title: Inefficient TLR4/MD-2 heterotetramerization by monophosphoryl lipid A.
abstract: Synthetic forms of E. coli monophosphoryl lipid A (sMLA) weakly activate the MyD88 (myeloid differentiation primary response protein) branch of the bifurcated TLR4 (Toll-like receptor 4) signaling pathway, in contrast to diphosphoryl lipid A (sDLA), which is a strong activator of both branches of TLR4. sMLA's weak MyD88 signaling activity is apparent downstream of TLR4/MyD88 signaling as we show that sMLA, unlike sDLA, is unable to efficiently recruit the TNF receptor-associated factor 6 (TRAF6) to the Interleukin-1 receptor-associated kinase 1 (IRAK1). This reduced recruitment of TRAF6 explains MLA's lower MAPK (Mitogen Activated Protein Kinase) and NF-κB activity. As further tests of sMLA's ability to activate TLR4/Myeloid differentiation factor 2 (MD-2), we used the antibody MTS510 as an indicator for TLR4/MD-2 heterotetramer formation. Staining patterns with this antibody indicated that sMLA does not effectively drive heterotetramerization of TLR4/MD-2 when compared to sDLA. However, a F126A mutant of MD-2, which allows lipid A binding but interferes with TLR4/MD-2 heterotetramerization, revealed that while sMLA is unable to efficiently form TLR4/MD-2 heterotetramers, it still needs heterotetramer formation for the full extent of signaling it is able to achieve. Monophosphoryl lipid A's weak ability to form TLR4/MD-2 heterotetramers was not restricted to synthetic E. coli type because cells exposed to a biological preparation of S. minnesota monophosphoryl lipid A (MPLA) also showed reduced TLR4/MD-2 heterotetramer formation. The low potency with which sMLA and MPLA drive heterotetramerization of TLR4/MD-2 contributes to their weak MyD88 signaling activities.
authors: Institute for Cellular Therapeutics, University of Louisville School of Medicine, Louisville, Kentucky, United States of America.
authors: Carolyn R
authors: CR
authors: Casella
authors: Thomas C
authors: TC
authors: Mitchell
mesh_terms: D000818:Animals; D057809:HEK293 Cells; D006801:Humans; D053592:Interleukin-1 Receptor-Associated Kinases / Q000378:metabolism; D008050:Lipid A / Q000031:analogs & derivatives* / Q000494:pharmacology; D051218:Lymphocyte Antigen 96 / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D050505:Mutant Proteins / Q000378:metabolism; D053594:Myeloid Differentiation Factor 88 / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D055503:Protein Multimerization / Q000187:drug effects*; D021381:Protein Transport / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects; D048029:TNF Receptor-Associated Factor 6 / Q000378:metabolism; D051197:Toll-Like Receptor 4 / Q000378:metabolism*
chemical_list: D008050:Lipid A; C411728:Ly96 protein, mouse; D051218:Lymphocyte Antigen 96; D050505:Mutant Proteins; D053594:Myeloid Differentiation Factor 88; D016328:NF-kappa B; D010710:Phosphates; D048029:TNF Receptor-Associated Factor 6; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4; D053592:Interleukin-1 Receptor-Associated Kinases; C048436:monophosphoryl lipid A
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