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Production of recombinant human trimeric CD137L (4-1BBL). Cross-linking is essential to its T cell co-stimulation activity.
J Biol Chem. 2005 Dec 16; 280(50):41472-81.JB

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+Show Affiliations

INSERM U601, 9 quai Moncousu, 44035 Nantes Cedex, France.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

16204238

Citation

Rabu, Catherine, et al. "Production of Recombinant Human Trimeric CD137L (4-1BBL). Cross-linking Is Essential to Its T Cell Co-stimulation Activity." The Journal of Biological Chemistry, vol. 280, no. 50, 2005, pp. 41472-81.
Rabu C, Quéméner A, Jacques Y, et al. Production of recombinant human trimeric CD137L (4-1BBL). Cross-linking is essential to its T cell co-stimulation activity. J Biol Chem. 2005;280(50):41472-81.
Rabu, C., Quéméner, A., Jacques, Y., Echasserieau, K., Vusio, P., & Lang, F. (2005). Production of recombinant human trimeric CD137L (4-1BBL). Cross-linking is essential to its T cell co-stimulation activity. The Journal of Biological Chemistry, 280(50), 41472-81.
Rabu C, et al. Production of Recombinant Human Trimeric CD137L (4-1BBL). Cross-linking Is Essential to Its T Cell Co-stimulation Activity. J Biol Chem. 2005 Dec 16;280(50):41472-81. PubMed PMID: 16204238.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Production of recombinant human trimeric CD137L (4-1BBL). Cross-linking is essential to its T cell co-stimulation activity. AU - Rabu,Catherine, AU - Quéméner,Agnès, AU - Jacques,Yannick, AU - Echasserieau,Klara, AU - Vusio,Patricia, AU - Lang,François, Y1 - 2005/10/04/ PY - 2005/10/6/pubmed PY - 2006/2/8/medline PY - 2005/10/6/entrez SP - 41472 EP - 81 JF - The Journal of biological chemistry JO - J. Biol. Chem. VL - 280 IS - 50 N2 - 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. SN - 0021-9258 UR - https://www.unboundmedicine.com/medline/citation/16204238/Production_of_recombinant_human_trimeric_CD137L__4_1BBL___Cross_linking_is_essential_to_its_T_cell_co_stimulation_activity_ L2 - http://www.jbc.org/cgi/pmidlookup?view=long&pmid=16204238 DB - PRIME DP - Unbound Medicine ER -