Purification of recombinant human cPLA2 gamma and identification of C-terminal farnesylation, proteolytic processing, and carboxymethylation by MALDI-TOF-TOF analysis.
Purification of recombinant human cPLA2 gamma and identification of C-terminal farnesylation, proteolytic processing, and carboxymethylation by MALDI-TOF-TOF analysis.
复制标题
通过 MALDI-TOF-TOF 分析纯化重组人 cPLA2 gamma 并鉴定 C 末端法尼基化、蛋白水解加工和羧甲基化。
DOI：
10.1021/bi034611q
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发表时间：
2003
期刊：
影响因子：
2.9
通讯作者：
Gross,RichardW
中科院分区：
文献类型：
--
作者：
Jenkins,ChristopherM;Han,Xianlin;Yang,Jingyue;Mancuso,DavidJ;Sims,HaroldF;Muslin,AnthonyJ;Gross,RichardW
胞浆型磷脂酶A2γ（cPLA 2 γ）是一种钙离子非依赖性的膜结合型磷脂酶A2，具有一个C-末端异戊烯基序（-CCLA），其共价结构不能仅从一级序列推断。因此，我们在Sf 9细胞中过表达含有N-末端His标签的人cPLA 2 γ（（His）6cPLA 2 γ），并通过连续固定化金属亲和和Mono Q柱层析定量溶解和纯化酶。最终的制备物在SDS-PAGE/银染后显示为单一的61 kDa条带，具有高溶血磷脂酶活性（50 μmol min-1 mg-1），并且被棕榈酰辅酶A抑制，但不水解。在不存在他汀类药物的情况下，用[3 H]-甲羟戊酸内酯对重组人cPLA 2 γ进行放射性标记，随后用雷尼镍裂解异戊二烯基，结果表明该酶仅被法尼基化，而不是香叶基香叶基化。通过基质辅助激光解吸/电离飞行时间/飞行时间（MALDI/TOF-TOF）质谱分析CNBr消化的cPLA 2 γ，证明Cys-538处存在法尼基部分，Cys 538 − Cys 539键断裂，以及所得C末端异戊二烯化半胱氨酸的羧甲基化。总的来说，这些结果描述了重组cPLA 2 γ的溶解和纯化至同质，并将cPLA 2 γ鉴定为法尼基化蛋白，其经历至少三个连续的翻译后修饰，这可能促进其靶向和与其膜底物的相互作用。
Cytosolic phospholipase A2γ (cPLA2γ) is a calcium-independent, membrane-associated phospholipase A2that possesses a C-terminal prenylation motif (-CCLA) whose covalent structure cannot be deduced from the primary sequence alone. Accordingly, we overexpressed human cPLA2γ containing an N-terminal His tag ((His)6cPLA2γ) in Sf9 cells and quantitatively solubilized and purified the enzyme by sequential immobilized metal affinity and Mono Q column chromatographies. The final preparation appeared as a single 61 kDa band after SDS−PAGE/silver-staining, possessed high lysophospholipase activity (50 μmol min-1mg-1), and was inhibited by, but did not hydrolyze, palmitoyl-CoA. Radiolabeling of recombinant human cPLA2γ with [3H]-mevalonolactone in the absence of statins and subsequent cleavage of prenyl groups with Raney nickel revealed that the enzyme is only farnesylated and is not geranylgeranylated. Analysis of CNBr-digested cPLA2γ by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI/TOF-TOF) mass spectrometry demonstrated the presence of a farnesyl moiety at Cys-538, cleavage of the Cys538−Cys539bond, and carboxymethylation of the resultant C-terminal prenylated cysteine. Collectively, these results describe the solubilization and purification of recombinant cPLA2γ to homogeneity and identify cPLA2γ as a farnesylated protein that undergoes at least three sequential posttranslational modifications that likely facilitate its targeting and interactions with its membrane substrates.