Mechanism of pattern recognition by TLR2-TLR6 heterodimer

다국어 초록 (Multilingual Abstract)

TLR2 initiates potent immune responses by recognizing diacylated and triacylated lipopeptides. Its ligand specificity is controlled by whether it heterodimerizes with TLR1 or TLR6. In present study, we found three major factors contribute to the ligand specificity of TLR2-TLR1/6 heterodimers. First, the lipid channel of TLR6 has two phenylalanines, which block the lipid channel for amide bound lipid chain of triacylated lipopeptides. Simultaneous mutation of these phenylalanines made TLR2-TLR6 fully responsive not only to diacylated but also to triacylated lipopeptides. Second, through the alanine mutation exprimens of human TLR6, Asn314, Phe319, Tyr325, Thr326, Cys348, Gln362, Asp367, Phe370, Cys373 and Lys390 are found to be important residues in the ligand induced activation of TLR2/TLR6 heterodimerization. Furthermore, PE-DTPA, a phosphatidylethanolamine derivative, forms a stable complex with TLR2, but it cannot induce downstream signaling. We found that TLR2 cannot form heterodimers with TLR1 and TLR6 by binding PE-DTPA. This data demonstrates that two lipid chains are required but not enough for TLR2 activation. Collectively, a precise interaction pattern of the head group of ligand is essential for a robust immune response by TLR2 heterodimers.

목차 (Table of Contents)

• I. INTRODUCTION 1
• II. MATERIALS AND METHODS 14
• 1. Cell culture 14
• 2. Plasmids 14
• 3. Reverse transcriptase (RT)-PCR analysis 15
• 4. Cloning of human interleukin 8 (IL-8) Promoter 15
• 5. Human TLR1, TLR2 and TLR6 wild type cloning and TLR6 Point
• mutant constructs 16
• 6. Transient transfection 17
• 7. Stimulation and luciferase assays 17
• 8. Surface Plasmon Resonance (SPR) analysis 18
• 9. SDS-PAGE gel 19
• III. RESULTS 24
• 1. Alanine mutagenesis of LRR11-15 of human TLR6 24
• 2. Mutants of human TLR6, F343M and F365L restore IL-8 activity
• with tri-acylated lipopeptide, Pam3CSK4 37
• 3. TLR2-TLR1/6 complex cannot lead to activate IL-8 reporter
• by PE-DTPA 39
• IV. DISCUSSION 58
• V. REFERENCES 65
• VI. SUMMARY in KOREAN 75