An isoleucine-zipper motif enhances costimulation of human soluble trimeric GITR ligand.

Glucocorticoid-induced tumor-necrosis factor receptor (GITR) and its ligand, GITRL, play significant roles in regulating immune responses. It is clear that human soluble GITRL (hsGITRL) transduces signal activity through multiple oligomerization states. To develop human soluble trimeric GITRL protein as a potential therapeutic target, we explored the link of the isoleucine-zipper (ILZ) motif to the N-terminus of the human soluble GITRL with two leucine sequences. hsGITRL, with the ILZ motif (ILZ-hsGITRL), was firstly expressed in Escherichia coli, which exhibited a predominant trimer when identified by Sephadex G-100 filtration and non-reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The significantly higher biological activity of the ILZ-hsGITRL compared with hsGITRL was confirmed by CD4(+) T proliferation, interferon-gamma (IFN-gamma) secretion and binding activity assay. To reveal and compare the underlying mechanisms, the level of extracellular signal-regulated kinase-1/2 (ERK1/2) phosphorylation was examined, indicating that ILZ-hsGITRL induced more persistent and stronger ERK1/2 activation than hsGITRL. In conclusion, the incorporation of an ILZ motif could markedly improve the costimulation of hsGITRL.