MD-2 Is Necessary for the Toll-Like Receptor 4 Protein To Undergo Glycosylation Essential for Its Translocation to the Cell Surface

Abstract

MD-2 has been reported to be required for the translocation of the Toll-like receptor 4 (TLR4) to the cell surface. However, the mechanism by which MD-2 promotes TLR4 translocation is unknown. We identified the presence of two forms of TLR4 with different molecular masses (approximately 110 and 130 kDa) when TLR4 was expressed together with MD-2. Expressing TLR4 alone produced only the 110-kDa form. Using a membrane-impermeable biotinylation reagent, we found that only the 130-kDa form of TLR4 was expressed on the cell surface. When a cellular extract prepared from cells expressing TLR4 and MD-2 was treated with N-glycosidase, the two forms of TLR4 converged into a single band whose size was smaller than the 110-kDa form of TLR4. Mutation of TLR4 at Asn 526 or Asn 575 resulted in the disappearance of the 130-kDa form and prevented TLR4 from being expressed on the cell surface without affecting the ability of TLR4 to associate with MD-2. These results indicate that TLR4 is able to undergo multiple glycosylations without MD-2 but that the specific glycosylation essential for cell surface expression requires the presence of MD-2.

Carbon Monoxide Suppresses Membrane Expression of TLR4 via Myeloid Differentiation Factor-2 in βTC3 Cells

“…Studies have shown that the binding of TLR4 to MD-2 is not sufficient for the translocation of TLR4 to the membrane surface; the glycosylation of Asn 526 or Asn 575 is necessary for translocation (16). We thus evaluated whether the glycosylation of TLR4 is required for its membrane trafficking.…”

“…It has been reported that TLR4 can undergo multiple glycosylations without MD-2 but the specific glycosylation essential for cell surface transport requires the presence of MD-2 (16,38). To elucidate whether MD-2 is required for the glycosylation of TLR4 and its consequent membrane trafficking in bTC3 cells, we separated glycosylated and unglycosylated TLR4 using WGA agarose in cells in which MD-2 was knocked down by siRNA.…”

“…However, how TLR4 is distributed and regulated in b cells and how CO exposure suppresses TLR4 activation in those cells remain largely unknown. Two forms of TLR4 exist in the transfected human embryonic kidney 293 cells: the immature form with a molecular mass of 110 kDa and the mature form with a molecular mass of 130 kDa (16). Immature TLR4 resides in the endoplasmic reticulum (ER) and the Golgi apparatus and migrates to the cell membrane after maturation, a process that requires glycosylation at Asn 526 or Asn 575 (16).…”

“…Two forms of TLR4 exist in the transfected human embryonic kidney 293 cells: the immature form with a molecular mass of 110 kDa and the mature form with a molecular mass of 130 kDa (16). Immature TLR4 resides in the endoplasmic reticulum (ER) and the Golgi apparatus and migrates to the cell membrane after maturation, a process that requires glycosylation at Asn 526 or Asn 575 (16). The molecular mechanisms by which TLR4 stays in the Golgi/ER and migrates to the cell surface are currently unknown.…”

Isolation of Monomeric and Dimeric Secreted MD-2

“…The monomeric E⅐MD-2 complex is necessary and sufficient to induce TLR4-dependent cell activation by endotoxin (4,6,10). MD-2 associates noncovalently with the N-terminal ectodomain of TLR4 and plays a pivotal role not only in TLR4 activation but also in trafficking of TLR4 to the cell surface (7,(11)(12)(13). MD-2 also likely interacts transiently with CD14 facilitating transfer of endotoxin monomer from CD14 to MD-2 and, at high molar excess of CD14, reverse transfer of endotoxin from MD-2 to CD14 (14).…”