Tools to Study Paucimannose N-Glycans, C-Mannose, and O-Mannose in Human Leukocytes

• Applicant: Dr. Hendrikus Hans Bakker
• Subject Area: Biochemistry ; Structural Biology ; Cell Biology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 545519849

In animals, we can distinguish three types of protein linked glycans that terminate in mannose. C-mannose and O-mannose consist of a single mannose residue directly linked to the protein, whereas N-glycans may have up to nine mannoses linked by two N-acetylglucosamine residues to the protein. Small N-glycans with up to three mannoses are called paucimannose glycans. They are formed by enzymatic degradation of larger N-glycans and typically found on lysosomal proteins. Some cell types, in particular neutrophils and monocytes, have lysosome like organelles called granules that also contain paucimannose proteins. These proteins are secreted or delivered into phagosomes to combat infections. Neutrophils are the most abundant human white blood cells, and the first line of defense against pathogens. Mannoses play various roles in host-pathogen interactions and immunology, but the exact role of the small mannose terminating glycans is not known. Currently, only a limited number of abundant proteins have been shown to carry paucimannose N-glycans (e.g. myeloperoxidase) or C-mannose (e.g. complement factors). In previous work, we have identified the mannose binding bacterial lectins BC2L-A and FimH as useful tools to study mannosylated glycans. BCL2-A has been used to enrich and identify new C- and O-mannosylated proteins, whereas FimH allowed crystallization of a paucimannosylated protein, in which electron density of the glycan moiety was clearly visible. We now wish to use these lectins to identify paucimannosylated proteins in neutrophils and monocytes and have, to this end, established a protocol that eliminate peptides with high mannose glycans, but leaves paucimannose. The paucimannosidic peptides can then, together with the O-and C-mannosylated peptides, be captured by the lectins and analyzed by mass spectrometry. Furthermore, we will apply co-crystallization of the lectins with azurocidin, which is one of the major paucimannose protein secreted by neutrophils and determine the importance of paucimannose for bacterial killing. BC2L-A and FimH will be compared for their ability to capture C-mannose, O-mannose, and paucimannose glycopeptides to develop methods that allow more distinctive isolation of these type of glycans.

• DFG Programme: Research Grants
• International Connection: France
• Partner Organisation: Agence Nationale de la Recherche / The French National Research Agency
• Cooperation Partner: Julie Bouckaert, Ph.D.