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.
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.
Sato
Takashi
T
Glycogene Function Team of Research Center for Glycoscience (RCG), National Institute of Advanced Industrial Science and Technology (AIST), Tukuba, Ibaraki, Japan.
Sato
Maiko
M
Kiyohara
Katsue
K
Sogabe
Maki
M
Shikanai
Toshihide
T
Kikuchi
Norihiro
N
Togayachi
Akira
A
Ishida
Hiroyasu
H
Ito
Hiromi
H
Kameyama
Akihiko
A
Gotoh
Masanori
M
Narimatsu
Hisashi
H
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D018076:DNA, Complementary / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D004926:Escherichia coli / Q000235:genetics; D005643:Fucose / Q000378:metabolism; D005647:Fucosyltransferases / Q000096:biosynthesis; D005700:Galactosyltransferases; D005964:Glucosyltransferases / Q000032:analysis / Q000737:chemistry* / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D019700:Thrombospondin 1 / Q000737:chemistry / Q000378:metabolism*
D018076:DNA, Complementary; D011134:Polysaccharides; D011994:Recombinant Proteins; D019700:Thrombospondin 1; D005643:Fucose; C514116:B3GLCT protein, human; D005647:Fucosyltransferases; D005700:Galactosyltransferases; D005964:Glucosyltransferases; C020570:1,3-beta-glucan synthase