C-mannosylation of R-spondin3 regulates its secretion and activity of Wnt/β-catenin signaling in cells

R-spondin3 (Rspo3) is a secreted protein, which acts as an agonist of canonical Wnt/β-catenin signaling that plays an important role in embryonic development and homeostasis. In this study, we focused on C-mannosylation, a unique type of glycosylation, of human Rspo3. Rspo3 has two putative C-mannosylation sites at Trp¹⁵³ and Trp¹⁵⁶; however, it had been unclear whether these sites are C-mannosylated or not. We demonstrated that Rspo3 was C-mannosylated at both Trp¹⁵³ and Trp¹⁵⁶ by mass spectrometry. Using C-mannosylation-defective Rspo3 mutant-overexpressing cell lines, we found that C-mannosylation of Rspo3 promotes its secretion and activates Wnt/β-catenin signaling.