title: 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.
authors: Department of Molecular Internal Medicine, Medical Clinic and Polyclinic II, University of Wuerzburg, Wuerzburg 97070, Germany.
authors: D
authors: D
authors: Berg
authors: M
authors: M
authors: Lehne
authors: N
authors: N
authors: Müller
authors: D
authors: D
authors: Siegmund
authors: S
authors: S
authors: Münkel
authors: W
authors: W
authors: Sebald
authors: K
authors: K
authors: Pfizenmaier
authors: H
authors: H
authors: Wajant
mesh_terms: D000818:Animals; D003432:Cross-Linking Reagents / Q000494:pharmacology; D053222:Fas Ligand Protein / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D019169:Jurkat Cells; D051379:Mice; D050505:Mutant Proteins / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012995:Solubility / Q000187:drug effects; D013329:Structure-Activity Relationship; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000737:chemistry* / Q000378:metabolism*; D019063:Tenascin / Q000378:metabolism
chemical_list: D003432:Cross-Linking Reagents; D053222:Fas Ligand Protein; D050505:Mutant Proteins; D011993:Recombinant Fusion Proteins; D053221:TNF-Related Apoptosis-Inducing Ligand; D019063:Tenascin