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

Tsuchiya,Miyu; Niwa,Yuki; Simizu,Siro

doi:10.3892/ol.2016.4425

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

Authors:
- Miyu Tsuchiya
- Yuki Niwa
- Siro Simizu
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Affiliations: Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Yokohama 223‑8522, Japan
Published online on: April 7, 2016 https://doi.org/10.3892/ol.2016.4425
Pages: 3279-3286
Copyright: © Tsuchiya et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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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.

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