Die Rolle von Proteinglykosylierungen und Kohlenhydratrezeptoren bei der Kommunikation von Makrophagen und Neutrophilen während einer Mycobacterium tuberculosis Infektion
Biochemie
Zellbiologie
Zusammenfassung der Projektergebnisse
Tuberculosis (TB) is one of the deadliest infectious diseases worldwide. The causing agent, Mycobacterium tuberculosis (M.tb) can invade host cells, such as macrophages, by manipulating the immune response and is able to survive within cells meant to eliminate pathogenic intruders. Surprisingly, not all infected individuals show symptoms for an active infection (5-15%). In recent years evidence points towards a crucial involvement of macrophages and neutrophils in the eventually establishing chronic inflammation that is beneficial for the long-term survival of the pathogen. Relatively little is known about the exact communication and coordination between cells of the immune system while infection. However, often it is realized through conjugated carbohydrates (glycans) on carriers such as proteins (glycoproteins) and specialized receptors on cell surfaces. The mechanisms coordinating the response during a M.tb infections are poorly characterized. Based on the known crucial importance of glycans in immune-related processes in general and their involvement in communication processes between cells, we set out to investigate these fascinating structures, particularly on macrophages during M.tb infections but also other cell types and key proteins of the innate immune system. Due to limited access to untreated M.tb-infected tissue samples from real patients, ex vivo infected primary cell specimens have become a valuable and excepted alternative for investigations. The desired macrophages can be obtained by granulocyte macrophage colonystimulating factor (GM-CSF)-treatment of monocytes of peripheral blood, subsequently used for M.tb infections. In order to obtain deep glycomic and detailed site-specific structural information of protein glycosylation in parallel with information gained by other biochemical investigation techniques, it was first necessary to improve the sensitivity of the used glycomics analytical platform. The introduction of an organic solvent stream via a Tee-piece after the column separation demonstrated that the supplement of either of the three organic modifier tested (methanol, isopropanol and acetonitrile) improved the glycomics analysis for both, N- and O-glycans released from several standard glycoproteins. The new, ultrasensitive method was subsequently used to elucidate the glycan changes happening during the GM-CSF-initiated CD14+ monocyte-to-macrophage transition with a longitudinal sampling regime at day 0, 1, 3, 5, and 7 and resulted in the most comprehensive map of N- and O-glycans so far. Mannose-terminating glycans consisting of oligomannosidic (30-35%) and the less common paucimannosidic (22-31%) N-glycan types along with highly branched α2,3-/6-sialylated bi- and tri-antennary complex-type N-glycans variably decorated with α1,6-(core) fucosylation (28-39%) were identified within the monocyte-macrophage differentiation. Glycans carrying unsubstituted core 1 and 2 structures were observed for O-glycans. Both glycomes appeared relatively stable over the 7 day transition. However, lectin flow cytometry indicated a temporal-spacial regulation of cell surface glycosylation rather than the whole glycome. The project also paved the way to further gain knowledge of particular glycome changes happening on ex vivo generated macrophages infected with M.tb after the monocyte-tomacrophage transition.
Projektbezogene Publikationen (Auswahl)
- Post-Column Make-up Flow (PCMF) Enhances the Performance of Capillary-Flow PGC-LC-MS/MS-Based Glycomics. Anal Chem (2019)
Hinneburg, H. et al.
(Siehe online unter https://doi.org/10.1021/acs.analchem.8b05720) - Protein Paucimannosylation is an Enriched N-glycosylation Signature of Human Cancers. Proteomics, (2019).2
Chatterjee, S., … Hinneburg, H.,… et al.
(Siehe online unter https://doi.org/10.1002/pmic.201900010) - (2020): High-resolution longitudinal N- and O-glycoprofiling of human monocyte-to-macrophage transition. In: Glycobiology 30 (9), S. 679–694
Hinneburg, Hannes; Pedersen, Jessica L.; Bokil, Nilesh J.; Pralow, Alexander; Schirmeister, Falko; Kawahara, Rebeca; Rapp, Erdmann; Saunders, Bernadette M.; Thaysen-Andersen, Morten
(Siehe online unter https://doi.org/10.1093/glycob/cwaa020)
