Abstract
Progranulin (PGRN) is a secreted anti-inflammatory protein which can be processed by neutrophil proteases to various granulins. It has been reported that at least a significant portion of the anti-inflammatory effects of PGRN is due to direct high affinity binding to tumor necrosis factor receptor-1 (TNFR1) and TNFR2 and inhibition of tumor necrosis factor (TNF)-induced TNFR1/2 signaling. Two studies failed to reproduce the interaction of TNFR1 and TNFR2 with PGRN, but follow up reports speculated that this was due to varying experimental circumstances and/or the use of PGRN from different sources. However, even under consideration of these speculations, there is still a striking discrepancy in the literature between the concentrations of PGRN needed to inhibit TNF signaling and the concentrations required to block TNF binding to TNFR1 and TNFR2. While signaling events induced by 0.2–2 nM of TNF have been efficiently inhibited by low, near to equimolar concentrations (0.5–2.5 nM) of PGRN in various studies, the reported inhibitory effects of PGRN on TNF-binding to TNFR1/2 required a huge excess of PGRN (100–1,000-fold). Therefore, we investigated the effect of PGRN on TNF binding to TNFR1 and TNFR2 in highly sensitive cellular binding studies. Unlabeled TNF inhibited >95% of the specific binding of a Gaussia princeps luciferase (GpL) fusion protein of TNF to TNFR1 and TNFR2 and blocked binding of soluble GpL fusion proteins of TNFR1 and TNFR2 to membrane TNF expressing cells to >95%, too. Purified PGRN, however, showed in both assays no effect on TNF–TNFR1/2 interaction even when applied in huge excess. To rule out that tags and purification- or storage-related effects compromise the potential ability of PGRN to bind TNF receptors, we directly co-expressed PGRN, and as control TNF, in TNFR1- and TNFR2-expressing cells and looked for binding of GpL-TNF. While expression of TNF strongly inhibited binding of GpL-TNF to TNFR1/2, co-expression of PGRN had not effect on the ability of the TNFR1/2-expressing cells to bind TNF.
Highlights
Tumor necrosis factor-alpha (TNF) is a pleiotropic cytokine which has crucially implicated in a variety of immunoregulatory processes in innate and adaptive immunity, but has manifold roles in the control of tissue homeostasis [1]
In surface plasmon resonance (SPR) experiments, in which PGRN binding to the soluble monomeric extracellular domains of tumor necrosis factor receptor-1 (TNFR1) and TNF receptor-2 (TNFR2) monomers adsorbed to a sensor chip has been investigated, Tang et al identified PGRN as a high affinity ligand of TNFR1 and TNFR2 with KDvalues of 1.77 and 1.52 nM [7]
Since PGRN was reported to act as a competitive inhibitor of sTNF binding, we evaluated the ability of recombinant purified PGRN to inhibit binding of a Gaussia princeps luciferase (GpL) fusion protein of soluble TNF (GpL-TNF) to TNFR1 and TNFR2
Summary
Tumor necrosis factor-alpha (TNF) is a pleiotropic cytokine which has crucially implicated in a variety of immunoregulatory processes in innate and adaptive immunity, but has manifold roles in the control of tissue homeostasis [1]. TNF is initially expressed as a trimeric type II transmembrane protein (memTNF) from which a soluble trimeric molecule (sTNF) is released by cleavage by the protease TNF converting enzyme (TACE) [1]. Both sTNF and memTNF bind with high affinity to two types of receptors, tumor necrosis factor receptor-1 (TNFR1) and TNF receptor-2 (TNFR2). The crystallographic structures of TNF in complex with the ectodomain of TNFR2 and of LTα in complex with the ectodomain of TNFR1 have been solved Both structures show that three molecules of TNFR1 or TNFR2 bind into the three grooves formed by three protomers of a ligand trimer [3, 4]. Five TNF-neutralizing biologicals have been approved, there are still enormous preclinical and clinical efforts to develop new drugs (antibodies, ligand mutants, small-molecules) inhibiting TNF, TNFR1, or TNFR2
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