Abstract
R-spondin 1 (Rspo1) plays an essential role in stem cell biology by potentiating Wnt signaling activity. Despite the fact that Rspo1 holds therapeutic potential for a number of diseases, its biogenesis is not fully elucidated. All Rspo proteins feature two amino-terminal furin-like repeats, which are responsible for Wnt signal potentiation, and a thrombospondin type 1 (TSR1) domain that can provide affinity towards heparan sulfate proteoglycans. Using chemical inhibitors, deglycosylase and site-directed mutagenesis, we found that human Rspo1 and Rspo3 are both N-glycosylated at N137, a site near the C-terminus of the furin repeat 2 domain, and Rspo2 is N-glycosylated at N160, a position near the N-terminus of TSR1 domain. Elimination of N-glycosylation at these sites affects their accumulation in media but have no effect on the ability towards heparin. Introduction of the N-glycosylation site to Rspo2 mutant at the position homologous to N137 in Rspo1 restored full glycosylation and rescued the accumulation defect of nonglycosylated Rspo2 mutant in media. Similar effect can be observed in the N137 Rspo1 or Rspo3 mutant engineered with Rspo2 N-glycosylation site. The results highlight the importance of N-glycosylation at these two positions in efficient folding and secretion of Rspo family. Finally, we further showed that human Rspo1 is subjected to endoplasmic reticulum (ER) quality control in N-glycan-dependent manner. While N-glycan of Rspo1 plays a role in its intracellular stability, it had little effect on secreted Rspo1. Our findings provide evidence for the critical role of N-glycosylation in the biogenesis of Rspo1.
Highlights
R-spondins belong to a family of four secreted proteins capable of potentiating Wnt signaling activity
The link between Rspos and Wnt signaling was originally identified by the co-expression of Wnt and Rspos in the dorsal neural tube during embryonic development [1] and later characterized in Xenopus, where Rspo2 was identified as an activator of canonical Wnt signaling [2]
All members of the Rspo family contain an N-terminal signal peptide (SP), two furin-repeat domains that are rich in cysteine residues (FR1 and FR2; called cysteine-rich domain, CRD), one thrombospondin type 1 domain (TSR1), and a C-terminal region enriched with positively charged amino acids (CT)
Summary
R-spondins (roof plate-specific spondins, Rspos) belong to a family of four secreted proteins capable of potentiating Wnt signaling activity. N-glycans on nascent glycoproteins play an essential role in protein secretion, as they affect protein folding, provide ligands for lectin chaperones, contribute to quality control surveillance in the ER and mediate transit and selective protein targeting throughout the secretory pathway [26,33]. Based on the amino acid sequence, human Rspo contains one potential N-glycosylation consensus sequence (sequon) at position Asn137 (N137), located at the C-terminus of the FR2. This sequon is highly conserved in most Rspo family members, including Rspo and Rspo. While N-glycan of Rspo plays a role in its intracellular stability, it had little effect on stability of secreted Rspo
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