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PNAS 110 (12): 4714-4719

Copyright © 2013 by the National Academy of Sciences.


BIOLOGICAL SCIENCES / MEDICAL SCIENCES

TLR4–MD-2 complex is negatively regulated by an endogenous ligand, globotetraosylceramide

Yuji Kondoa, Kazutaka Ikedab, Noriyo Tokudaa, Chiaki Nishitanic, Umeharu Ohtod, Sachiko Akashi-Takamurae, Yasutomo Itof, Makoto Uchikawag, Yoshio Kurokic, Ryo Taguchih, Kensuke Miyakee, Qing Zhanga, Keiko Furukawaa,h, and Koichi Furukawaa,1

aDepartment of Biochemistry II, and fDivision for Medical Research Engineering, Nagoya University Graduate School of Medicine, Nagoya 466-0065, Japan; bInstitute for Advanced Biosciences, Keio University, Tsuruoka 997-0052, Japan; cDepartment of Biochemistry, School of Medicine, Sapporo Medical University, Sapporo 060-8556, Japan; dLaboratory of Protein Structural Biology, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033, Japan; eDivision of Infectious Genetics, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan; g Japanese Red Cross Tokyo Blood Center, Tokyo 150-0012, Japan; and hDepartment of Biomedical Sciences and Health Science Hills, College of Life and Health Sciences, Chubu University, Kasugai 487-8501, Japan

Edited* by Sen-itiroh Hakomori, Pacific Northwest Research Institute, Seattle, WA, and approved February 11, 2013 (received for review October 23, 2012)

Abstract: Although endogenous ligands for Toll-like receptor (TLR)4–myeloid differentiation factor 2 (MD2) have not been well-understood, we here report that a globo-series glycosphingolipid, globotetraosylceramide (Gb4), attenuates the toxicity of lipopolysaccharides (LPSs) by binding to TLR4–MD-2. Because α1,4-galactosyltransferase (A4galt)-deficient mice lacking globo-series glycosphingolipids showed higher sensitivity to LPS than wild-type mice, we examined mechanisms by which globo-series glycosphingolipids attenuate LPS toxicity. Cultured endothelial cells lacking A4galt showed higher expression of LPS-inducible genes upon LPS treatment. In turn, introduction of A4galt cDNA resulted in the neo expression of Gb4, leading to the reduced expression of LPS-inducible genes. Exogenous Gb4 induced similar effects. As a mechanism for the suppressive effects of Gb4 on LPS signals, specific binding of Gb4 to the LPS receptor TLR4–MD-2 was demonstrated by coprecipitation of Gb4 with recombinant MD-2 and by native PAGE. A docking model also supported these data. Taken together with colocalization of TLR4–MD-2 with Gb4 in lipid rafts after LPS stimulation, it was suggested that Gb4 competes with LPS for binding to TLR4–MD-2. Finally, administration of Gb4 significantly protected mice from LPS-elicited mortality. These results suggest that Gb4 is an endogenous ligand for TLR4–MD-2 and is capable of attenuating LPS toxicity, indicating the possibility for its therapeutic application in endotoxin shock.

Key Words: innate immunity • sepsis


Author contributions: Y. Kondo, Y. Kuroki, R.T., K.M., Keiko Furukawa, and Koichi Furukawa designed research; Y. Kondo, K.I., N.T., C.N., U.O., S.A.-T., Y.I., M.U., and Q.Z. performed research; Y. Kondo, K.I., Y. Kuroki, R.T., and K.M. analyzed data; and Y. Kondo, Keiko Furukawa, and Koichi Furukawa wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1218508110/-/DCSupplemental.

1To whom correspondence should be addressed. E-mail: koichi{at}med.nagoya-u.ac.jp.



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