Identification of Tumor Necrosis Factor (TNF) Amino Acids Crucial for Binding to the Murine p75 TNF Receptor and Construction of Receptor-selective Mutants

Abstract

The bioactivity of tumor necrosis factor (TNF) is mediated by two TNF receptors (TNF-Rs), more particularly TNF-RI and TNF-RII. Although human TNF (hTNF) and murine TNF (mTNF) are very homologous, hTNF binds only to mTNF-RI. By measuring the binding of a panel of mTNF/hTNF chimeras to both mTNF-R, we pinpointed the TNF region that mediates the interaction with mTNF-RII. Using site-specific mutagenesis, we identified amino acids 71-73 and 89 as the main interacting residues. Mutein hTNF-S71D/T72Y/H73⌬/T89E interacts with both types of mTNF-R and is active in CT6 cell proliferation assays mediated by mTNF-RII. Mutein mTNF-D71S/Y72T/⌬73H/E89T binds to mTNF-RI only and is no longer active on CT6 cells. However, the L929s cytotoxicity of this mutein (an effect mediated by mTNF-RI triggering) was also 100-fold lower than that of wild-type mTNF due to enhanced dissociation during incubation at subnanomolar concentrations. The additional mutation of amino acid 102, resulting in the mutein mTNF-D71S/Y72T/⌬73H/E89T/P102Q, restored the trimer stability, which led to an enhanced specific activity on L929s cells. Hence the specific activity of a TNF species is governed not only by its receptor binding characteristics but also by its trimer stability after incubation at subnanomolar concentrations. In conclusion, the mutation of TNF amino acids 71-73, 89, and 102 is sufficient to obtain a mTNF mutein selective for mTNF-RI and a hTNF mutein that, unlike wild-type hTNF, also acts on mTNF-RII. Tumor necrosis factor (TNF)1 is a pleiotropic cytokine with a wide range of biological activities including cytotoxicity, immune cell proliferation, and mediation of inflammatory responses (1-3). It exerts both direct and indirect antitumor effects on a number of tumors in vivo. However, its therapeutic application in the treatment of tumors is severely hampered by pathogenic side effects such as hypotension and liver toxicity (4). The multiple biological effects of TNF are mediated by two cell surface TNF receptors (TNF-Rs), namely TNF-RI and TNF-RII (5, 6). These receptors bind in the groove regions between TNF subunits; hence one trimeric TNF molecule binds three receptor molecules (7). Clustering of surface-bound receptors initiates a signaling cascade in the cell. Moreover, receptorspecific muteins of hTNF have been obtained by mutating amino acids located at the intersubunit grooves (8 -10).Although murine TNF (mTNF) and human TNF (hTNF) are 79% identical at the amino acid level, hTNF interacts with mTNF-RI but not with mTNF-RII. Due to the high homology, chimeric TNF genes can be obtained by exchanging homologous regions between the hTNF and mTNF genes. Bacterial expression of such in-frame chimeric genes results in chimeric TNF subunits that are able to trimerize into bioactive molecules. These chimeric proteins are subsequently used to localize the epitopes for species-specific TNF monoclonal antibodies (11).We created and characterized different mTNF/hTNF chimeras to identify the region(s) responsible for interaction with mTNF-RI...

Differential effects of peripheral and brain tumor necrosis factor on inflammation, sickness, emotional behavior and memory in mice

“…Using IP human TNF, we could provide supportive evidence for TNF peripheral-brain passage: human and murine TNF protein structures are similar (Brouckaert et al, 1986;Kuprash et al, 1999) and should exhibit similar peripheral-brain passage (Banks et al, 1995), although human TNF exhibits higher binding to TNF receptor 1 (Ameloot et al, 2001) which might enhance its passage. A single ip TNF injection increased whole brain Tnf mRNA and TNF protein expression at 1 h post-injection (Qin et al, 2007).…”

Targeted Bioactivity of Membrane-Anchored TNF by an Antibody-Derived TNF Fusion Protein

“…Nevertheless, it is well known from the literature that tumor-bearing animals are more susceptible to TNF-mediated kachexia (38). The animal model was not designed primarily to demonstrate antitumor efficacy, because rhTNF fails to induce murine TNF-R2 signaling (39), but the cooperative signaling through both TNF-Rs represents the therapeutic principle of the anti-FAP-TNF dimer, exerting its antitumor activity in a membrane TNF-like manner. Therefore, drug-related therapeutic events should be expected via TNF-R1 (35).…”

The Design and Characterization of Receptor-selective APRIL Variants

“…Receptor-selective TNF family ligands have been previously generated by employing rational design-or directed evolutionbased methods. Rational design approaches have been previously used to design receptor-selective TNF-␣ variants (35) and TRAIL variants (22,23,26,36). Directed evolution methods employing phage display were also used to generate receptorselective TRAIL variants (37), a receptor-selective TNF␣ antagonist (38), and a LIGHT/LT␤ variant that does not bind DcR3 (39).…”