Protein O-Fucosyltransferase 2 Adds O-Fucose to Thrombospondin Type 1 Repeats

Department of Biochemistry and Cell Biology, Institute for Cell and Developmental Biology, Stony Brook University, Stony Brook, New York 11794-5215, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 05/2006; 281(14):9393-9. DOI: 10.1074/jbc.M511975200
Source: PubMed


O-Fucose is an unusual form of glycosylation found on epidermal growth factor-like (EGF) repeats and thrombospondin type 1 repeats (TSRs) in many secreted and transmembrane proteins. Recently O-fucose on EGF repeats was shown to play important roles in Notch signaling. In contrast, physiological roles for O-fucose on TSRs are unknown. In the accompanying paper (Luo, Y., Nita-Lazar, A., and Haltiwanger, R. S. (2006) J. Biol. Chem. 281, 9385-9392), we demonstrated that an enzyme distinct from protein O-fucosyltransferase 1 adds O-fucose to TSRs. A known homologue of O-fucosyltransferase 1 is putative protein O-fucosyltransferase 2. The cDNA sequence encoding O-fucosyltransferase 2 was originally identified during a data base search for fucosyltransferases in Drosophila. Like O-fucosyltransferase 1, O-fucosyltransferase 2 is conserved from Caenorhabditis elegans to humans. Although O-fucosyltransferase 2 was assumed to be another protein O-fucosyltransferase, no biochemical characterization existed supporting this contention. Here we show that RNAi-mediated reduction of the O-fucosyltransferase 2 message significantly decreased TSR-specific O-fucosyltransferase activity in Drosophila S2 cells. We also found that O-fucosyltransferase 2 is predominantly localized in the endoplasmic reticulum compartment of these cells. Furthermore, we expressed recombinant Drosophila O-fucosyltransferase 2 and showed that it O-fucosylates TSRs but not EGF repeats in vitro. These results demonstrate that O-fucosyltransferase 2 is in fact a TSR-specific O-fucosyltransferase.

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    • "The family GT68 contains protein accessions sharing sequence identities with protein O-fucosyltransferases (POFUT2) characterized in Drosophila and human [62], [63]. These enzymes transfer an α-l-fucose residue from GDP-fucose to a conserved serine or threonine residue in thrombospondin type 1 repeats (TSRs). "
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    • "Reversed-phase HPLC retention time comparison of these samples and a fucose standard indicated that the source of the 146 Da modification on L3 peptide was fucose. observed on O-fucosylated TSRs in mammalian cells (Hofsteenge et al. 2001; Gonzalez de et al. 2002; Luo, Nita- Lazar, et al. 2006), the modification by a single O-fucose monosaccharide has been observed on EGF-like repeats as illustrated by the example of factor VII, where O-fucose is present as a monosaccharide (Bjoern et al. 1991). We must note that O-fucose on another EGF domain containing molecule of the clotting cascade, factor IX, is observed as the full-length tetrasaccharide mentioned above (Nishimura et al. 1992). "
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    ABSTRACT: We describe the characterization of an O-fucosyl modification to a serine residue on the light chain of a recombinant, human IgG1 molecule expressed in Chinese hamster ovary (CHO) cells. Cation exchange chromatography (CEX) and hydrophobic interaction chromatography (HIC) were used to isolate a Fab population which was 146 Da heavier than the expected mass. Isolated Fab fragments were treated with a reducing agent to facilitate mass spectrometric analysis of the reduced light chain (LC) and fragment difficult (Fd). An antibody light chain with a net addition of 146 Da was detected by mass spectrometric analysis of the modified Fab. A light chain tryptic peptide in complementarity determining region-1 (CDR-1) was subsequently identified with a net addition of 146 Da by a peptide map. Results from a nanospray infusion of the modified peptide into a linear ion trap mass spectrometer with electron transfer dissociation (ETD) functionality indicated that the modified residue was a serine at position 30 in the light chain. Acid hydrolysis of the modified tryptic peptide followed by fluorescent labeling with 2-aminoanthranilic acid (2AA) and HPLC comparison with monosaccharide standards confirmed the presence of fucose on the light chain peptide. The presence of O-fucose on an antibody has not been previously reported. Currently, O-fucose has been described as occurring on mammalian proteins with amino acid sequence motifs associated with epidermal growth factor (EGF)-like repeats or thrombospondin type 1 repeats (TSRs). The amino acid sequence around the modified Ser in the IgG1 molecule does not conform to any known O-fucosylation sequence motif and thus is the first description of this type of modification on a nonconsensus sequence in a mammalian protein.
    Full-text · Article · Nov 2008 · Glycobiology
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    • "A homologue of Pofut1 known as protein O-fucosyltransferase 2 (Pofut2) (Luo et al., 2002, Luo et al., 2006a, Luo et al., 2006b) adds a fucose in α-linkage to a serine or threonine within TSRs containing the consensus sequence: C-X-X-[S/T]-C-X-X-G (between the first and second cysteines for class 1 TSRs, second and third for class 2) (Hofsteenge et al., 2001, Ricketts et al, 2007, Wang et al., 2007) (Figure 1D). This fucosylation event also appears to occur in the ER despite the lack of an obvious ER localization sequence in the Pofut2 sequence (Luo et al., 2006a). The O-fucose can be elongated by a β1,3-glucosyltransferase (β3GlcT) to the disaccharide, Glc-β1,3-Fuc (Kozma et al., 2006, Sato et al, 2006). "
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    Preview · Article · Nov 2008 · The international journal of biochemistry & cell biology
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