C-mannosylation supports folding and enhances stability of thrombospondin repeats

• Published in: eLife Vol. 8
• Main Authors: Shcherbakova, Aleksandra, Preller, Matthias, Taft, Manuel H, Pujols, Jordi, Ventura, Salvador, Tiemann, Birgit, Buettner, Falk Fr, Bakker, Hans
• Format: Journal Article
• Language: English
• Published: England eLife Science Publications, Ltd 23.12.2019; eLife Sciences Publications Ltd; eLife Sciences Publications, Ltd
• Subjects: Analysis; Arginine; Biochemistry and Chemical Biology; C-mannosylation; Cell Biology; Denaturation; Endoplasmic reticulum; glycosylation; Hydrogen; Hydrogen bonding; Insects; Low temperature; Mass spectrometry; Molecular dynamics; Protein denaturation; Protein folding; protein stability; Proteins; Scientific imaging; Temperature; Thrombospondin; thrombospondin type 1 repeats; Tryptophan; tryptophan-arginine ladder; C. elegans; cell biology; C-mannosylation; chemical biology; biochemistry; thrombospondin type 1 repeats; glycosylation; protein folding; D. melanogaster; protein stability; tryptophan-arginine ladder
• ISSN: 2050-084X; 2050-084X
• DOI: 10.7554/elife.52978

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.