Glycobiology Advance Access published online on August 9, 2006
Glycobiology, doi:10.1093/glycob/cwl035
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1 Glycogene Function Team of Research Center for Glycoscience (RCG), National Institute of Advanced Industrial Science and Technology (AIST), Open Space Laboratory Central-2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568
* To whom correspondence should be addressed. 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; i.e. on epidermal growth factor-like domains, and on thrombospondin type 1 repeat (TSR) domains. We report here the molecular cloning and characterization of a novel * These authors contributed equally to this work and both should be considered as first authors
Received May 11, 2006
Revised July 28, 2006
Accepted August 2, 2006
Article
Molecular Cloning and Characterization of a Novel Human
Takashi Sato 1 *, Maiko Sato 1 *, Katsue Kiyohara 1, Maki Sogabe 1, Toshihide Shikanai 2, Norihiro Kikuchi 2, Akira Togayachi 1, Hiroyasu Ishida 1, Hiromi Ito 1, Akihiko Kameyama 1, Masanori Gotoh 3, and Hisashi Narimatsu 1 *
1,3-Glucosyltransferase, 3Glc-T, Which is Localized at the Endoplasmic Reticulum and Glucosylates O-linked Fucosylglycan on Thrombospondin Type 1 Repeat Domain
2 Glycogene Function Team of Research Center for Glycoscience (RCG), National Institute of Advanced Industrial Science and Technology (AIST), Open Space Laboratory Central-2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568; Mitsui Knowledge Industry Co., Ltd., Honcho 1-Chome, Nakano-ku, Tokyo 164-8721
3 Glycogene Function Team of Research Center for Glycoscience (RCG), National Institute of Advanced Industrial Science and Technology (AIST), Open Space Laboratory Central-2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568; GlycoGene, Inc., Open Space Laboratory Central-2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568
Hisashi Narimatsu, E-mail: h.narimatsu{at}aist.go.jp
Abstract 
1,3 glucosyltransferase that synthesizes a Glc1,3Fuc
- structure on the TSR domain. We found a novel glycosyltransferase gene with 3GT motifs in databases. The recombinant enzyme expressed in human embryonic kidney 293T cells exhibited glucosyltransferase activity toward fucose (Fuc) --para-nitro-phenyl(pNp). Thin layer chromatography analysis revealed that the product of the recombinant enzyme migrated to the same position as did the product of endogenous 3Glc-T of CHO cells. The two products could be digested by -glucosidase from almond and by exo-1,3--glucanase from Trichoderma sp. These results strongly suggested that the product has the structure of Glc1-3Fuc. Therefore, we named this novel enzyme 1,3 glucosyltransferase (3Glc-T). Immunostaining revealed that FLAG-Tagged 3Glc-T is an enzyme residing in the endoplasmic reticulum via retention signal, "REEL", which is a KDEL-like sequence, at the C-terminus. The TSR domain expressed in E. coli was first fucosylated by the recombinant protein O-fucosyltransferase 2, after which it became an acceptor substrate for the recombinant 3Glc-T, which could apparently transfer Glc to the fucosylated TSR domain. Our results suggest that a novel glycosyltransferase, 3Glc-T, contributes to elongation of O-fucosylglycan, and that this occurs specifically on TSR domains.3GT motif/O-fucose/thrombospondin/TSR.
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