PMID: 11706042

 

    Legend: Sugar

Title : MD-2 and TLR4 N-linked glycosylations are important for a functional lipopolysaccharide receptor

Abstract :

1. The lipopolysaccharide (LPS) receptor is a multi-protein complex that consists of at least three proteins , CD14 , TLR4 , and MD-2
2. Because each of these proteins is glycosylated, we have examined the functional role of N-linked carbohydrates of both MD-2 and TLR4
3. We demonstrate that MD-2 contains 2 N-glycosylated sites at positions Asn(26) and Asn(114) , whereas the amino-terminal ectodomain of human TLR4 contains 9 N-linked glycosylation sites
4. Site-directed mutagenesis studies showed that cell surface expression of MD-2 did not depend on the presence of either N-linked site , whereas in contrast, TLR4 mutants carrying substitutions in Asn(526) or Asn(575) failed to be transported to the cell surface
5. Using a UV-activated derivative of Re595 LPS ( ASD-Re595 LPS ) in cross-linking assays, we demonstrated a critical role of MD-2 and TLR4 carbohydrates in LPS cross-linking to the LPS receptor
6. The ability of the various glycosylation mutants to support cell activation was also evaluated in transiently transfected HeLa cells
7. The double mutant of MD-2 failed to support LPS-induced activation of an interleukin-8 ( IL-8 ) promoter-driven luciferase reporter to induce IL-8 secretion or to activate amino-terminal c-Jun kinase ( JNK )
8. Similar results were observed with TLR4 mutants lacking three or more N-linked glycosylation sites
9. Surprisingly, the reduction in activation resulting from expression of the Asn mutants of MD-2 and TLR4 can be partially reversed by co-expression with CD14
10. This suggests that the functional integrity of the LPS receptor depends both on the surface expression of at least three proteins , CD14 , MD-2 , and TLR4 , and that N-linked sites of both MD-2 and TLR4 are essential in maintaining the functional integrity of this receptor