N-glycosylation of R-spondin1 at Asn137 negatively regulates its secretion and Wnt/β-catenin signaling-enhancing activity

Miyu Tsuchiya, Yuki Niwa, Siro Simizu

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

N-glycosylation is a post-translational protein modification with a wide variety of functions. It has been predicted that R-spondin1 (RSPO1) is N-glycosylated, although this remains unknown. The present study identified that RSPO1 was N-glycosylated at Asn137, and that N-glycosylation of RSPO1 negatively influenced its secretion and enhancing effect on Wnt/β-catenin signaling. In vitro treatment with peptide-N-glycosidase F increased the electrophoretic mobility of RSPO1. Furthermore, treatment of wild-type (wt) RSPO1-overexpressing HT1080 cells with tunicamycin (TM), which inhibits N-glycosylation, resulted in a significant reduction in the molecular weight of RSPO1. However, TM treatment had no effect in the RSPO1 mutant whereby the Asn137 residue was replaced by Gln (N137Q). These results demonstrated for the first time that RSPO1 is N-glycosylated at Asn137. RSPO1 is a secreted protein that has Wnt/β-catenin signaling-enhancing activity and is expected to have therapeutic applications. The role of N-glycosylation in RSPO1 was evaluated by conducting comparative experiments with wt and N137Q RSPO1, which revealed that the N137Q mutant increased the secretion and Wnt/β-catenin signaling-enhancing effect of RSPO1, compared with wt RSPO1. These results suggest that N-glycosylation of RSPO1 has a negative influence on its secretion and Wnt/β-catenin signaling-enhancing effect.

Original languageEnglish
Pages (from-to)3279-3286
Number of pages8
JournalOncology Letters
Volume11
Issue number5
DOIs
Publication statusPublished - 2016 May 1

Fingerprint

Catenins
Glycosylation
Tunicamycin
Wnt Proteins
Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase
Post Translational Protein Processing
Molecular Weight

Keywords

  • Glycosylation
  • Heparin-binding protein
  • N-linked glycosylation
  • Protein secretion
  • R-spondin1
  • Wnt signaling

ASJC Scopus subject areas

  • Oncology
  • Cancer Research

Cite this

N-glycosylation of R-spondin1 at Asn137 negatively regulates its secretion and Wnt/β-catenin signaling-enhancing activity. / Tsuchiya, Miyu; Niwa, Yuki; Simizu, Siro.

In: Oncology Letters, Vol. 11, No. 5, 01.05.2016, p. 3279-3286.

Research output: Contribution to journalArticle

@article{e67a34c2b87d4de1b31603e837594a15,
title = "N-glycosylation of R-spondin1 at Asn137 negatively regulates its secretion and Wnt/β-catenin signaling-enhancing activity",
abstract = "N-glycosylation is a post-translational protein modification with a wide variety of functions. It has been predicted that R-spondin1 (RSPO1) is N-glycosylated, although this remains unknown. The present study identified that RSPO1 was N-glycosylated at Asn137, and that N-glycosylation of RSPO1 negatively influenced its secretion and enhancing effect on Wnt/β-catenin signaling. In vitro treatment with peptide-N-glycosidase F increased the electrophoretic mobility of RSPO1. Furthermore, treatment of wild-type (wt) RSPO1-overexpressing HT1080 cells with tunicamycin (TM), which inhibits N-glycosylation, resulted in a significant reduction in the molecular weight of RSPO1. However, TM treatment had no effect in the RSPO1 mutant whereby the Asn137 residue was replaced by Gln (N137Q). These results demonstrated for the first time that RSPO1 is N-glycosylated at Asn137. RSPO1 is a secreted protein that has Wnt/β-catenin signaling-enhancing activity and is expected to have therapeutic applications. The role of N-glycosylation in RSPO1 was evaluated by conducting comparative experiments with wt and N137Q RSPO1, which revealed that the N137Q mutant increased the secretion and Wnt/β-catenin signaling-enhancing effect of RSPO1, compared with wt RSPO1. These results suggest that N-glycosylation of RSPO1 has a negative influence on its secretion and Wnt/β-catenin signaling-enhancing effect.",
keywords = "Glycosylation, Heparin-binding protein, N-linked glycosylation, Protein secretion, R-spondin1, Wnt signaling",
author = "Miyu Tsuchiya and Yuki Niwa and Siro Simizu",
year = "2016",
month = "5",
day = "1",
doi = "10.3892/ol.2016.4425",
language = "English",
volume = "11",
pages = "3279--3286",
journal = "Oncology Letters",
issn = "1792-1074",
publisher = "Spandidos Publications",
number = "5",

}

TY - JOUR

T1 - N-glycosylation of R-spondin1 at Asn137 negatively regulates its secretion and Wnt/β-catenin signaling-enhancing activity

AU - Tsuchiya, Miyu

AU - Niwa, Yuki

AU - Simizu, Siro

PY - 2016/5/1

Y1 - 2016/5/1

N2 - N-glycosylation is a post-translational protein modification with a wide variety of functions. It has been predicted that R-spondin1 (RSPO1) is N-glycosylated, although this remains unknown. The present study identified that RSPO1 was N-glycosylated at Asn137, and that N-glycosylation of RSPO1 negatively influenced its secretion and enhancing effect on Wnt/β-catenin signaling. In vitro treatment with peptide-N-glycosidase F increased the electrophoretic mobility of RSPO1. Furthermore, treatment of wild-type (wt) RSPO1-overexpressing HT1080 cells with tunicamycin (TM), which inhibits N-glycosylation, resulted in a significant reduction in the molecular weight of RSPO1. However, TM treatment had no effect in the RSPO1 mutant whereby the Asn137 residue was replaced by Gln (N137Q). These results demonstrated for the first time that RSPO1 is N-glycosylated at Asn137. RSPO1 is a secreted protein that has Wnt/β-catenin signaling-enhancing activity and is expected to have therapeutic applications. The role of N-glycosylation in RSPO1 was evaluated by conducting comparative experiments with wt and N137Q RSPO1, which revealed that the N137Q mutant increased the secretion and Wnt/β-catenin signaling-enhancing effect of RSPO1, compared with wt RSPO1. These results suggest that N-glycosylation of RSPO1 has a negative influence on its secretion and Wnt/β-catenin signaling-enhancing effect.

AB - N-glycosylation is a post-translational protein modification with a wide variety of functions. It has been predicted that R-spondin1 (RSPO1) is N-glycosylated, although this remains unknown. The present study identified that RSPO1 was N-glycosylated at Asn137, and that N-glycosylation of RSPO1 negatively influenced its secretion and enhancing effect on Wnt/β-catenin signaling. In vitro treatment with peptide-N-glycosidase F increased the electrophoretic mobility of RSPO1. Furthermore, treatment of wild-type (wt) RSPO1-overexpressing HT1080 cells with tunicamycin (TM), which inhibits N-glycosylation, resulted in a significant reduction in the molecular weight of RSPO1. However, TM treatment had no effect in the RSPO1 mutant whereby the Asn137 residue was replaced by Gln (N137Q). These results demonstrated for the first time that RSPO1 is N-glycosylated at Asn137. RSPO1 is a secreted protein that has Wnt/β-catenin signaling-enhancing activity and is expected to have therapeutic applications. The role of N-glycosylation in RSPO1 was evaluated by conducting comparative experiments with wt and N137Q RSPO1, which revealed that the N137Q mutant increased the secretion and Wnt/β-catenin signaling-enhancing effect of RSPO1, compared with wt RSPO1. These results suggest that N-glycosylation of RSPO1 has a negative influence on its secretion and Wnt/β-catenin signaling-enhancing effect.

KW - Glycosylation

KW - Heparin-binding protein

KW - N-linked glycosylation

KW - Protein secretion

KW - R-spondin1

KW - Wnt signaling

UR - http://www.scopus.com/inward/record.url?scp=84961601826&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84961601826&partnerID=8YFLogxK

U2 - 10.3892/ol.2016.4425

DO - 10.3892/ol.2016.4425

M3 - Article

AN - SCOPUS:84961601826

VL - 11

SP - 3279

EP - 3286

JO - Oncology Letters

JF - Oncology Letters

SN - 1792-1074

IS - 5

ER -