Abstract
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 (KD: 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.
Highlights
IntroductionThe visceral adipose tissue derived serine protease inhibitor (vaspin, SERPINA12 according to the serpin nomenclature [1]) was first identified in the Otsuka Long-Evans Tokushima fatty (OLETF) rat type 2 diabetes model [2]
The visceral adipose tissue derived serine protease inhibitor was first identified in the Otsuka Long-Evans Tokushima fatty (OLETF) rat type 2 diabetes model [2]
We investigated the interaction of vaspin with two other components of the cell surface, phosphatidylinositol phosphates (PtdInsP) and polyphosphates
Summary
The visceral adipose tissue derived serine protease inhibitor (vaspin, SERPINA12 according to the serpin nomenclature [1]) was first identified in the Otsuka Long-Evans Tokushima fatty (OLETF) rat type 2 diabetes model [2]. Vaspin serum levels are significantly higher in patients with obesity and type 2 diabetes [3,4] and multiple lines of evidence indicate a compensatory and counteracting role in obesity-related disorders and diseases (reviewed in [5]). Vaspin administration improves glucose tolerance and adipose tissue inflammation in obese mice [6,7]. Vaspin was shown to exhibit anti-apoptotic and anti-atherogenic potential in endothelial and smooth muscle cells [11,12,13] as well as anti-inflammatory effects in adipocytes [14,15] and skin [16]. Transgenic overexpression of vaspin protects mice from diet-induced adipose tissue inflammation while knockout mice exhibit deterioration of metabolic functions in obesity [17]. Further anti-inflammatory effects in the liver have been in part associated with vaspins interaction with the ER chaperon GRP78 [17]
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