Molecular cloning and expression of GalNAc alpha 2,6-sialyltransferase.
Molecular cloning and expression of GalNAc alpha 2,6-sialyltransferase.
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GalNAc α 2,6-唾液酸转移酶的分子克隆和表达。
DOI：
10.1016/s0021-9258(17)42272-1
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发表时间：
1994
期刊：
影响因子：
--
通讯作者：
S. Tsuji
中科院分区：
文献类型：
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作者：
N. Kurosawa;T. Hamamoto;Y. Lee;T. Nakaoka;N. Kojima;S. Tsuji
编码GalNAc α 2,6-唾液酸转移酶(EC 2.4.99.3)的cDNA克隆已经使用从先前克隆的酶的保守氨基酸序列获得的序列信息从鸡胚cDNA文库中分离出来。 cDNA序列包含编码566个氨基酸的开放阅读框，推导的氨基酸序列与鸡胚Galβ1,4GlcNAcα2,6-唾液酸转移酶的氨基酸序列有12%的同一性。该酶的一级结构表明了一个假定的结构域结构，与其他糖基转移酶一样，由一个短的 NH2 末端胞质结构域、一个信号膜锚定结构域、一个蛋白水解敏感的茎区域和一个大的 COOH 末端活性结构域组成。通过构建重组唾液酸转移酶证实了该酶的身份，其中 NH2 末端部分（232 个氨基酸残基）被免疫球蛋白信号序列取代。该重组体在 COS-7 细胞中的表达导致具有催化活性且可溶形式的酶分泌到培养基中。表达的酶仅对脱唾液酸粘蛋白和(脱唾液酸)胎球蛋白表现出活性，对测试的其他糖蛋白和糖脂底物没有检测到显着活性。从用该酶重新唾液酸化的脱唾液酸粘蛋白获得的 14C-唾液酸化二醇与 NeuAc α 2,6-GalNAc-ol 和 GlcNAc beta 1,3(NeuAc α 2,6) GalNAc-ol 相同。合成的 GalNAc-SerNAc 也可作为 α 2,6-唾液酸化的受体。这些结果清楚地表明所表达的酶是GalNAcα2,6-唾液酸转移酶。
cDNA clones encoding GalNAc alpha 2,6-sialyltransferase (EC 2.4.99.3) have been isolated from chick embryo cDNA libraries using sequence information obtained from the conserved amino acid sequence of the previously cloned enzymes. The cDNA sequence included an open reading frame coding for 566 amino acids, and the deduced amino acid sequence showed 12% identity with that of Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase from chick embryo. The primary structure of this enzyme suggested a putative domain structure, like that in other glycosyltransferases, consisting of a short NH2-terminal cytoplasmic domain, a signal-membrane anchor domain, a proteolytically sensitive stem region, and a large COOH-terminal active domain. The identity of this enzyme was confirmed by the construction of a recombinant sialyltransferase in which the NH2-terminal part (232 amino acid residues) was replaced with the immunoglobulin signal sequence. The expression of this recombinant in COS-7 cells resulted in secretion of a catalytically active and soluble form of the enzyme into the medium. The expressed enzyme exhibited activity toward only asialomucin and (asialo)fetuin, no significant activity being detected toward the other glycoprotein and glycolipid substrates tested. 14C-Sialylated glycols obtained from asialomucin re-sialylated with this enzyme were identical to NeuAc alpha 2,6-GalNAc-ol and GlcNAc beta 1,3(NeuAc alpha 2,6) GalNAc-ol. Synthetic GalNAc-SerNAc also served as an acceptor for alpha 2,6-sialylation. These results clearly showed that the expressed enzyme is GalNAc alpha 2,6-sialyltransferase.