Enforced covalent trimerization increases the activity of the TNF ligand family members TRAIL and CD95L

Abstract

Variants of human TRAIL (hTRAIL) and human CD95L (hCD95L), encompassing the TNF homology domain (THD), interact with the corresponding receptors and stimulate CD95 and TRAILR2 signaling after cross-linking. The murine counterparts (mTRAIL, mCD95L) showed no or only low receptor binding and were inactive/poorly active after cross-linking. The stalk region preceding the THD of mCD95L conferred secondary aggregation and restored CD95 activation in the absence of cross-linking. A corresponding variant of mTRAIL, however, was still not able to activate TRAIL death receptors, but gained good activity after cross-linking. Notably, disulfide-bonded fusion proteins of the THD of mTRAIL and mCD95L with a subdomain of the tenascin-C (TNC) oligomerization domain, which still assembled into trimers, efficiently interacted with their cognate cellular receptors and robustly stimulated CD95 and TRAILR2 signaling after secondary cross-linking. Introduction of the TNC domain also further enhanced the activity of THD encompassing variants of hTRAIL and hCD95L. Thus, spatial fixation of the N-terminus of the THD appears necessary in some TNF ligands to ensure proper receptor binding. This points to yet unanticipated functions of the stalk and/or transmembrane region of TNF ligands for the functionality of these molecules and offers a broadly applicable option to generate recombinant soluble ligands of the TNF family with superior activity. Ligands of the tumor necrosis factor (TNF) family fulfill crucial roles in the immune system, but have also been implicated in the development of epithelial and endothelial structures. 1 TNF family ligands are primarily expressed as trimeric type II transmembrane proteins and are often processed into soluble variants that are also organized as trimers. 1,2 Although shedding of some TNF ligands does not interfere with their capability to activate their corresponding receptors and might be even important for their physiological function, other TNF ligands become inactivated by proteolytic processing. 2 Soluble TNF ligands that are not or only poorly active still interact with their cognate receptors. For example, the soluble forms of TNF, CD95L, TRAIL, and CD40L interact with TNFR2, CD95, TRAILR2, and CD40, respectively, but do not or only poorly activate signaling by these receptors. [3][4][5][6] Notably, inactive or poorly active soluble TNF ligands can be converted into highly active molecules by artificially increasing their avidity. For example, soluble flag-tagged variants of TNF, CD95L, TRAIL, and CD40L stimulate robust signaling by TNFR2, CD95, TRAILR2, and CD40, respectively, provided they were cross-linked with the Flag-specific mAb M2. Likewise, hexameric and dodecameric fusion proteins of soluble CD95L and soluble CD40L as well as non-specifically aggregated preparations of TNF ligands produced in E. coli display high activity. [6][7][8] The structural hallmark of the ligands of the TNF family is the carboxy-terminal 'TNF homology domain', which is part of both the transmembrane and s...

Tetravalent Antibody–scTRAIL Fusion Proteins with Improved Properties

“…Recently, we further demonstrated that conversion of the monovalent scFv-scTRAIL fusion protein into a bivalent diabodyscTRAIL fusion protein-exhibiting two antigen-binding sites and two scTRAIL moieties-further increased cytotoxic activity against tumor cells without increasing cytotoxicity toward normal cells (11). This is most likely due to the fact that the dimeric scTRAIL fusion proteins are capable of mimicking the activity of membrane-displayed, that is, multivalent, TRAIL, leading to increased receptor signal complex formation and activation (12,13). The dimerization of scTRAIL in the diabody-scTRAIL fusion protein is mediated by the antibody moiety, thus combining targeting and dimerization in the same module.…”

Studies of Binding of Tumor Necrosis Factor (TNF)-like Weak Inducer of Apoptosis (TWEAK) to Fibroblast Growth Factor Inducible 14 (Fn14)

“…Using the flanking EcoRI (5Ј) and XbaI (3Ј) sites, the TWEAK domain-containing DNA fragment of FLAG-TWEAK-pCR3 was subcloned in pCR3 derivatives with an N-terminal GpL-FLAG epitope-TNC or a LeaderhIgG1(Fc)-FLAG epitope encoding cassette to allow expression of secretable GpL-FLAG-TNC-TWEAK and Fc-FLAG-TWEAK. TNC refers to the trimerization domain of chicken tenascin C, which serves to stabilize the trimeric assembly of ligands of the TNF family and thus reduces formation of inactive misfolded protein species (26,27). The G. princeps luciferase (GpL)-encoding DNA fragment used was a synthetic product of the complete reading frame of the GpL gene with human codon usage.…”

“…1, A-C). We further included a small trimerization domain from tenascin C (ϳ3 kDa) to stabilize the trimeric assembly of TWEAK and to minimize the formation of inactive misfolded ligand species (26,27). After 4 -6 days of cultivation, cell culture supernatants of Hek293 cells stably transfected with expression plasmids encoding secretable GpL-FLAG-TNC-TWEAK and FLAG-TWEAK contained typically 40 -80 g of the recombinant protein per 15-cm cell culture Petri dish.…”

Signaling Active CD95 Receptor Molecules Trigger Co-translocation of Inactive CD95 Molecules into Lipid Rafts

“…36), Metridia longa luciferase (MlL), secreted alkaline phosphatase (SEAP), yellow fluorescent protein (YFP), and an O 6 -alkylguanine-DNA-alkyltransferase-based SNAP tag as reporter domains. We recently observed that the integrity of the trimeric nature of some soluble TNF ligands benefits from the N-terminal fusion of the small (ϳ3 kDa) trimerization domain of tenascin-C (37,38). We, therefore, included this domain also in our fusion proteins between the reporter domain (including a FLAG tag) and the THD.…”