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
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
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DOI：
10.1093/glycob/cwl035
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发表时间：
2006-12-01
期刊：
影响因子：
4.3
通讯作者：
Narimatsu, Hisashi
中科院分区：
文献类型：
--
作者：
Sato, Takashi;Sato, Maiko;Narimatsu, Hisashi
蛋白质O-连接的岩藻糖基化是一种不寻常的糖基化，与许多重要的生物学功能，如Notch信号相关。已报道了半胱氨酸结蛋白上合成O-岩藻糖基聚糖的两种岩藻糖基化途径，即表皮生长因子样（EGF样）结构域和血小板反应蛋白1型重复（TSR）结构域。我们在这里报告了一种新型β 1，3-葡萄糖基转移酶（β 3Glc-T）的分子克隆和表征，该转移酶在TSR结构域上合成Glc β 1，3 Fuc α结构。我们在数据库中发现了一个新的具有β 1，3-糖基转移酶（β 3GT）基序的糖基转移酶基因。在人胚肾293 T（HEK 293 T）细胞中表达的重组酶表现出对岩藻糖-α-对硝基苯基（Fuc α-pNp）的葡糖基转移酶活性。薄层层析（TLC）分析显示重组酶的产物迁移到与中国仓鼠卵巢（CHO）细胞的内源性β 3Glc-T的产物相同的位置。这两种产物均能被杏仁β-葡萄糖苷酶和木霉外切1，3-β-葡聚糖酶消化，表明其结构为Glc β 1-3Fuc。因此，我们将这种新型酶命名为β 3Glc-T。免疫染色显示，FLAG标记的β 3Glc-T是一种酶，通过滞留信号“REEL”（其为C末端的KDEL样序列）驻留在内质网（ER）中。在大肠杆菌中表达的TSR结构域首先被重组蛋白O-fucosyltransferase 2（POFUT 2）岩藻糖基化，然后它成为重组β 3Glc-T的受体底物，该受体底物可以明显地将Glc转移到岩藻糖基化的TSR结构域。我们的研究结果表明，一种新的糖基转移酶，β 3Glc-T，有助于延长的O-岩藻糖基聚糖，这是发生在TSR结构域。
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.