Enzymatic characterization of human alpha1,3-fucosyltransferase Fuc-TVII synthesized in a B cell lymphoma cell line.
The human alpha1,3-fucosyltransferase, Fuc-TVII, a key enzyme in the biosynthesis of selectin ligands, was expressed as a soluble protein-A chimeric form in a human B cell lymphoma cell line, Namalwa KJM-1, and purified using IgG-Sepharose. The enzymatic properties of recombinant soluble Fuc-TVII were then examined. Its enzyme activity was highest at pH 7.5, and the presence of 25 mM Mn2+ was required for full activity. Fuc-TVII exhibits an acceptor specificity restricted to alpha2,3-sialylated type 2 oligosaccharides, and the apparent Km values for alpha2,3-sialyl lacto-N-neotetraose and GDP-fucose were 3.08 mM and 16.4 microM, respectively. The inhibitory effects of various nucleotides on the activity of Fuc-TVII reflected its donor specificity for the nucleotide portion of GDP. Fuc-TVII was demonstrated to be useful for the synthesis of a sialyl Lewis x hexasaccharide from lacto-N-neotetraose in combination with an alpha2, 3-sialyltransferase, ST3Gal IV. Polyethylene glycols enhanced the thermal stability of Fuc-TVII, leading to increased formation of the reaction product.
Shinoda
K
K
Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 3-6-6, Asahi-machi, Machida-shi, Tokyo 194, Japan.
Morishita
Y
Y
Sasaki
K
K
Matsuda
Y
Y
Takahashi
I
I
Nishi
T
T
D002051:Burkitt Lymphoma / Q000201:enzymology* / Q000473:pathology; D002384:Catalysis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005647:Fucosyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009844:Oligosaccharides / Q000096:biosynthesis; D011092:Polyethylene Glycols / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000378:metabolism; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D004791:Enzyme Inhibitors; D009844:Oligosaccharides; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D011092:Polyethylene Glycols; D005647:Fucosyltransferases; C031692:galactoside 3-fucosyltransferase; D005033:Ethylmaleimide