Trimerization of murine TNF ligand family member LIGHT increases the cytotoxic activity against the FM3A mammary carcinoma cell line

Abstract

LIGHT is a member of the tumor necrosis factor ligand superfamily, which plays important roles in inflammatory and immune responses. LIGHT forms a membrane-anchored homotrimeric complex on the cell surface and is often processed as a soluble protein. Recombinant soluble human LIGHT produced by mammalian cells or Escherichia coli is functional at nanomolar concentrations. However, there is little information about the biological activity of mouse LIGHT (mLIGHT) because of the difficulty in producing bioactive soluble mLIGHT. In this study, recombinant trimeric soluble mLIGHT, or Foldon-mLIGHT, was produced by fusing mLIGHT with the trimerization domain foldon from bacteriophage T4 fibritin. Foldon-mLIGHT was secreted from 293F cells as a 68-kDa trimeric protein. The recombinant protein potently inhibited the growth of the FM3A mouse mammary carcinoma cell line with an IC(50) of 77 pM; however, the monomer or dimer forms of mLIGHT produced by E. coli or mammalian cell systems showed weak or no inhibitory activity. These data clearly indicated that trimerization of soluble mLIGHT is essential for its biological activity.

Therapeutic Blockade of LIGHT Interaction With Herpesvirus Entry Mediator and Lymphotoxin β Receptor Attenuates In Vivo Cytotoxic Allogeneic Responses

“…Taken together, the binding results indicate that in the mouse system, LIGHT indeed binds the relevant receptors HVEM and LTβR, which is in agreement with and extends conclusions of previous reports describing the binding interactions between LIGHT and its natural receptors (7,31). …”

“…We and others have evaluated a classical approach of fusing the extracellular region of mouse LIGHT either to Flag, the Fc fragment of the immunoglobulin heavy chain, or to an isoleucine zipper for the multimerization of the molecule (32), but none of these engineered genetic constructs succeeded in generating a protein with detectable binding affinity for mouse HVEM and LTβR. This was possible through a strategy that consisted of fusing the extracellular region of mouse LIGHT to a Flag-Foldon tag at the C-terminal site of this type II transmembrane protein (7). …”

“…It can even augment antitumor activity directly (7) or indirectly through enhancing CTL activity against tumor cells (4). In line with the costimulatory activity of LIGHT, constitutive transgenic expression of LIGHT under the control of a T cell-specific promoter led to chronic inflammation of mucosal tissues (8,9).…”

Applying γ‐Substituted Prolines in the Foldon Peptide: Polarity Contradicts Preorganization

“…In the last 15 years the foldon domain has been successfully used for tagging different natural trimeric structures, with examples including the stabilization of collagen‐mimicking peptides in the context of collagen research,8, 9 and the application of the foldon motif in viral structures and in interaction proteins such as the adenovirus fiber shaft fragment,10 the HSV‐1 gB ectodomain,11 and the rabies virus glycoprotein ectodomain,12 as well as its use in antibodies to improve their binding affinity and neutralizing potency 13. Besides increasing the stabilities of trimeric structures or functionality improvements, it is also possible to overcome the insolubility of systems by using the foldon motif 14…”

Selective Blockade of Herpesvirus Entry Mediator–B and T Lymphocyte Attenuator Pathway Ameliorates Acute Graft-versus-Host Reaction

“…Flag-tagged soluble human LIGHT (hereafter, Flag-shLIGHT) and Flag-Foldon–tagged soluble murine LIGHT (from now on, Flag-Foldon-smLIGHT) (provided by C.F. Ware, La Jolla, CA, and Y. Shintani, Osaka, Japan, respectively) were used for the binding experiments ( 25 ).…”