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International Immunology Advance Access published online on June 25, 2009
International Immunology, doi:10.1093/intimm/dxp059
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© The Japanese Society for Immunology. 2009. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Mutational analysis of Cys88 of Toll-like receptor 4 highlights the critical role of MD-2 in cell surface receptor expression

Chiaki Nishitani1,2,*, Motoko Takahashi1,2,*, Hiroaki Mitsuzawa1,2,3, Takeyuki Shimizu1,2, Shigeru Ariki1,2, Norio Matsushima4 and Yoshio Kuroki1,2

1 Department of Biochemistry, Sapporo Medical University School of Medicine, Sapporo, Japan
2 CREST, Japan Science and Technology, Kawaguchi, Japan
3 Department of Otolaryngology, Sapporo Medical University School of Medicine, Sapporo, Japan
4 School of Health Sciences, Sapporo Medical University, Sapporo, Japan

Correspondence to: C. Nishitani; E-mail: nchiaki{at}sapmed.ac.jp

The role of MD-2 in cell surface expression of Toll-like receptor (TLR) 4 has been controversial. The purposes of this study were to characterize the N-glycan of TLR4 and to investigate the roles of MD-2 in N-linked glycosylation and cell surface expression of TLR4. Lectin blot and cell surface biotinylation revealed that TLR4 exhibited the 110 kDa protein with high mannose type N-glycans and the 130 kDa protein with complex type N-glycans and that only the 130 kDa TLR4 with complex type N-glycans was expressed on the cell surface. The cells transfected with a mutant TLR4C88A alone expressed only the 110 kDa TLR4 with a high mannose type N-glycan, which did not appear on the cell surface. However, TLR4C88A acquired complex type N-glycans and was expressed on the cell surface when MD-2 was co-transfected. The amount of the 130 kDa TLR4C88A with complex type N-glycans expressed on the cell surface depended on that of MD-2 transfected. {alpha}-Mannosidase II inhibitor blocked the processing N-glycans to complex type, but TLR4 with high mannose type appeared on the cell surface, suggesting that TLR4 is destined to locate on the cell surface before processing N-glycans from a high mannose type to a complex type. From these results, we conclude that MD-2 is critical for cell surface expression of TLR4C88A. This study provides evidence that MD-2 possesses potential ability to play an essential role in cell surface expression of TLR4.

Keywords: innate immunity, LPS, MD-2, N-linked glycosylation, Toll-like receptor

* These authors contributed equally to this study.

Transmitting editor: S. Koyasu

Received 10 March 2009, accepted 22 May 2009.


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