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

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