C‑mannosylation of R‑spondin2 activates Wnt/β‑catenin signaling and migration activity in human tumor cells

R-spondin2 (Rspo2), one of the four members of the R-spondin family of proteins, has agonistic activity in the Wnt/β-catenin signaling pathway, and it is associated with normal development, as well as disease, such as cancer. The present study focused on the C-mannosylation of Rspo2, which is a novel and unique type of glycosylation that occurs via a C-C linkage between the tryptophan residue and an α-mannose. Although Rspo2 has two putative C-mannosylation sites at residues Trp¹⁵⁰ and Trp¹⁵³, it had not been reported to date whether these sites are C-mannosylated. Firstly, results from mass spectrometry demonstrated that Rspo2 was C-mannosylated at the Trp¹⁵⁰ and Trp¹⁵³ residues. Notably, while this C-mannosylation of Rspo2 resulted in increased extracellular secretion in human fibrosarcoma HT1080 cells, in other human tumor cell lines it inhibited secretion. However, C-mannosylation had consistent effects on the activation of Wnt/β-catenin signaling in PANC1 and MDA-MB-231 cells, as well as HT1080 cells. Furthermore, overexpression of wild-type Rspo2 significantly increased the migratory ability of A549 and HT1080 cells, whereas overexpression of a C-mannosylation-defective mutant enhanced migration to a lesser degree. These results suggested that C-mannosylation of Rspo2 may promote cancer progression and that the inhibition of C-mannosylation may serve as a potential novel therapeutic approach for cancer therapy.