Multiple splice variants of the human calcium-independent phospholipase A2 and their effect on enzyme activity
Multiple splice variants of the human calcium-independent phospholipase A2 and their effect on enzyme activity
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DOI：
10.1074/jbc.273.1.207
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发表时间：
1998-01-02
影响因子：
4.8
通讯作者：
Kennedy, BP
中科院分区：
文献类型：
--
作者：
Larsson, PKA;Claesson, HE;Kennedy, BP
最近，从中国仓鼠卵巢细胞以及小鼠和大鼠中克隆了一种新的Ca2+非依赖性磷脂酶a (2) (iPLA(2))，该酶含有一个c端脂肪酶基元和8个n端锚蛋白重复序列。在这篇报告中，我们描述了人类iPLA(2) cDNA的克隆及其在b细胞中的表达，并表明iPLA(2)基因经历了广泛的选择性剪接，产生了多种异构体，这有助于控制iPLA(2)活性的新机制。全长cDNA克隆编码一个806个氨基酸的蛋白，计算分子量为88 kDa，该蛋白包含一个脂肪酶基序，GXSXG和锚蛋白重复序列，如鼠和啮齿动物形式的酶所描述的，但在8个锚蛋白重复序列的最后一个额外的54个氨基酸丰富的插入（残基395-449）。此外，至少三个额外的异构体很可能是由于选择性剪接被鉴定。其中一个在表达序列标签数据库中作为部分cDNA存在，与iPLA(2)相似，但在脂肪酶活性位点之后终止，另外两个异构体只包含iPLA(2)锚蛋白重复序列（锚蛋白-iPLA(2)-1和-2），锚蛋白重复序列参与蛋白质-蛋白质相互作用，因为纯化的iPLA(2)酶以270-350 kDa的多聚体复合物存在。仅锚蛋白-iPLA(2)序列的表达表明它们也可能与iPLA(2)寡聚物相互作用，并可能调节PLA(2)的活性。将人iPLA(2) cDNA转染到COS细胞中，细胞裂解液中钙非依赖性PLA(2)活性显著增加，而转染锚蛋白-iPLA(2)-1 cDNA后，未观察到高于背景的活性。然而,co-transfection ankyrin-iPLA(2) 1和iPLA(2)互补导致活动减少2倍与iPLA(2),类似的co-transfection ankyrin-iPLA (2) 1 cDNA cPLA (2) cDNA没有影响解放军(2)活动,这些结果表明,ankyrin-iPLA(2)序列可以作为负调节iPLA(2)活动的可变剪接iPLA(2)基因可以直接影响酶活性的衰减。
Recently, the cloning of a novel Ca2+-independent phospholipase A(2) (iPLA(2)) from Chinese hamster ovary cells as well as from mouse and rat sources containing a C-terminal lipase motif and eight N-terminal ankyrin repeats has been described. In this report we describe the cloning of the human iPLA(2) cDNA and its expression in B-cells and show that the iPLA(2) gene undergoes extensive alternative splicing generating multiple isoforms that contribute to a novel mechanism to control iPLA(2) activity. The full-length cDNA clone encodes a 806-amino acid protein with a calculated molecular mass of 88 kDa, The protein contains a lipase motif, GXSXG, and ankyrin repeats, as described for the hamster and rodent forms of the enzyme but has an additional 54-amino acid proline rich insertion in the last of the eight ankyrin repeats (residues 395-449). Furthermore, at least three additional isoforms most likely due to alternative splicing were identified. One that is present as a partial cDNA in the expressed sequence tag data base is similar to iPLA(2) but terminates just after the lipase active site, and two other-isoforms contain only the iPLA(2) ankyrin repeat sequence (ankyrin-iPLA(2)-1 and -2), Ankyrin repeats are involved in protein-protein interactions and because the purified iPLA(2) enzyme exists as a multimeric complex of 270-350 kDa, the expression of just the ankyrin-iPLA(2) sequence suggested that these may also interact with the iPLA(2) oligomeric complexes and perhaps modulate PLA(2) activity. Transfection of the human iPLA(2) cDNA into COS cells resulted in a substantial increase in calcium-independent PLA(2) activity in cell lysate, No activity above background was observed following ankyrin-iPLA(2)-1 cDNA transfection. However, co-transfection of the ankyrin-iPLA(2)-1 and the iPLA(2) cDNAs resulted in a 2-fold reduction in activity compared with iPLA(2) alone, A similar co-transfection of ankyrin-iPLA(2)-1 cDNA with the cPLA(2) cDNA had no effect on PLA(2) activity, These results suggest that the ankyrin-iPLA(2) sequence can function as a negative regulator of iPLA(2) activity and that the alternative splicing of the iPLA(2) gene can have a direct effect on the attenuation of enzyme activity.