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This Article Full Text Full Text (PDF) All Versions of this Article: jimmunol.0901544v1 183/8/5138    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Google Scholar Articles by Vasl, J. Articles by Jerala, R. PubMed PubMed Citation Articles by Vasl, J. Articles by Jerala, R.

Novel Roles of Lysines 122, 125, and 58 in Functional Differences between Human and Murine MD-2¹

Joica Val,^* Alja Oblak,^* Theresa L. Gioannini, Jerrold P. Weiss, and Roman Jerala^2*

^*Department of Biotechnology, National Institute of Chemistry, Hajdrihova 19, 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; and Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, Slovenia

The MD-2/TLR4 complex provides a highly robust mechanism for recognition and response of mammalian innate immunity to Gram-negative bacterial endotoxins. Despite overall close structural and functional similarity, human (h) and murine (m) MD-2 show several species-related differences, including the ability of hMD-2, but not mMD-2, to bind endotoxin (E) in the absence of TLR4. Wild-type mMD-2 can support TLR4-dependent cell activation by E only when mMD-2 and mTLR4 are coexpressed in the same cell. However, replacement of Glu122, Leu125, and/or Asn58 of mMD-2 with the corresponding residues (lysines) of hMD-2 was sufficient to yield soluble extracellular MD-2 that reacted with monomeric E · sCD14 complex to form extracellular monomeric E · MD-2 that activated cells expressing TLR4 without MD-2. Moreover, in contrast to wild-type mMD-2, double and triple mMD-2 mutants also supported E-triggered signaling in combination with human TLR4. Conversely, a K125L mutant of hMD-2 reacted with E · CD14 and activated TLR4 only when coexpressed with TLR4, and not when secreted without TLR4. These findings reveal novel roles of lysines 122, 125, and 58 in human MD-2 that contribute to the functional differences between human and murine MD-2 and, potentially, to differences in the sensitivity of humans and mice to endotoxin.

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 The project was supported by grants from the Slovenian research agency (J1-9795, J1-2271, P4-0176), a bilateral Slovenian-USA collaborative grant, and grants from the National Institute of Allergy and Infectious Disease (AI05732; to J.W.) and the Veterans’ Administration (to T.L.G.).

² Address correspondence and reprint requests to Prof. Roman Jerala, National Institute of Chemistry, Hajdrihova 19, PO Box 660, Ljubljana, Slovenia. E-mail address: roman.jerala{at}ki.si

³ Abbreviations used in this paper: E, endotoxin; LOS, lipooligosaccharide; LBP, LPS-binding protein; s, soluble; HEK, human embryonic kidney; HSA, human serum albumin; wt, wild type; RLA, relative luciferase activity; TLR4_ecd, TLR4 ectodomain.