N-Glycosylation of Human R-Spondin 1 Is Required for Efficient Secretion and Stability but Not for Its Heparin Binding Ability

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.

A novel function of R-spondin1 in regulating estrogen receptor expression independent of Wnt/β-catenin signaling

“…While RSPO1 induced the wild type promoter-luciferase in a dose-dependent manner, it could not activate the reporter with CRE mutations ( TGcCAgCA ) ( Figure 6e ), rendering its specificity. In addition, we co-expressed ESR1 -luciferase with RSPO1-FL (full length), RSPO1-N137Q (a mutated form with compromising secretion) ( Chang et al, 2016 ), RSPO1 F110A/F106A (unable to bind LGR4) ( Wang et al, 2013 ), and RSPO1 R66A/Q71A (binds to LGRs but is unable to amplify Wnt signaling) ( Xie et al, 2013 ). RSPO1-FL and RSPO1 R66A/Q71A were able to activate the luciferase activities, but RSPO1-N137Q and RSPO1 F110A/F106A could not ( Figure 6f ), suggesting the secretion of Rspo1 and its association to LGR4 are critical for Esr1 transcription.…”

Syndecan-1 promotes Wnt/β-catenin signaling in multiple myeloma by presenting Wnts and R-spondins

“…All 4 R-spondins have a common TSP domain, which mediates glycosaminoglycan binding. [59][60][61] To further study the functional role of R-spondin interaction with HS, we employed a mutant R-spondin (R-spondin-ΔTSP)lacking the TSP domain and C-terminal basic amino acid-rich domain. As a control, we used R-spondin lacking the basic amino acid-rich domain only (R-spondin-ΔAA).…”

“…All 4 R-spondins contain a TSP domain that can bind to HSPGs/ heparin. 60,61,65 Deletion of the TSP domain of R-spondin resulted in a reduced capacity to promote Wnt signaling in MM. Activation of Wnt signaling by this TSP domain-deficient R-spondin mutant was markedly reduced and was no longer dependent on cell-surface HS expression.…”

Intrinsic disorder in spondins and some of their interacting partners

“…For example, it was shown that the Nglycosylation of human Rspo1 at Asn137 (which is a residue located at the C-terminus of the Fu2 domain of this protein and is predicted to be disordered, being characterized by the mean disorder score of 0.53) is required for the efficient secretion and stability of this protein, but does not play a role in interaction of Rspo1 with heparin sulfate proteoglycans. 62 A single phosphorylation site (Thr253) is located within the disordered C-tail of human Rspo1. Since this site is positioned within one of the MoRFs, it is likely that it plays a role in regulation of the disorder-based interactivity of this protein.…”

“…In fact, similar to Rspo1, human Rspo2 can undergo Nglycosylation, which is important for secretion and stability of this protein. 62 Curiously, although Nglycosylation of Rspo2 was shown to occur at Asn160, a position near the N-terminus of TSR1 domain, it did not affect the heparin binding capability of this protein, 62 a rather unexpected outcome since the TSR domain is known to be responsible for interaction with heparin sulfate proteoglycans (HSPGs). 55 Therefore, it seems that similar to the functional importance of long IDPRs on the intracellular side for mediating protein-protein interactions and/or PTMs, extracellular proteins might use their long IDPRs to carry PTM sites and sites of protein-protein interactions.…”