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J. Biol. Chem., Vol. 281, Issue 50, 38322-38329, December 15, 2006

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Toll-like Receptor 4 Region Glu²⁴–Lys⁴⁷ Is a Site for MD-2 Binding

IMPORTANCE OF CYS²⁹ AND CYS^40*

Chiaki Nishitani, Hiroaki Mitsuzawa, Hitomi Sano, Takeyuki Shimizu, Norio Matsushima^¶, and Yoshio Kuroki¹

From the Department of Biochemistry, School of Medicine and ^¶School of Health Sciences, Sapporo Medical University, Sapporo 060-8556, Japan and CREST, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan

Toll-like receptor 4 (TLR4) is a signaling receptor for lipopolysaccharide (LPS), but its interaction with MD-2 is required for efficient responses to LPS. Previous studies with deletion mutants indicate a critical role of the amino-terminal TLR4 region in interaction with MD-2. However, it is uncertain which region in the TLR4 molecule directly binds to MD-2. The purpose of this study was to determine a critical stretch of primary sequence in the TLR4 region that directly binds MD-2 and is critical for LPS signaling. The synthetic TLR4 peptide corresponding to the TLR4 region Glu²⁴–Lys⁴⁷ directly binds to recombinant soluble MD-2 (sMD-2). The TLR4 peptide inhibited the binding of a recombinant soluble form of the extracellular TLR4 domain (sTLR4) to sMD-2 and significantly attenuated LPS-induced NF-B activation and IL-8 secretion in wild type TLR4-transfected cells. Reduction and S-carboxymethylation of sTLR4 abrogated its association with sMD-2. The TLR4 mutants, TLR4^C29A, TLR4^C40A, and TLR4^C29A,C40A, were neither co-precipitated with MD-2 nor expressed on the cell surface and failed to transmit LPS signaling. These results demonstrate that the TLR4 region Glu²⁴–Lys⁴⁷ is a site for MD-2 binding and that Cys²⁹ and Cys⁴⁰ within this region are critical residues for MD-2 binding and LPS signaling.

Received for publication, July 20, 2006 , and in revised form, October 5, 2006.

^* This work was supported in part by a Grant-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture, Japan. 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 To whom correspondence should be addressed: Dept. of Biochemistry, Sapporo Medical University School of Medicine, South-1 West-17, Chuo-ku, Sapporo 060-8556, Japan. Tel.: 81-11-611-2111; Fax: 81-11-611-2236; E-mail: kurokiy{at}sapmed.ac.jp.

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