Identification of a GDP-L-fucose:polypeptide fucosyltransferase and enzymatic addition of O-linked fucose to EGF domains.
An assay of GDP-fucose:polypeptide fucosyltransferase has been established. The enzyme catalyzes the reaction that attaches fucose through an O-glycosidic linkage to a conserved serine or threonine residue in EGF domains. The assay uses recombinant human factor VII EGF-1 domain as acceptor substrate and GDP-fucose as donor substrate. Synthetic peptides with sequences taken from five proteins previously shown to contain O-linked fucose (Harris and Spellman, 1993; Glycobiology, 3, 219-224) did not serve as efficient acceptor substrates. These synthetic peptides did not compromise complete EGF domains and did not contain all six cysteine residues that define the EGF structure. Therefore, the enzyme appears to require more than just a consensus primary sequence and likely requires that the EGF domain disulfide bonds be properly formed. The enzymatic reaction showed linear dependency of its activity on time, amount of enzyme, and substrates. Although the enzyme did not exhibit an absolute requirement for Mn2+, enzymatic activity did increase ten fold in the presence of 50 mM MnCl2. The in vitro glycosylation reaction resulted in complete conversion of the acceptor substrate to glycosylated product, and characterization of the purified product by electrospray mass spectrometry revealed that one fucose was added onto the polypeptide. Most of the enzymatic activity was found to be in the soluble fraction of CHO cell homogenates. However, when enzyme was prepared from rat liver in the presence of protease inhibitors, 37% of the activity was recovered by Triton X-100 extraction of the membrane particles after extensive aqueous washes. The result suggests that the enzyme is probably a membrane protein and, by analogy with other glycosyltransferases, probably has a 'stem' region that is very susceptible to proteolysis.
Wang
Y
Y
Department of Pharmacokinetics and Metabolism, Genentech, Inc., South San Francisco, CA 94080, USA.
Lee
G F
GF
Kelley
R F
RF
Spellman
M W
MW
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004815:Epidermal Growth Factor / Q000737:chemistry / Q000378:metabolism*; D005643:Fucose / Q000378:metabolism*; D005647:Fucosyltransferases / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D051381:Rats
D008565:Membrane Proteins; D005643:Fucose; D004815:Epidermal Growth Factor; D005647:Fucosyltransferases; C105148:polypeptide fucosyltransferase