Glycobiology, 2000, Vol. 10, No. 9 931-939
© 2000 Oxford University Press
The C-terminal N-glycosylation sites of the human 
1,3/4-fucosyltransferase III, -V, and -VI (hFucTIII, -V and -VI) are necessary for the expression of full enzyme activity
Laboratory of Molecular Diagnostics, Department of Clinical Biochemistry, Skejby University Hospital, Brendstrupgaardsvej, 8200 Aarhus N, Denmark, 2Institute of Human Genetics, University of Aarhus, 8000 Aarhus C, Denmark, and 3Research Unit for Molecular Medicine, University of Aarhus, Skejby University Hospital, Brendstrupgaardsvej, 8200 Aarhus N, Denmark
The 
1,3/4-fucosyltransferases are involved in the synthesis of fucosylated cell surface glycoconjugates. Human 1,3/4-fucosyltransferase III, -V, and -VI (hFucTIII, -V, and -VI) contain two conserved C-terminal N-glycosylation sites (hFucTIII: Asn154 and Asn185; hFucTV: Asn167 and Asn198; and hFucTVI: Asn153 and Asn184). In the present study, we have analyzed the functional role of these potential N-glycosylation sites, laying the main emphasis on the sites in hFucTIII. Tunicamycin treatment completely abolished hFucTIII enzyme activity while castanospermine treatment diminished hFucTIII enzyme activity to 
40% of the activity of the native enzyme. To further analyze the role of the conserved N-glycosylation sites in hFucTIII, -V, and -VI, we made a series of mutant genomic DNAs in which the asparagine residues in the potential C-terminal N-glycosylation sites were replaced by glutamine. Subsequently, the hFucTIII, -V, and -VI wild type and the mutants were expressed in COS-7 cells. All the mutants exhibited lower enzyme activity than the wild type and elimination of individual sites had different effects on the activity. The mutations did not affect the protein level of the mutants in the cells, but reduced the molecular mass as predicted. Kinetic analysis of hFucTIII revealed that lack of glycosylation at Asn185 did not change the Km values for the oligosaccharide acceptor and the nucleotide sugar donor. The present study demonstrates that hFucTIII, -V, and -VI require N-glycosylation at the two conserved C-terminal N-glycosylation sites for expression of full enzyme activity.
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