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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

Co-IP of FXR1P and CMAS in HEK293T cells. Cells were transfected with pCMV-HA-FXR1 and pCMV-Myc-CNS, then lysates were subjected IP using anti-HA or anti-Myc mouse monoclonal antibodies, normal mouse IgG used as a negative control. The IP components of the assay and whole cell lysate were examined by western blot analysis with anti-HA and anti-Myc antibodies. IP, immunoprecipitation; IgG, immunoglobulin G; HA, hemagglutinin tag; Myc, v-myc avian myelocytomatosis viral oncogene homolog; CMAS, CMP-N-acetylneuraminic acid synthetase; FXR1P, fragile X related 1; IB, immunoblot.
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f1-mmr-14-02-1501: Co-IP of FXR1P and CMAS in HEK293T cells. Cells were transfected with pCMV-HA-FXR1 and pCMV-Myc-CNS, then lysates were subjected IP using anti-HA or anti-Myc mouse monoclonal antibodies, normal mouse IgG used as a negative control. The IP components of the assay and whole cell lysate were examined by western blot analysis with anti-HA and anti-Myc antibodies. IP, immunoprecipitation; IgG, immunoglobulin G; HA, hemagglutinin tag; Myc, v-myc avian myelocytomatosis viral oncogene homolog; CMAS, CMP-N-acetylneuraminic acid synthetase; FXR1P, fragile X related 1; IB, immunoblot.

Mentions: To verify the direct interaction between FXR1P and CMAS, two recombinant vectors, pCMV-HA-FXR1 and pCMV-Myc-CMAS, were constructed and the results of sequencing analysis suggested that the inserts were in-frame and the restriction sites were correct. Subsequently, pCMV-Myc-CMAS was co-transfected with pCMV-HA-FXR1 into HEK293T cells, and cell extracts were immunoprecipitated using the polyclonal anti-HA antibody or anti-Myc antibody, and protein A/G agarose beads. Following precipitation with anti-HA antibody, CMAS-Myc was detected using anti-Myc antibody and FXR1P-HA using anti-HA antibody. Furthermore, following precipitation with the anti-Myc antibody, FXR1P-HA was detected by anti-HA antibody (Fig. 1) and CMAS-Myc was detected by anti-Myc antibody (Fig. 1). Additionally, a negative control was performed using IgG (Fig. 1). The results indicated that the demonstrated interaction between FXR1P and CMAS is not because of nonspecific binding with IgG.


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)

Co-IP of FXR1P and CMAS in HEK293T cells. Cells were transfected with pCMV-HA-FXR1 and pCMV-Myc-CNS, then lysates were subjected IP using anti-HA or anti-Myc mouse monoclonal antibodies, normal mouse IgG used as a negative control. The IP components of the assay and whole cell lysate were examined by western blot analysis with anti-HA and anti-Myc antibodies. IP, immunoprecipitation; IgG, immunoglobulin G; HA, hemagglutinin tag; Myc, v-myc avian myelocytomatosis viral oncogene homolog; CMAS, CMP-N-acetylneuraminic acid synthetase; FXR1P, fragile X related 1; IB, immunoblot.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4940058&req=5

f1-mmr-14-02-1501: Co-IP of FXR1P and CMAS in HEK293T cells. Cells were transfected with pCMV-HA-FXR1 and pCMV-Myc-CNS, then lysates were subjected IP using anti-HA or anti-Myc mouse monoclonal antibodies, normal mouse IgG used as a negative control. The IP components of the assay and whole cell lysate were examined by western blot analysis with anti-HA and anti-Myc antibodies. IP, immunoprecipitation; IgG, immunoglobulin G; HA, hemagglutinin tag; Myc, v-myc avian myelocytomatosis viral oncogene homolog; CMAS, CMP-N-acetylneuraminic acid synthetase; FXR1P, fragile X related 1; IB, immunoblot.
Mentions: To verify the direct interaction between FXR1P and CMAS, two recombinant vectors, pCMV-HA-FXR1 and pCMV-Myc-CMAS, were constructed and the results of sequencing analysis suggested that the inserts were in-frame and the restriction sites were correct. Subsequently, pCMV-Myc-CMAS was co-transfected with pCMV-HA-FXR1 into HEK293T cells, and cell extracts were immunoprecipitated using the polyclonal anti-HA antibody or anti-Myc antibody, and protein A/G agarose beads. Following precipitation with anti-HA antibody, CMAS-Myc was detected using anti-Myc antibody and FXR1P-HA using anti-HA antibody. Furthermore, following precipitation with the anti-Myc antibody, FXR1P-HA was detected by anti-HA antibody (Fig. 1) and CMAS-Myc was detected by anti-Myc antibody (Fig. 1). Additionally, a negative control was performed using IgG (Fig. 1). The results indicated that the demonstrated interaction between FXR1P and CMAS is not because of nonspecific binding with IgG.

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