{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,23]],"date-time":"2026-01-23T12:07:49Z","timestamp":1769170069143,"version":"3.49.0"},"reference-count":52,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2016,6,14]],"date-time":"2016-06-14T00:00:00Z","timestamp":1465862400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"the National Science Council, Taiwan","award":["NSC-101-2311-B-040-001"],"award-info":[{"award-number":["NSC-101-2311-B-040-001"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>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.<\/jats:p>","DOI":"10.3390\/ijms17060937","type":"journal-article","created":{"date-parts":[[2016,6,14]],"date-time":"2016-06-14T11:12:12Z","timestamp":1465902732000},"page":"937","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":12,"title":["N-Glycosylation of Human R-Spondin 1 Is Required for Efficient Secretion and Stability but Not for Its Heparin Binding Ability"],"prefix":"10.3390","volume":"17","author":[{"given":"Chiung-Fang","family":"Chang","sequence":"first","affiliation":[{"name":"Department of Life Sciences, National Chung Hsing University, Taichung 40227, Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Li-Sung","family":"Hsu","sequence":"additional","affiliation":[{"name":"Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung 40201, Taiwan"},{"name":"Clinical Laboratory, Chung Shan Medical University Hospital, Taichung 40201, Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Chieh-Yu","family":"Weng","sequence":"additional","affiliation":[{"name":"Department of Medical Laboratory and Biotechnology, Chung Shan Medical University, Taichung 40201, Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Chih-Kai","family":"Chen","sequence":"additional","affiliation":[{"name":"Department of Medical Laboratory and Biotechnology, Chung Shan Medical University, Taichung 40201, Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shu-Ying","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Medical Laboratory and Biotechnology, Chung Shan Medical University, Taichung 40201, Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yi-Hwa","family":"Chou","sequence":"additional","affiliation":[{"name":"Department of Medical Laboratory and Biotechnology, Chung Shan Medical University, Taichung 40201, Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yan-Yu","family":"Liu","sequence":"additional","affiliation":[{"name":"Department of Medical Laboratory and Biotechnology, Chung Shan Medical University, Taichung 40201, Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zi-Xiu","family":"Yuan","sequence":"additional","affiliation":[{"name":"Department of Medical Laboratory and Biotechnology, Chung Shan Medical University, Taichung 40201, Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Wen-Ying","family":"Huang","sequence":"additional","affiliation":[{"name":"Department of Medical Laboratory and Biotechnology, Chung Shan Medical University, Taichung 40201, Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7849-9137","authenticated-orcid":false,"given":"Ho","family":"Lin","sequence":"additional","affiliation":[{"name":"Department of Life Sciences, National Chung Hsing University, Taichung 40227, Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yau-Hung","family":"Chen","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Tamkang University, Tamsui, New Taipei City 25137, Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7221-2334","authenticated-orcid":false,"given":"Jen-Ning","family":"Tsai","sequence":"additional","affiliation":[{"name":"Clinical Laboratory, Chung Shan Medical University Hospital, Taichung 40201, Taiwan"},{"name":"Department of Medical Laboratory and Biotechnology, Chung Shan Medical University, Taichung 40201, Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2016,6,14]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"51","DOI":"10.1016\/j.bbaexp.2003.10.009","article-title":"R-spondin, a novel gene with thrombospondin type 1 domain, was expressed in the dorsal neural tube and affected in Wnts mutants","volume":"1676","author":"Kamata","year":"2004","journal-title":"Biochim. 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