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.

Fluorescently Labeled Substrates for Monitoring α1,3‐Fucosyltransferase IX Activity

“…The reaction mixtures were examined by using HPTLC (Figure 2A,C). Evaluation of the spot intensities revealed a reaction rate of about 15 pmol h −1 for ATTO 550 labeled GDP‐ L ‐fucose 1 (Figure 2B), which is a factor of around a 1000 less than for the unlabeled substrate, and about 25 nmol h −1 for the ATTO‐labeled acceptor substrate 2 b (Figure 2D), which is in the same range as for the unlabeled N ‐acetyllactosamine 4…”

“…Biochemical procedures : Cloning of the HF9 gene containing vector pFastTHF9, production of recombinant bacmid bacTHF9, cell culture experiments, as well as the production and purification of a recombinant, truncated variant of human fucosyltransferase IX (HF9) were performed according to Seelhorst et al 4…”

“…Recently we reported on the comparison of different expression strategies for α1,3‐FucT IX in order to find conditions suitable for the production of high amounts of the soluble recombinant enzyme 3. We produced a truncated variant of human fucosyltransferase IX containing the soluble extracellular catalytic domain (HF9) by applying a baculovirus expression system in insect cells and obtained reasonable amounts of the enzyme 4. However, detailed information about the reaction mechanism of hFucT IX is still lacking.…”

“…However, detailed information about the reaction mechanism of hFucT IX is still lacking. So far we have elucidated the importance of the possible glycosylation state of putative glycosylation sites in the activity of the enzyme by analyzing corresponding variants 4. Murray et al.…”

Tagging Glycoproteins with Fluorescently Labeled GDP‐Fucoses by Using α1,3‐Fucosyltransferases

“…Biochemical procedures : For standard biochemical procedures such as protein production and purification, activity assays, and concentration determinations see previous publications 5b. 11 FucT V and FucT VII were purchased from R&D Systems (Minneapolis, US). Fetuin and asialofetuin were from Sigma–Aldrich, and recombinant gp130 was from Conaris (Kiel, Germany).…”