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Volume 272, Number 17, Issue of April 25, 1997 pp. 11613-11621
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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

(Received for publication, December 23, 1996)

Júlia Costa Dagger , Eckart Grabenhorst Dagger , Manfred Nimtz and Harald S. Conradt Dagger

From the Departments of Dagger  Protein Glycosylation and  Molecular and Instrumental Structural Research, Gesellschaft für Biotechnologische Forschung, Mascheroder Weg 1, D-38124 Braunschweig, Germany

Stable BHK-21 cell lines were constructed expressing the Golgi membrane-bound form and two secretory forms of the human alpha 1,3/4-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 Galbeta 1-3GlcNAc motif. The acceptor oligosaccharide in bovine asialofetuin was identified as the Man-3 branched triantennary isomer with one Galbeta 1-3GlcNAc. The type II motif Galbeta 1-4GlcNAc in bi-, tri-, or tetraantennary neutral or alpha 2-3/alpha 2-6 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.


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