CMP‑N‑acetylneuraminic acid synthetase interacts with fragile X related protein 1

Ma,Yun; Tian,Shuai; Wang,Zongbao; Wang,Changbo; Chen,Xiaowei; Li,Wei; Yang,Yang; He,Shuya

doi:10.3892/mmr.2016.5438

CMP‑N‑acetylneuraminic acid synthetase interacts with fragile X related protein 1

Authors:
- Yun Ma
- Shuai Tian
- Zongbao Wang
- Changbo Wang
- Xiaowei Chen
- Wei Li
- Yang Yang
- Shuya He
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Affiliations: Department of Biochemistry & Biology, University of South China, Hengyang, Hunan 421001, P.R. China, Hunan Province Cooperative Innovation Center for Molecular Target New Drug Study, Institute of Pharmaceutical and Biological Sciences, University of South China, Hengyang, Hunan 421001, P.R. China
Published online on: June 23, 2016 https://doi.org/10.3892/mmr.2016.5438
Pages: 1501-1508
Copyright: © Ma et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Fragile X mental retardation protein (FMRP), fragile X related 1 protein (FXR1P) and FXR2P are the members of the FMR protein family. These proteins contain two KH domains and a RGG box, which are characteristic of RNA binding proteins. The absence of FMRP, causes fragile X syndrome (FXS), the leading cause of hereditary mental retardation. FXR1P is expressed throughout the body and important for normal muscle development, and its absence causes cardiac abnormality. To investigate the functions of FXR1P, a screen was performed to identify FXR1P‑interacting proteins and determine the biological effect of the interaction. The current study identified CMP‑N‑acetylneuraminic acid synthetase (CMAS) as an interacting protein using the yeast two‑hybrid system, and the interaction between FXR1P and CMAS was validated in yeast using a β‑galactosidase assay and growth studies with selective media. Furthermore, co‑immunoprecipitation was used to analyze the FXR1P/CMAS association and immunofluorescence microscopy was performed to detect expression and intracellular localization of the proteins. The results of the current study indicated that FXR1P and CMAS interact, and colocalize in the cytoplasm and the nucleus of HEK293T and HeLa cells. Accordingly, a fragile X related 1 (FXR1) gene overexpression vector was constructed to investigate the effect of FXR1 overexpression on the level of monosialotetrahexosylganglioside 1 (GM1). The results of the current study suggested that FXR1P is a tissue‑specific regulator of GM1 levels in SH‑SY5Y cells, but not in HEK293T cells. Taken together, the results initially indicate that FXR1P interacts with CMAS, and that FXR1P may enhance the activation of sialic acid via interaction with CMAS, and increase GM1 levels to affect the development of the nervous system, thus providing evidence for further research into the pathogenesis of FXS.

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1	Vincent A, Heitz D, Petit C, Kretz C, Oberlé I and Mandel JL: Abnormal pattern detected in fragile-X patients by pulsed-field gel electrophoresis. Nature. 349:624–626. 1991. View Article : Google Scholar : PubMed/NCBI
2	Verkerk AJ, Pieretti M, Sutcliffe JS, Fu YH, Kuhl DP, Pizzuti A, Reiner O, Richards S, Victoria MF, Zhang FP, et al: Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome. Cell. 65:905–914. 1991. View Article : Google Scholar : PubMed/NCBI
3	Kremer EJ, Pritchard M, Lynch M, Yu S, Holman K, Baker E, Warren ST, Schlessinger D, Sutherland GR and Richards RI: Mapping of DNA instability at the fragile X to a trinucleotide repeat sequence p(CCG)n. Science. 252:1711–1714. 1991. View Article : Google Scholar : PubMed/NCBI
4	Siomi MC, Siomi H, Sauer WH, Srinivasan S, Nussbaum RL and Dreyfuss G: FXR1, an autosomal homolog of the fragile X mental retardation gene. EMBO J. 14:2401–2408. 1995.PubMed/NCBI
5	Zhang Y, O'Connor JP, Siomi MC, Srinivasan S, Dutra A, Nussbaum RL and Dreyfuss G: The fragile X mental retardation syndrome protein interacts with novel homologs FXR1 and FXR2. EMBO J. 14:5358–5366. 1995.PubMed/NCBI
6	Ashley CT Jr, Wilkinson KD, Reines D and Warren ST: FMR1 protein: Conserved RNP family domains and selective RNA binding. Science. 262:563–566. 1993. View Article : Google Scholar : PubMed/NCBI
7	Siomi H, Siomi MC, Nussbaum RL and Dreyfuss G: The protein product of the fragile X gene, FMR1, has characteristics of an RNA-binding protein. Cell. 74:291–298. 1993. View Article : Google Scholar : PubMed/NCBI
8	Brown V, Small K, Lakkis L, Feng Y, Gunter C, Wilkinson KD and Warren ST: Purified recombinant Fmrp exhibits selective RNA binding as anintrinsic property of the fragile X mental retardation protein. J Biol Chem. 273:15521–15527. 1998. View Article : Google Scholar : PubMed/NCBI
9	Darnell JC, Jensen KB, Jin P, Brown V, Warren ST and Darnell RB: Fragile X mental retardation protein targets G quartet mRNAs important for neuronal function. Cell. 107:489–499. 2001. View Article : Google Scholar : PubMed/NCBI
10	Schaeffer C, Bardoni B, Mandel JL, Ehresmann B, Ehresmann C and Moine H: The fragile X mental retardation protein binds specifically to its mRNA via a purine quartet motif. EMBO J. 20:4803–4813. 2001. View Article : Google Scholar : PubMed/NCBI
11	Eberhart DE, Malter HE, Feng Y and Warren ST: The fragile X mental retardation protein is a ribonucleoprotein containing both nuclear localization and nuclear export signals. Hum Mol Genet. 5:1083–1091. 1996. View Article : Google Scholar : PubMed/NCBI
12	Eberhart DE and Warren ST: The molecular basis of fragile X syndrome. Cold Spring Harb Symp Quant Biol. 61:679–687. 1996. View Article : Google Scholar : PubMed/NCBI
13	Winograd C and Ceman S: Fragile X family members have important and non-overlapping functions. Biomol Concepts. 2:343–352. 2011. View Article : Google Scholar : PubMed/NCBI
14	Siomi MC, Zhang Y, Siomi H and Dreyfuss G: Specific sequences in the fragile X syndrome protein FMR1 and the FXR proteins mediate their binding to 60S ribosomal subunits and the interactions among them. Mol Cell Biol. 16:3825–3832. 1996. View Article : Google Scholar : PubMed/NCBI
15	Ceman S, Brown V and Warren ST: Isolation of an FMRP-associated messenger ribonucleoprotein particle and identification of nucleolin and the fragile X-related proteins as components of the complex. Mol Cell Biol. 19:7925–7932. 1999. View Article : Google Scholar : PubMed/NCBI
16	Tamanini F, Willemsen R, van Unen L, Bontekoe C, Galjaard H, Oostra BA and Hoogeveen AT: Differential expression of FMR1, FXR1 and FXR2 proteins in human brain and testis. Hum Mol Genet. 6:1315–1322. 1997. View Article : Google Scholar : PubMed/NCBI
17	Bakker CE, de Diego Otero Y, Bontekoe C, Raghoe P, Luteijn T, Hoogeveen AT, Oostra BA and Willemsen R: Immunocytochemical and biochemical characterization of FMRP, FXR1P, and FXR2P in the mouse. Exp Cell Res. 258:162–170. 2000. View Article : Google Scholar : PubMed/NCBI
18	Tamanini F, Van Unen L, Bakker C, Sacchi N, Galjaard H, Oostra BA and Hoogeveen AT: Oligomerization properties of fragile-X mental-retardation protein (FMRP) and the fragile-X-related proteins FXR1P and FXR2P. Biochem J. 343:517–523. 1999. View Article : Google Scholar : PubMed/NCBI
19	Kirkpatrick LL, Mcllwain KA and Nelson DL: Alternative splicing in the murine and human FXR1 genes. Genomics. 59:193–202. 1999. View Article : Google Scholar : PubMed/NCBI
20	Mientjes EJ, Willemsen R, Kirkpatrick LL, Nieuwen-huizen IM, Hoogeveen-Westerveld M, Verweij M, Reis S, Bardoni B, Hoogeveen AT, Oostra BA and Nelson DL: Fxr1 knockout mice show a striated muscle phenotype: Implications for Fxr1p function in vivo. Hum Mol Genet. 13:1291–1302. 2004. View Article : Google Scholar : PubMed/NCBI
21	Khandjian EW, Bardoni B, Corbin F, Sittler A, Giroux S, Heitz D, Tremblay S, Pinset C, Montarras D, Rousseau F and Mandel J: Novel isoforms of the fragile X related protein FXR1P are expressed during myogenesis. Hum Mol Genet. 7:2121–2128. 1998. View Article : Google Scholar : PubMed/NCBI
22	Huot ME, Bisson N, Davidovic L, Mazroui R, Labelle Y, Moss T and Khandjian EW: The RNA-binding protein fragile X-related 1 regulates somite formation in Xenopus laevis. Mol Biol Cell. 16:4350–4361. 2005. View Article : Google Scholar : PubMed/NCBI
23	Van't Padje S, Chaudhry B, Severijnen LA, van der Linde HC, Mientjes EJ, Oostra BA and Willemsen R: Reduction in fragile X related 1 protein causes cardiomyopathy and muscular dystrophy in zebrafish. J Exp Biol. 212:2564–2570. 2009. View Article : Google Scholar : PubMed/NCBI
24	Davidovic L, Sacconi S, Bechara EG, Delplace S, Allegra M, Desnuelle C and Bardoni B: Alteration of expression of muscle specific isoforms of the fragile X related protein 1 (FXR1P) in facioscapulohumeral muscular dystrophy patients. J Med Genet. 45:679–685. 2008. View Article : Google Scholar : PubMed/NCBI
25	Khera TK, Dick AD and Nicholson LB: Fragile X-related protein FXR1 controls post-transcriptional suppression of lipopolysaccharide-induced tumour necrosis factor-alpha production by transforming growth factor-beta1. FEBS J. 277:2754–2765. 2010. View Article : Google Scholar : PubMed/NCBI
26	Zarnescu DC and Gregorio CC: Fragile hearts: New insights into translational control in cardiac muscle. Trends Cardiovasc Med. 23:275–281. 2013. View Article : Google Scholar : PubMed/NCBI
27	Blech-Hermoni Y and Ladd AN: RNA binding proteins in the regulation of heart development. Int J Biochem Cell Biol. 45:2467–2478. 2013. View Article : Google Scholar : PubMed/NCBI
28	Ma Y, Wang C, Li B, Qin L, Su J, Yang M and He S: Bcl-2-associated transcription factor 1 interacts with fragile X-related protein 1. Acta Biochim Biophys Sin (Shanghai). 46:119–127. 2014. View Article : Google Scholar
29	Huang X, Qiu YL, Tan JY, Qiao YF and Li M: Effect of UBE2C overexpression on the proliferation of 293Tcell line. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 27:634–636. 2011.In Chinese. PubMed/NCBI
30	Chen Y, Sittler A, Yu M, Bardoni B and Wu G: Screening of proteins interact with FMR1 by yeast two-hybrid system. Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 20:173–178. 1998.In Chinese.
31	Mockli N and Auerbach D: Quantitative beta-galactosidase assay suitable for high-throughput applications in the yeast two-hybrid system. Biotechniques. 36:872–876. 2004.PubMed/NCBI
32	Zou J, Mi L, Yu XF and Dong J: Interaction of 14–3-3σ with KCMF1 suppresses the proliferation and colony formation of human colon cancer stem cells. World J Gastroenterol. 19:3770–3780. 2013. View Article : Google Scholar : PubMed/NCBI
33	Zheng Y, Zhang LP, Jia XQ and Wang HY: Construction of a yeast two-hybrid cDNA library from the human testis. Zhonghua Nan Ke Xue. 18:310–313. 2012.In Chinese. PubMed/NCBI
34	Ma Y, He S, Yang Y, Chen Q, Xiao W, Li B, Jiao S and Fu X: Ferritin, heavy polypeptide 1 interacts with fragile X-related protein 1. Neural Regen Res. 6:790–796. 2011.
35	Duan Z, Chen J, He L, Xu H, Li Q, Hu S and Liu X: Matrix protein of Newcastle disease virus interacts with avian nucleophosmin B23.1 in HEK293T cells. Wei Sheng Wu Xue Bao. 53:730–736. 2013.In Chinese. PubMed/NCBI
36	Yuan SF, Shi CH, Yan W, Yao Q, Li NL, Wang T, Wang L and Zhang YQ: Construction and expression of eukaryotic coexpression plasmid containing human MUC1 gene and GM-CSF gene. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 23:18–20. 242007.In Chinese.
37	Abitbol M, Menini C, Delezoide AL, Rhyner T, Vekemans M and Mallet J: Nucleus basalis magnocellularis and hippo-campus are the major sites of FMR-1 expression in the human fetal brain. Nat Genet. 4:147–153. 1993. View Article : Google Scholar : PubMed/NCBI
38	Dictenberg JB, Swanger SA, Antar LN, Singer RH and Bassell GJ: A direct role for FMRP in activity-dependent dendritic mRNA transport links filopodial-spine morphogenesis to fragile X syndrome. Dev Cell. 14:926–939. 2008. View Article : Google Scholar : PubMed/NCBI
39	Bassell GJ and Warren ST: Fragile X syndrome: Loss of local mRNA regulation alters synaptic development and function. Neuron. 60:201–214. 2008. View Article : Google Scholar : PubMed/NCBI
40	Cook D, Sanchez-Carbente Mdel R, Lachance C, Radzioch D, Tremblay S, Khandjian EW, DesGroseillers L and Murai KK: Fragile X related protein 1 clusters with ribosomes and messenger RNAs at a subset of dendritic spines in the mouse hippocampus. PLoS One. 6:e261202011. View Article : Google Scholar : PubMed/NCBI
41	Agulhon C, Blanchet P, Kobetz A, Marchant D, Faucon N, Sarda P, Moraine C, Sittler A, Biancalana V, Malafosse A and Abitbol M: Expression of FMR1, FXR1, and FXR2 genes in human prenatal tissues. J Neuropathol Exp Neurol. 58:867–880. 1999. View Article : Google Scholar : PubMed/NCBI
42	Guo D, Rajamäki ML and Valkonen J: Protein-protein interactions: The yeast two-hybrid system. Methods Mol Biol. 451:421–439. 2008. View Article : Google Scholar : PubMed/NCBI
43	Monti M, Orrù S, Pagnozzi D and Pucci P: Interaction proteomic. Biosci Rep. 25:45–56. 2005. View Article : Google Scholar : PubMed/NCBI
44	Horsfall LE, Nelson A and Berry A: Identification and characterization of important residues in the catalytic mechanism of CMP-Neu5Ac synthetase from Neisseria meningitides. FEBS J. 277:2779–2790. 2010. View Article : Google Scholar : PubMed/NCBI
45	Qasba PK, Ramakrishnan B and Boeggeman E: Substrate-induced conformational changes in glycosyltransferases. Trends Biochem Sci. 30:53–62. 2005. View Article : Google Scholar : PubMed/NCBI
46	Samuels NM, Gibson BW and Miller SM: Investigation of the kinetic mechanism of cytidine 5′-monophosphate N-acetylneuraminic acid synthetase from Haemophilus ducreyi with new insights on rate-limiting steps from product inhibition analysis. Biochemistry. 38:6195–6203. 1999. View Article : Google Scholar : PubMed/NCBI
47	Sánchez-Felipe L, Villar E and Muñoz-Barroso I: α2-3- and α2-6-N-linked sialic acids allow efficient interaction of Newcastle Disease Virus with target cells. Glycoconj J. 29:539–549. 2012. View Article : Google Scholar
48	Gurnida DA, Rowan AM, Idjradinata P, Muchtadi D and Sekarwana N: Association of complex lipids containing gangli-osides with cognitive development of 6-month-old infants. Early Hum Dev. 88:595–601. 2012. View Article : Google Scholar : PubMed/NCBI
49	Hirano-Sakamaki W, Sugiyama E, Hayasaka T, Ravid R, Setou M and Taki T: Alzheimer's disease is associated with disordered localization of ganglioside GM1 molecular species in the human dentate gyrus. FEBS Lett. 589:3611–3616. 2015. View Article : Google Scholar : PubMed/NCBI
50	Sommer BR, Cohen BM, Satlin A, Cole JO, Jandorf L and Dorsey F: Changes in tardive dyskinesia symptoms in elderly patients treated with ganglioside GM1 or placebo. J Geriatr Psychiatry Neurol. 7:234–237. 1994. View Article : Google Scholar : PubMed/NCBI
51	Ma Y, Qin L, Don X, Li B, Wang C, Xu C, Wang S and He S: Biological effect of the interaction between mental retardation related protein (FXR1P) and CMAS. Prog Biochem Biophys. 40:1124–1131. 2013.