Mutational analysis of Cys 88 of Toll-like receptor 4 highlights the critical role of MD-2 in cell surface receptor expression

Author： Nishitani Chiaki Takahashi Motoko Mitsuzawa Hiroaki Shimizu Takeyuki Ariki Shigeru Matsushima Norio Kuroki Yoshio

Publisher： Oxford University Press

ISSN： 1460-2377

Source： International Immunology, Vol.21, Iss.8, 2009-08, pp. : 925-934

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Abstract

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 TLR4^C88A alone expressed only the 110 kDa TLR4 with a high mannose type N-glycan, which did not appear on the cell surface. However, TLR4^C88A acquired complex type N-glycans and was expressed on the cell surface when MD-2 was co-transfected. The amount of the 130 kDa TLR4^C88A with complex type N-glycans expressed on the cell surface depended on that of MD-2 transfected. -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 TLR4^C88A. This study provides evidence that MD-2 possesses potential ability to play an essential role in cell surface expression of TLR4.