OBJECTIVETo compare the substrate specificity of three murine GDP fucose: beta-galactoside alpha1,2-fucosyltransferases (alpha1,2-FT).

METHODSThree members of MFUT- I, -II and -III, coding for a alpha1,2-FT, a GDP-fucose, were cloned from a cDNA of murine small intestine by reverse transcription-polymerase chain reaction. The coding regions were ligated into mammalian expression vector pcDNA 3.1 (pcDNA3.1-MFUT-I, pcDNA3.1-MFUT- II , and pcDNA3.1-MFUT- III) and were transiently transfected into COS-7 cells using a cellphect transfection kit. Then the cells were analyzed for expression and function of alpha1,2-FT and the substrate specificity of three alpha1,2-FT was compared.

RESULTSMFUT- I, -II, and -III exhibited sequence homology with human H (77%), Se (79%), and Sec1 (75%) genes, respectively. COS-7 cells transfected with pcDNA3.1-MFUT- I and pcDNA3.1-MFUT- II showed alpha1,2-FT activity, but no activity was detected in COS-7 cells transfected with pcDNA3.1- MFUT-III. MFUT- II showed alpha1,2-FT activity with both asialo-monosialoteterahexosyl ganglioside (GA1) and monosialoteterahexosyl ganglioside (GM1) as substrates to produce fucosyl GA1(FGA1) and fucosyl GM1(FGM1), respectively, but MFUT- I only showed alpha1,2-FT activity with GA1. The relative activity of MFUT- II with GA1 was 80-90-folds higher compared with MFUT- I, and the relative activity of MFUT- II with GA1 was 10-20-folds higher than that of GM1. The fucosyltransferase encoded by the MFUT- II gene showed the enzyme activity not only responsible for the synthesis of type 4-H antigens FGA1 and FGM1, but also responsible for the synthesis of type 1-H and 2-H antigens with lactotetraosylceramide and neolactotetraosylceramide as substrates.

CONCLUSIONMFUT- II is the main alpha1,2-FT in mouse and MFUT- II can product type 4-H antigen FGA1 and FGM1, but MFUT- I only synthesizes FGA1. MFUT-III has no alpha1,2-FT activity.

Animals ; Antigens, Bacterial ; biosynthesis ; COS Cells ; Cercopithecus aethiops ; Cloning, Molecular ; Fucosyltransferases ; chemistry ; genetics ; metabolism ; Gangliosides ; metabolism ; Mice ; Substrate Specificity ; Transfection

OBJECTIVE: To investigate the regulatory role of α-1, 6-fucosyltransferase (FUT8) in TGF-β1-induced proliferation, migration and fibrosis of human embryonic lung fibroblasts (MRC-5 cells) and explore the underlying molecular mechanism. METHODS: C57/BL6 mice were randomized into 4 groups for treatment with saline (control group), bleomycin, bleomycin+sh-NC or bleomycin+sh-FUT8, and pulmonary fibrosis was observed using Masson staining.MRC-5 cells were transfected with si-NC, FUT8 siRNA (si-FUT8), or both si-FUT8 and a galectin-3(Gal-3) overexpression plasmid (pcDNA3.1-Gal) prior to TGF-β1 treatment, and the changes in cell proliferation and migration were assessed using CCK-8 assay, BrdU assay, and wound healing assay; the changes in the expression levels of α-SMA, collagen I (COLIA1) and extracellular matrix fibronectin (FN) were detected with real-time quantitative PCR (RT-qPCR) and Western blotting.The interaction of FUT8 and Gal-3 was tested using coimmunoprecipitation (Co-IP) assay, and the effect of FUT8 silencing on Gal-3 and FAK/Akt signaling pathways was analyzed. RESULTS: FUT8 knockdown significantly reduced bleomycin-induced extracellular collagen deposition in the lung tissues of the mice.Silencing FUT8 obviously inhibited cell proliferation (P < 0.05) and migration mediated by TGF-β1.FUT8 knockdown down-regulated the mRNA and protein levels of α-SMA, COLIA1 and FN (P < 0.05) in the cells.Coimmunoprecipitation analysis showed that FUT8 interacted with Gal-3.Silencing FUT8 significantly down-regulated Gal-3 expression and inhibited the activation of the FAK/Akt signaling pathway (P < 0.05).Overexpression of Gal-3 obviously reversed the effects of FUT8 silencing on cell proliferation, migration and fibrosis (P < 0.05). CONCLUSION FUT8 regulates TGF-β1-induced proliferation, migration and fibrosis of MRC-5 cells by modulating Gal-3 expression, in which the FAK/Akt pathway may play a role.
Animals ; Bleomycin/metabolism* ; Fibroblasts/metabolism* ; Fibrosis ; Fucosyltransferases/metabolism* ; Galectin 3/genetics* ; Humans ; Lung/metabolism* ; Mice ; Proto-Oncogene Proteins c-akt/metabolism* ; Transforming Growth Factor beta1/metabolism*

OBJECTIVE: To investigate distribution specificity of human fucosyltransferase 5 (FUT5) as well as its expression and localization in spermatids. METHODS: Human semen, vaginal swab, saliva and venous blood from healthy individuals were collected. The spermatids were isolated and the spermatid membrane protein was then extracted. Expression levels of FUT5 from human spermatid membrane, seminal plasma, vaginal fluid, saliva and serum were detected by immunoblotting technique. The expression and localization of FUT5 in spermatids were analyzed by immunofluorescent method. RESULTS: Immunoblotting technique showed that FUT5 was expressed on spermatid membranes and in serum, but not in seminal plasma, vaginal fluid and saliva. The expressed FUT5 on spermatids was mostly localized on head of spermatids by fluorescent microscopy, suggesting that there was certain amount of FUT5 on human spermatid membrane, and the spermatids might be isolated from mixed stains with vaginal fluid by antigen-antibody reaction. CONCLUSION Human FUT5 shows a characteristic distribution specificity, and this feature may be used for identification of mixed stain involved in criminal sexual offence in future forensic practice.
Cell Membrane/metabolism* ; Female ; Fluorescent Antibody Technique/methods* ; Forensic Genetics/methods* ; Fucosyltransferases/metabolism* ; Humans ; Immunoblotting ; Male ; Saliva/metabolism* ; Semen/metabolism* ; Spermatids/metabolism* ; Vagina/metabolism*

Antigens, Tumor-Associated, Carbohydrate ; metabolism ; Fucosyltransferases ; metabolism ; Humans ; Lewis X Antigen ; metabolism ; Neoplasms ; immunology ; pathology ; Neoplastic Stem Cells ; immunology ; Stage-Specific Embryonic Antigens ; metabolism

OBJECTIVE: To explore the possible molecular mechanism of Ikaros regulation on FUT4 expression by analyzing the correlation of the functional state of Ikaros with level of FUT4 expression, so as to provide the theoretical basis for personalized treatment in children with ALL. METHODS: The subtypes of Ikaros were identified by nested PCR and sequencing. The expression level of FUT4 was detected by quantitative PCR and analyzed by ΔΔCt method in the early stage of treatment, remission and relapse of ALL. RESULTS: Ik1 and Ik2 were the main functional subtypes, and the dominant negative Ikaros was Ik6; the Ik6 was detected in 23 patients with ALL. It was found that 2.73% patients expressing Ik6 alone and 18.18% patients with heterozygous expression were detected. The expression of FUT4 in the newly diagnosed ALL was higher than that in the control group, and the functional Ikaros negatively correlated with the FUT4 expression(r=-0.6329). CONCLUSION Dominant negative Ikaros closely correlated with the relapse of acute lymphoblastic leukemia in children. The functional Ikaros negatively correlated with FUT4 expression. Ikaros inhibit the transcriptional activity of FUT4, that may be the molecular mechanism of Ikaros regulating the expression of FUT4.
Acute Disease ; Child ; Fucosyltransferases ; metabolism ; Humans ; Ikaros Transcription Factor ; metabolism ; Lewis X Antigen ; metabolism ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; Protein Isoforms ; Recurrence

OBJECTIVETo establish the eukaryotic cell expression system for the alpha-1,2 fucosyltransferase gene FUT1 293C to T and 658C to T mutations and explore the mechanism of FUT1 mutations resulting in the reduced expression of H antigen.

METHODSGenomic DNAs were extracted from individuals with para-Bombay phenotype and full coding region of FUT1 was amplified. The amplification fragments were ligated with pcDNA3.1 plasmid to construct the recombinant expression vectors. The recombinant plasmids were transfected into the COS-7 cells using lipofectamine transfection reagent and stable expression screening was performed. FUT1 mRNA expression was determined by real-time quantitative PCR. The activity of enzyme was measured by high performance liquid chromatography. Expressed protein was identified by SDS-PAGE and Western blotting.

RESULTSpcDNA3.1-FUT1 wildtype, pcDNA3.1-FUT1 293C to T and pcDNA3.1-FUT1 658C to T recombinant vectors were constructed, respectively. COS-7 cells with stable expression of FUT1 were obtained through recombinant plasmid transfection and screening with G418. The FUT1 mRNA level of transfected cells with 293C to T and 658C to T recombinant vectors reached 97.10% and 104.74% of the wildtype FUT1 transfected cell. A specific protein band with about 46 000 was confirmed in the transfected cell lysates by SDS-PAGE electrophoresis and Western blotting with 6×His Tag antibody. The wildtype FUT1 transfected cell lysates can catalyze the enzymatic reaction, while the enzyme activity of cell lysates from 293C to T and 658C to T were abolished.

CONCLUSIONThe results suggested that the 293C to T and 658C to T mutations of FUT1 gene did not affect the RNA and protein expression levels, but the enzyme activity of cells with FUT1 mutations was significantly decreased which resulted in the reduced expressin of H antigen.

Animals ; COS Cells ; Cercopithecus aethiops ; Fucosyltransferases ; genetics ; metabolism ; Gene Expression Regulation ; Genetic Vectors ; genetics ; Mutant Proteins ; genetics ; metabolism ; RNA, Messenger ; genetics

OBJECTIVETo transfect human alpha1, 2-fucosyltransferase (alpha1, 2-FT) gene to ovarian cancer cell line RMG-I and investigate the antigenic expression change of Lewis y and the other related oligosaccharides.

METHODSThe expression vector pcDNA3.1(-)-HFUT-H was constructed by polymerase chain reaction (PCR) to clone human alpha1, 2-FT gene coding region. The alpha1, 2-FT gene stable high-expression cell line RMG-I-H was established by transfecting pcDNA3.1(-)-HFUT-H to ovarian cell line RMG-I. The change of alpha1, 2-FT activity in the cell line before and after the tranfection was confirmed by the determination of enzymatic activity. The changes of cell lipid and glucolipid, especially the change of type II oligosaccharide, in the cell line before and after the transfection was determined by Thin-Layer Chromatography (TLC) and TLC immunostaining method, respectively.

RESULTSThe H-1 antigen and Lewis y antigen were obviously increased in the cell line RMG-1-H, especially the latter one, which was 20 times higher than before, and the type I saccharide chain Lewis b was decreased significantly. The main lipid components on the cell membrane, cholesterol and phosphatides, showed no change in the cell lines before and after the transfection, and the neutral glycolipid also showed no obvious change.

CONCLUSIONSThe transfection of alpha1, 2-FT gene can increase the activity of alpha1, 2-FT in the cell line RMG-I and mainly increase the expression of Lewis y antigen simultaneously. The construction of RMG-I Lewis y high expression cell line provides a cell model for further study on the relationship between Lewis y antigen and biological behaviors in the ovarian cancer.

Cell Line, Tumor ; Chromatography, Thin Layer ; Female ; Fucosyltransferases ; genetics ; physiology ; Humans ; Lewis Blood-Group System ; metabolism ; Ovarian Neoplasms ; metabolism ; Transfection ; methods

OBJECTIVETo explore the strategies which reduce the amount of xenoantigen Galalpha1,3Gal.

METHODSHuman alpha-galactosidase gene and alpha1,2-fucosyltransferase gene were transferred into cultured porcine vascular endothelial cells PEDSV.15 and human alpha-galactosidase transgenic mice were produced. The Galalpha1,3Gal on the cell surface and susceptibility of cells to human antibody-mediated lysis were analyzed.

RESULTSHuman alpha-galactosidase gene alone reduced 78% of Galalpha1,3Gal on PEDSV.15 cell surface while human alpha-galactosidase combined with alpha1,2-fucosyltransferase genes removed Galalpha1,3Gal completely. Decrease of Galalpha1,3Gal could reduce susceptibility of cells to human antibody-mediated lysis, especially during co-expression of alpha-galactosidase gene and alpha1,2-fucosyltransferase gene. RT-PCR indicated positive human alpha-galactosidase gene expression in all organs of positive human alpha-galactosidase transgenic F1 mice including heart, liver, kidney, lung, and spleen, the amount of Galalpha1,3Gal antigens on which was reduced largely. 58% of spleen cells from F1 mice were destroyed by complement-mediated lysis compared with 24% of those from normal mice.

CONCLUSIONSHuman alpha-galactosidase gene and alpha1,2-fucosyltransferase gene effectively reduce the expression of Galalpha1,3Gal antigens on endothelial cell surface and confers resistance to human serum-mediated cytolysis. The expression of human alpha-galactosidase in mice can also eliminate the Galalpha1,3Gal antigens in most tissues and decrease the susceptibility of spleen cells to human serum-mediated cytolysis.

Animals ; Antigens, Heterophile ; metabolism ; Cell Death ; Cells, Cultured ; Disaccharides ; metabolism ; Endothelial Cells ; metabolism ; Fucosyltransferases ; genetics ; metabolism ; Graft Rejection ; genetics ; Humans ; Mice ; Mice, Transgenic ; Spleen ; cytology ; Swine ; Transfection ; alpha-Galactosidase ; genetics ; metabolism

OBJECTIVETo explore the effect of α -1,2 fucosyltransferase (FUT1) gene 682A> G and 547_552delAG mutations on the expression of FUT1 mRNA and activity of α -1,2 fucosyltransferase.

METHODSRecombinant expression vectors of FUT1 682A> G and FUT1 547_552delAG were constructed and transfected into COS-7 cells for stable expression screening. Expression of FUT1 mRNA was determined using real-time quantitative PCR. The activity of FUT1 was measured with high-performance liquid chromatography.

RESULTSStably transfected COS-7 cells with wild type FUT1, FUT1 682A> G and FUT1 547_552delAG were respectively obtained. The FUT1 mRNA level of transfected cells with 682A> G and 547_552delAG recombination vectors have measured 101.69% and 102.79% compared with that of wild type FUT1 transfected cells. A specific protein band with about 46 kD was confirmed in the 682A> G transfected cell lysates by SDS-PAGE electrophoresis and Western blotting with 6× His Tag antibody. Similar protein was not identified in the 547_552delAG cells lysates. Enzymes activity of FUT1 682A> G has measured 61.01% compared with wild type FUT1 protein, whilst the activity of FUT1 547_557delAG was completely abolished.

CONCLUSIONFUT1 682A> G and 547_552delAG mutations do not affect the transcript efficiency, although various mutations have different impact on the enzyme's activity.

Animals ; Base Sequence ; Blotting, Western ; COS Cells ; Cercopithecus aethiops ; Chromatography, High Pressure Liquid ; DNA Mutational Analysis ; Fucosyltransferases ; genetics ; metabolism ; Humans ; Mutation ; Recombinant Proteins ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo investigate the influence of Lewis y antigen on the gene expression of partial drug resistance associated proteins in human ovarian cancer cell line RMG-I-H.

METHODSRT-PCR was used to determine the gene expressions of partial drug resistance associated proteins in RMG-I-H cell line transfected with alpha1, 2-fucosyltransferases gene and RMG-I cell line, as well as in RMG-I-H treated with or without anti-Lewis y monoclonal antibody at the concentration of 10 micro/g/ml. The immunocytochemical method was used to detect the expression of P-glycoprotein (P-gp) in RMG-I and RMG-I-H cell lines. RMG-I and RMG-I-H cells were transplanted into nude mice and the expression of P-gp in the tissues was measured by immunohistochemistry.

RESULTSThe mRNA expressions of protein kinase C-alpha (PKC-alpha), topoismerase I ( Topo I ), multidrug resistance-associated protein-1 (MRP-1), and MRP-2 were significantly higher in RMG-I-H cells than those in RMG-I cells (0.46 +/- 0.02 vs. 0.27 +/- 0.05, 0.82 +/- 0.08 vs. 0.52 +/- 0.04, 0.66 +/- 0.07 vs. 0.34 +/- 0.12, and 0.44 +/- 0.08 vs. 0.23 +/- 0.05; all P < 0.05). However, the mRNA expression of multi-drug resistance 1 (MDR-1) was significantly lower in RMG-I-H cells than that in RMG-I cells (0.26 +/- 0.05 vs. 0.45 +/- 0.08, P < 0.05). The P-gp level increased in RMG-I-H cells compared with that in RMG-I cells both in vivo and in vitro (P < 0.05). Expressions of MDR-1, MRP-1, MRP-2, PKC-alpha, and Topo I mRNA decreased by the time in RMG-I-H cells treated with anti-Lewis y monoclonal antibody (all P < 0.05), while mRNA expressions of those genes in the control group did not statistically change (P > 0.05). In addition, MDR-1, MRP-1, MRP-2, PKC-alpha, and Topo I mRNA expressions were significantly lower in RMG-I-H cells treated with anti-Lewis y monoclonal antibody than those in the control group at 6 hours (all P < 0.05) and the inhibition ratios were 48.55%, 77.50%, 70.18%, 45.86%, and 46.13%, respectively.

CONCLUSIONThe Lewis y antigen of the human ovarian cancer cell surface is closely correlated with the regulation on the gene expression of partial drug resistance associated proteins.

ATP-Binding Cassette, Sub-Family B, Member 1 ; genetics ; metabolism ; Animals ; Cell Line ; Cell Line, Tumor ; DNA Topoisomerases, Type I ; genetics ; metabolism ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; genetics ; Female ; Fucosyltransferases ; Gene Expression ; Gene Expression Regulation ; physiology ; Gene Expression Regulation, Neoplastic ; physiology ; Humans ; Lewis Blood-Group System ; physiology ; Mice ; Mice, Nude ; Multidrug Resistance-Associated Proteins ; genetics ; metabolism ; Ovarian Neoplasms ; Transfection