Molecular cloning and characterization of a novel human β1,3-glucosyltransferase, which is localized at the endoplasmic reticulum and glucosylates O-linked fucosylglycan on thrombospondin type 1 repeat domain

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 beta 1,3-glucosyltransferase (beta 3Glc-T) that synthesizes a Glc beta 1,3Fuc alpha- structure on the TSR domain. We found a novel glycosyltransferase gene with beta 1,3-glycosyltransferase (beta 3GT) motifs in databases. The recombinant enzyme expressed in human embryonic kidney 293T (HEK293T) cells exhibited glucosyltransferase activity toward fucose-alpha-para-nitrophenyl (Fuc alpha-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 beta 3Glc-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 Glc beta 1-3Fuc. Therefore, we named this novel enzyme beta 3Glc-T. Immunostaining revealed that FLAG-tagged beta 3Glc-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 beta 3Glc-T, which could apparently transfer Glc to the fucosylated TSR domain. Our results suggest that a novel glycosyltransferase, beta 3Glc-T, contributes to the elongation of O-fucosylglycan and that this occurs specifically on TSR domains.