Both STT3A- and STT3B-containing OST complexes are involved in the asparagine-linked glycosylation of human R-SPONDIN 3

Both STT3A- and STT3B-containing OST complexes are involved in the asparagine-linked glycosylation of human R-SPONDIN 3

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) atN137 on RSPO1 affects intracellular stability and secretion efficiency. In the present study, we focused onRSPO3 as it has multiple N-linked glycan chains.Methods: The N-glycosylation sites on RSPO3 were explored by site-directed mutagenesis and Westernblot analysis. The identification of the N-glycosyltransferase(s) responsible for RSPO3 N-glycosylationwas 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 abrogationof N-glycosylation at N36 impaired its secretion, loss of N-glycan at N194 enhanced its secretion. Theseresults 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 isSTT3B-dependent using specific siRNA knockdown.Conclusion: Based on the results, STT3A- and STT3B-associated OST complexes differentiallyglycosylate RSPO3 at multiple sites.