[Substrate specificity of three murine GDP-fucose: beta-galactoside alpha1,2-fucosyltransferases].
OBJECTIVE
To compare the substrate specificity of three murine GDP fucose: beta-galactoside alpha1,2-fucosyltransferases (alpha1,2-FT).


METHODS
Three 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.


RESULTS
MFUT- 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.


CONCLUSIONS
MFUT- 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.
Lin
Bei
B
Department of Obstetrics and Gynecology, the Second Affiliated Hospital of China Medical University, Shenyang 110004, China. linbei88@hotmail.com
Makiko
Saito
S
Masao
Iwamori
I
D000818:Animals; D000942:Antigens, Bacterial / Q000096:biosynthesis; D019556:COS Cells; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D005647:Fucosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005732:Gangliosides / Q000378:metabolism; D051379:Mice; D013379:Substrate Specificity; D014162:Transfection; D000097631:Galactoside 2-alpha-L-fucosyltransferase
D000942:Antigens, Bacterial; D005647:Fucosyltransferases; D005732:Gangliosides; D000097631:Galactoside 2-alpha-L-fucosyltransferase; C022863:H antigen