Purpose: R-spondins (RSPOs) play a pivotal role in stem cell development by potentiating Wnt signaling. We previously studied the biogenesis of RSPOs and found that asparagine-linked glycan (N-glycan) at N137 on RSPO1 affects intracellular stability and secretion efficiency. In the present study, we focused on RSPO3 as it has multiple N-linked glycan chains. Methods: The N-glycosylation sites on RSPO3 were explored by site-directed mutagenesis and Western blot analysis. The identification of the N-glycosyltransferase(s) responsible for RSPO3 N-glycosylation was approached by specific siRNA knockdown. Results: In addition to N137, two glycosylation sites at positions N36 and N194 were identified. Elimination of glycosylation at these sites led to opposing effects on RSPO3 secretion. While abrogation of N-glycosylation at N36 impaired its secretion, loss of N-glycan at N194 enhanced its secretion. These results highlight the differential roles of N-glycan at these two positions in the secretion of RSPO3. Finally, we showed that N137 is an STT3A-dependent site and N-glycosylation at N36 and N194 is STT3B-dependent using specific siRNA knockdown. Conclusion: Based on the results, STT3A- and STT3B-associated OST complexes differentially glycosylate RSPO3 at multiple sites.
