Novel Roles in Human MD-2 of Phenylalanines 121 and 126 and Tyrosine 131 in Activation of Toll-like Receptor 4 by Endotoxin

Athmane Teghanemt,Fabio Re,Polonca Prohinar,Richard Widstrom,Theresa L Gioannini,Jerrold P Weiss

doi:10.1074/jbc.m705994200

Abstract

Potent mammalian cell activation by Gram-negative bacterial endotoxin requires sequential protein-endotoxin and protein-protein interactions involving lipopolysaccharide-binding protein, CD14, MD-2, and Toll-like receptor 4 (TLR4). TLR4 activation requires simultaneous binding of MD-2 to endotoxin (E) and the ectodomain of TLR4. We now describe mutants of recombinant human MD-2 that bind TLR4 and react with E.CD14 but do not support cellular responsiveness to endotoxin. The mutants F121A/K122A MD-2 and Y131A/K132A MD-2 react with E.CD14 only when co-expressed with TLR4. Single mutants K122A and K132A each react with E.CD14 +/- TLR4 and promote TLR4-dependent cell activation by endotoxin suggesting that Phe(121) and Tyr(131) are needed for TLR4-independent transfer of endotoxin from CD14 to MD-2 and also needed for TLR4 activation by bound E.MD-2. The mutant F126A MD-2 reacts as well as wild-type MD-2 with E.CD14 +/- TLR4. E.MD-2(F126A) binds TLR4 with high affinity (K(d) approximately 200 pm) but does not activate TLR4 and instead acts as a potent TLR4 antagonist, inhibiting activation of HEK/TLR4 cells by wild-type E.MD-2. These findings reveal roles of Phe(121) and Tyr(131) in TLR4-independent interactions of human MD-2 with E.CD14 and, together with Phe(126), in activation of TLR4 by bound E.MD-2. These findings strongly suggest that the structural properties of E.MD-2, not E alone, determine agonist or antagonist effects on TLR4.

Highlights

Potent mammalian cell activation by Gram-negative bacterial endotoxin requires sequential protein-endotoxin and proteinprotein interactions involving lipopolysaccharide-binding protein, CD14, MD-2, and Toll-like receptor 4 (TLR4)
Presence of Monomeric E1⁄7sCD14 during Secretion of MD-2 from HEK293T Cells Results in High Yields of Bioactive E1⁄7MD-2— We have previously demonstrated that bioactive monomeric endotoxin1⁄7MD-2 can be efficiently generated by incubation of monomeric endotoxin1⁄7sCD14 with conditioned medium harvested from insect cells expressing and secreting recombinant human MD-2 [7]
Kennedy et al [17] had previously shown that recombinant human MD-2 secreted into serum-free culture medium lost activity in a time (24 h) and temperature (37 °C)-dependent manner unless serum (LBP and sCD14) and LPS were present in the medium

Summary

Introduction

Potent mammalian cell activation by Gram-negative bacterial endotoxin requires sequential protein-endotoxin and proteinprotein interactions involving lipopolysaccharide-binding protein, CD14, MD-2, and Toll-like receptor 4 (TLR4). The potency of [3H]LOS1⁄7MD-2 derived from MD-2F121A/K122A and MD-2Y131A/K132A) and, for comparison, recombinant MD-2 produced by HEK293T cells or by insect wild-type MD-2, were co-expressed and secreted with the (High Five) cells was essentially the same (Fig. 1D), confirming TLR4 ectodomain into a culture medium “spiked” with the stability of mammalian cell-derived recombinant MD-2 [3H]LOS1⁄7sCD14.

Results

Conclusion