Structure-function Relationship Of The Extracellular Domain Of Toll-like Receptor 2

The innate immune system has evolved as the first line of defense against invading microorganisms. Accumulating evidence suggests that TLR molecules are involved in signalling receptor complexes which recognise components of Gram-positive and Gram-negative bacteria and mycobacteria. In Drosophila, the Toll family of proteins is responsible for the recognition of bacteria and fungi. In mammals, Toll-like receptors (TLRs) are able to recognize and respond to microbial pathogens.To date, ten members of the TLR family have been identified in humans, and several of them appear to recognize specific microbial products, including lipopolysaccharide, bacterial lipoproteins, peptidoglycan, ssRNA, flagellin and bacterial DNA. The TLRs share the IL-1R cytoplasmic signaling cascade but are distinguished by their extracellular leucine-rich repeat (LRR) structure. The pathway to NF-kappa B activation is essential in innate immune cell signaling via TLRs.Recent findings have defined the effect of TLR ligation on host defense. In the case of sepsis and septic shock, however, an exaggerated systemic response may, in fact, contribute to the morbidity and mortality associated with overwhelming infections. Therefore, these findings have provided a framework for determining how TLRs may by used to therapeutically modulate immune responses to infection.Toll-like receptor 2 (TLR2) is a member of the expanding TLR family involved in innate immunity and functions as a pattern recognition receptor for diverse bacterial products. And in the TLR2-mediated responses to some ligands, there appears to exist a functional interaction between the receptors TLR2, and TLR6 (or TLR1).Zhang Lin. Structure-function Relationship of the Extracellular Domain of Toll-like Receptor 2In order to investigate the role of the extracellular leucine-rich repeat-containing domain of TLR2 in the TLR2-mediated signaling pathway, mammalian expression plasmids of the extracellular domain of TLR2 and its two fagments are to be constructed. With integration with wet molecular biology, an Internet-based structure-function analysis of the extracellular domain of TLR2 is also to be performed.The cDNA fragment encoding complete extracellular domain was isolated by semi-nested RT-PCR with the total RNA from HL-60 cells, as templates, linked into pGEM-T Easy vector, transformed into E. coli JM109. Identified by sequencing, the construct of the ectodomain was referred to as pGEM-Tex. In turn, the gene in pGEM-Tex was inserted into pcDNA3 vector. Then two fragments amplified from the pGEM-Tex construct were inserted into pGEM-T Easy vector and pcDNA3, respectively. These constructs were named as pGEM-NTex, pGEM-CTex, pcDNA-Tex, pcDNA-NTex and pcDNA-CTex, respectively. Using electrophoretic mobility shift assay (EMSA), we observed these three constructs effects on the Peptidoglycan (PGN)-induced TLR2-mediated Nuclear Factor kappa B (NF-kB) nuclear translocation in THP-1 cells.On the other hand, an internet-based structure-function analysis of the extracellular domain of TLR2 was performed for further insights into this LRR domain. Several protein prediction tools all from Internet, including 3D-PSSM, SPDBV, Rasmol, WHAT IF, QSE and GRAMM, were intergrated to model the three dimensional structure of the ectodomain of TLR2, and to explore its structure-function relationship in signalling. In a similar manner, furthermore, models for CD 14 and MD-2 were also built and analysed. And electrostatic potentials of the threaded ectodomain models of human TLR1-6 directly from the 3D-PSSM Server were computed for comparision.The result of sequencing indicated that the sequences of our cDNA is identical with the corresponding parts of the mRNA for human TLR2 in GenBank?except 4 nucleotides, and with a homology of 0.998. The EMSA analysis shows a significant reduction of NF-kB activity in pcDNA-Tex or pcDNA-NTex-transfected THP-1 cells within 2 h of incubation with PGN. In contrast, NF-kB activity neither in pcDNA-NTex-transfected nor in pcDNAS-transfected...