title: Identification of DPY19L3 as the C-mannosyltransferase of R-spondin1 in human cells.
abstract: R-spondin1 (Rspo1) is a secreted protein that enhances Wnt signaling, which has crucial functions in embryonic development and several cancers. C-mannosylation is a rare type of glycosylation and might regulate secretion, protein-protein interactions, and enzymatic activity. Although human Rspo1 contains 2 predicted C-mannosylation sites, C-mannosylation of Rspo1 has not been reported, nor have its functional effects on this protein. In this study, we demonstrate by mass spectrometry that Rspo1 is C-mannosylated at W(153) and W(156). Using Lec15.2 cells, which lack dolichol-phosphate-mannose synthesis activity, and mutant Rspo1-expressing cells that replace W(153) and W(156) by alanine residues, we observed that C-mannosylation of Rspo1 is required for its secretion. Further, the enhancement of canonical Wnt signaling by Rspo1 is regulated by C-mannosylation. Recently DPY19 was reported to be a C-mannosyltransferase in Caenorhabditis elegans, but no C-mannosyltransferases have been identified in any other organism. In gain- and loss-of-function experiments, human DPY19L3 selectively modified Rspo1 at W(156) but not W(153) based on mass spectrometry. Moreover, knockdown of DPY19L3 inhibited the secretion of Rspo1. In conclusion, we identified DPY19L3 as the C-mannosyltransferase of Rspo1 at W(156) and found that DPY19L3-mediated C-mannosylation of Rspo1 at W(156) is required for its secretion.
authors.affiliation: Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Yokohama 223-8522, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Niwa
authors.affiliation: Biomolecular Characterization Unit, RIKEN Center for Sustainable Resource Science, Wako 351-0198, Japan.
authors.forename: Takehiro
authors.initials: T
authors.lastname: Suzuki
authors.affiliation: Biomolecular Characterization Unit, RIKEN Center for Sustainable Resource Science, Wako 351-0198, Japan.
authors.forename: Naoshi
authors.initials: N
authors.lastname: Dohmae
authors.affiliation: Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Yokohama 223-8522, Japan simizu@applc.keio.ac.jp.
authors.forename: Siro
authors.initials: S
authors.lastname: Simizu
mesh_terms: D055785:Gene Knockdown Techniques; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D008364:Mannosyltransferases / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D019699:Thrombospondins / Q000235:genetics / Q000378:metabolism*; D014364:Tryptophan / Q000378:metabolism; D060449:Wnt Signaling Pathway; D000098907:R-Spondins
chemical_list: D008364:Mannosyltransferases; D019699:Thrombospondins; D014364:Tryptophan; C503243:RSPO1 protein, human; D000098907:R-Spondins; C000612280:DPY19L3 protein, human