Investigating membrane-binding properties of lipoxygenases using surface plasmon resonance.
Investigating membrane-binding properties of lipoxygenases using surface plasmon resonance.
复制标题
使用表面等离子体共振研究脂氧合酶的膜结合特性。
DOI：
10.1016/j.bbrc.2023.05.066
复制
发表时间：
2023
影响因子：
3.1
通讯作者：
Garcia,BrandonL
中科院分区：
文献类型：
--
作者：
Rohlik,DeniseL;Patel,Ethan;Gilbert,NathanielC;Offenbacher,AdamR;Garcia,BrandonL
脂氧合酶（LOX）催化多不饱和脂肪酸的氧化并合成氧化脂素产物，其驱动植物和动物中重要的细胞信号传导过程。虽然已经有间接的证据提出了哺乳动物LOX与膜的相互作用，LOX-膜相互作用的分子细节的定量研究是缺乏的。在这里，我们模仿生物膜使用表面等离子体共振（SPR）传感器芯片衍生的2-D平面亲脂性锚（2D LP）捕获脂质体的不同磷脂组合物，自组装成脂质双层的SPR芯片。传感器芯片表面，然后用于研究模型LOX酶的膜结合特性。SPR结合试验显示可重现的和稳定的脂质体捕获到传感器芯片表面，允许详细表征LOX-膜相互作用。我们的研究表明，珊瑚8 R-LOX和人15-LOX-2的膜结合活性的钙依赖性。此外，我们的数据证实了这些LOX酶中的每一种中的关键膜插入环残基对于膜结合活性的重要性。利用模型植物和人类LOX的实验揭示了膜结合特异性的差异。我们的研究建立并验证了一个强大的基于SPR的平台，使用2D LP传感器芯片，允许在不同的实验条件下详细研究LOX-膜相互作用，包括改变膜组成。总的来说，这项研究提高了我们对LOX-膜相互作用特性的整体理解，并且我们基于SPR的方法具有未来用于开发基于LOX的疗法的潜力。
Lipoxygenases (LOXs) catalyze the oxidation of polyunsaturated fatty acids and synthesize oxylipin products that drive important cellular signaling processes in plants and animals. While there has been indirect evidence presented for the interaction of mammalian LOXs with membranes, a quantitative study of the molecular details of LOX-membrane interactions is lacking. Here, we mimicked biological membranes using surface plasmon resonance (SPR) sensor chips derivatized with 2-D planar lipophilic anchors (2D LP) to capture liposomes of varying phospholipid compositions that self-assemble into lipid bilayers on the SPR chip. The sensor chip surfaces were then used to investigate the membrane-binding properties of model LOX enzymes. SPR binding assays displayed reproducible and stable liposome capture to the sensor chip surface that allowed for the detailed characterization of LOX-membrane interactions. Our studies demonstrate a calcium-dependence for the membrane binding activities of coral 8R-LOX and human 15-LOX-2. Furthermore, our data confirm the importance of key membrane insertion loop residues in each of these LOX enzymes for membrane binding activity. Experiments utilizing model plant and human LOXs reveal differences in membrane-binding specificities. Our study establishes and validates a robust SPR-based platform using 2D LP sensor chips that allows for the detailed study of LOX-membrane interactions under different experimental conditions, including altered membrane compositions. Collectively, this investigation improves our overall understanding of LOX-membrane interaction properties, and our SPR-based approach holds potential for future use in the development of LOX-based therapeutics.