Abstract B135: Secretion of R-spondin1 is regulated by C-mannosylation.

Abstract

Abstract Background and Objective: R-spondin (Rspo) family proteins (Rspo1-4) activate canonical Wnt/β-catenin signaling and noncanonical Wnt/PCP signalling, and are related to differentiation, tissue development, and sex determination. It is reported that Rspos bound to Wnt receptor (Frizzled8), Wnt co-receptor (LRP6), and LGR4-6, but the mechanism which Rspos activate Wnt signaling remains unclear. Rspo1, a Wnt signal agonist, contains one thrombospondin type-1 repeats (TSR-1) domain. TSR-1 domain frequently involves Trp-Xaa-Xaa-Trp/Cys motif (Xaa represents any amino acids), which is the consensus sequence of protein C-mannosylation. Although several TSR-1 containing proteins have been reported to be C-mannosylated, it is not reported whether Rspo1 is C-mannosylated or not. In this study, we investigated whether Rspo1 is C-mannosylated, and the role of C-mannosylation on Rspo1 functions. Results: We established Rspo1-overexpressing HT1080 cells, and purified recombinant Rspo1 protein from conditioned medium of the cells. Using mass spectrometric analyses, we revealed that Rspo1 is C-mannosylated at both Trp153 and Trp156. Replacement of mannosylated Trp to Ala residues inhibited secretion of Rspo1, indicating that C-mannosylation of Rspo1 is involved in the secretion. It is reported that Rspo1 synergistically activates canonical Wnt/β-catenin signaling in the presence of Wnt3a. In C-mannosylated tryptophan residues-defective Rspo1-expressing cells, Wnt3a did not activate promoter activity of TCF. Therefore, it is suggested that C-mannosylation of Rspo1 might be a new target of cancer treatment. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B135. Citation Format: Yuki Niwa, Siro Simizu. Secretion of R-spondin1 is regulated by C-mannosylation. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B135.