| 1. |
- Zuntini, Alexandre R., et al.
(författare)
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Phylogenomics and the rise of the angiosperms
- 2024
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Ingår i: NATURE. - 0028-0836 .- 1476-4687. ; 629, s. 843-850
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Tidskriftsartikel (refereegranskat)abstract
- Angiosperms are the cornerstone of most terrestrial ecosystems and human livelihoods(1,2). A robust understanding of angiosperm evolution is required to explain their rise to ecological dominance. So far, the angiosperm tree of life has been determined primarily by means of analyses of the plastid genome(3,4). Many studies have drawn on this foundational work, such as classification and first insights into angiosperm diversification since their Mesozoic origins(5-7). However, the limited and biased sampling of both taxa and genomes undermines confidence in the tree and its implications. Here, we build the tree of life for almost 8,000 (about 60%) angiosperm genera using a standardized set of 353 nuclear genes(8). This 15-fold increase in genus-level sampling relative to comparable nuclear studies(9) provides a critical test of earlier results and brings notable change to key groups, especially in rosids, while substantiating many previously predicted relationships. Scaling this tree to time using 200 fossils, we discovered that early angiosperm evolution was characterized by high gene tree conflict and explosive diversification, giving rise to more than 80% of extant angiosperm orders. Steady diversification ensued through the remaining Mesozoic Era until rates resurged in the Cenozoic Era, concurrent with decreasing global temperatures and tightly linked with gene tree conflict. Taken together, our extensive sampling combined with advanced phylogenomic methods shows the deep history and full complexity in the evolution of a megadiverse clade.
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| 2. |
- Miller, R. L., et al.
(författare)
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Shotgun ion mobility mass spectrometry sequencing of heparan sulfate saccharides
- 2020
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- Despite evident regulatory roles of heparan sulfate (HS) saccharides in numerous biological processes, definitive information on the bioactive sequences of these polymers is lacking, with only a handful of natural structures sequenced to date. Here, we develop a "Shotgun" Ion Mobility Mass Spectrometry Sequencing (SIMMS2) method in which intact HS saccharides are dissociated in an ion mobility mass spectrometer and collision cross section values of fragments measured. Matching of data for intact and fragment ions against known values for 36 fully defined HS saccharide structures (from di- to decasaccharides) permits unambiguous sequence determination of validated standards and unknown natural saccharides, notably including variants with 3O-sulfate groups. SIMMS2 analysis of two fibroblast growth factor-inhibiting hexasaccharides identified from a HS oligosaccharide library screen demonstrates that the approach allows elucidation of structure-activity relationships. SIMMS2 thus overcomes the bottleneck for decoding the informational content of functional HS motifs which is crucial for their future biomedical exploitation. Heparan sulfates (HS) contain functionally relevant structural motifs, but determining their monosaccharide sequence remains challenging. Here, the authors develop an ion mobility mass spectrometry-based method that allows unambiguous characterization of HS sequences and structure-activity relationships.
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| 3. |
- Antonelli, Alexandre, 1978, et al.
(författare)
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Settling a family feud: a high-level phylogenomic framework for the Gentianales based on 353 nuclear genes and partial plastomes
- 2021
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Ingår i: American Journal of Botany. - : Wiley. - 0002-9122 .- 1537-2197. ; 108:7, s. 1143-1165
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Tidskriftsartikel (refereegranskat)abstract
- Premise: Comprising five families that vastly differ in species richness—ranging from Gelsemiaceae with 13 species to the Rubiaceae with 13,775 species—members of the Gentianales are often among the most species-rich and abundant plants in tropical forests. Despite considerable phylogenetic work within particular families and genera, several alternative topologies for family-level relationships within Gentianales have been presented in previous studies. Methods: Here we present a phylogenomic analysis based on nuclear genes targeted by the Angiosperms353 probe set for approximately 150 species, representing all families and approximately 85% of the formally recognized tribes. We were able to retrieve partial plastomes from off-target reads for most taxa and infer phylogenetic trees for comparison with the nuclear-derived trees. Results: We recovered high support for over 80% of all nodes. The plastid and nuclear data are largely in agreement, except for some weakly to moderately supported relationships. We discuss the implications of our results for the order’s classification, highlighting points of increased support for previously uncertain relationships. Rubiaceae is sister to a clade comprising (Gentianaceae + Gelsemiaceae) + (Apocynaceae + Loganiaceae). Conclusions: The higher-level phylogenetic relationships within Gentianales are confidently resolved. In contrast to recent studies, our results support the division of Rubiaceae into two subfamilies: Cinchonoideae and Rubioideae. We do not formally recognize Coptosapelteae and Luculieae within any particular subfamily but treat them as incertae sedis. Our framework paves the way for further work on the phylogenetics, biogeography, morphological evolution, and macroecology of this important group of flowering plants.
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| 4. |
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| 5. |
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| 6. |
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| 7. |
- Gupta, Kallol, et al.
(författare)
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The role of interfacial lipids in stabilizing membrane protein oligomers
- 2017
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 541:7637, s. 421-424
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Tidskriftsartikel (refereegranskat)abstract
- Oligomerization of membrane proteins in response to lipid binding has a critical role in many cell-signalling pathways(1) but is often difficult to define(2) or predict(3). Here we report the development of a mass spectrometry platform to determine simultaneously the presence of interfacial lipids and oligomeric stability and to uncover how lipids act as key regulators of membrane-protein association. Evaluation of oligomeric strength for a dataset of 125 alpha-helical oligomeric membrane proteins reveals an absence of interfacial lipids in the mass spectra of 12 membrane proteins with high oligomeric stability. For the bacterial homologue of the eukaryotic biogenic transporters (LeuT(4), one of the proteins with the lowest oligomeric stability), we found a precise cohort of lipids within the dimer interface. Delipidation, mutation of lipid-binding sites or expression in cardiolipin-deficient Escherichia coli abrogated dimer formation. Molecular dynamics simulation revealed that cardiolipin acts as a bidentate ligand, bridging across subunits. Subsequently, we show that for the Vibrio splendidus sugar transporter SemiSWEET(5), another protein with low oligomeric stability, cardiolipin shifts the equilibrium from monomer to functional dimer. We hypothesized that lipids are essential for dimerization of the Na+/H+ antiporter NhaA from E. coli, which has the lowest oligomeric strength, but not for the substantially more stable homologous Thermus thermophilus protein NapA. We found that lipid binding is obligatory for dimerization of NhaA, whereas NapA has adapted to form an interface that is stable without lipids. Overall, by correlating interfacial strength with the presence of interfacial lipids, we provide a rationale for understanding the role of lipids in both transient and stable interactions within a range of a-helical membrane proteins, including G-protein-coupled receptors.
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| 8. |
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| 9. |
- Liu, Y., et al.
(författare)
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The minimum information required for a glycomics experiment (MIRAGE) project: improving the standards for reporting glycan microarray-based data
- 2017
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 0959-6658 .- 1460-2423. ; 27:4, s. 280-284
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Tidskriftsartikel (refereegranskat)abstract
- MIRAGE (Minimum Information Required for A Glycomics Experiment) is an initiative that was created by experts in the fields of glycobiology, glycoanalytics and glycoinformatics to produce guidelines for reporting results from the diverse types of experiments and analyses used in structural and functional studies of glycans in the scientific literature. As a sequel to the guidelines for sample preparation (Struwe et al. 2016, Glycobiology, 26: 907-910) and mass spectrometry data (Kolarich et al. 2013, Mol. Cell Proteomics, 12: 991-995), here we present the first version of guidelines intended to improve the standards for reporting data from glycan microarray analyses. For each of eight areas in the workflow of a glycan microarray experiment, we provide guidelines for the minimal information that should be provided in reporting results. We hope that the MIRAGE glycan microarray guidelines proposed here will gain broad acceptance by the community, and will facilitate interpretation and reproducibility of the glycan microarray results with implications in comparison of data from different laboratories and eventual deposition of glycan microarray data in international databases.
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| 10. |
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| 11. |
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| 12. |
- Söderblom, T., et al.
(författare)
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Alarming spread of vancomycin resistant enterococci in Sweden since 2007
- 2010
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Ingår i: Eurosurveillance. - 1025-496X .- 1560-7917. ; 15:29
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Tidskriftsartikel (refereegranskat)abstract
- The total number of persons infected or colonised with vancomycin-resistant enterococci mandatorily reported to the Swedish Institute for Infectious Disease Control increased dramatically during 2007 and 2008. During a period of twenty months from 1 July 2007 to 28 February 2009, a total of 760 cases were reported compared with 194 cases reported during the entire period from 2000 to 2006. This rise was mainly attributed to a wide dissemination of vancomycin resistant enterococci which started in a number of hospitals in Stockholm in the autumn of 2007 and was followed by dissemination in various healthcare facilities (hospitals and homes for the elderly) in a further two Swedish counties in 2008. The majority of the cases (97%) were acquired in Sweden and among these, healthcare-acquired E. faecium vanB dominated (n=634). The majority of these isolates had identical or closely related pulsed-field gel electrophoresis patterns indicating clonal dissemination in the affected counties. The median minimum inhibitory concentration of vancomycin was 32 mg/L (ranging from 4 to > 128 mg/L) and of teichoplanin 0.12 mg/L (ranging from 0.06 to 0.25 mg/L). Particular emphasis was placed on countermeasures such as screening, contact tracing, cleaning procedures, education in accurate use of infection control practices as well as increasing awareness of hygiene among patients and visitors. With these measures the dissemination rate decreased substantially, but new infections with the E. faecium vanB strain were still detected.
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| 13. |
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| 14. |
- York, W. S., et al.
(författare)
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MIRAGE: The minimum information required for a glycomics experiment
- 2014
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 0959-6658 .- 1460-2423. ; 24:5, s. 402-406
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Tidskriftsartikel (refereegranskat)abstract
- The MIRAGE (minimum information required for a glycomics experiment) initiative was founded in Seattle, WA, in November 2011 in order to develop guidelines for reporting the qualitative and quantitative results obtained by diverse types of glycomics analyses, including the conditions and techniques that were applied to prepare the glycans for analysis and generate the primary data along with the tools and parameters that were used to process and annotate this data. These guidelines must address a broad range of issues, as glycomics data are inherently complex and are generated using diverse methods, including mass spectrometry (MS), chromatography, glycan array-binding assays, nuclear magnetic resonance (NMR) and other rapidly developing technologies. The acceptance of these guidelines by scientists conducting research on biological systems in which glycans have a significant role will facilitate the evaluation and reproduction of glycomics experiments and data that is reported in scientific journals and uploaded to glycomics databases. As a first step, MIRAGE guidelines for glycan analysis by MS have been recently published (Kolarich D, Rapp E, Struwe WB, Haslam SM, Zaia J., et al. 2013. The minimum information required for a glycomics experiment (MIRAGE) project - Improving the standards for reporting mass spectrometry-based glycoanalytic data. Mol. Cell Proteomics. 12:991-995), allowing them to be implemented and evaluated in the context of real-world glycobiology research. In this paper, we set out the historical context, organization structure and overarching objectives of the MIRAGE initiative.
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| 15. |
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| 16. |
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| 17. |
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| 18. |
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| 19. |
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| 20. |
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| 21. |
- Garbe, Julia, et al.
(författare)
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EndoE from Enterococcus faecalis Hydrolyzes the Glycans of the Biofilm Inhibiting Protein Lactoferrin and Mediates Growth.
- 2014
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:3
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Tidskriftsartikel (refereegranskat)abstract
- Glycosidases are widespread among bacteria. The opportunistic human pathogen Enterococcus faecalis encodes several putative glycosidases but little is known about their functions. The identified endo-β-N-acetylglucosaminidase EndoE has activity on the N-linked glycans of the human immunoglobulin G (IgG). In this report we identified the human glycoprotein lactoferrin (hLF) as a new substrate for EndoE. Hydrolysis of the N-glycans from hLF was investigated using lectin blot, UHPLC and mass spectrometry, showing that EndoE releases major glycoforms from this protein. hLF was shown to inhibit biofilm formation of E. faecalis in vitro. Glycans of hLF influence the binding to E. faecalis, and EndoE-hydrolyzed hLF inhibits biofilm formation to lesser extent than intact hLF indicating that EndoE prevents the inhibition of biofilm. In addition, hLF binds to a surface-associated enolase of E. faecalis. Culture experiments showed that the activity of EndoE enables E. faecalis to use the glycans derived from lactoferrin as a carbon source indicating that they could be used as nutrients in vivo when no other preferred carbon source is available. This report adds important information about the enzymatic activity of EndoE from the commensal and opportunist E. faecalis. The activity on the human glycoprotein hLF, and the functional consequences with reduced inhibition of biofilm formation highlights both innate immunity functions of hLF and a bacterial mechanism to evade this innate immunity function. Taken together, our results underline the importance of glycans in the interplay between bacteria and the human host, with possible implications for both commensalism and opportunism.
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| 22. |
- Hayes, Jerrard M, et al.
(författare)
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Fc Gamma Receptor Glycosylation Modulates the Binding of IgG Glycoforms : A Requirement for Stable Antibody Interactions
- 2014
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 13:12, s. 5471-5485
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Tidskriftsartikel (refereegranskat)abstract
- FcγRs play a critical role in the immune response following recognition of invading particles and tumor associated antigens by circulating antibodies. In the present study we investigated the role of FcγR glycosylation in the IgG interaction and observed a stabilizing role for receptor N-glycans. We performed a complete glycan analysis of the recombinant FcγRs (FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIaPhe158/Val158, and FcγRIIIb) expressed in human cells and demonstrate that receptor glycosylation is complex and varied between receptors. We used surface plasmon resonance to establish binding patterns between rituximab and all receptors. Complex binding was observed for FcγRIa and FcγRIIIa. The IgG-FcγR interaction was further investigated using a combination of kinetic experiments and enzymatically deglycosylated FcγRIa and FcγRIIIaPhe158/Val158 receptors in an attempt to determine the underlying binding mechanism. We observed that antibody binding levels decreased for deglycosylated receptors, and at the same time, binding kinetics were altered and showed a more rapid approach to steady state, followed by an increase in the antibody dissociation rate. Binding of rituximab to deglycosylated FcγRIIIaPhe158 was now consistent with a 1:1 binding mechanism, while binding of rituximab to FcγRIIIaVal158 remained heterogeneous. Kinetic data support a complex binding mechanism, involving heterogeneity in both antibody and receptor, where fucosylated and afucosylated antibody forms compete in receptor binding and in receptor molecules where heterogeneity in receptor glycosylation plays an important role. The exact nature of receptor glycans involved in IgG binding remains unclear and determination of rate and affinity constants are challenging. Here, the use of more extended competition experiments appear promising and suggest that it may be possible to determine dissociation rate constants for high affinity afucosylated antibodies without the need to purify or express such variants. The data described provide further insight into the complexity of the IgG-FcγR interaction and the influence of FcγR glycosylation.
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| 23. |
- Hochberg, Georg K A, et al.
(författare)
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Structural principles that enable oligomeric small heat-shock protein paralogs to evolve distinct functions.
- 2018
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Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 359:6378, s. 930-935
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Tidskriftsartikel (refereegranskat)abstract
- Oligomeric proteins assemble with exceptional selectivity, even in the presence of closely related proteins, to perform their cellular roles. We show that most proteins related by gene duplication of an oligomeric ancestor have evolved to avoid hetero-oligomerization and that this correlates with their acquisition of distinct functions. We report how coassembly is avoided by two oligomeric small heat-shock protein paralogs. A hierarchy of assembly, involving intermediates that are populated only fleetingly at equilibrium, ensures selective oligomerization. Conformational flexibility at noninterfacial regions in the monomers prevents coassembly, allowing interfaces to remain largely conserved. Homomeric oligomers must overcome the entropic benefit of coassembly and, accordingly, homomeric paralogs comprise fewer subunits than homomers that have no paralogs.
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| 24. |
- Jin, Chunsheng, et al.
(författare)
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Separation of Isomeric O-Glycans by Ion Mobility and Liquid Chromatography-Mass Spectrometry
- 2019
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 91:16, s. 10604-10613
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Tidskriftsartikel (refereegranskat)abstract
- Glycosylation is one of the most important post-translational modifications essential for modulating biological functions on cellular surfaces and within cells. Glycan structures are not predictable from the genome since their biosynthesis is nontemplate driven and subject to multiple sequential and competitive glycosyltransferases/glycosidases. From a structural viewpoint, their analysis presents a particular challenge in terms of sensitivity and structural characterization. Porous graphitized carbon liquid chromatography coupled mass spectrometry (PGCLC-MS) is arguably the gold-standard for the structural characterization of glycoconjugates, especially complex mixtures typical in biological samples. This high performance is due in large part to chromatographic separation of isomers and the information delivered by collision induced fragmentation of each glycan in the mass spectrometer. More recently, ion mobility mass spectrometry (IM-MS) has emerged as an effective tool for gas-phase separation of isomeric oligosaccharides that has been demonstrated with small oligosaccharides and N-glycans. Here, we present a direct comparison of the IM- and LC-separation of O-glycans from porcine gastric and human salivary mucins. Our results identify structures, which are resolved by PGCLC and/or IM, validating the combination of the two methods. Taken together, the incorporation of both techniques into a single platform would be powerful and undoubtedly valuable for determining the full glycome of unknown samples.
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| 25. |
- Larsson, Sofie, et al.
(författare)
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A microsimulation model projecting the health care costs for resistance to antibacterial drugs in Sweden
- 2019
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Ingår i: European journal of public health. - : Oxford University Press (OUP). - 1464-360X .- 1101-1262. ; 29:3, s. 392-396
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Previous studies have shown that increasing antibacterial resistance (ABR) globally will cause extensive morbidity, deaths and escalated health care costs. METHODS: To project economic consequences of resistance to antibacterial drugs for the Swedish health care sector, we used an individual-based microsimulation model, SESIM. Health care consumption was represented as increased numbers of hospital days, outpatient visits and contact tracing for individuals getting clinical infections or becoming asymptomatic carriers. The risk of contracting a resistant bacterium was calculated using the incidence of mandatorily notifiable ABR in Sweden. RESULTS: We estimate accumulated additional health care costs attributable to notifiable ABR from 2018 until 2030 to EUR 406 million and EUR 1, 503 million until 2050. Until 2030 the largest proportion, more than EUR 247 million (EUR 958 million until 2050), was due to ESBL, followed by methicillin resistant Staphylococcus aureus, carbapenemase-producing Enterobacteriaceae, vancomycin-resistant Enterococci and penicillin non-susceptible Pneumococci which incurred costs of EUR 128 million (EUR 453 million, 2050), EUR 15 million (EUR 58 million, 2050), EUR 13 million (EUR 28 million, 2050) and EUR 2 million (EUR 6 million, 2050), respectively. CONCLUSIONS: Projections concerning the future costs of ABR can be used to guide priorities and distribution of limited health care resources. Our estimates imply that costs in Sweden will have doubled by 2030 and increased more than 4-fold by 2050 if present trends continue and infection control practices remain unchanged. Still, indirect societal costs and costs for non-notifiable resistance remain to be added. © The Author(s) 2018. Published by Oxford University Press on behalf of the European Public Health Association.
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| 26. |
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| 27. |
- Rojas-Macias, Miguel A., 1979, et al.
(författare)
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Towards a standardized bioinformatics infrastructure for N- and O-glycomics
- 2019
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- The mass spectrometry (MS)-based analysis of free polysaccharides and glycans released from proteins, lipids and proteoglycans increasingly relies on databases and software. Here, we review progress in the bioinformatics analysis of protein-released N- and O-linked glycans (N-and O-glycomics) and propose an e-infrastructure to overcome current deficits in data and experimental transparency. This workflow enables the standardized submission of MS-based glycomics information into the public repository UniCarb-DR. It implements the MIRAGE (Minimum Requirement for A Glycomics Experiment) reporting guidelines, storage of unprocessed MS data in the GlycoPOST repository and glycan structure registration using the GlyTouCan registry, thereby supporting the development and extension of a glycan structure knowledgebase.
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| 28. |
- Sjögren, Jonathan, et al.
(författare)
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EndoS2 is a unique and conserved enzyme of serotype M49 group A Streptococcus that hydrolyses N-linked glycans on IgG and α1-acid glycoprotein
- 2013
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Ingår i: Biochemical Journal. - 0264-6021. ; 455:1, s. 107-118
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Tidskriftsartikel (refereegranskat)abstract
- Many bacteria have evolved ways to interact with glycosylation functions of the immune system of their hosts. Streptococcus pyogenes [GAS (group A Streptococcus)] secretes the enzyme EndoS that cleaves glycans on human IgG and impairs the effector functions of the antibody. The ndoS gene, encoding EndoS, has, until now, been thought to be conserved throughout the serotypes. However, in the present study, we identify EndoS2, an endoglycosidase in serotype M49 GAS strains. We characterized EndoS2 and the corresponding ndoS2 gene using sequencing, bioinformatics, phylogenetic analysis, recombinant expression and LC–MS analysis of glycosidic activity. This revealed that EndoS2 is present exclusively, and highly conserved, in serotype M49 of GAS and is only 37% identical with EndoS. EndoS2 showed endo-β-N-acetylglucosaminidase activity on all N-linked glycans of IgG and on biantennary and sialylated glycans of AGP (α1-acid glycoprotein). The enzyme was found to act only on native IgG and AGP and to be specific for free biantennary glycans with or without terminal sialylation. GAS M49 expression of EndoS2 was monitored in relation to carbohydrates present in the culture medium and was linked to the presence of sucrose. We conclude that EndoS2 is a unique endoglycosidase in serotype M49 and differs from EndoS of other GAS strains by targeting both IgG and AGP. EndoS2 expands the repertoire of GAS effectors that modify key glycosylated molecules of host defence.
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| 29. |
- Skoog, G., et al.
(författare)
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Repeated nationwide point-prevalence surveys of antimicrobial use in Swedish hospitals: data for actions 2003-2010
- 2016
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Ingår i: Eurosurveillance. - : EUR CENTRE DIS PREVENTION & CONTROL. - 1560-7917 .- 1025-496X. ; 21:25, s. 13-21
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Tidskriftsartikel (refereegranskat)abstract
- This study sought to analyse antimicrobial pressure, indications for treatment, and compliance with treatment recommendations and to identify possible problem areas where inappropriate use could be improved through interventions by the network of the local Swedish Strategic Programme Against Antibiotic Resistance (Strama) groups. Five point-prevalence surveys were performed in between 49 and 72 participating hospitals from 2003 to 2010. Treatments were recorded for 19 predefined diagnosis groups and whether they were for community-acquired infection, hospital-acquired infection, or prophylaxis. Approximately one-third of inpatients were treated with antimicrobials. Compliance with guidelines for treatment of community-acquired pneumonia with narrow-spectrum penicillin was 17.0% during baseline 2003-2004, and significantly improved to 24.2% in 2010. Corresponding figures for quinolone use in uncomplicated cystitis in women were 28.5% in 2003-2004, and significantly improved, decreasing to 15.3% in 2010. The length of surgical prophylaxis improved significantly when data for a single dose and 1 day were combined, from 56.3% in 2003-2004 to 66.6% in 2010. Improved compliance was possibly the effect of active local feedback, repeated surveys, and increasing awareness of antimicrobial resistance. Strama groups are important for successful local implementation of antimicrobial stewardship programs in Sweden.
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| 31. |
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| 32. |
- Tjondro, Harry C., et al.
(författare)
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Hyper-truncated Asn355- And Asn391-glycans modulate the activity of neutrophil granule myeloperoxidase
- 2021
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 296
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 33. |
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