C-Mannosylation of Thryptophan Residues - Characterization of Mammalian Enzymes and Functional and Structural Aspect of the Modification on Thrombospondin Type 1 Repeats

• Applicant: Dr. Hendrikus Hans Bakker
• Subject Area: Biochemistry
• Term: from 2014 to 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 263740516

C-Mannosylation of the WXXW motif in proteins is a unique type of glycosylation typically occurring on thrombospondin type 1 repeats and type I cytokine receptors. By the recent identification of C. elegans DPY-19 as the C-mannosyltransferase, we could show that C-mannosylation and N-glycosylation are not only functionally related, but that the C-mannosyltransferase and the oligosaccharyltransferase (the enzyme responsible for N-glycosylation) are structurally related proteins as well. Whereas only one form of the enzyme is found in C. elegans, there are four homologs of DPY-19 in mammals (DPY19L1 - 4). Most notably, mutations in DPY19L2 are the main cause of globozoospermia, a rare congenital disorder causing male infertility. The current proposal has two objectives. In the first part of the proposal the focus lays on the structural and functional consequences of C-mannosylation. For these studies, the already known target proteins of C. elegans will be used. Inspired by the fact that the C. elegans mutant dpy-19 is temperature sensitive, functional studies will be carried out that are directed at the influence of C-mannosylation on temperature dependent folding properties in the endoplasmic reticulum and on thermostability of the proteins. Another ambition is to establish methods to purify and separate mannosylated and non-mannosylated thrombospondin repeats, required for future structural studies. A second goal is to establish the activity of the four mammalian homologs of DPY-19. The putative mouse C-mannosyltransferases will be cloned and expressed in the C-mannosyltransferase negative Drosophila S2 cell line. Enzymatic activity will be determined using different synthetic peptides and by co-expression of putative acceptor proteins, representing the different types of known C-mannosylated proteins, and a sperm specific protein that is potentially mannosylated by DPL19L2.

• DFG Programme: Research Grants
• Participating Persons: Professor Dr. Falk Büttner; Professor Dr. Dietmar J. Manstein