N-glycosylation of human R-spondin 1 is required for efficient secretion and stability but not for its heparin binding ability

Chiung Fang Chang, Li Sung Hsu, Chieh Yu Weng, Chih Kai Chen, Shu Ying Wang, Yi Hwa Chou, Yan Yu Liu, Zi Xiu Yuan, Wen Ying Huang, Ho Lin, Yau Hung Chen, Jen Ning Tsai

Research output: Contribution to journalArticle

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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.

Original languageEnglish (US)
Article number937
JournalInternational Journal of Molecular Sciences
Volume17
Issue number6
DOIs
StatePublished - Jun 14 2016

Fingerprint

heparins
Glycosylation
secretions
Heparin
biological evolution
Furin
endoplasmic reticulum
mutagenesis
Polysaccharides
stem cells
quality control
Cytology
inhibitors
folding
Thrombospondin 1
affinity
elimination
sulfates
Heparan Sulfate Proteoglycans
Mutagenesis

Keywords

  • N-glycosylation
  • R-spondin 1
  • Secretion
  • Stability
  • Wnt signaling

ASJC Scopus subject areas

  • Physical and Theoretical Chemistry
  • Organic Chemistry
  • Spectroscopy
  • Inorganic Chemistry
  • Catalysis
  • Molecular Biology
  • Computer Science Applications

Cite this

N-glycosylation of human R-spondin 1 is required for efficient secretion and stability but not for its heparin binding ability. / Chang, Chiung Fang; Hsu, Li Sung; Weng, Chieh Yu; Chen, Chih Kai; Wang, Shu Ying; Chou, Yi Hwa; Liu, Yan Yu; Yuan, Zi Xiu; Huang, Wen Ying; Lin, Ho; Chen, Yau Hung; Tsai, Jen Ning.

In: International Journal of Molecular Sciences, Vol. 17, No. 6, 937, 14.06.2016.

Research output: Contribution to journalArticle

Chang, CF, Hsu, LS, Weng, CY, Chen, CK, Wang, SY, Chou, YH, Liu, YY, Yuan, ZX, Huang, WY, Lin, H, Chen, YH & Tsai, JN 2016, 'N-glycosylation of human R-spondin 1 is required for efficient secretion and stability but not for its heparin binding ability', International Journal of Molecular Sciences, vol. 17, no. 6, 937. https://doi.org/10.3390/ijms17060937
Chang, Chiung Fang ; Hsu, Li Sung ; Weng, Chieh Yu ; Chen, Chih Kai ; Wang, Shu Ying ; Chou, Yi Hwa ; Liu, Yan Yu ; Yuan, Zi Xiu ; Huang, Wen Ying ; Lin, Ho ; Chen, Yau Hung ; Tsai, Jen Ning. / N-glycosylation of human R-spondin 1 is required for efficient secretion and stability but not for its heparin binding ability. In: International Journal of Molecular Sciences. 2016 ; Vol. 17, No. 6.
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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.",
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AU - Chang, Chiung Fang

AU - Hsu, Li Sung

AU - Weng, Chieh Yu

AU - Chen, Chih Kai

AU - Wang, Shu Ying

AU - Chou, Yi Hwa

AU - Liu, Yan Yu

AU - Yuan, Zi Xiu

AU - Huang, Wen Ying

AU - Lin, Ho

AU - Chen, Yau Hung

AU - Tsai, Jen Ning

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AB - 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.

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