Abstract
Previous studies demonstrated importance of C-mannosylation for efficient protein secretion. To study its impact on protein folding and stability, we analyzed both C-mannosylated and non-C-mannosylated thrombospondin type 1 repeats (TSRs) of netrin receptor UNC-5. In absence of C-mannosylation, UNC-5 TSRs could only be obtained at low temperature and a significant proportion displayed incorrect intermolecular disulfide bridging, which was hardly observed when C-mannosylated. Glycosylated TSRs exhibited higher resistance to thermal and reductive denaturation processes, and the presence of C-mannoses promoted the oxidative folding of a reduced and denatured TSR in vitro. Molecular dynamics simulations supported the experimental studies and showed that C-mannoses can be involved in intramolecular hydrogen bonding and limit the flexibility of the TSR tryptophan-arginine ladder. We propose that in the endoplasmic reticulum folding process, C-mannoses orient the underlying tryptophan residues and facilitate the formation of the tryptophan-arginine ladder, thereby influencing the positioning of cysteines and disulfide bridging.
Highlights
Protein glycosylation is a major form of co- and post-translational modification, affecting the majority of secreted and cell-surface proteins
C-mannosylation of the TSRs was obtained by co-expression of the C. elegans DPY-19 C-mannosyltransferase
We compared non- and C-mannosylated UNC-5 TSRs and were able to show that C-mannoses added on the first two tryptophans of the WXXWXXW motif affect the dynamics of a TSR and play an important role in its folding, stability and secretion
Summary
Protein glycosylation is a major form of co- and post-translational modification, affecting the majority of secreted and cell-surface proteins. It can influence the folding of a protein as well as its physical properties, activity and ability to interact with other macromolecules, playing an important role in a great variety of cellular processes (Varki, 2017). C-mannosylation is presently one of the less well characterized glycosylation types. It takes place in the endoplasmic reticulum (ER), presumably co-translationally (Doucey et al, 1998; Krieg et al, 1998), and is performed by C-mannosyltransferase enzymes of the DPY(dumpy)-19 family (Buettner et al, 2013; Niwa et al, 2016; Shcherbakova et al, 2017).
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