Molecular cloning and enzymatic characterization of a UDP-GalNAc:GlcNAcβ-R β1,4-N-acetylgalactosaminyltransferase from Caenorhabditis elegans
Molecular cloning and enzymatic characterization of a UDP-GalNAc:GlcNAcβ-R β1,4-N-acetylgalactosaminyltransferase from Caenorhabditis elegans
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DOI：
10.1074/jbc.m206112200
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发表时间：
2002-09-20
影响因子：
4.8
通讯作者：
Cummings, RD
中科院分区：
文献类型：
--
作者：
Kawar, ZS;Van Die, I;Cummings, RD
动物糖蛋白和糖脂的聚糖中常见的末端结构是乳糖胺序列 Galbeta4GlcNAc-R（LacNAc 或 LN）。存在于脊椎动物和无脊椎动物糖缀合物中的替代序列是 GalNAcbeta4GlcNAc-R（LacdiNAc 或 LDN）。虽然编码负责 LN 合成的 beta4GalTs 的基因已被报道，但负责 LDN 合成的 beta4GalNAcT 尚未鉴定。在这里，我们报告了秀丽隐杆线虫中编码 UDP-GalNAc:GlcNAcbeta-R beta1,4-N-乙酰半乳糖胺基转移酶 (Cebeta4GalNAcT) 的基因的鉴定，该基因合成 LDN 结构。 Cebeta4GalNAcT 是 beta4GalT 家族的成员，其 cDNA 预计编码 383 个氨基酸的 2 型膜糖蛋白。在 CHO-Lec8 细胞中表达的可溶性、表位标记的 Cebeta4GalNAcT 重组形式使用 UDP-GalNAc（而非 UDPGal）作为多种受体底物的供体具有活性，这些受体底物在 N-和 O-聚糖类型结构中含有末端 β-连接的 GlcNAc。产物的LDN结构通过共色谱法、真实标准品和H-1 NMR波谱进行验证。此外，表达 Cebeta4GalNAcT 的中国仓鼠卵巢 CHO-Lec8 和 CHO-Lec2 细胞在内源性糖蛋白 N-聚糖上获得了 LDN 决定簇，表明该酶作为真正的 beta4GalNAcT 在哺乳动物细胞中具有活性。这种新型酶的鉴定和可用性应该会增强我们对含有 LDN 的糖复合物的结构和功能的理解。
A common terminal structure in glycans from animal glycoproteins and glycolipids is the lactosamine sequence Galbeta4GlcNAc-R (LacNAc or LN). An alternative sequence that occurs in vertebrate as well as in invertebrate glycoconjugates is GalNAcbeta4GlcNAc-R (LacdiNAc or LDN). Whereas genes encoding beta4GalTs responsible for LN synthesis have been reported, the beta4GalNAcT(s) responsible for LDN synthesis has not been identified. Here we report the identification of a gene from Caenorhabditis elegans encoding a UDP-GalNAc:GlcNAcbeta-R beta1,4-N-acetylgalactosaminyltransferase (Cebeta4GalNAcT) that synthesizes the LDN structure. Cebeta4GalNAcT is a member of the beta4GalT family, and its cDNA is predicted to encode a 383-amino acid type 2 membrane glycoprotein. A soluble, epitope-tagged recombinant form of Cebeta4GalNAcT expressed in CHO-Lec8 cells was active using UDP-GalNAc, but not UDPGal, as a donor toward a variety of acceptor substrates containing terminal beta-linked GlcNAc in both N- and O-glycan type structures. The LDN structure of the product was verified by co-chromatography with authentic standards and H-1 NMR spectroscopy. Moreover, Chinese hamster ovary CHO-Lec8 and CHO-Lec2 cells expressing Cebeta4GalNAcT acquired LDN determinants on endogenous glycoprotein N-glycans, demonstrating that the enzyme is active in mammalian cells as an authentic beta4GalNAcT. The identification and availability of this novel enzyme should enhance our understanding of the structure and function of LDN-containing glycoconjugates.