C‑mannosylation of R‑spondin2 activates Wnt/β‑catenin signaling and migration activity in human tumor cells

  • Authors:
    • Hayato Mizuta
    • Kenta Kuga
    • Takehiro Suzuki
    • Yuki Niwa
    • Naoshi Dohmae
    • Siro Simizu
  • View Affiliations

  • Published online on: April 1, 2019     https://doi.org/10.3892/ijo.2019.4767
  • Pages: 2127-2138
Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Abstract

R‑spondin2 (Rspo2), one of the four members of the R‑spondin family of proteins, has agonistic activity in the Wnt/β‑catenin signaling pathway, and it is associated with normal development, as well as disease, such as cancer. The present study focused on the C‑mannosylation of Rspo2, which is a novel and unique type of glycosylation that occurs via a C‑C linkage between the tryptophan residue and an α‑mannose. Although Rspo2 has two putative C‑mannosylation sites at residues Trp150 and Trp153, it had not been reported to date whether these sites are C‑mannosylated. Firstly, results from mass spectrometry demonstrated that Rspo2 was C‑mannosylated at the Trp150 and Trp153 residues. Notably, while this C‑mannosylation of Rspo2 resulted in increased extracellular secretion in human fibrosarcoma HT1080 cells, in other human tumor cell lines it inhibited secretion. However, C‑mannosylation had consistent effects on the activation of Wnt/β‑catenin signaling in PANC1 and MDA‑MB‑231 cells, as well as HT1080 cells. Furthermore, overexpression of wild‑type Rspo2 significantly increased the migratory ability of A549 and HT1080 cells, whereas overexpression of a C‑mannosylation‑defective mutant enhanced migration to a lesser degree. These results suggested that C‑mannosylation of Rspo2 may promote cancer progression and that the inhibition of C‑mannosylation may serve as a potential novel therapeutic approach for cancer therapy.

References

1 

Kamata T, Katsube K, Michikawa M, Yamada M, Takada S and Mizusawa H: R-spondin, a novel gene with thrombospondin type 1 domain, was expressed in the dorsal neural tube and affected in Wnts mutants. Biochim Biophys Acta. 1676:51–62. 2004. View Article : Google Scholar : PubMed/NCBI

2 

Kazanskaya O, Glinka A, del Barco Barrantes I, Stannek P, Niehrs C and Wu W: R-Spondin2 is a secreted activator of Wnt/β-catenin signaling and is required for Xenopus myogenesis. Dev Cell. 7:525–534. 2004. View Article : Google Scholar : PubMed/NCBI

3 

de Lau WB, Snel B and Clevers HC: The R-spondin protein family. Genome Biol. 13:2422012. View Article : Google Scholar : PubMed/NCBI

4 

Kim KA, Zhao J, Andarmani S, Kakitani M, Oshima T, Binnerts ME, Abo A, Tomizuka K and Funk WD: R-Spondin proteins: A novel link to β-catenin activation. Cell Cycle. 5:23–26. 2006. View Article : Google Scholar

5 

Carmon KS, Gong X, Lin Q, Thomas A and Liu Q: R-spondins function as ligands of the orphan receptors LGR4 and LGR5 to regulate Wnt/β-catenin signaling. Proc Natl Acad Sci USA. 108:11452–11457. 2011. View Article : Google Scholar

6 

de Lau W, Barker N, Low TY, Koo BK, Li VSW, Teunissen H, Kujala P, Haegebarth A, Peters PJ, van de Wetering M, et al: Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling. Nature. 476:293–297. 2011. View Article : Google Scholar : PubMed/NCBI

7 

Glinka A, Dolde C, Kirsch N, Huang YL, Kazanskaya O, Ingelfinger D, Boutros M, Cruciat CM and Niehrs C: LGR4 and LGR5 are R-spondin receptors mediating Wnt/β-catenin and Wnt/PCP signalling. EMBO Rep. 12:1055–1061. 2011. View Article : Google Scholar : PubMed/NCBI

8 

Hao HX, Xie Y, Zhang Y, Charlat O, Oster E, Avello M, Lei H, Mickanin C, Liu D, Ruffner H, et al: ZNRF3 promotes Wnt receptor turnover in an R-spondin-sensitive manner. Nature. 485:195–200. 2012. View Article : Google Scholar : PubMed/NCBI

9 

Anastas JN and Moon RT: WNT signalling pathways as therapeutic targets in cancer. Nat Rev Cancer. 13:11–26. 2013. View Article : Google Scholar

10 

Cisternas P, Henriquez JP, Brandan E and Inestrosa NC: Wnt signaling in skeletal muscle dynamics: Myogenesis, neuromuscular synapse and fibrosis. Mol Neurobiol. 49:574–589. 2014. View Article : Google Scholar

11 

van Amerongen R and Berns A: Knockout mouse models to study Wnt signal transduction. Trends Genet. 22:678–689. 2006. View Article : Google Scholar : PubMed/NCBI

12 

Kawakami Y, Rodriguez Esteban C, Raya M, Kawakami H, Martí M, Dubova I and Izpisúa Belmonte JC: Wnt/β-catenin signaling regulates vertebrate limb regeneration. Genes Dev. 20:3232–3237. 2006. View Article : Google Scholar : PubMed/NCBI

13 

Nusse R and Clevers H: Wnt/β-Catenin signaling, disease, and emerging therapeutic modalities. Cell. 169:985–999. 2017. View Article : Google Scholar : PubMed/NCBI

14 

Bell SM, Schreiner CM, Wert SE, Mucenski ML, Scott WJ and Whitsett JA: R-spondin 2 is required for normal laryngeal-tracheal, lung and limb morphogenesis. Development. 135:1049–1058. 2008. View Article : Google Scholar : PubMed/NCBI

15 

Aoki M, Kiyonari H, Nakamura H and Okamoto H: R-spondin2 expression in the apical ectodermal ridge is essential for outgrowth and patterning in mouse limb development. Dev Growth Differ. 50:85–95. 2008. View Article : Google Scholar

16 

Abed É, Chan TF, Delalandre A, Martel-Pelletier J, Pelletier JP and Lajeunesse D: R-spondins are newly recognized players in osteoarthritis that regulate Wnt signaling in osteoblasts. Arthritis Rheum. 63:3865–3875. 2011. View Article : Google Scholar : PubMed/NCBI

17 

Han XH, Jin YR, Seto M and Yoon JK: A WNT/β-catenin signaling activator, R-spondin, plays positive regulatory roles during skeletal myogenesis. J Biol Chem. 286:10649–10659. 2011. View Article : Google Scholar : PubMed/NCBI

18 

Wu C, Qiu S, Lu L, Zou J, Li WF, Wang O, Zhao H, Wang H, Tang J, Chen L, et al: RSPO2-LGR5 signaling has tumour-suppressive activity in colorectal cancer. Nat Commun. 5:31492014. View Article : Google Scholar : PubMed/NCBI

19 

Yin X, Yi H, Wang L, Wu W, Wu X and Yu L: R-spondin 2 promotes proliferation and migration via the Wnt/β-catenin pathway in human hepatocellular carcinoma. Oncol Lett. 14:1757–1765. 2017. View Article : Google Scholar : PubMed/NCBI

20 

Ilmer M, Boiles AR, Regel I, Yokoi K, Michalski CW, Wistuba II, Rodriguez J, Alt E and Vykoukal J: RSPO2 enhances canonical Wnt signaling to confer stemness-associated traits to susceptible pancreatic cancer cells. Cancer Res. 75:1883–1896. 2015. View Article : Google Scholar : PubMed/NCBI

21 

Krieg J, Hartmann S, Vicentini A, Gläsner W, Hess D and Hofsteenge J: Recognition signal for C-mannosylation of Trp-7 in RNase 2 consists of sequence Trp-x-x-Trp. Mol Biol Cell. 9:301–309. 1998. View Article : Google Scholar : PubMed/NCBI

22 

Julenius K: NetCGlyc 1.0: Prediction of mammalian C-mannosylation sites. Glycobiology. 17:868–876. 2007. View Article : Google Scholar : PubMed/NCBI

23 

Hofsteenge J, Müller DR, de Beer T, Löffler A, Richter WJ and Vliegenthart JFG: New type of linkage between a carbohydrate and a protein: C-glycosylation of a specific tryptophan residue in human RNase Us. Biochemistry. 33:13524–13530. 1994. View Article : Google Scholar : PubMed/NCBI

24 

Niwa Y and Simizu S: C-mannosylation: Previous studies and future research perspectives. Trends Glycosci Glycotechnol. 30:E231–E238. 2018. View Article : Google Scholar

25 

Krieg J, Gläsner W, Vicentini A, Doucey MA, Löffler A, Hess D and Hofsteenge J: C-Mannosylation of human RNase 2 is an intracellular process performed by a variety of cultured cells. J Biol Chem. 272:26687–26692. 1997. View Article : Google Scholar : PubMed/NCBI

26 

Morishita S, Suzuki T, Niwa Y, Dohmae N and Simizu S: Dpy-19 like 3-mediated C-mannosylation and expression levels of RPE-spondin in human tumor cell lines. Oncol Lett. 14:2537–2544. 2017. View Article : Google Scholar : PubMed/NCBI

27 

Otani K, Niwa Y, Suzuki T, Sato N, Sasazawa Y, Dohmae N and Simizu S: Regulation of granulocyte colony-stimulating factor receptor-mediated granulocytic differentiation by C-mannosylation. Biochem Biophys Res Commun. 498:466–472. 2018. View Article : Google Scholar : PubMed/NCBI

28 

Sasazawa Y, Sato N, Suzuki T, Dohmae N and Simizu S: C-Mannosylation of thrombopoietin receptor (c-Mpl) regulates thrombopoietin-dependent JAK-STAT signaling. Biochem Biophys Res Commun. 468:262–268. 2015. View Article : Google Scholar : PubMed/NCBI

29 

Goto Y, Niwa Y, Suzuki T, Dohmae N, Umezawa K and Simizu S: C-mannosylation of human hyaluronidase 1: Possible roles for secretion and enzymatic activity. Int J Oncol. 45:344–350. 2014. View Article : Google Scholar : PubMed/NCBI

30 

Niwa Y, Suzuki T, Dohmae N and Simizu S: Identification of DPY19L3 as the C-mannosyltransferase of R-spondin1 in human cells. Mol Biol Cell. 27:744–756. 2016. View Article : Google Scholar : PubMed/NCBI

31 

Perez-Vilar J, Randell SH and Boucher RC: C-Mannosylation of MUC5AC and MUC5B Cys subdomains. Glycobiology. 14:325–337. 2004. View Article : Google Scholar : PubMed/NCBI

32 

Ihara Y, Manabe S, Ikezaki M, Inai Y, Matsui ISL, Ohta Y, Muroi E and Ito Y: C-Mannosylated peptides derived from the thrombospondin type 1 repeat interact with Hsc70 to modulate its signaling in RAW264.7 cells. Glycobiology. 20:1298–1310. 2010. View Article : Google Scholar : PubMed/NCBI

33 

Buettner FF, Ashikov A, Tiemann B, Lehle L and Bakker H: C. elegans DPY-19 is a C-mannosyltransferase glycosylating thrombospondin repeats. Mol Cell. 50:295–302. 2013. View Article : Google Scholar : PubMed/NCBI

34 

Shcherbakova A, Tiemann B, Buettner FFR and Bakker H: Distinct C-mannosylation of netrin receptor thrombospondin type 1 repeats by mammalian DPY19L1 and DPY19L3. Proc Natl Acad Sci USA. 114:2574–2579. 2017. View Article : Google Scholar

35 

Fujiwara M, Kato S, Niwa Y, Suzuki T, Tsuchiya M, Sasazawa Y, Dohmae N and Simizu S: C-mannosylation of R-spondin3 regulates its secretion and activity of Wnt/β-catenin signaling in cells. FEBS Lett. 590:2639–2649. 2016. View Article : Google Scholar : PubMed/NCBI

36 

Dong X, Liao W, Zhang L, Tu X, Hu J, Chen T, Dai X, Xiong Y, Liang W, Ding C, et al: RSPO2 suppresses colorectal cancer metastasis by counteracting the Wnt5a/Fzd7-driven nonca-nonical Wnt pathway. Cancer Lett. 402:153–165. 2017. View Article : Google Scholar : PubMed/NCBI

37 

Tsuchiya M, Niwa Y and Simizu S: N-glycosylation of R-spondin1 at Asn137 negatively regulates its secretion and Wnt/β-catenin signaling-enhancing activity. Oncol Lett. 11:3279–3286. 2016. View Article : Google Scholar : PubMed/NCBI

38 

Miyazaki S, Sasazawa Y, Mogi T, Suzuki T, Yoshida K, Dohmae N, Takao K and Simizu S: Identification of seco-clavi-lactone B as a small-molecule actin polymerization inhibitor. FEBS Lett. 590:1163–1173. 2016. View Article : Google Scholar : PubMed/NCBI

39 

Miyazaki I, Simizu S, Ichimiya H, Kawatani M and Osada H: Robust and systematic drug screening method using chemical arrays and the protein library: Identification of novel inhibitors of carbonic anhydrase II. Biosci Biotechnol Biochem. 72:2739–2749. 2008. View Article : Google Scholar : PubMed/NCBI

40 

Kawahara R, Niwa Y and Simizu S: Integrin β1 is an essential factor in vasculogenic mimicry of human cancer cells. Cancer Sci. 109:2490–2496. 2018. View Article : Google Scholar : PubMed/NCBI

41 

Niwa Y, Suzuki T, Dohmae N and Simizu S: O-Fucosylation of CCN1 is required for its secretion. FEBS Lett. 589:3287–3293. 2015. View Article : Google Scholar : PubMed/NCBI

42 

Yasukagawa T, Niwa Y, Simizu S and Umezawa K: Suppression of cellular invasion by glybenclamide through inhibited secretion of platelet-derived growth factor in ovarian clear cell carcinoma ES-2 cells. FEBS Lett. 586:1504–1509. 2012. View Article : Google Scholar : PubMed/NCBI

43 

Simizu S, Umezawa K, Takada M, Arber N and Imoto M: Induction of hydrogen peroxide production and Bax expression by caspase-3(-like) proteases in tyrosine kinase inhibitor-induced apoptosis in human small cell lung carcinoma cells. Exp Cell Res. 238:197–203. 1998. View Article : Google Scholar : PubMed/NCBI

44 

Matsuki W, Miyazaki S, Yoshida K, Ogura A, Sasazawa Y, Takao KI and Simizu S: Synthesis and evaluation of biological activities of vibsanin A analogs. Bioorg Med Chem Lett. 27:4536–4539. 2017. View Article : Google Scholar : PubMed/NCBI

45 

Veeman MT, Slusarski DC, Kaykas A, Louie SH and Moon RT: Zebrafish prickle, a modulator of noncanonical Wnt/Fz signaling, regulates gastrulation movements. Curr Biol. 13:680–685. 2003. View Article : Google Scholar : PubMed/NCBI

46 

Willert K, Brown JD, Danenberg E, Duncan AW, Weissman IL, Reya T, Yates JR III and Nusse R: Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature. 423:448–452. 2003. View Article : Google Scholar : PubMed/NCBI

47 

Komai K, Niwa Y, Sasazawa Y and Simizu S: Pirin regulates epithelial to mesenchymal transition independently of Bcl3-Slug signaling. FEBS Lett. 589:738–743. 2015. View Article : Google Scholar : PubMed/NCBI

48 

Ishida K, Wierzba MK, Teruya T, Simizu S and Osada H: Novel heparan sulfate mimetic compounds as antitumor agents. Chem Biol. 11:367–377. 2004. View Article : Google Scholar : PubMed/NCBI

49 

Nam JS, Turcotte TJ, Smith PF, Choi S and Yoon JK: Mouse cristin/R-spondin family proteins are novel ligands for the Frizzled 8 and LRP6 receptors and activate β-catenin-dependent gene expression. J Biol Chem. 281:13247–13257. 2006. View Article : Google Scholar : PubMed/NCBI

50 

Hirschberg K, Miller CM, Ellenberg J, Presley JF, Siggia ED, Phair RD and Lippincott-Schwartz J: Kinetic analysis of secretory protein traffic and characterization of golgi to plasma membrane transport intermediates in living cells. J Cell Biol. 143:1485–1503. 1998. View Article : Google Scholar : PubMed/NCBI

51 

Komekado H, Yamamoto H, Chiba T and Kikuchi A: Glycosylation and palmitoylation of Wnt-3a are coupled to produce an active form of Wnt-3a. Genes Cells. 12:521–534. 2007. View Article : Google Scholar : PubMed/NCBI

52 

Hendee K, Wang LW, Reis LM, Rice GM, Apte SS and Semina EV: Identification and functional analysis of an ADAMTSL1 variant associated with a complex phenotype including congenital glaucoma, craniofacial, and other systemic features in a three-generation human pedigree. Hum Mutat. 38:1485–1490. 2017. View Article : Google Scholar : PubMed/NCBI

53 

Wang LW, Leonhard-Melief C, Haltiwanger RS and Apte SS: Post-translational modification of thrombospondin type-1 repeats in ADAMTS-like 1/punctin-1 by C-mannosylation of tryptophan. J Biol Chem. 284:30004–30015. 2009. View Article : Google Scholar : PubMed/NCBI

54 

Li JS, Cui L, Rock DL and Li J: Novel glycosidic linkage in Aedes aegypti chorion peroxidase: N-mannosyl tryptophan. J Biol Chem. 280:38513–38521. 2005. View Article : Google Scholar : PubMed/NCBI

55 

Brunner F, Wirtz W, Rose JKC, Darvill AG, Govers F, Scheel D and Nürnberger T: A β-glucosidase/xylosidase from the phytopathogenic oomycete, Phytophthora infestans. Phytochemistry. 59:689–696. 2002. View Article : Google Scholar : PubMed/NCBI

56 

Moremen KW, Tiemeyer M and Nairn AV: Vertebrate protein glycosylation: Diversity, synthesis and function. Nat Rev Mol Cell Biol. 13:448–462. 2012. View Article : Google Scholar : PubMed/NCBI

57 

Doucey MA, Hess D, Cacan R and Hofsteenge J: Protein C-mannosylation is enzyme-catalysed and uses dolichyl-phosphate-mannose as a precursor. Mol Biol Cell. 9:291–300. 1998. View Article : Google Scholar : PubMed/NCBI

58 

Anand M, Rush JS, Ray S, Doucey MA, Weik J, Ware FE, Hofsteenge J, Waechter CJ and Lehrman MA: Requirement of the Lec35 gene for all known classes of mono-saccharide-P-dolichol-dependent glycosyltransferase reactions in mammals. Mol Biol Cell. 12:487–501. 2001. View Article : Google Scholar : PubMed/NCBI

Related Articles

Journal Cover

June 2019
Volume 54 Issue 6

Print ISSN: 1019-6439
Online ISSN:1791-2423

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
APA
Mizuta, H., Kuga, K., Suzuki, T., Niwa, Y., Dohmae, N., & Simizu, S. (2019). C‑mannosylation of R‑spondin2 activates Wnt/β‑catenin signaling and migration activity in human tumor cells. International Journal of Oncology, 54, 2127-2138. https://doi.org/10.3892/ijo.2019.4767
MLA
Mizuta, H., Kuga, K., Suzuki, T., Niwa, Y., Dohmae, N., Simizu, S."C‑mannosylation of R‑spondin2 activates Wnt/β‑catenin signaling and migration activity in human tumor cells". International Journal of Oncology 54.6 (2019): 2127-2138.
Chicago
Mizuta, H., Kuga, K., Suzuki, T., Niwa, Y., Dohmae, N., Simizu, S."C‑mannosylation of R‑spondin2 activates Wnt/β‑catenin signaling and migration activity in human tumor cells". International Journal of Oncology 54, no. 6 (2019): 2127-2138. https://doi.org/10.3892/ijo.2019.4767