title: Production of recombinant human trimeric CD137L (4-1BBL). Cross-linking is essential to its T cell co-stimulation activity.
abstract: The interaction between 4-1BB ligand (CD137L), a member of the tumor necrosis factor superfamily, and its receptor 4-1BB provides a co-stimulatory signal for T lymphocyte proliferation and survival. However, the structure of 4-1BBL has not been thoroughly investigated, and none of the human recombinant 4-1BBL molecules available have been described as capable of co-stimulating T cells. The present work provides a model of the three-dimensional structure of the tumor necrosis factor homology domain of 4-1BBL and describes the production of a recombinant human soluble 4-1BBL whose originality lies in that it contains the whole extracellular tail preceding the tumor necrosis factor homology domain and an AviTag peptide (AviTag-4-1BBL) allowing enzymatic biotinylation and multimerization via streptavidin. We provide evidence that this chimeric protein exists as a homotrimer, whereas commercial FLAG-tagged 4-1BBL does not. This resulted in a much higher affinity for 4-1BB (1.2 nM) as compared with FLAG-4-1BBL (55.2 nM). We demonstrate that the single extracellular cysteine residue in the tail (Cys-51) could form a disulfide bond, both in our recombinant protein and in physiologically expressed 4-1BBL. The mutation of this cysteine residue exerted no effect on trimerization but increased the dissociation rate of AviTag-4-1BBL from 4-1BB. In its soluble form, AviTag-4-1BBL did not stimulate purified T cells but dramatically inhibited proliferation of peripheral blood mononuclear cells stimulated with anti-CD3 mAb. In contrast, a very significant co-stimulatory effect was observed on purified T cells once AviTag-4-1BBL was immobilized onto streptavidin beads. In addition, we show that the cross-linking of two trimeric AviTag-4-1BBL molecules was the minimum step required to elicit significant costimulatory activity.
authors.affiliation: INSERM U601, 9 quai Moncousu, 44035 Nantes Cedex, France.
authors.forename: Catherine
authors.initials: C
authors.lastname: Rabu
authors.forename: Agnès
authors.initials: A
authors.lastname: Quéméner
authors.forename: Yannick
authors.initials: Y
authors.lastname: Jacques
authors.forename: Klara
authors.initials: K
authors.lastname: Echasserieau
authors.forename: Patricia
authors.initials: P
authors.lastname: Vusio
authors.forename: François
authors.initials: F
authors.lastname: Lang
mesh_terms: D053330:4-1BB Ligand; D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000737:chemistry*; D019921:Biotinylation; D015153:Blotting, Western; D049109:Cell Proliferation; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004330:Drosophila; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007313:Insecta; D007963:Leukocytes, Mononuclear / Q000166:cytology; D008958:Models, Molecular; D015233:Models, Statistical; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017475:Receptors, Nerve Growth Factor / Q000737:chemistry*; D018124:Receptors, Tumor Necrosis Factor / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D019809:Streptavidin / Q000737:chemistry; D020349:Surface Plasmon Resonance; D013601:T-Lymphocytes / Q000166:cytology / Q000378:metabolism*; D013997:Time Factors; D053261:Tumor Necrosis Factor Receptor Superfamily, Member 9; D048069:Tumor Necrosis Factors / Q000737:chemistry* / Q000378:metabolism
chemical_list: D053330:4-1BB Ligand; D015703:Antigens, CD; D003432:Cross-Linking Reagents; D018076:DNA, Complementary; D004220:Disulfides; D010455:Peptides; D017475:Receptors, Nerve Growth Factor; D018124:Receptors, Tumor Necrosis Factor; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C505714:TNFRSF9 protein, human; C505974:TNFSF9 protein, human; D053261:Tumor Necrosis Factor Receptor Superfamily, Member 9; D048069:Tumor Necrosis Factors; D019809:Streptavidin; D003545:Cysteine