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ica Va
l*
,
* Department of Biotechnology, National Institute of Chemistry, Ljubljana, Slovenia;
Inflammation Program, Department of Microbiology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242; and
Veterans Affairs Medical Center, Iowa City, IA 52246
MD-2 is an essential component of endotoxin (LPS) sensing, binding LPS independently and when bound to the ectodomain of the membrane receptor TLR4. Natural variation of proteins involved in the LPS-recognition cascade such as the LPS-binding protein, CD14, and TLR4, as well as proteins involved in intracellular signaling downstream of LPS binding, affect the cellular response to endotoxin and host defense against bacterial infections. We now describe the functional properties of two nonsynonymous coding polymorphisms of MD-2, G56R and P157S, documented in HapMap. As predicted from the MD-2 structure, the P157S mutation had little or no effect on MD-2 function. In contrast, the G56R mutation, located close to the LPS-binding pocket, significantly decreased cellular responsiveness to LPS. Soluble G56R MD-2 showed markedly reduced LPS binding that was to a large degree rescued by TLR4 coexpression or presence of TLR4 ectodomain. Thus, cells that express TLR4 without MD-2 and whose response to LPS depends on ectopically produced MD-2 were most affected by expression of the G56R variant of MD-2. Coexpression of wild-type and G56R MD-2 yielded an intermediate phenotype with responses to LPS diminished to a greater extent than that resulting from expression of the D299G TLR4 polymorphic variant.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This research was supported by the Slovenian Research Agency and by the Slovenian-USA bilateral collaborative grant and, in part, by U.S. Public Health Service Grant AI59372 (to J.P.W.) and a Veterans’ Administration Merit Review grant (to T.L.G.).
2 Address correspondence and reprint requests to Dr. Roman Jerala, National Institute of Chemistry, Hajdrihova 19, PO Box 660, Ljubljana 1000, Slovenia. E-mail address: roman.jerala{at}ki.si
3 Abbreviations used in this paper: LOS, lipooligosaccharides; agg, aggregate; cm, conditioned medium; ECD, ectodomain; HSA, human serum albumin; LBP, LPS-binding protein; RLA, relative luciferase activity; s, soluble; S-LPS, smooth LPS; TBSTT, TBS containing 0.05% Tween 20 and 0.2% Triton X-100; wt, wild type.
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