Protein-Lipid Interactions and the Influence of Cellular Lipid Environments on Glycosylation Processes

C-mannosylation of tryptophan (W) residues of proteins is an animal-specific glycosylation process that occurs in the endoplasmic reticulum (ER). It uses dolichol-phosphate-mannose as sugar donor, which is also required for N-glycosylation and O-mannosylation. Virtually nothing is known about the C-mannosylation process in the ER and the function of C-mannosylation of individual proteins. Only recently, we identified the gene encoding the C-mannosyltransferase. As it is related to the enzyme responsible for N-glycosylation, mechanistic and functional resemblances might exist between the two processes. C-mannose is found on a specific target sequence (WxxW), often occurring as a double motif (WxxWxxW) in which all tryptophans can be C-mannosylated.

Most invertebrates have one C-mannosyltransferase gene, named dpy-19 after the dumpy phenotype of the C. elegans mutant, but mammals have four homologous enzymes. We have shown that mammals require two enzymes, DPY19L1 and DPY19L3, to fully mannosylate all three tryptophans of the WxxWxxW motif. DPY19L1 uses the first two tryptophans and DPY19L3 is responsible for the modification of the third tryptophan (Figure).  In FOR 2509, we will investigate the C-mannosylation process in the ER. Is it happening co- or post-translationally, are there cofactors involved, and how is it related to N-glycosylation and O-mannosylation? We have indications that C-mannosylation aids the folding and stability of proteins, which provides an explanation for the temperature-dependent dpy-19 phenotype in C. elegans . We will further study the function of C-mannosylation for individual proteins after biosynthesis by biophysical methods and functional assays and prove whethermultiple C-mannosesprovide a recognition motif that is similar to mannoses of N-glycans or O-mannoses.


Team

Britta Brügger (PI), has earned her PhD in Frankfurt and is interested in understanding role of lipids in regulating protein functions.

Rainer Beck has earned his PhD in Heidelberg and is specialized in biochemical reconstitution systems and supports the project in all aspects dealing with the analysis of enzyme activities.

Frauke Kikul received her training in Chemistry at Vienna university. In her PhD thesis she is investigating the interplay of lipid and cellular glycosylation reactions in CDG model systems.

Timo Sachsenheimer is a specialist in mass spectrometric analysis of lipids and supports the project with MS analysis and in establishing workflow for the analysis of glycosylation intermediates.

Alexia Herrmann (MTA) and Iris Leibrecht (CTA) are providing technical support for the project.

 

For more information on Britta Brügger’s lab visit her website.


Collaborations

We are collaborating with the P1 Bakker, P6 Schwalbe, P7 Sinning and P8 Strahl on the molecular characterization of glycosylation machineries, and with P3 Büttner, P9 Thiel and P10 Wittbrodt on analyses addressing the effects of defective glycosylation in CDG models. Reconstitution experiments are performed in close collaboration with P7 Sinning and P8 Strahl. Mass spectrometric analysis of CDG models are performed using material provided by P3 Büttner, P8 Strahl, P9 Thiel and P10 Wittbrodt. With P4 Rapp and P5 Ruppert we collaborate on mass spectrometric approaches to determine protein and lipid alterations under CDG conditions.