LRP1 is the cell-surface endocytosis receptor for vaspin in adipocytes.

Vaspin is a serine protease inhibitor that protects against adipose tissue inflammation and insulin resistance, two key drivers of adipocyte dysfunction and metabolic disorders in obesity. Inhibition of target proteases such as KLK7 has been shown to reduce adipose tissue inflammation in obesity, while vaspin binding to cell surface GRP78 has been linked to reduced obesity-induced ER stress and insulin resistance in the liver. However, the molecular mechanisms by which vaspin directly affects cellular processes in adipocytes remain unknown. Using fluorescently labeled vaspin, we found that vaspin is rapidly internalized by mouse and human adipocytes, but less efficiently by endothelial, kidney, liver, and neuronal cells. Internalization occurs by active, clathrin-mediated endocytosis, which is dependent on vaspin binding to the LRP1 receptor, rather than GRP78 as previously thought. This was demonstrated by competition experiments and RNAi-mediated knock-down in adipocytes and by rescuing vaspin internalization in LRP1-deficient Pea13 cells after transfection with a functional LRP1 minireceptor. Vaspin internalization is further increased in mature adipocytes after insulin-stimulated translocation of LRP1. Although vaspin has nanomolar affinity for LRP1 clusters II-IV, binding to cell surface heparan sulfates is required for efficient LRP1-mediated internalization. Native, but not cleaved vaspin, and also vaspin polymers are efficiently endocytosed, and ultimately targeted for lysosomal degradation. Our study provides mechanistic insight into the uptake and degradation of vaspin in adipocytes, thereby broadening our understanding of its functional repertoire. We hypothesize the vaspin-LRP1 axis to be an important mediator of vaspin effects not only in adipose tissue but also in other LRP1-expressing cells.

Development of a synthesis strategy for sulfamethoxazole derivatives and their coupling with hydrogel microparticles.

Sulfonamides were the first synthetic antibiotics broadly applied in veterinary and human medicine. Their increased use over the last few decades and limited technology to degrade them after entering the sewage system have led to their accumulation in the environment. A new hydrogel microparticle based biosensing application for sulfonamides is developed to overcome existing labour-intensive, and expensive detection methods to analyse and quantify their environmental distribution. This biosensing assay is based on the soft colloidal probe principle and requires microparticle functionalization strategies with target molecules. In this study, we developed a step-wise synthesis approach for sulfamethoxazole (SMX) derivatives in high yield, with SMX being one of the most ubiquitous sulfonamide antibiotics. After de novo synthesis of the SMX derivative, two coupling schemes to poly(ethylene glycol) (PEG) hydrogel microparticles bearing maleimide and thiol groups were investigated. In one approach, we coupled a cysteamine linker to a carboxyl group at the SMX derivative allowing for subsequent binding via the thiol-functionality to the maleimide groups of the microparticles in a mild, high-yielding thiol-ene "click" reaction. In a second approach, an additional 1,11-bis(maleimido)-3,6,9-trioxaundecane linker was coupled to the cysteamine to target the hydrolytically more stable thiol-groups of the microparticles. Successful PEG microparticle functionalization with the SMX derivatives was proven by IR spectroscopy and fluorescence microscopy. SMX-functionalized microparticles will be used in future applications for sulfonamide detection as well as for pull-down assays and screenings for new sulfomethoxazole binding targets.

Membrane Phospholipids and Polyphosphates as Cofactors and Binding Molecules of SERPINA12 (vaspin).

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 ß-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.