Modification of epidermal growth factor-like repeats with O-fucose: Molecular cloning and expression of a novel GDP-fucose protein O-fucosyltransferase

Yang Wang, Li Shao, Shaolin Shi, Reed J. Harris, Michael W. Spellman, Pamela Stanley, Robert S. Haltiwanger

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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 β1,3-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.

Original languageEnglish (US)
Pages (from-to)40338-40345
Number of pages8
JournalJournal of Biological Chemistry
Volume276
Issue number43
StatePublished - Oct 26 2001

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Guanosine Diphosphate Fucose
Fucosyltransferases
Fucose
Cloning
Molecular Cloning
Epidermal Growth Factor
Cricetulus
Ovary
Proteins
Complementary DNA
Genes
Expressed Sequence Tags
Chromosomes
Gene Library
Chromosomes, Human, Pair 20
Ligands
Amino Acids
Gene encoding
Chromosomes, Human, Pair 2
Centromere

ASJC Scopus subject areas

  • Biochemistry

Cite this

Modification of epidermal growth factor-like repeats with O-fucose : Molecular cloning and expression of a novel GDP-fucose protein O-fucosyltransferase. / Wang, Yang; Shao, Li; Shi, Shaolin; Harris, Reed J.; Spellman, Michael W.; Stanley, Pamela; Haltiwanger, Robert S.

In: Journal of Biological Chemistry, Vol. 276, No. 43, 26.10.2001, p. 40338-40345.

Research output: Contribution to journalArticle

Wang, Yang ; Shao, Li ; Shi, Shaolin ; Harris, Reed J. ; Spellman, Michael W. ; Stanley, Pamela ; Haltiwanger, Robert S. / Modification of epidermal growth factor-like repeats with O-fucose : Molecular cloning and expression of a novel GDP-fucose protein O-fucosyltransferase. In: Journal of Biological Chemistry. 2001 ; Vol. 276, No. 43. pp. 40338-40345.
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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 β1,3-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.",
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T2 - Molecular cloning and expression of a novel GDP-fucose protein O-fucosyltransferase

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AU - Shao, Li

AU - Shi, Shaolin

AU - Harris, Reed J.

AU - Spellman, Michael W.

AU - Stanley, Pamela

AU - Haltiwanger, Robert S.

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