Biology subjects

Thaysen-Andersen, M.

Publications and source records attributed to Thaysen-Andersen, M..

5 recordsLinked to original sources

bioRxiv2020

An improved comprehensive strategy for deep and quantitative N-glycomics based on optimization of sample preparation, isotope-based data quality control and quantification, new N-glycan libraries and new algorithms

Withdrawal statement"The authors have withdrawn their manuscript because the authors need to re-organize the data and writing, meanwhile more experimental evidence from human cellular N-glycome will be added to support their viewpoints. In this preprint, only one human cell line, Acute Promyelocytic Leukemia (APL) cells, was deeply investigated to show the N-glycome landscape. To provide the shared features of human N-glycome, more human cells should be investigated. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author."

biochemistry↗

bioRxiv2020

Mapping the SARS-CoV-2 spike glycoprotein-derived peptidome presented by HLA class II on dendritic cells

Understanding and eliciting protective immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an urgent priority. To facilitate these objectives, we have profiled the repertoire of human leukocyte antigen class II (HLA-II)-bound peptides presented by HLA-DR diverse monocyte-derived dendritic cells pulsed with SARS-CoV-2 spike (S) protein. We identify 209 unique HLA-II-bound peptide sequences, many forming nested sets, which map to sites throughout S including glycosylated regions. Comparison of the glycosylation profile of the S protein to that of the HLA-II-bound S peptides revealed substantial trimming of glycan residues on the latter, likely introduced during antigen processing. Our data also highlight the receptor-binding motif in S1 as a HLA-DR-binding peptide-rich region. Results from this study have application in vaccine design, and will aid analysis of CD4+ T cell responses in infected individuals and vaccine recipients.

immunology↗

bioRxiv2020

Hyper-Truncated N-Acetyl-β-D-Glucosamine Decorations Augment the Activity and Inhibition Potential 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 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.

biochemistry↗

bioRxiv2020

Integrated glycoproteomics identifies a role of N-glycosylation and galectin-1 on myogenesis and muscle development

ABSTRACTMany cell surface and secreted proteins are modified by the covalent addition of glycans that play an important role in the development of multicellular organisms. These glycan modifications enable communication between cells and the extracellular matrix via interactions with specific glycan-binding lectins and the regulation of receptor-mediated signaling. Aberrant protein glycosylation has been associated with the development of several muscular diseases suggesting essential glycan- and lectin-mediated functions in myogenesis and muscle development but our molecular understanding of the precise glycans, catalytic enzymes and lectins involved remain only partially understood. Here, we quantified dynamic remodeling of the membrane-associated proteome during a time-course of myogenesis in cell culture. We observed wide-spread changes in the abundance of several important lectins and enzymes facilitating glycan biosynthesis. Glycomics-based quantification of released N-linked glycans confirmed remodeling of the glycome consistent with the regulation of glycosyltransferases and glycosidases responsible for their formation including a previously unknown di-galactose-to-sialic acid switch supporting a functional role of these glycoepitopes in myogenesis. Furthermore, dynamic quantitative glycoproteomic analysis with multiplexed stable isotope labelling and analysis of enriched glycopeptides with multiple fragmentation approaches identified glycoproteins modified by these regulated glycans including several integrins and growth factor receptors. Myogenesis was also associated with the regulation of several lectins most notably the up-regulation of galectin-1 (LGALS1). CRISPR/Cas9-mediated deletion of Lgals1 inhibited differentiation and myotube formation suggesting an early functional role of galectin-1 in the myogenic program. Importantly, similar changes in N-glycosylation and the up-regulation of galectin-1 during postnatal skeletal muscle development were observed in mice. Treatment of new-born mice with recombinant adeno-associated viruses to overexpress galectin-1 in the musculature resulted in enhanced muscle mass. Our data form a valuable resource to further understand the glycobiology of myogenesis and will aid the development of intervention strategies to promote healthy muscle development or regeneration.Competing Interest StatementThe authors have declared no competing interest.AbbreviationsAAV adeno-associated virusANOVAanalysis of variationCBDscarbohydrate binding domainsCDGscongenital disorders of glycosylationCRISPRclustered regularly interspaced short palindromic repeatsCIDcollisional induced dissociationDTTdithiothreitolECMextracellular matrixEThcDelectron transfer dissociation with higher collisional dissociation supplemental activationFBSfetal bovine serumFDRfalse discovery rateFAformic acidGBPsglycan-binding proteinsHCDhigher collisional dissociationHILIChydrophilic interaction liquid chromatographyMCSmultiple cloning siteMeCNacetonitrileNMJneuromuscular junctionsPCAprincipal component analysisPGCporous graphitized carbonPSMpeptide spectral matchTEABtriethylammonium bicarbonateTFAtrifluoroacetic acidTMTtandem mass tagsView Full Text

biochemistry↗

bioRxiv2020

Glycan analysis of human neutrophil granules implicates a maturation-dependent glycosylation machinery

Protein glycosylation is essential to trafficking and immune functions of human neutrophils. During granulopoeisis in the bone marrow, distinct neutrophil granules are successively formed. Distinct receptors and effector proteins, many of which are glycosylated, are targeted to each type of granule according to their time of expression, a process called targeting-by-timing. Therefore, these granules are time capsules reflecting different times of maturation that can be used to understand how glycosylation evolves during granulopoiesis. Herein, neutrophil subcellular granules were fractionated by Percoll density gradient centrifugation and N- and O-glycans present in each compartment were analyzed by liquid chromatography and tandem mass spectrometry. We found abundant paucimannosidic N-glycans and lack of O-glycans in early-formed azurophil granules (AG), whereas later-formed specific and gelatinase granules (SG and GG) contained complex N- and O-glycans with remarkably elongated N-acetyllactosamine repeats with Lewis-x and sialyl-Lewis-x epitopes. Many glycans identified are unique to neutrophils and their complexity increased progressively from AG to SG and then to GG, suggesting temporal changes in the glycosylation machinery indicative of glycosylation-by-timing during granulopoiesis. In summary, this comprehensive neutrophil granule glycome map, the first of its kind, highlights novel granule-specific glycosylation features and is a crucial first step towards a better understanding of the mechanisms regulating protein glycosylation during neutrophil granulopoiesis and a more detailed understanding of neutrophil biology and function.

biochemistry↗