Localization, purification and specificity of the full-length membrane-bound form of human recombinant alpha 1,3/4-fucosyltransferase from BHK-21B cells.
Fucosyltransferase III [galactoside 3(4)-L-fucosyltransferase; EC 2.4.1.65] (FT3) is a Golgi type II membrane protein that catalyses the synthesis of fucosylated Lewis motifs that are associated with cell-adhesion events and are differentially expressed during cell differentiation. In the present work, the full-length membrane bound form of FT3 has been expressed in baby hamster kidney cells. The enzyme has been found in the trans-Golgi and trans-Golgi network (TGN) of the transfected cells, where it appeared as monomers and dimers, but not as oligomers with high molecular masses. Therefore oligomerization is not the basis for correct localization of FT3 in the Golgi. The enzyme has been purified, with a final yield of 2% and a total purification of 2900-fold, by DEAE-Sepharose, SP-Sepharose, GDP-Fractogel and Superdex 200 chromatography. The purified enzyme showed a clear preference for the Gal beta 3GlcNAc motif in oligosaccharides conjugated with the hydrophobic tail (CH(2))(3)-NHCO-(CH(2))(5)-NH-biotin. Substitution of galactose with alpha 2-linked fucose or alpha 2,3-linked N-acetylneuraminic acid yielded a 1.9-fold increase or a 43% decrease in activity respectively. The enzyme showed no activity towards asialofetuin, a glycoprotein containing the Gal beta 3GlcNAc acceptor motif. Therefore it has been concluded that the membrane-bound form of FT3 is present in the Golgi and the TGN in an equilibrium of monomers<-->dimers, which might fucosylate glycans from glycolipids, but not from glycoproteins.
Sousa
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VL
Instituto de Tecnologia Química e Biológica, Avenida da República, Apartado 127, 2780-901 Oeiras, Portugal.
Costa
M T
MT
Palma
A S
AS
Enguita
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F
Costa
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J
D000818:Animals; D002478:Cells, Cultured; D006224:Cricetinae; D005647:Fucosyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D006056:Golgi Apparatus / Q000201:enzymology / Q000378:metabolism*; D006801:Humans; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection
D011994:Recombinant Proteins; D005647:Fucosyltransferases; C019818:3-galactosyl-N-acetylglucosaminide 4-alpha-L-fucosyltransferase