Tetramerization domain of human butyrylcholinesterase is at the C-terminus
Tetramerization domain of human butyrylcholinesterase is at the C-terminus
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DOI：
10.1042/bj3270747
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发表时间：
1997-11-01
影响因子：
4.1
通讯作者：
Lockridge, O
中科院分区：
文献类型：
--
作者：
Blong, RM;Bedows, E;Lockridge, O
人血清中的丁酰胆碱酯酶（BChE）主要由四聚体组成。然而，在中国仓鼠卵巢 (CHO) 细胞中表达的重组 BChE 由大约 1 个组成。 55% 二聚体、10-30% 四聚体和 15-40% 单体。为了确定单体种类的起源，我们将FLAG表位（表位标签，氨基酸序列DYKDDDDK）添加到酶的C末端，并在CHO细胞中表达BChE-FLAG。我们发现分泌的活性单体失去了 FLAG 表位，表明这些单体是通过 C 末端二聚体或四聚体的蛋白水解产生的。为了估计可以从 C 末端删除而不失去 BChE 活性的氨基酸数量，我们表达了删除突变体。我们发现从 C 末端删除多达 50 个氨基酸会产生活性单体，但删除 51 个氨基酸会破坏 BChE 活性并导致无活性的蛋白质保留在细胞内。从 N 末端删除 8 个或更多氨基酸也会导致细胞内保留无活性的蛋白质。单体BChE具有野生型K-m和K-cat值（丁酰硫代胆碱为8μM和24000min(-1)）并且显示出底物激活。 Cys-571-->Ala 突变体虽然不能形成链间二硫键，但具有与重组野生型 BChE 几乎相同数量的四聚体 BChE。这些结果支持以下结论：BChE 的四聚化结构域位于 C 末端，在末端 50 个氨基酸内，并且链间二硫键对于四聚化不是必需的。分子模型表明，四聚化结构域是一个四螺旋束，通过七个保守芳香族氨基酸的相互作用来稳定。
Butyrylcholinesterase (BChE) in human serum consists predominantly of tetramers. Recombinant BChE, however, expressed in Chinese hamster ovary (CHO) cells, consists of approx. 55% dimers, 10-30% tetramers and 15-40% monomers. To determine the origin of the monomer species we added the FLAG epitope (epitope tag, amino acid sequence DYKDDDDK) to the C-terminus of the enzyme, and expressed BChE-FLAG in CHO cells. We found that secreted, active monomers had lost their FLAG epitope, suggesting that the monomers were made by proteolysis of dimers or tetramers at the C-terminus. To estimate the number of amino acids that could be deleted from the C-terminus without losing BChE activity, we expressed deletion mutants. We found that deletion of up to 50 amino acids from the C-terminus yielded active monomers, but that deletion of 51 amino acids destroyed BChE activity and caused the inactive protein to remain within the cell. Deletion of eight or more amino acids from the N-terminus also resulted in inactive protein that remained inside the cell. Monomeric BChE had wild-type K-m and K-cat values (8 mu M and 24000 min(-1) for butyrylthiocholine) and showed substrate activation. The Cys-571-->Ala mutant, though incapable of forming the interchain disulphide bond, had nearly the same amount of tetrameric BChE as recombinant wild-type BChE. These results support the conclusion that the tetramerization domain of BChE is at the C-terminus, within the terminal 50 amino acids, and that the interchain disulphide bond is not essential for tetramerization. Molecular modelling suggested that the tetramerization domain was a four-helix bundle, stabilized by interactions of seven conserved aromatic amino acids.