Mutational analysis of Cys(88) of Toll-like receptor 4 highlights the critical role of MD-2 in cell surface receptor expression.
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. 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 TLR4(C88A). This study provides evidence that MD-2 possesses potential ability to play an essential role in cell surface expression of TLR4.
Nishitani
Chiaki
C
Department of Biochemistry, Sapporo Medical University School of Medicine, Sapporo, Japan. nchiaki@sapmed.ac.jp
Takahashi
Motoko
M
Mitsuzawa
Hiroaki
H
Shimizu
Takeyuki
T
Ariki
Shigeru
S
Matsushima
Norio
N
Kuroki
Yoshio
Y
D002460:Cell Line; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004252:DNA Mutational Analysis; D006031:Glycosylation; D006801:Humans; D051218:Lymphocyte Antigen 96 / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D011134:Polysaccharides / Q000378:metabolism; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism*
D051218:Lymphocyte Antigen 96; D011134:Polysaccharides; D051197:Toll-Like Receptor 4; D003545:Cysteine