Monitoring the glycosylation network by quantitative mass spectrometry to study its functional and structural organization

• Applicant: Dr. Marcin Luzarowski, since 12/2023
• Subject Area: Biochemistry
• Term: since 2017
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 289991887

In mammals protein N-glycosylation occurs in the endoplasmic reticulum (ER) by the oligosaccharyltransferase complex, which transfers a complex oligosaccharide structure from the lipid-anchor dolichol en bloc to the N-glycosylation site of a protein. Protein O-mannosylation and protein C-mannosylation also start in the ER. These processes are mediated by the protein O-mannosyltransferase and C-mannosyltransferases complex, respectively, both using dolichol monophosphate-activated mannose (Dol-P-Man) as a substrate. For the synthesis of the dolichol oligosaccharide precursor, which consists of 14 sugar residues, a complex biosynthetic pathway is necessary, in which twelve ALG (asparagine- linked glycosylation) enzymes are involved. Textbook knowledge suggests a liner pathway, which is tightly controlled due to the importance of this posttranslational modification.ProteomicsDB is a mass spectrometry based database of human proteins, which contains normalized absolute amounts of each identified protein in a given cell line or tissue. Unexpectedly, the reported ALG levels differ by several orders of magnitude dependent on cell type or tissue.In this project we aim to build a mass spectrometry based, high-throughput method, called multi-reaction-monitoring (MRM), for the sensitive and reliable monitoring of the enzymes necessary for dolichol oligosaccharide precursor production and transfer to the N-glycosylation site. The method will also contain the transferases for O- and C-mannosylation as well as glycosylated peptides and their un-glycosylated counterparts, to monitor the effect of different ALK levels on the process.. From the data obtained we will calculate absolute amounts of each protein included in the MRM assay.Measuring the expression levels, turn over rates and changes upon inhibitor treatment, we expect to get information upon the structural organization of the different pathways and the interplay between the different types of glycosylation. Changes during the differentiation of stem cells will give insight into the way, how these pathways are adjusted to the different cell types. Analyzing fibroblast from patients, we will focus on the question how the protein levels, compared to wt fibroblasts, are affected by the functional or structural loss of a member of the ALG enzymes, namely ALG1.

• DFG Programme: Research Units
• Subproject of: FOR 2509: The concert of dolichol-based glycosylation: from molecules to disease models
• Ehemaliger Antragsteller: Dr. Thomas Ruppert, until 11/2023