Screening And Identification Of Functions Site And Inhibitor Peptides Of Lipopolysaccharide Binding Protein From A Phage Random 12-mer Peptide Library

Lipopolysaccharide binding protein (LBP) is a 60 kD acute phase glycoprotein capable of binding the LPS. In many species, including human, LBP is present in normal serum at <0.5μg/ml, rising to 50μg/ml 24 hours after induction of an acute phase response. LBP seems to play a critical role in regulating host responses to Lipopolysaccharide(LPS). LBP has a concentration-dependent dual role in the pathogenesis of gram negative sepsis: Low concentrations of LBP recognizes bacterial LPS and transfers it to CD14, thereby enhancing host cell stimulation, eventually resulting in pathogenic states such as septic shock. High concentrations of LBP was shown to detoxify LPS by transferring LPS into HDL particles , diminished LPS transfer activity and inhibited LPS-mediated cytokine release. LBP significantly decreased mortality rate in LPS-challenged mice, as well as in a murine model of bacteremia. The functions of LBP may depend on different binding domain. When one function site of LBP bind to LPS, LBP accelerated internalization and detoxification of LPS. When other function site of LBP bind to LPS, LBP greatly enhances the activity of LPS. The objective of the present study was to determine the functions site of LBP, and to screen and identify the bioactive peptides that could inhibit the function of LBP of enhancement of LPS-dependent activation of inflammatory cells from phage random 12-mer peptide library.Methods:Phage display technique was employed in our study. After 4 sequential rounds ofbiopanning to a phage random 12-mer peptide library, some phage clones which compete for LBP binding to LPS were identified. They were selected randomly and amplified .The binding activity and competitive inhibition activity of these phage clones were investigated by ELISA. The postive clones were further explored functional properties. Inhibition of LBP enhancement of LPS-induced peripheral blood mononuclear cells (PBMC) tumor necrosis factor alpha (TNFα) secretion of these phage clones were examed by ELISA. Then inhibition of LBP accelerateion of LPS internalization of these phage clones were texted by flow cytometry (FCM). Single stranded genomic DNA from the postive clones were extracted for sequencing as described in manual, and then were sequenced. The peptide sequences were deduced from DNA sequences of these phage clones. Through Blast, the peptide sequences were compared with LBP for homology analysis. The bioactive peptide of inhibition of LBP enhancement of LPS-dependent activation of PBMC was synthesized by the solid-phage method, and antiinflammatory role of the peptide was texted.Results:1. Using LPS as target, 4 rounds of biopanning were performed as described in methods. The bound phages were eluted by the glycine solution at the first two rounds, and were competitively eluted by LBP at the third and fourth round. The phage clones which compete for LBP binding to LPS were concentrated.2. 100 phage clones were selected randomly and amplified . 72 phage clones had higher affinity to LPS, and 16 phage clones of these clones could inhibit the binding of LBP to LPS significantly.3. 9 phage clones of the 16 phage clones could inhibite the function of LBP enhancement of LPS-induced PBMC TNFαsecretion, and they all could not inhibit the function of LBP accelerateion of LPS internalization. The core peptide sequence of 9 phage clones was WKXRKXFXKXXG.. By homology analysis, one higher homologous sequence was found in LBP 91-102 aa WKVRKSFFKLQG.. The 91-102 aa of LBP could be inflammatory function site.4. 3 of the16 phage clones could inhibit the function of LBP accelerateion of LPS internalization, and they all had not any effects on the function of LBP enhancement of LPS-dependent activation of PBMC. They had a consensus peptide sequence FHTRWNYWPYLH. FHTRWNYWPYLH could mimic the antiinflammation domain of LBP.5. The synthetic peptide with the sequence of WKVRKSFFKLQG-NH2 could inhibit the function of LBP enhancement of LPS-induced PBMC TNFαsecretion significantly.Conclusions:The 91-102 aa WKVRK...