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J Biol Chem, Vol. 273, Issue 47, 30985-30994, November 20, 1998

In Vivo Specificity of Human 1,3/4-Fucosyltransferases III-VII in the Biosynthesis of Lewis^X and Sialyl Lewis^X Motifs on Complex-type N-Glycans
COEXPRESSION STUDIES FROM BHK-21 CELLS TOGETHER WITH HUMAN -TRACE PROTEIN

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

From  Protein Glycosylation, Gesellschaft für Biotechnologische Forschung mbH, D-38124 Braunschweig, Germany and ^§ Instituto de Tecnologia Química e Biológica, P-2780 Oeiras, Portugal

Each of the five human 1,3/4-fucosyltransferases (FT3 to FT7) has been stably expressed in BHK-21 cells together with human -trace protein (-TP) as a secretory reporter glycoprotein. In order to study their in vivo properties for the transfer of peripheral Fuc onto N-linked complex-type glycans, detailed structural analysis was performed on the purified glycoprotein. All fucosyltransferases were found to peripherally fucosylate 19-52% of the diantennary -TP N-glycans, and all enzymes were capable of synthesizing the sialyl Lewis^X (sLe^x) motif. However, each enzyme produced its own characteristic ratio of sLe^x/Le^x antennae as follows: FT7 (only sLe^x), FT3 (14:1), FT5 (3:1), FT6 (1.1:1), and FT4 (1:7). Fucose transfer onto -TP N-glycans was low in FT3 cells (11% of total antennae), whereas the values for FT7, FT5, FT4, and FT6 cells were 21, 25, 35, and 47%, respectively. FT3, FT4, FT5, and FT7 transfer preponderantly one Fuc per diantennary N-glycan. FT4 preferentially synthesizes di-Le^x on asialo diantennary N-glycans and mono-Le^x with monosialo chains. In contrast, FT6 forms mostly 1,3-difucosylated chains with no, one, or two NeuAc residues. FT3, FT4, and FT6 were proteolytically cleaved and released into the culture medium in significant amounts, whereas FT7 and FT5 were found to be largely resistant toward proteolysis. Studies on engineered soluble variants of FT6 indicate that these forms do not significantly contribute to the in vivo fucose transfer activity of the enzyme when expressed at activity levels comparable to those obtained for the wild-type Golgi form of FT6 in the recombinant host cells.

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Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.

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