title: N-glycosylations of human α1,3-fucosyltransferase IX are required for full enzyme activity.
abstract: Human α1,3-fucosyltransferase IX catalyzes the transfer of l-fucose from guanosine diphosphate-β-L-fucose to N-acetyllactosamine, generating a Lewis X epitope, and is thereby involved in the synthesis of fucosylated cell surface glycoconjugates. It contains three putative N-glycosylation sites (Asn62, Asn101 and Asn153). The current study considers the functional role of these potential N-glycosylations within the enzyme. We produced truncated variants of human fucosyltransferase IX containing the soluble extracellular catalytic domain. To analyze the relevance of each N-glycosylation site, several genomic mutant DNAs encoding a glutamine (Gln/Q) instead of the asparagine residue were created prosperously using site-directed mutagenesis and subsequently expressed in Spodoptera frugiperda cells applying a baculovirus expression system. After production and purification of these variants of human FucT IX, the wild-type (wt) enzyme and the variants were characterized regarding their activity and kinetic properties. The variants showed lower activity than the wt FucT, whereas the individual N-glycosylation sites had different effects on the enzyme activity and kinetic parameters. While the single variant N62Q still showed ∼60% of wt activity and N101Q retained ∼30% activity, replacement of Asn153 by glutamine led to an almost complete loss of enzymatic activity. The same could be observed for variants missing two or more putative N-glycosylation sites, which indicated the importance of N-glycosylation for enzyme stability and activity.
authors.affiliation: Hamburg University, MIN-Faculty, Department of Chemistry, Institute for Biochemistry and Molecular Biology, Martin-Luther-King-Platz 6, 20146 Hamburg, Germany.
authors.forename: Katrin
authors.initials: K
authors.lastname: Seelhorst
authors.forename: Christina
authors.initials: C
authors.lastname: Stacke
authors.forename: Patrick
authors.initials: P
authors.lastname: Ziegelmüller
authors.forename: Ulrich
authors.initials: U
authors.lastname: Hahn
mesh_terms: D000818:Animals; D020134:Catalytic Domain; D005647:Fucosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D017384:Sequence Deletion; D061987:Sf9 Cells; D018411:Spodoptera
chemical_list: C519500:FUT9 protein, human; D005647:Fucosyltransferases