Background/Purpose: We previously reported that Progranulin (PGRN) bound to TNF receptors (TNFR) and was therapeutic in inflammatory arthritis (Tang, W., et al, Science, 2011). PGRN is highly cysteine-rich glycoprotein, and contains a number of internal disulfide bonds, which are critical for maintaining the proper folding and confirmation of this protein. The objective of this project is 1) to determine whether PGRN also form a complex with TNFR in immune cells, and whether PGRN affects the binding of TNFα to immune cells, 2) to compare the effects of folding of PGRN on its binding to TNFR and Sortilin, a neuronal receptor known to interact with PGRN as well, and 3) to dissect and identify the domains of TNFR required for PGRN interaction.

Methods: Co-Immunoprecipitation (CoIP) assay and TNF-α blocking assay with flow cytometry were performed to examine the association of PGRN and TNFR in immune cells and its inhibition of TNF binding to cell surface; Various protein-protein interaction assays, including Solid phase binding, Surface Plasmon Resonance, yeast-two hybrid and GST pull-down, were performed to compare the binding activity of various sources of PGRN, to determine the effects of folding of PGRN to TNFR, and to identify the domains of TNFR responsible for binding to PGRN; Differentiation of bone marrow-derived macrophages were used to measure the biological activity of PGRN with ELISA and real-time PCR.

Results: Our previous study showed that PGRN bound to TNFR in chondrocytes. Here we found that PGRN also associated with TNFR2 in splenocytes in a Co-IP assay, and PGRN blocked the binding of TNFα to splenocytes in a dose-dependent manner.

Proper folding and modification of PGRN appear to be essential for its binding to TNFR, as DTT treatment, which is known to disturb the formation of disulfide bonds in PGRN and in turn affects its folding, abolished its binding to TNFR. Interestingly, the binding of PGRN to Sortilin was actually enhanced by DTT treatment. This is probably due to the fact that the C-terminal last three amino acids (DLL) of PGRN, known to be required for Sortilin binding, are exposed in the unfolded PGRN and become more easily accessible to Sortilin. Additionally, in vitro direct protein interaction assays, including Solid phase and Surface Plasmon Resonance, and cell-based functional assays revealed 1) that some commercial PGRNs are of poor quality, and that 2) the selection of chips in Surface Plasmon Resonance is important for demonstrating the high affinity binding of PGRN to TNFR.

Yeast two hybrid assays with numerous deletion mutants of TNFR extracellular domain demonstrated that CRD2 and CRD3 of TNFR are important for the interaction with PGRN, similar to its binding to their canonical ligand TNFα. This finding was confirmed with in vitro GST pulldown and solid phase assay with recombinant proteins.

Conclusion: These findings provide the molecular basis underlying PGRN-mediated anti-inflammatory activity in various autoimmune diseases and conditions.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/progranulin-directly-binds-to-the-crd-2-and-crd3-of-tnfr-extracellular-domains/