In vitro alpha1-3 or alpha1-4 fucosylation of type I and II oligosaccharides with secreted forms of recombinant human fucosyltransferases III and VI.
Transgalactosylation of chitobiose and chitotriose employing beta-galactosidase from bovine testes yielded mixtures with beta1-3 linked galactose (type I) and beta1-4 linked galactose (type II) in a final ratio of 1:1 for the tri- and 1:1.4 for the tetrasaccharide. After 24 h incubations of the two purified oligosaccharide mixtures with large amounts (20-fold increase compared with standard conditions) of human alpha1,3/4-fucosyltransferase III (FucT III), the type I tri-/tetrasaccharides were completely converted to the Lewis(a) structure, whereas approximately 10% fucosylation of the type II isomers to the Lewis(x) oligosaccharides was observed in long-term incubations. Employing large amounts of human alpha1,3-fucosyltransferase VI (FucT VI), the type I trisaccharide substrate was exclusively fucosylated at the proximal O-4 substituted N-acetylglucosamine (GlcNAc) (20%) whereas almost all of the type II isomers was converted to the corresponding Lewis(x) product. 45% of the type I tetrasaccharide was fucosylated at the second GlcNAc solely by FucT VI. The type II isomer was almost completely alpha1-3 fucosylated to yield the Lewisx derivative with traces of a structure that contained an additional fucose at the reducing GlcNAc. The results obtained in the present study employing high amounts of enzyme confirmed our previous results that FucT III acts preponderantly as a beta1-4 fucosyltransferase onto GlcNAc in vitro. Human FucT VI attaches fucose exclusively in an alpha1-3 linkage to 4-substituted GlcNAc in vitro and does not modify any 3-substituted GlcNAc to yield Lewis(a) oligosaccharides. With 8-methoxycarbonyloctyl glycoside acceptors used under standard conditions, FucT III acts exclusively on the type I and FucT VI only on the type II derivative. With lacto-N-tetraose, lacto-N-fucopentraose I, or LS-tetrasaccharide as substrates, FucT III modified the 3-substituted GlcNAc and the reducing glucose; FucT VI recognized only lacto-N-neotetraose as a substrate.
Gesellschaft für Biotechnologische Forschung, Braunschweig, Germany.
M
M
Nimtz
E
E
Grabenhorst
U
U
Gambert
J
J
Costa
V
V
Wray
M
M
Morr
J
J
Thiem
H S
HS
Conradt
D002240:Carbohydrate Sequence; D002460:Cell Line; D004187:Disaccharides / Q000378:metabolism; D005647:Fucosyltransferases / Q000235:genetics / Q000378:metabolism*; D005690:Galactose / Q000378:metabolism; D006801:Humans; D007983:Lewis Blood Group Antigens / Q000096:biosynthesis; D008745:Methylation; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection; D014312:Trisaccharides / Q000378:metabolism
D004187:Disaccharides; D007983:Lewis Blood Group Antigens; D009844:Oligosaccharides; D011994:Recombinant Proteins; D014312:Trisaccharides; C041161:chitotriose; C032438:chitobiose; D005647:Fucosyltransferases; D005690:Galactose