Vickers P J, Adam M, Charleson S, Abramovitz M, O'Neill G, Mancini J A
Department of Pharmacology, Merck Frosst Centre for Therapeutic Research, Pointe Claire-Dorva, Quebec, Canada.
J Lipid Mediat. 1993 Mar-Apr;6(1-3):31-42.
5-Lipoxygenase-activating protein (FLAP) plays an essential role in cellular leukotriene (LT) synthesis and represents the target of three classes of LT biosynthesis inhibitors. We have taken three approaches to localize regions of FLAP involved in the binding of these inhibitors. A comparison of the amino acid sequences of FLAP from eight mammalian species identifies regions of the protein which are highly conserved and consequently may be involved in functional and inhibitor binding properties of the protein. Conversely, amino acids not conserved amongst these species are unlikely to play an essential role in inhibitor binding. Immunoprecipitation of peptide fragments of FLAP cross-linked to photoaffinity analogues of LT biosynthesis inhibitors following site-specific peptide cleavage indicates that the inhibitor attachment site is amino-terminal to 72Trp. Taken together, the cross-species analysis and photoaffinity labelling studies suggest a region within the first hydrophilic loop of FLAP which may be important for inhibitor binding. Site-directed mutagenesis of human FLAP followed by the analysis of FLAP mutants in a radioligand binding assay was used to more accurately define critical amino acid residues within this region. Mutagenesis studies reveal that mutants containing deletions of amino acids in regions of FLAP not conserved between species retain the ability to specifically bind inhibitors. Furthermore, mutants containing deletions in a highly conserved region of the protein (residues 42-61) do not bind inhibitors. These studies have therefore localized specific amino acids of FLAP which are essential for inhibitor binding. The roles that these amino acids play in inhibitor binding and may play in 5-LO activation is under investigation.
5-脂氧合酶激活蛋白（FLAP）在细胞白三烯（LT）合成中起关键作用，是三类LT生物合成抑制剂的作用靶点。我们采用了三种方法来定位FLAP中与这些抑制剂结合相关的区域。对来自八个哺乳动物物种的FLAP氨基酸序列进行比较，确定了该蛋白中高度保守的区域，因此这些区域可能与蛋白的功能及抑制剂结合特性有关。相反，这些物种中不保守的氨基酸不太可能在抑制剂结合中起关键作用。在进行位点特异性肽段切割后，对与LT生物合成抑制剂的光亲和类似物交联的FLAP肽段进行免疫沉淀，结果表明抑制剂附着位点在72位色氨酸的氨基端。综合来看，跨物种分析和光亲和标记研究表明FLAP的第一个亲水环内的一个区域可能对抑制剂结合很重要。通过对人FLAP进行定点诱变，然后在放射性配体结合试验中分析FLAP突变体，以更准确地确定该区域内的关键氨基酸残基。诱变研究表明，在物种间不保守的FLAP区域中含有氨基酸缺失的突变体仍具有特异性结合抑制剂的能力。此外，在蛋白高度保守区域（第42 - 61位残基）含有缺失的突变体不结合抑制剂。因此，这些研究确定了FLAP中对抑制剂结合至关重要的特定氨基酸。这些氨基酸在抑制剂结合中以及可能在5-脂氧合酶（5-LO）激活中所起的作用正在研究中。
