makhubu ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Drug Discovery ◽  
Protein Interaction ◽  
Drug Target ◽  
Native Mass Spectrometry ◽  
Cancer Drug ◽  
Protein Protein Interaction ◽  
Cancer Drug Discovery ◽  
First Time

This communication discusses for the first time, the use of mass spectrometry as a platform for screening for PPI inhibitors, without protein tethering or labeling. Furthermore, in the context of cancer drug discovery, this study demonstrates the ligandability and therefore the potential druggability of HOP, whose PPI with HSP90 has been routinely discussed as a difficult to drug target of substantial potential.

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