Institute of Biochemistry, Faculty of Life Sciences, Leipzig University, 04103 Leipzig, Germany.
Institute of Bioanalytical Chemistry, Center for Biotechnology and Biomedicine, Leipzig University, 04103 Leipzig, Germany.
Molecules. 2020 Apr 24;25(8):1992. doi: 10.3390/molecules25081992.
Visceral adipose tissue derived serine protease inhibitor (vaspin) is a member of the serpin family and has been shown to have beneficial effects on glucose tolerance, insulin stability as well as adipose tissue inflammation, parameters seriously affected by obesity. Some of these effects require inhibition of target proteases such as kallikrein 7(KLK7) and many studies have demonstrated vaspin-mediated activation of intracellular signaling cascades in various cells and tissues. So far, little is known about the exact mechanism how vaspin may trigger these intracellular signaling events. In this study, we investigated and characterized the interaction of vaspin with membrane lipids and polyphosphates as well as their potential regulatory effects on serpin activity using recombinant vaspin and KLK7 proteins and functional protein variants thereof. Here, we show for the first time that vaspin binds to phospholipids and polyphosphates with varying effects on KLK7 inhibition. Vaspin binds strongly to monophosphorylated phosphatidylinositol phosphates (PtdInsP) with no effect on vaspin activation. Microscale thermophoresis (MST) measurements revealed high-affinity binding to polyphosphate 45 (K: 466 ± 75 nM) and activation of vaspin in a heparin-like manner. Furthermore, we identified additional residues in the heparin binding site in β-sheet A by mutating five basic residues resulting in complete loss of high-affinity heparin binding. Finally, using lipid overlay assays, we show that these residues are additionally involved in PtdInsP binding. Phospholipids play a major role in membrane trafficking and signaling whereas polyphosphates are procoagulant and proinflammatory agents. The identification of phospholipids and polyphosphates as binding partners of vaspin will contribute to the understanding of vaspins involvement in membrane trafficking, signaling and beneficial effects associated with obesity.
内脏脂肪组织来源的丝氨酸蛋白酶抑制剂（vaspin）是丝氨酸蛋白酶抑制剂家族的一员，已被证明对葡萄糖耐量、胰岛素稳定性以及脂肪组织炎症具有有益作用，这些参数受肥胖的严重影响。其中一些作用需要抑制靶蛋白酶，如激肽释放酶 7（KLK7），许多研究已经证明了 vaspin 在各种细胞和组织中激活细胞内信号级联的作用。到目前为止，人们对 vaspin 如何引发这些细胞内信号事件的具体机制知之甚少。在这项研究中，我们使用重组 vaspin 和 KLK7 蛋白及其功能蛋白变体，研究并表征了 vaspin 与膜脂质和多磷酸盐的相互作用，以及它们对丝氨酸蛋白酶抑制剂活性的潜在调节作用。在这里，我们首次表明 vaspin 与磷脂和多磷酸盐结合，对 KLK7 抑制有不同的影响。vaspin 与单磷酸化磷脂酰肌醇磷酸酯（PtdInsP）强烈结合，对 vaspin 激活没有影响。微量热泳（MST）测量显示与多磷酸盐 45（K：466±75 nM）具有高亲和力结合，并以肝素样方式激活 vaspin。此外，我们通过突变五个碱性残基，在β-折叠 A 中鉴定了肝素结合位点中的其他残基，导致高亲和力肝素结合完全丧失。最后，使用脂质覆盖测定法，我们表明这些残基还参与了 PtdInsP 的结合。磷脂在膜运输和信号转导中起主要作用，而多磷酸盐是促凝和促炎剂。鉴定磷脂和多磷酸盐作为 vaspin 的结合伴侣将有助于理解 vaspin 参与膜运输、信号转导以及与肥胖相关的有益作用。
