Glycobiology Advance Access originally published online on August 9, 2006
Glycobiology 2006 16(12):1194-1206; doi:10.1093/glycob/cwl035
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Molecular cloning and characterization of a novel human ß1,3-glucosyltransferase, which is localized at the endoplasmic reticulum and glucosylates O-linked fucosylglycan on thrombospondin type 1 repeat domain
3 Glycogene Function Team of Research Center for Glycoscience (RCG), National Institute of Advanced Industrial Science and Technology (AIST), and
4 GlycoGene, Inc., Open Space Laboratory Central-2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan; and
5 Mitsui Knowledge Industry Co., Ltd., Honcho 1-Chome, Nakano-ku, Tokyo 164-8721, Japan
2 To whom correspondence should be addressed; e-mail: h.narimatsu{at}aist.go.jp
Received on May 11, 2006; revised on July 28, 2006; accepted on August 2, 2006
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 ß1,3-glucosyltransferase (ß3Glc-T) that synthesizes a Glcß1,3Fuc
- structure on the TSR domain. We found a novel glycosyltransferase gene with ß1,3-glycosyltransferase (ß3GT) motifs in databases. The recombinant enzyme expressed in human embryonic kidney 293T (HEK293T) cells exhibited glucosyltransferase activity toward fucose--para-nitrophenyl (Fuc-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 ß3Glc-T of Chinese hamster ovary (CHO) cells. The two products could be digested by ß-glucosidase from almond and by exo-1,3-ß-glucanase from Trichoderma sp. These results strongly suggested that the product has the structure of Glcß1-3Fuc. Therefore, we named this novel enzyme ß3Glc-T. Immunostaining revealed that FLAG-tagged ß3Glc-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 ß3Glc-T, which could apparently transfer Glc to the fucosylated TSR domain. Our results suggest that a novel glycosyltransferase, ß3Glc-T, contributes to the elongation of O-fucosylglycan and that this occurs specifically on TSR domains.
Key words: glucosyltransferase / glycosyltransferase / O-fucose / thrombospondin / TSR
The nucleotide sequence reported in this article has been registered in the GenBank/EBI Data Bank with accession number AB101481 for human and AB253762 for mouse.
1 These authors contributed equally to the work and both should be considered as first authors.
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