Isolation of Monomeric and Dimeric Secreted MD-2

Abstract

Potent cell activation by endotoxin requires sequential protein-endotoxin and protein-protein interactions involving lipopolysaccharide-binding protein, CD14, MD-2, and Toll-like receptor 4 (TLR4). MD-2 plays an essential role by bridging endotoxin (E) recognition initiated by lipopolysaccharide-binding protein and CD14 to TLR4 activation by presenting endotoxin as a monomeric E⅐MD-2 complex that directly and potently activates TLR4. Secreted MD-2 (sMD-2) exists as a mixture of monomers and multimers. Published data suggest that only MD-2 monomer can interact with endotoxin and TLR4 and support cell activation, but the apparent instability of MD-2 has thwarted efforts to more fully separate and characterize the individual species of sMD-2. We have taken advantage of the much greater stability of sMD-2 in insect culture medium to fully separate sMD-2 monomer from dimer by gel sieving chromatography. At low nanomolar concentrations, the sMD-2 monomer, but not dimer, reacted with a monomeric complex of E⅐sCD14 to form monomeric E⅐MD-2 and activate HEK293/ TLR4 cells. The monomer, but not dimer, also reacted with the ectodomain of TLR4 with an affinity comparable with the picomolar affinity of E⅐MD-2. These findings demonstrate directly that the monomeric form of sMD-2 is the active species both for reaction with E⅐CD14 and TLR4, as needed for potent endotoxin-induced TLR4 activation.Invasion by Gram-negative bacteria (GNB) 3 is specifically detected and responded to by mammals through mobilization of the innate immune system. In many strains and species of GNB, this process depends on host recognition of and response to the unique complex glycolipid, endotoxin (lipooligosaccharide (LOS) or lipopolysaccharide (LPS)), that comprises much of the outer leaflet of the outer membrane of GNB (1, 2). Minute amounts of endotoxin (E), presented either as an integral part of the outer membrane of GNB or as large aggregates of extracted and purified endotoxin, can stimulate pro-inflammatory responses (2-5). This sensitivity depends upon an ordered series of endotoxin-protein and protein-protein interactions that include the host proteins LPS-binding protein (LBP), membrane-bound or soluble (s)CD14, secreted or TLR4-associated MD-2, and TLR4 (6 -8). Engagement of endotoxin-rich membranes or isolated endotoxin aggregates by LBP facilitates the extraction of endotoxin monomers by CD14 to form monomeric E⅐CD14 complexes that are the most efficient substrate for transfer of endotoxin to MD-2 (4, 6, 9). The monomeric E⅐MD-2 complex is necessary and sufficient to induce TLR4-dependent cell activation by endotoxin (4, 6, 10). MD-2 associates noncovalently with the N-terminal ectodomain of TLR4 and plays a pivotal role not only in TLR4 activation but also in trafficking of TLR4 to the cell surface (7, 11-13). MD-2 also likely interacts transiently with CD14 facilitating transfer of endotoxin monomer from CD14 to MD-2 and, at high molar excess of CD14, reverse transfer of endotoxin from MD-2 to CD14 (14). Simultaneous engagement by MD-2...

High-affinity caspase-4 binding to LPS presented as high molecular mass aggregates or in outer membrane vesicles

“…Because of the amphipathic properties of endotoxin, endotoxin monomers do not exist stably in isolated form in aqueous environments but rather as a complex with a (host) protein that can extract an endotoxin monomer from an interface (e.g., CD14) or receive an endotoxin monomer that is bound to another protein molecule (e.g., MD-2 and MD-2/TLR4 from endotoxin m :CD14 and LOS m :albumin). 1,14,21,24 To test whether caspase-4 shows that interactive ability as well, we measured by co-capture assay interaction of [ 14 C]-LOS:sCD14 with His-tagged caspase-4. As shown in Figure 1H, caspase-4 neither binds to nor extracts LOS from LOS:sCD14.…”

Purified monomeric ligand.MD-2 complexes reveal molecular and structural requirements for activation and antagonism of TLR4 by Gram-negative bacterial endotoxins

“…4): i) “agonistindependent” interactions that are manifest irrespective of the ligand bound to wt MD-2 or even in the absence of bound ligand, reflecting the likely role of these sites and interactions in the genesis of MD-2/TLR4 heterodimers by cells producing both TLR4 and MD-2; and ii) “agonist-dependent” interactions between neighboring ligand.MD-2.TLR4 ternary complexes in which the MD-2 ligand acts with MD-2 as a TLR4 agonist (e.g., hexaacylated endotoxin.MD-2). The very similar dose-dependent inhibition of LOS.MD-2[ 125 I] binding by the various ligand.MD-2 complexes and by unlabeled monomeric MD-2 (39; Fig. 3), strongly suggests that the high affinity (pM) TLR4 binding of each of the ligand.MD-2 complexes corresponds to agonist-independent MD-2-TLR4 interactions within an individual ligand.MD-2/TLR4 ternary complex (i.e., intra -ternary complex interactions).…”

Emerging Principles Governing Signal Transduction by Pattern-Recognition Receptors: Table 1.

“…Thus, several proteins facilitate LPS-induced signal transduction by TLR4. These include LPS-binding protein (LBP), albumin, CD14, and MD2 (Gioannini et al 2004; Prohinar et al 2007; Teghanemt et al 2007, 2008; Resman et al 2009; Esparza et al 2012). LBP is capable of binding LPS from the outer membrane of Gram-negative bacteria by a process facilitated by albumin (Gioannini et al 2002).…”