Stable expression of the Golgi form and secretory variants of human fucosyltransferase III from BHK-21 cells. Purification and characterization of an engineered truncated form from the culture medium.
Stable BHK-21 cell lines were constructed expressing the Golgi membrane-bound form and two secretory forms of the human alpha1, 3/4-fucosyltransferase (amino acids 35-361 and 46-361). It was found that 40% of the enzyme activity synthesized by cells transfected with the Golgi form of the fucosyltransferase was constitutively secreted into the medium. The corresponding enzyme detected by Western blot had an apparent molecular mass similar to those of the truncated secretory forms. The secretory variant (amino acids 46-361) was purified by a single affinity-chromatography step on GDP-Fractogel resin with a 20% final recovery. The purified enzyme had a unique NH2 terminus and contained N-linked endo H sensitive carbohydrate chains at its two glycosylation sites. The fucosyltransferase transferred fucose to the O-4 position of GlcNAc in small oligosaccharides, glycolipids, glycopeptides, and glycoproteins containing the type I Galbeta1-3GlcNAc motif. The acceptor oligosaccharide in bovine asialofetuin was identified as the Man-3 branched triantennary isomer with one Galbeta1-3GlcNAc. The type II motif Galbeta1-4GlcNAc in bi-, tri-, or tetraantennary neutral or alpha2-3/alpha2-6 sialylated oligosaccharides with or without N-acetyllactosamine repeats and in native glycoproteins were not modified. The soluble forms of fucosyltransferase III secreted by stably transfected cells may be used for in vitro synthesis of the Lewisa determinant on carbohydrates and glycoproteins, whereas Lewisx and sialyl-Lewisx structures cannot be synthesized.
Costa
J
J
Department of Protein Glycosylation, Gesellschaft für Biotechnologische Forschung, Mascheroder Weg 1, D-38124 Braunschweig, Germany.
Grabenhorst
E
E
Nimtz
M
M
Conradt
H S
HS
D000818:Animals; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000096:biosynthesis*; D001212:Asialoglycoproteins / Q000737:chemistry; D002240:Carbohydrate Sequence; D002417:Cattle; D002478:Cells, Cultured; D006224:Cricetinae; D060748:Fetuins; D005647:Fucosyltransferases / Q000235:genetics / Q000378:metabolism*; D014644:Genetic Variation; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000166:cytology; D007983:Lewis Blood Group Antigens; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis; D017384:Sequence Deletion; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D000509:alpha-Fetoproteins / Q000737:chemistry
D015295:Antigens, Tumor-Associated, Carbohydrate; D001212:Asialoglycoproteins; D060748:Fetuins; D007983:Lewis Blood Group Antigens; D009844:Oligosaccharides; D011994:Recombinant Proteins; D000509:alpha-Fetoproteins; C017029:asialofetuin; D005647:Fucosyltransferases; C019818:3-galactosyl-N-acetylglucosaminide 4-alpha-L-fucosyltransferase