Title :
MD-2 and
TLR4 N-linked glycosylations are important for a functional lipopolysaccharide receptor
Abstract :
- The lipopolysaccharide (LPS) receptor is a multi-protein complex that consists of at least three proteins , CD14 , TLR4 , and MD-2
- Because each of these proteins is glycosylated, we have examined the functional role of N-linked carbohydrates of both MD-2 and TLR4
- 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
- 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
- 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
- The ability of the various glycosylation mutants to support cell activation was also evaluated in transiently transfected HeLa cells
- 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 )
- Similar results were observed with TLR4 mutants lacking three or more N-linked glycosylation sites
- 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
- 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
Output (sent_index, trigger,
protein,
sugar,
site):
- 1. multi-protein, , multi, The lipopolysaccharide (LPS) receptor, -
- 2. MD-2, , MD-2, N-linked carbohydrates, -
- 2. TLR4, , TLR4, N-linked carbohydrates, -
- 2. glycosylated, , proteins, -, -
- 3. N-glycosylated, , -, -, sites
- 3. glycosylation, , -, -, sites
- 3. positions, , -, -, positions Asn(26) and Asn(114)
- 8. glycosylation, , -, -, sites
Output(Part-Of) (sent_index,
protein,
site):
- 10. MD-2, sites
- 10. TLR4, sites
- 3. MD-2, sites
- 3. TLR4, ectodomain
- 3. TLR4, sites
*Output_Site_Fusion* (sent_index,
protein,
sugar,
site):
- 3. -, -, positions Asn(26) and Asn(114)