Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Defining the protein N- and O-glycosylation associated with human monocyte-to-macrophage transition (#882)

Hannes Hinneburg 1 , Morten Thaysen-Andersen 1 , Erdmann Rapp 2 3 , Alexander Pralow 3 , Nilesh Bokil 4 , Jessica Pedersen 4 , Rebeca Sakuma 1 , Falko Schirmeister 5 , Bernadette Saunders 4 6
  1. Macquarie University, NSW, Australia
  2. glyXera GmbH, Magdeburg, Germany
  3. Max Planck Institute for Dynamics of Complex Technical Systems, MPI Magdeburg, Magdeburg, Germany
  4. Faculty of Science, University of Technology, Sydney, NSW, Australia
  5. None, Berlin, Germany
  6. Centennary Institute, Sydney, NSW, Australia

Monocytes are abundant mononuclear precursors to macrophages, immune cell populations essential for our innate immune system. Protein glycosylation is recognised to impact the function of monocytes and macrophages, but the exact glycophenotypes underpinning the monocyte-to-macrophage transition remain undocumented. Herein, we use label-free PGC-LC-MS/MS-based glycomics to determine the protein N- and O-glycosylation associated with human monocyte-to-macrophage transition. Primary CD14+ monocytes from blood of three healthy donors were differentiated and polarised ex vivo to classical M1-macrophages using a conventional seven-day GM-CSF differentiation protocol with longitudinal sampling at day 0, 1, 3, 5 and 7. Accompanying confocal microscopy and proteomics were used to confirm the expected morphological changes and the down-regulation of known monocyte-specific proteins including myeloperoxidase, neutrophil elastase, and cathepsin G and altered protein pathways impacted by macrophage maturation. Glycomics surprisingly demonstrated that monocytes and macrophages display a similar glyco-phenotype comprising predominantly paucimannosidic (Man1-3GlcNAc2Fuc0-1) (22-31%) and oligomannosidic (Man5-9GlcNAc2) (29-35%) N-glycans as well as complex α2,3- and α2,6-sialylated bi- and tri-antennary N-glycans with and without core fucosylation (31-43%). Relatively large donor-specific N-glycome variations were observed possibly reflecting a considerable physiology-dependent heterogeneity of monocytes and macrophages. Only few consistent N-glycome changes were observed to correlate with the monocyte-to-macrophage transition across all donors including a decrease in core fucosylation and a slightly reduced expression of mannose-terminating N-glycans (paucimannosidic/oligomannosidic) on mature macrophages. The less heterogeneous O-glycome also largely remained unchanged over the seven-day macrophage maturation, findings that were supported by a largely unchanged N- and O-glycosylation machinery as evaluated by quantitative proteomics. This high-resolution system-wide map of the protein N- and O-glycome associated with healthy monocyte-to-macrophage transition, the most detailed to date, aids our understanding of the molecular makeup pertaining to key innate immune cells and forms an essential reference library of value to future glycoimmunological studies involving human monocytes and macrophages.