cDNA cloning and expression of bovine UDP-N-acetylglucosamine:: α1,3-D-mannoside β1,4-N-acetylglucosaminyltransferase IV
cDNA cloning and expression of bovine UDP-N-acetylglucosamine:: α1,3-D-mannoside β1,4-N-acetylglucosaminyltransferase IV
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DOI：
10.1074/jbc.273.19.11556
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发表时间：
1998-05-08
影响因子：
4.8
通讯作者：
Takeuchi, M
中科院分区：
文献类型：
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作者：
Minowa, MT;Oguri, S;Takeuchi, M
UDP-N-乙酰葡糖胺：α 1，3-D-甘露糖苷β 1，4-N-乙酰葡糖胺转移酶（GnT-IV）是生产三触角和四触角Asn连接糖链的必需酶之一。最近，我们成功地从牛小肠中纯化了GnT-IV。根据纯化的牛GnT-IV酶的部分氨基酸序列，从牛小肠中克隆了其cDNA。开放阅读框长1，605个碱基对，该序列在COS-7细胞中瞬时表达时产生GnT-IV活性。虽然推导的氨基酸序列与其他已知的N-乙酰氨基葡萄糖基转移酶（GnTs）没有任何显著的同源性，但疏水性图谱显示典型的II型跨膜蛋白结构，这是许多糖基转移酶所共有的。纯化的GnT-IV的N-末端氨基酸序列分析显示，在纯化过程中，包括跨膜区在内的92个氨基酸被截短。在三个潜在的N-糖基化位点中，Asn-458在纯化的酶中实际上被糖基化，尽管该N-糖基化位点可以被消除而不降低GnT-IV活性。在N和C末端的连续缺失证明GnT-IV的催化结构域位于酶的中心区域。GnT-IV mRNA水平与酶活性在各种牛组织测试。
UDP-N-acetulglucosamine:alpha 1,3-D-mannoside beta 1,4-N-acetylglucosaminyltransferase (GnT-IV) is one of the essential enzymes in the production of tri-and tetra-antennary Asn-linked sugar chains. Recently, we have successfully purified GnT-IV from bovine small intestine. Based on the partial amino acid sequence of the purified bovine GnT-IV enzyme, its cDNA has been cloned from bovine small intestine. The open reading frame is 1,605 base pairs long, and this sequence produced GnT-IV activity on transient expression in COS-7 cells. Although the deduced amino acid sequence does not have any significant homology with other known N-acetylglucosaminyltransferases (GnTs), the hydrophobicity profile showed a typical type II transmembrane protein structure, which is common to many glycosyltransferases, N-terminal amino acid sequencing of the purified GnT-IV revealed that 92 amino acids, including a transmembrane region, were truncated during purification. Of the three potential N-glycosylation sites Asn-458 was actually glycosylated in the purified enzyme, although this N-glycosylation site could be abolished without any reduction in GnT-IV activity. Serial deletions at both the N and C termini proved that the catalytic domain of GnT-IV is located in the central region of the enzyme. The GnT-IV mRNA level correlated with enzymatic activity in the various bovine tissues tested.