Directed Evolution Of An LBP/CD14 Inhibitory Peptide And Its Anti-endotoxin Activity

BackgroundLipopolysaccharide(LPS) is the main component of the outer wall of gram-negative bacteria and is one of the major causes of acute lung injury(ALI). An LPS polymer can bind to CD14, which is located on the surfaces of macrophages, monocytes, and neutrophils, only after it is depolymerized into monomers by LPS-binding protein(LBP) and forms an LBP/LPS complex, that activates the LPS inflammatory signaling pathway. LBP and its ligand CD14 are located upstream of the signaling pathway for LPS-induced inflammation. In addition to amplifying the LPS-mediated inflammatory signaling pathway via CD14, LBP can transport LPS to highdensity, and thereby clear LPS from the circulation. Blocking LBP and CD14 binding might prevent LPS-induced inflammation. In previous studies, we obtained a peptide analog(MP12) for the LBP/CD14 binding site and showed that this peptide analog had anti-endotoxin activity. In this study, we used in vitro directed evolution for this peptide analog to improve its in vivo and in vitro anti-endotoxin activity.Methods1. We used error-prone PCR(ep-PCR) and induced mutations in the C-terminus of LBP and attached the PCR products to T7 phages to establish a mutant phage display library. The positive clones that competed with LBP for CD14 binding was obtained by screening. The polypeptide obtained in this experiment was designated P1. Another polypeptide containing the MP12 sequence produced during another synthesis was designated MP12. The anti-endotoxin activities of these two polypeptides were compared.2. U937 was cultured. Effects of polypeptides P1 and MP12 on LPS-inducedTNF-? expression at the m RNA and the protein level were compared by RT-PCR and ELISA,respectively. And effects of polypeptides P1 and MP12 on LPS-induced NF-?B activity were compared by EMSA.3. Effects of polypeptides P1 and MP12 on arterial oxygen partial pressure(Pa O2), oxygenation index(PaO2/Fi O2), pathological semi-quantitative scoring and pulmonary vascular permeability(PMVP) with LPS induced acute lung injury rats were observed.Results1.11 positive clones were obtained from among target phages. Sequencing showed that 9 positive clones had a threonine(T) to methionine(M) mutation in amino acid 287 of LBP. Based on these results, we synthesized an artificial polypeptide containing amino acids 252–291 of LBP, which included the mutation site noted above;this was designated P1. Its sequence was: FHRNHRSPVTLLAAVMSLPEEHNKMVYFAISDYVFNMASL.We also synthesized another polypeptide, designated MP12, which contained amino acids 252–291 of LBP. The only difference was that the sequence of amino acids 252–263 was substituted with FHRWPTWPLPSP, which was obtained in our previous experiments. Its polypeptide sequence was:FHRWPTWPLPSPAAVMSLPEEHNKMVYFAISDYVFNTASL.2. LPS treatment significantly increased the TNF-? expression at the mRNA and the protein level in U937 cells and NF-?B activity, and LBP significantly enhanced that(P<0.01). Polypeptides P1 and MP12 significantly reduced LBP/LPS-induced TNF-? expression and NF-?B activity(P<0.01). Polypeptide P1 inhibition of LBP/LPS-induced TNF-? m RNA expression and NF-?B activity was greater than that by MP12(P<0.05).Compared to polypeptide MP12, polypeptide P1 significantly inhibited LPS-induced TNF-? expression at the protein level by ELISA(P<0.01).3. After LPS injection, PaO2 and Pa O2/FiO2 decreased significantly, while lung pathology scores and PMVP increased significantly(P<0.01). Polypeptide P1 and MP12 improved Pa O2 and Pa O2/Fi O2, lung pathology scores and PMVP(P<0.01). Compared to MP12, P1 significantly improved Pa O2, Pa O2/Fi O2, lung pathology scores(P<0.05) and PMVP(P<0.01) in LPS-induced ALI rats.Conclusion1.By in vitro directed evolution of peptide analogs for the LBP/CD14 binding site, we established a new polypeptide(P1) with a threonine(T)-to-methionine(M) mutation in amino acid 287 of LBP, which could block the LBP site for binding to CD14.2.This polypeptide had high anti-endotoxin activity in vitro and in vivo.3. We found that amino acid 287 may play an important role in LBP binding to CD14. This finding provided additional insights for realizing the structure and function of LBP, which would aid in developing a more ideal strategy for anti-endotoxin treatments.