title: Modification of epidermal growth factor-like repeats with O-fucose. Molecular cloning and expression of a novel GDP-fucose protein O-fucosyltransferase.
abstract: The O-fucose modification is found on epidermal growth factor-like repeats of a number of cell surface and secreted proteins. O-Fucose glycans play important roles in ligand-induced receptor signaling. For example, elongation of O-fucose on Notch by the beta1,3-N-acetylglucosaminyltransferase Fringe modulates the ability of Notch to respond to its ligands. The enzyme that adds O-fucose to epidermal growth factor-like repeats, GDP-fucose protein O-fucosyltransferase (O-FucT-1), was purified previously from Chinese hamster ovary (CHO) cells. Here we report the isolation of a cDNA that encodes human O-FucT-1. A probe deduced from N-terminal sequence analysis of purified CHO O-FucT-1 was used to screen a human heart cDNA library and expressed sequence tag and genomic data bases. The cDNA contains an open reading frame encoding a protein of 388 amino acids with a predicted N-terminal transmembrane sequence typical of a type II membrane orientation. Likewise, the mouse homolog obtained from an expressed sequence tag and 5'-rapid amplification of cDNA ends of a mouse liver cDNA library encodes a type II transmembrane protein of 393 amino acids with 90.4% identity to human O-FucT-1. Homologs were also found in Drosophila and Caenorhabditis elegans with 41.2 and 29.4% identity to human O-FucT-1, respectively. The human gene (POFUT1) is on chromosome 20 between PLAGL2 and KIF3B, near the centromere at 20p11. The mouse gene (Pofut1) maps near Plagl2 on a homologous region of mouse chromosome 2. POFUT1 gene transcripts were expressed in all tissues examined, consistent with the widespread localization of the modification. Expression of a soluble form of human O-FucT-1 in insect cells yielded a protein of the predicted molecular weight with O-FucT-1 kinetic and enzymatic properties similar to those of O-FucT-1 purified from CHO cells. The identification of the gene encoding protein O-fucosyltransferase I now makes possible mutational strategies to examine the functions of the unusual O-fucose post-translational modification.
authors.affiliation: Department of Pharmacokinetics and Metabolism, Genentech, Inc., South San Francisco, California 94080, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Wang
authors.forename: L
authors.initials: L
authors.lastname: Shao
authors.forename: S
authors.initials: S
authors.lastname: Shi
authors.forename: R J
authors.initials: RJ
authors.lastname: Harris
authors.forename: M W
authors.initials: MW
authors.lastname: Spellman
authors.forename: P
authors.initials: P
authors.lastname: Stanley
authors.forename: R S
authors.initials: RS
authors.lastname: Haltiwanger
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D016466:CHO Cells / Q000201:enzymology; D003001:Cloning, Molecular; D006224:Cricetinae; D004815:Epidermal Growth Factor / Q000378:metabolism*; D005647:Fucosyltransferases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis; D020449:Repetitive Sequences, Amino Acid; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera / Q000166:cytology; D014018:Tissue Distribution
chemical_list: D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; D005647:Fucosyltransferases; C105148:polypeptide fucosyltransferase