The exon-3-encoded domain of IL-15ralpha contributes to IL-15 high-affinity binding and is crucial for the IL-15 antagonistic effect of soluble IL-15Ralpha.
We previously showed that a natural soluble form of interleukin-15 (IL-15) Ralpha corresponding to the full-length ectodomain of IL-15Ralpha behaved as a potent antagonist of IL-15 action through IL-15Ralpha/beta/gamma, whereas a recombinant soluble IL-15Ralpha sushi domain did not, but instead acted as an agonist of IL-15 action through IL-15Rbeta/gamma. In order to determine precisely the molecular basis governing these antagonistic versus agonistic actions, we compared the binding properties and biological effects of recombinant soluble IL-15Ralpha (sIL-15Ralpha) species containing the sushi domain and different remaining parts of the ectodomain. We first demonstrate that the exon-3-encoded domain and, more particularly, its N-terminal 13-amino-acid (aa) peptide are important, in addition to the adjacent exon-2-encoded sushi domain, for the stabilization of the high-affinity IL-15.IL-15Ralpha complex by slowing down its dissociation rate and by contributing to about 10-20% of the free energy of interaction. We next show that all sushi-containing sIL-15Ralpha are agonists on IL-15Rbeta/gamma, coordinately increasing IL-15 binding and IL-15-induced proliferation. Their agonistic potencies are proportional to their respective affinities for IL-15. We then show that the antagonistic effect of sIL-15Ralpha in the context of IL-15Ralpha/beta/gamma is due to the 13-aa peptide that creates a sterical constraint impeding the binding of the sIL-15Ralpha.IL-15 complex to the membrane-anchored IL-15Ralpha/beta/gamma. In the frame of the soluble IL-15Ralpha sushi domain-IL-15 fusion protein that contains the 13-aa peptide, this constraint is alleviated as a result of a conformational effect due to the covalent linking of the 13-aa peptide to the N-terminus of IL-15. The soluble IL-15Ralpha sushi domain-IL-15 fusion protein is therefore able to bind and activate both the IL-15Rbeta/gamma and the IL-15Ralpha/beta/gamma receptors.
Bouchaud
Grégory
G
INSERM, U892, Centre de Recherche en Cancérologie Nantes/Angers, Groupe Cytokines et Récepteurs, Nantes, France; Université de Nantes, IFR26, Nantes, France. bouchaud-g@univ-nantes.fr
Garrigue-Antar
Laure
L
Solé
Véronique
V
Quéméner
Agnès
A
Boublik
Yvan
Y
Mortier
Erwan
E
Perdreau
Harmonie
H
Jacques
Yannick
Y
Plet
Ariane
A
D001667:Binding, Competitive / Q000187:drug effects; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D005091:Exons / Q000235:genetics*; D006801:Humans; D019409:Interleukin-15 / Q000378:metabolism* / Q000494:pharmacology; D053720:Interleukin-15 Receptor alpha Subunit / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D053646:Interleukin-2 Receptor beta Subunit / Q000378:metabolism; D008954:Models, Biological; D050505:Mutant Proteins / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012995:Solubility / Q000187:drug effects; D013329:Structure-Activity Relationship
D019409:Interleukin-15; D053720:Interleukin-15 Receptor alpha Subunit; D053646:Interleukin-2 Receptor beta Subunit; D050505:Mutant Proteins; D011993:Recombinant Fusion Proteins