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

Ma Y, Tian S, Wang Z, Wang C, Chen X, Li W, Yang Y, He S - Mol Med Rep (2016)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry & Biology, University of South China, Hengyang, Hunan 421001, P.R. China.

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.

No MeSH data available.


Related in: MedlinePlus

Effect of different expression levels of the FXR1 gene on the concentration of GM1. (A) SH-SY5Y cells transfected with pcDNA3.1(-)-FXR1, (B) HEK293T cells transfected with pcDNA3.1(-)-FXR1, then lysed and enzyme-linked immunosorbent assay was performed. The GM1 concentration was detected by ND 2000c trace UV spectrophotometer. In SH-SY5Y cells, the GM1 concentration was not significantly increased when comparing the empty vector group with normal group, but there was a significant increase in the FXR1 gene overexpression group compared with the normal group (n=6). *P<0.05 vs. normal group. There were no significant differences in GM1 concentration in HEK293T cells among all three groups (P>0.05, n=6). GM1, monosialotetrahexosylganglioside; FXR1P, fragile X related 1.
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f4-mmr-14-02-1501: Effect of different expression levels of the FXR1 gene on the concentration of GM1. (A) SH-SY5Y cells transfected with pcDNA3.1(-)-FXR1, (B) HEK293T cells transfected with pcDNA3.1(-)-FXR1, then lysed and enzyme-linked immunosorbent assay was performed. The GM1 concentration was detected by ND 2000c trace UV spectrophotometer. In SH-SY5Y cells, the GM1 concentration was not significantly increased when comparing the empty vector group with normal group, but there was a significant increase in the FXR1 gene overexpression group compared with the normal group (n=6). *P<0.05 vs. normal group. There were no significant differences in GM1 concentration in HEK293T cells among all three groups (P>0.05, n=6). GM1, monosialotetrahexosylganglioside; FXR1P, fragile X related 1.

Mentions: The current study demonstrated that FXR1P interacts with CMAS. CMAS catalyzes the synthesis of CMP-sialic acid, which is an activated form of sialic and the raw material of ganglioside synthesis. Accordingly, CMAS activity has been demonstrated to be indirectly associated with amount of the sialic acid converted to GM1 in cells. To estimate the effect of FXR1P overexpression on CMAS activity, recombinant vector pcDNA (−) 3.1-FXR1 was constructed and then the concentration of GM1 was measured in SH-SY5Y and HEK293T cells transfected with pcDNA3.1 (−)-FXR1. ELISAs demonstrated that the concentration of GM1 was significantly increased in SH-SY5Y cells transfected with recombinant vector compared with untransfected cells (P=0.016; Table II; Fig. 4A), whereas there was no significant difference between the empty vector-transfected SH-SY5Y cells compared with untransfected cells (Table II; Fig. 4A). However, there was no significant difference in GM1 concentration among the three groups of HEK293 cells (Fig. 4B). These results demonstrated that overexpression of FXR1 increases the concentration of GM1 in SH-SY5Y cells by promoting CMAS activity, but not in HEK293T cells (Table II; Fig. 4B).


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

Ma Y, Tian S, Wang Z, Wang C, Chen X, Li W, Yang Y, He S - Mol Med Rep (2016)

Effect of different expression levels of the FXR1 gene on the concentration of GM1. (A) SH-SY5Y cells transfected with pcDNA3.1(-)-FXR1, (B) HEK293T cells transfected with pcDNA3.1(-)-FXR1, then lysed and enzyme-linked immunosorbent assay was performed. The GM1 concentration was detected by ND 2000c trace UV spectrophotometer. In SH-SY5Y cells, the GM1 concentration was not significantly increased when comparing the empty vector group with normal group, but there was a significant increase in the FXR1 gene overexpression group compared with the normal group (n=6). *P<0.05 vs. normal group. There were no significant differences in GM1 concentration in HEK293T cells among all three groups (P>0.05, n=6). GM1, monosialotetrahexosylganglioside; FXR1P, fragile X related 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4940058&req=5

f4-mmr-14-02-1501: Effect of different expression levels of the FXR1 gene on the concentration of GM1. (A) SH-SY5Y cells transfected with pcDNA3.1(-)-FXR1, (B) HEK293T cells transfected with pcDNA3.1(-)-FXR1, then lysed and enzyme-linked immunosorbent assay was performed. The GM1 concentration was detected by ND 2000c trace UV spectrophotometer. In SH-SY5Y cells, the GM1 concentration was not significantly increased when comparing the empty vector group with normal group, but there was a significant increase in the FXR1 gene overexpression group compared with the normal group (n=6). *P<0.05 vs. normal group. There were no significant differences in GM1 concentration in HEK293T cells among all three groups (P>0.05, n=6). GM1, monosialotetrahexosylganglioside; FXR1P, fragile X related 1.
Mentions: The current study demonstrated that FXR1P interacts with CMAS. CMAS catalyzes the synthesis of CMP-sialic acid, which is an activated form of sialic and the raw material of ganglioside synthesis. Accordingly, CMAS activity has been demonstrated to be indirectly associated with amount of the sialic acid converted to GM1 in cells. To estimate the effect of FXR1P overexpression on CMAS activity, recombinant vector pcDNA (−) 3.1-FXR1 was constructed and then the concentration of GM1 was measured in SH-SY5Y and HEK293T cells transfected with pcDNA3.1 (−)-FXR1. ELISAs demonstrated that the concentration of GM1 was significantly increased in SH-SY5Y cells transfected with recombinant vector compared with untransfected cells (P=0.016; Table II; Fig. 4A), whereas there was no significant difference between the empty vector-transfected SH-SY5Y cells compared with untransfected cells (Table II; Fig. 4A). However, there was no significant difference in GM1 concentration among the three groups of HEK293 cells (Fig. 4B). These results demonstrated that overexpression of FXR1 increases the concentration of GM1 in SH-SY5Y cells by promoting CMAS activity, but not in HEK293T cells (Table II; Fig. 4B).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry & Biology, University of South China, Hengyang, Hunan 421001, P.R. China.

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.

No MeSH data available.


Related in: MedlinePlus