No abstract

Toll-like receptor 4 (TLR4) recognizes lipopolysaccharide (LPS). MD-2 is associated with TLR4 and imparts LPS responsiveness to it. Little is known, however, as to whether MD-2 directly regulates LPS recognition by TLR4. To address the issue, we took advantage of a species-specific pharmacology of lipid IVa, an analogue of lipid A. Lipid IVa acted agonistically on mouse (m) TLR4/MD-2 but not on human (h) TLR4/MD-2. Lipid IVa antagonized the agonistic effect of lipid A on hTLR4/MD-2. We examined the chimeric complex consisting of mTLR4 and hMD-2 to ask whether species specificity is conferred by TLR4 or MD-2. hMD-2 was clearly distinct from mMD-2 in the way of influencing LPS recognition by mTLR4. hMD-2 conferred on mTLR4 responsiveness to lipid A but not to lipid IVa. Moreover, lipid IVa acted as a lipid A antagonist on mTLR4 that is associated with hMD-2. Collectively, MD-2 directly influences the fine specificity of TLR4.

No abstract

Lipopolysaccharides (LPS, endotoxin) represent a major virulence factor of Gram-negative bacteria, which can cause septic shock in mammals, including man. The lipid anchor of LPS to the bacterial outer membrane, lipid A, exhibits a peculiar chemical structure, harbours the`endotoxic principle' of LPS and is also responsible for the expression of pathophysiological effects. Chemically modified lipid A can be endotoxically inactive, but may express strong antagonistic activity against endotoxically active LPS. By applying orientation measurements with attenuated total reflectance (ATR) infrared spectroscopy on hydrated lipid A samples, we show here that these different biological activities are directly correlated to the intrinsic conformation of lipid A. Bisphosphorylhexaacyl lipid A molecules with an asymmetric (4/2) distribution of the acyl chains linked to the diglucosamine backbone have a large tilt angle (. 45 8) of the diglucosamine backbone with respect to the membrane surface, a conical molecular shape (larger cross-section of the hydrophobic than the hydrophilic moiety), and are endotoxically highly active. Monophosphoryl hexaacyl lipid A has a smaller tilt angle, and the conical shape is less expressed in favour of a more cylindrical shape. This correlates with decreasing endotoxic activity. Pentaand tetraacyl lipid A or hexaacyl lipid A with a symmetric acyl chain distribution (3/3) have a small tilt angle (, 25 8) and a cylindrical shape and are endotoxically inactive, but may be antagonistic.Keywords: lipid A; endotoxic shape; IR ATR; agonism; antagonism.Lipopolysaccharides (LPS), as major amphiphilic components of the outer leaflet of the outer membrane of Gram-negative bacteria, exert in isolated form a variety of biological activities in mammals and are, thus, called endotoxins [1]. Chemically, LPS consists of a hydrophilic heteropolysaccharide, which is covalently linked to a hydrophobic lipid portion, termed lipid A, which anchors the molecule to the outer leaflet of the outer membrane. As free lipid A has been shown to be responsible for the biological activity of LPS in most in vitro and in vivo test systems, it has been termed the`endotoxic principle' of endotoxin [2]. The specific requirements for endotoxin to be biologically active are still only partly defined. It has been shown, however, that for the full expression of biological activity, lipid A must possess a particular chemical composition and primary structure similar to that found in enterobacterial strains. Thus, for example, lipid A from the biologically most potent LPS of the deep rough (Re) mutant strain Escherichia coli F515 consists of a b-1,6-linked d-glucosamine (GlcN) disaccharide carrying two negatively charged phosphates and six saturated fatty acids in a defined asymmetric distribution; four at the nonreducing and two at the reducing GlcN. Variation of this composition (such as a reduction of the number of charges or the number of acyl chains), a change in their distribution or degree of saturation results in a dramatic ...

No abstract