PMID:16899492

Citation	Sato, T, Sato, M, Kiyohara, K, Sogabe, M, Shikanai, T, Kikuchi, N, Togayachi, A, Ishida, H, Ito, H, Kameyama, A, Gotoh, M and Narimatsu, H (2006) Molecular cloning and characterization of a novel human beta1,3-glucosyltransferase, which is localized at the endoplasmic reticulum and glucosylates O-linked fucosylglycan on thrombospondin type 1 repeat domain. Glycobiology 16:1194-206
Abstract	Protein O-linked fucosylation is an unusual glycosylation associated with many important biological functions such as Notch signaling. Two fucosylation pathways synthesizing O-fucosylglycans have been reported on cystein-knotted proteins, that is, on epidermal growth factor-like (EGF-like) domains and on thrombospondin Type 1 repeat (TSR) domains. We report here the molecular cloning and characterization of a novel beta1,3-glucosyltransferase (beta3Glc-T) that synthesizes a Glcbeta1,3Fucalpha- structure on the TSR domain. We found a novel glycosyltransferase gene with beta1,3-glycosyltransferase (beta3GT) motifs in databases. The recombinant enzyme expressed in human embryonic kidney 293T (HEK293T) cells exhibited glucosyltransferase activity toward fucose-alpha-para-nitrophenyl (Fucalpha-pNp). Thin-layer chromatography (TLC) analysis revealed that the product of the recombinant enzyme migrated to the same position as did the product of endogenous beta3Glc-T of Chinese hamster ovary (CHO) cells. The two products could be digested by beta-glucosidase from almond and by exo-1,3-beta-glucanase from Trichoderma sp. These results strongly suggested that the product has the structure of Glcbeta1-3Fuc. Therefore, we named this novel enzyme beta3Glc-T. Immunostaining revealed that FLAG-tagged beta3Glc-T is an enzyme residing in the endoplasmic reticulum (ER) via retention signal, "REEL," which is a KDEL-like sequence, at the C-terminus. The TSR domain expressed in Escherichia coli was first fucosylated by the recombinant protein O-fucosyltransferase 2 (POFUT2), after which it became an acceptor substrate for the recombinant beta3Glc-T, which could apparently transfer Glc to the fucosylated TSR domain. Our results suggest that a novel glycosyltransferase, beta3Glc-T, contributes to the elongation of O-fucosylglycan and that this occurs specifically on TSR domains.
Links	PubMed Online version:10.1093/glycob/cwl035
Keywords	Amino Acid Sequence; Animals; Cells, Cultured; Cloning, Molecular; Cricetinae; Cricetulus; DNA, Complementary/genetics; Endoplasmic Reticulum/enzymology; Escherichia coli/genetics; Fucose/metabolism; Fucosyltransferases/biosynthesis; Glucosyltransferases/analysis; Glucosyltransferases/chemistry; Glucosyltransferases/genetics; Glycosylation; Humans; Molecular Sequence Data; Polysaccharides/metabolism; Protein Processing, Post-Translational; Protein Structure, Tertiary; Recombinant Proteins/biosynthesis; Recombinant Proteins/genetics; Recombinant Proteins/isolation & purification; Sequence Analysis, DNA; Substrate Specificity; Thrombospondin 1/chemistry; Thrombospondin 1/metabolism
edit table

Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
HUMAN:B3GT1		GO:0005783: endoplasmic reticulum	ECO:0000315:		C		Fig 6C	complete

References

See Help:References for how to manage references in GONUTS.