The Escherichia coli (E. coli) strains, DH5α and TOP10 (China Center for Type Culture Collection, Wuhan, China), were cultured in LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl, pH 7.0; Oxoid; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C. The Saccharomyces cerevisiae strains, Y187 and AH109, were cultured at 30°C in yeast extract peptone dextrose (YPD) medium (2% tryptone, 1% yeast extract, and 2% glucose; Oxoid; Thermo Fisher Scientific, Inc.) or SC medium (0.67% yeast nitrogen base without amino acids, 2% glucose) supplemented with an amino acid mixture without the indicated amino acids for selection. HeLa and HEK293T cells (Academy of Military Medical Sciences, Beijing, China) were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal calf serum (Zhejiang Tianhang Biotechnology Co., Ltd., Hangzhou, China) in a humidified 5% CO₂ incubator at 37°C. SH-SY5Y cells were cultured in DMEM:nutrient mixture F12 supplemented with 10% fetal calf serum in a humidified 5% CO₂ incubator at 37°C.

For two-hybrid screening, the full open reading frame of the human FXR1 (GenBank NM_005087.3) gene was subcloned into the pGBKT7 vector (28) (Clontech Laboratories, Inc., Mountainview, CA, USA) to construct pGBKT7-FXR1 (Table I), which was used as the bait plasmid. In the co-immunoprecipitation assay, the full open reading frames of the human FXR1 gene and human CMP-N-acetyl-neuraminic acid synthetase (CMAS) gene (GenBank NM_018686.4) were subcloned into the pCMV-HA vector (28) (Clontech Laboratories, Inc.) and pCMV-Myc vector (28) (Clontech Laboratories, Inc.) to construct the recombinant vectors, pCMV-HA-FXR1 and pCMV-Myc-CMAS. In the colocalization assay, the full open reading frame of the human FXR1 gene and human CMAS gene were subcloned into the pEGFP-N1 vector (28) (Clontech Laboratories, Inc.) and pDsRed-Monomer-N1 vector (28) (Clontech Laboratories, Inc.), respectively, to construct pEGFP-N1-FXR1 and pDsRed-Monomer-N1-CMAS (Table I). Additionally, the full open reading frame of the human FXR1 gene was inserted into the pcDNA3.1(−) vector (29) (Clontech Laboratories, Inc.) to generate the recombinant vector, pcDNA3.1(−)-FXR1 (Table I), which was transfected into SH-SY5Y cells using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) and biological effect of the interaction between FXR1P and CMAS was detected.

All procedures were performed out according to the yeast two-hybrid system manufacturer's instructions (Clontech Laboratories, Inc.). The yeast strain AH109 (Clontech Laboratories, Inc.) was first transformed with the pGBKT7-FXR1 recombinant plasmid, which was used as the bait to screen the library. The Human Fetal Brain Matchmaker cDNA library (Clontech Laboratories, Inc.) cloned into the vector pGADT7 was transformed into the yeast strain Y187, which was used as prey for two-hybrid screening (30). The yeast strain AH109-pGBKT7-FXR1 was mated with Y187 pre-transformed with the cDNA library and then cultured for 20 h in YPD adenine medium supplemented with kanamycin to produce diploid zygotes. The products were plated on leucine, tryptophan and histidine-free SD medium (SD/LTH) and incubated for 3 days at 30°C. After 3 days, colonies on individual SD/LTH plates were counted and the total number of transformants was calculated. To verify putative interactions between bait protein and prey proteins from the cDNA libraries, the colonies present on SD/LTH medium were collected and assayed for β-galactosidase (β-gal) activity (31). The β-gal activity assay was conducted using a colony-lift filter assay according to the manufacturer's instructions (Clontech Laboratories, Inc.). Subsequently, the library from the positive colonies (positive growth in SD/LTH media and positive β-gal activity) were extracted and digested with Hind III using a Qiagen Plasmid Midi and Maxi kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions. Plasmid fragment sizes were analyzed by 1% agarose gel electrophoresis to segregate colonies. Plasmid DNA purified from the positive yeast colonies was used to transform E. coli TOP10. Finally, the identity of the positive clones was determined by sequencing and Basic Local Alignment Search Tool searches.

The purified bait plasmid pGBKT7-FXR1 was transformed into the AH109 strain and was then cultured on tryptophan-free SD medium (SD/T) plates for toxicity and autonomous activation assays (32). The recombinant vector pGADT7-CMAS was transformed into the yeast strain Y187 and then plated on SD medium lacking leucine (SD/L) (33) (Oxoid; Thermo Fisher Scientific, Inc.). Subsequently, AH109-pGBKT7-FXR1 was mated to Y187-pGADT7-CMAS. The transformants were cultured in yeast peptone dextrose adenine medium for 20 h and then plated on the SD/LTH. The interaction between CMAS and FXR1P was confirmed following colony formation on the plates and β-gal assays.

HEK293T cells were maintained in DMEM medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 mg/ml streptomycin. At 70% confluency, HEK293T cells were seeded into 6-well plates with basal serum and antibiotic-free DMEM. According to manufacturer's protocols, the plasmids pCMV-HA-FXR1 and pCMV-Myc-CMAS were co-transfected into HEK293 cells with Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), and pCMV-HA-FXR1 and pCMV-Myc-CMAS were transfected as controls. After 5 h, the cells were cultured with DMEM containing 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin. After 48 h, the transfected cells were collected, and then lysed with RIPA lysis buffer containing protease inhibitor. The cell lysates were harvested and mixed with 2 µg anti-hemagglutinin (HA; CW0092; Beijing ComWin Biotech Co., Ltd., Beijing, China) or anti-v-myc avian myelocytomatosis viral oncogene homolog (Myc; CW0088; Beijing ComWin Biotech Co., Ltd.) antibody and placed on a shaker for overnight at 4°C. Subsequently, protein A/G agarose beads were added and then rocked at 4°C for 4 h. The pulled-down proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride membranes. The membranes were blocked with dried skim milk for 2 h at room temperature and the blot was probed with Myc monoclonal antibody (1:1,000) overnight at 4°C. The membranes were then incubated for 2 h at 4°C with horseradish peroxidase-conjugated goat anti-mouse antibody (1:1,000; CW0102; Beijing ComWin Biotech Co., Ltd.). Bands were visualized using an ECL Western Blotting Detection System (Shanghai Majorbio Bio-Pharm Technology Co., Ltd., Shanghai, China) and imaged (34).

HEK293T and HeLa cells were maintained in DMEM medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin. At 70% confluency, HEK293T and HeLa cells were plated in laser scanning confocal petri dish with basal serum- and antibiotic-free DMEM. The plasmids pEGFP-N1-FXR1 and pDsRed-Monomer-N1-CMAS were single-transfected and co-transfected into HEK293T cells and HeLa cells using Lipofectamine 2000 (35). After 5 h, the medium was then replaced with complete DMEM containing 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin. After 48 h, the transfected cells were collected and then washed twice with phosphate-buffered saline. Subsequently, 4% paraformaldehyde was used to fix the transfected cells and the nuclei were stained with 4′,6-diamidino-2-phenylindole (Beyotime Institute of Biotechnology, Nantong, China) for 10 min. A laser confocal microscope (Carl Zeiss AG, Oberkochen, Germany) was used to observe the localization and colocalization of CMAS and FXR1P in the cells.

HEK293T and SH-SY5Y cells were maintained in basal DMEM and DMEM/F12, respectively, lacking FBS and antibiotics. HEK293T and SH-SY5Y cells were divided into normal cell, empty vector and FXR1 overexpression group cells. The empty vector group and FXR1 overexpression group cells were transfected with the empty plasmid pcDNA3.1(−) and recombinant vector pcDNA3.1 (−)-FXR1, respectively, using Lipofectamine 2000 (36). Subsequently, cells from each group were collected and lysed. Lysates were centrifuged to remove cell debris (400 × g, 5 min, 4°C), and ELISA was performed by a human anti-GM1 ELISA kit (YM-L0587; Shanghai YuanMu Biological Technology Co., Ltd., Shanghai, China) according to the manufacturer's protocol. Measurements in all groups were repeated six times.

Data are presented as the mean ± standard deviation. The association among three groups was assessed by analysis of variance using SPSS software, version 19.0 (IBM SPSS, Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

The bait plasmid pGBKT7-FXR1 was constructed with complete human FXR1 and the prey plasmids pGADT7-cDNA were constructed with the complete Human Fetal Brain Matchmaker cDNA library. The results of sequencing analysis demonstrated that the inserts were in-frame and the restriction sites were correct. Subsequently, AH109-pGBKT7-FXR1 with Y187-pGADT7-cDNA transformants were mated on SD/LTH medium and β-gal assays were performed. In total, 10 positive colonies were formed on the SD/LTH medium and exhibited positive results in the β-gal assay. Following sequencing and analysis, a colony that produced a 434-amino acid protein with high identity with human CMAS (99% identity) was identified and used in the subsequent experiments.

To validate the interaction between the bait protein and prey protein from the cDNA library, Y187-pGADT7-cDNA colonies and AH109-pGBKT7-FXR1 were mated again and cultured on SD/LTH medium plates. Prey plasmid was recovered as described previously from colonies that continued to grow after 5 days on SD/LTH medium. Subsequently, the β-gal assay demonstrated a positive result. Additionally, no β-gal activity was observed in the yeast strain transformed with only the bait plasmid (pGBKT7-FXR1).

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.

To measure the intracellular interaction between FXR1P and CMAS, two recombinant eukaryotic expression vectors, pEGFP-N1-FXR1 and pDsRed-N1-CMAS, were constructed. pEGFP-N1-FXR1 contained a gene segment of FXR1 and pDsRed-N1-CMAS contained a gene segment of CMAS. All recombinant constructs were sequence-verified. HEK293T and HeLa cells were transfected with the constructs, and the expression and subcellular distribution of these proteins were analyzed by laser confocal microscopy.

HEK293T and HeLa cells transfected with pEGFP-N1-FXR1 exhibited strong cytoplasmic fluorescence, whereas transfection with pDsRed-N1-CMAS produced fluorescence in the nucleus (Fig. 2). Thus, the fusions to the N-terminus of EGFP or DsRed did not affect the fluorescence properties of the native protein, allowing the fusion protein to be correctly localized in vivo.

To evaluate the colocalization of FXR1P and CMAS, pEGFP-N1-FXR1 and pDsRed-N1-CMAS plasmids were transiently cotransfected into HEK293T and HeLa cells (Fig. 3). Transfection with pEGFP-N1-FXR1 produced green fluorescence, predominantly in the cytoplasm, whereas pDsRed-N1-CMAS transfection predominantly produced red fluorescence in the nucleus and less in the cytoplasm. The two polypeptides (pEGFP-N1-FXR1 and pDsRed-N1-CMAS) maintained intracellular localization, with yellow fluorescence demonstrating FXR1P and CMAS colocalization in the cytoplasm and the edge of the nucleus (Fig. 3). Typically, these genes are expressed in different parts of the cell, with no fluorescence overlap, but co-overexpression leads to the formation of oligomers in the cytoplasmic and nucleus, producing yellow fluorescence.

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).