Characterizing The Peptide Mimics Of Ligands For Human TLR-2

In Drosophila devoid of an adaptive immune system, Toll proteins play very important role to mediate innate immunity for resistance to bacteria and fungal infection. Toll-like receptors, the homologues of Drosophila Toll, were also found in mammalian and plant. Toll and Toll-like receptors all belong to the type I transmembrane protein. TLRs are characterized by extracellular leucine-rich repeat domains and intracellular signaling domain that shares homology with cytoplasmic sequences of the mammalian IL-1 receptor. Activation of most TLRs leads to recruitment of MyD88, MyD88 interacts with IL-1 receptor-associated kinase, leading to initiation of a signal transduction cascade culminating in nuclear translocation of NF-kB family members and altered gene expression, such as IL-1, TNFα, IL-6, IL-12 and other cytokines which play important role in inflammation and immune defence. Now 11 of mouse TLRs and 10 of human TLRs had been identified. Originally, TLRs were considered mediating recognition of pathogen-associated molecular patterns(PAMPs). In addition, TLRs could induce DC maturation, participate in immune tolerance, participate in apoptosis, mediate recognition of non-infection factors and et al, which have been considered as a linkage of innate and adaptive immunity.Among mammalian TLRs, TLR-2 is expressed very extensively, and mediates recognition of a wide range of microbial products. TLR-2 can be expressed in monocyte, macrophage, dendritic cell, γ/δT, Th1, Th2α/βT, especially expressed at high level in spleen and peripheral blood leukocyte; and also some intestinalepithelial cell lines, B cell lines, rat myocardial cells, brain, gingiva cells express TLR-2 at different level. TLR-2 mediates recognition of many PAMPs, also participates in recognition of endogenous ligands (such as repeat freeze-thawing necrosis cells, the products of ischemical reperfusion injury and et al),DC maturation, apoptosis,immune tolerance and autoimmune disearse,allergy, severe trauma, artherosclerosis,oxidative stress of cadiocyte, ischemical reperfusion injury and other pathological reaction. So TLR-2 is considered as a central pattern recognition receptor.As TLR-2 can recognize so many different ligands, moreover, human and mouse TLR-2 can recognize identical or homoplastic ligands, which suggests that TLR-2 may possess a special structure for recognition to many different ligands , and there might be conservative structure shared by different ligands. Therefore, we prepared TLR-2 extracellular domain by cloning and expression in CHO cell and E.Coli, and screened binding peptides of TLR-2 extracellular domain from random phage display peptide library. We attempt to find the characteristics of conservative structure of ligands, and explore the interactive mechanism between TLR-2 and ligands, as well as any possibility to find candidate epitope of vaccine or target for development of new drug.Part I Cloning and expression of human Toll-like receptor 21. Cloning and expression of human TLR-2 extracellular domain(TLR-2ED)Based on The sequence gene of human TLR-2 from Genebank, TLR-2 gene was cloned from peripheral blood monoclear cells using RT-PCR, and the pcDNA3.1+/TLR-2ED(A26-T588) recombinant plasmid was constructed. Using eukaryotic expression system, TLR-2ED protein was successfully expressed. TLR-2ED protein can bind with polyclonal antibody(TLR-2,Ab-l) immunized with TLR-2(179L-204I) synthetic peptide (Oncogen company). In addition, three short fragments TLR-2a(7M-225E), TLR-2b(185L-392L), TLR-2c(436E-595C) were cloned and expressed in prokaryotic expression system. pGEX4Tl/TLR-2a, pMBPc/TLR-2a, pET32a/TLR-2b and pET32a/TLR-2c recombinant plasmids were constructed. pGEX4Tl/TLR-2a, pMBPc/TLR-2a, pET32a/TLR-2c were expressed in E.coli. BL21, and TLR-2a/GST, TLR-2a/His and TLR-2c/His recombinant proteinswere purified from inclusion body. TLR-2a could bind with polyclonal antibody (TLR-2,Ab-l) and anti-His antibody; TLR-2c could react with anti-His antibody only. Above results indicated that TLR-2ED and TLR-2a were correctly expressed, and showed antigenicity of TLR-2; but TLR-2c protein was correctly expressed only, without antigen specificity.2. Cloning and eukaryotic expression of human TLR-2 whole length geneUsing RT-PCR , TLR-2 whole length gene was obtained and cloned into pcDNA3.1+, then the correct pcDNA3.1+/TLR-2 plasmid was transfected into CHO cells, and stable CHO cells expressing human TLR-2 gene were obtained through G418 selection. The results of RT-PCR and immunohistochemistry with commercial polyclonal antibody (TLR-2,Ab-l) indicated that TLR-2 gene and protein expressed in CHO cells.3. Preparation of antibody against different fragments of human TLR-2EDRabbits were immunized with CHO/TLR-2 cells or TLR-2a/His protein. The antisera of rabbit immunized with CHO/TLR-2 cells could react with TLR-2ED (A26-T588) protein(titer 1:3200), TLR-2a/GST, TLR-2a/His and TLR-2c/His protein(titer 1:800), mouse TLR-2 N terminal protein(titer 1:1600). The antisera of rabbit immunized with TLR-2a/His protein could react with TLR-2a/GST(titer 1:6400), mouse TLR-2N terminal protein(titer 1:3200), but could not react with TLR-2(A26-T588). These results indicated that CHO/TLR-2 cells induced the production of antibody to TLR-2 extracellular fragment, TLR-2a and TLR-2c recombinant protein were expressed corretly; and human TLR-2ED, TLR-2a/ (7M-225E) shared common epitope with mouse TLR-2N(13A-165I), which may provide a useful tool for the research of mouse TLR-2. Now only TL2.5 (monoclonal antibody to human TLR-2) could react with both mouse and human TLR-2. The antisera of rabbits immunized with TLR-2a/His rabbit can not bind with TLR-2ED(A26-T588) , which might be caused by different expression system.4. Identification on biological activity of recombinant human TLR-2 fragments.The biological activities of recombinant TLR-2 extracellular fragment identified by testing if binding with ligands of TLR-2 and stimulating the production of cytokines. The results indicated that TLR-2ED, TLR-2a and TLR-2c could bind with PGN, LTAand LPS, especially better in binding with PGN and LTA which were known as important ligands from Gram-positive bacteria to TLR-2. Unexpectedly, TLR-2c(436E-595C) nearly to cytomembrane could bind with three kinds of ligands, which suggested that the recognition by TLR-2 might depend on conformational epitope or domain.Part II Screening and identification of ligand-mimicpeptide of TLR-2 extracellular domain(A26-T588)In this part, the peptide mimics of ligands for TLR-2 were screened from C7C phage display peptide library and 12mer phage display peptide library by TLR-2ED(A26-T588) as target.1. Screening of linear peptide mimics of ligands for TLR-2. Through two independent screening of 12mer peptide library, thirty-three positive clones binding to TLR-ED protein were obtained. All of these positivity clones share a common sequence: G-G-S-G-T-S-R-T-P-I-L-G which was named as PI2-1. PI2-1 contains many micromolecule lyophobic amino acids, and the water solution of PI2-1 is weak acidity. The binding between phage PI2-1 to TLR-2ED protein could be inhibited by LPS from EcoliJ5 and PGN . Synthetic peptide Bio-P12-1 could bind with TLR-2ED protein and CHO/TLR-2 cells identified by ELISA and immunohistochemistry. In addition, Bio-P12-1 could stimulate THP-1/CD14 cells to secrete TNFa. But the binding of Bio-P12-1 with TLR-2(A26-T588) or CHO/TLR-2 cells was a little weak, which might be related to unstable of linear peptide in buffer or without combinatory recognition with TLR-1 and/or TLR-6. Besides above reasons, even if the sequence of PI2-1 is highly conservative, PI2-1 was a peptide mimics without lipid and saccharide structure which might influence the binding affinity of P12-1 to TLR-2.2. Screening of cycle peptide mimics of ligands for TLR-2. In order to obtain high affinity phage mimicry peptide, C7C phage display peptide library was also screened using TLR-2ED protein as target, twelve of positive clones were obtained. Nine of twelve positive clones showed the same DNA sequence which was deduced into amino acids sequence as C-K-K-S-G-K-P-L-C. One phage clone C7C-1 with C-K-K-S-G-K-P-L-C sequence was picked up for more identification.The binding of TLR-2(A26-T588) with positive phage clone C7C-1 could be inhibited by PGN and LTA with IC50 20|ng/ml and 60ug/ml, respectively. All these results suggested phage clone C7C-1 could be mimetic the binding epitope on PGN and LTA to TLR-2 extracellular fragment, and the disulfide bond would make the construction of cyclopeptide more stable in solution and more suitable to match with receptor. The synthetic peptide pC7C-l could bind with TLR-2ED and antisera of rabbits and mice immunized with peptide L7 which could be mimetic to epitope on PGN and LPS from different bacteria. Comparing these two peptide mimics to ligands of TLR-2, pC7C-l could bind with TLR-2ED and antisera against L7 better, which suggested pC7C-l with more conservative epitope shared by ligands of TLR-2. In bioactivities, P12-1 could induce production of TNFa by THP-1/CD14 cells, but pC7C-l cannot.