Abstract
BackgroundLPS-binding protein (LBP) and its ligand CD14 are located upstream of the signaling pathway for LPS-induced inflammation. 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.MethodsWe 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. We used both in vivo and in vitro experiments to compare the anti-endotoxin activities of a polypeptide designated P1 contained in a positive clone and MP12.Results11 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. Compared to polypeptide MP12, polypeptide P1 significantly inhibited LPS-induced TNF-α expression and NF-κB activity in U937 cells (P<0.05). Compared to MP12, P1 significantly improved arterial oxygen pressure, an oxygenation index, and lung pathology scores in LPS-induced ARDS rats (P<0.05).ConclusionBy 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. This polypeptide had high anti-endotoxin activity in vitro and in vivo, which suggested that amino acid 287 in the C-terminus of LBP may play an important role in LBP binding with CD14.
Highlights
Lipopolysaccharide (LPS) is the main component of the outer wall of gram-negative bacteria and is one of the major causes of acute respiratory distress syndrome (ARDS) [1]
LPS-binding protein (LBP) and its ligand CD14 play a key role in LPS identification and transmitting LPS inflammatory signals
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 LBP and forms an LBP/LPS complex, that activates the LPS inflammatory signaling pathway [2,3,4]
Summary
Lipopolysaccharide (LPS) is the main component of the outer wall of gram-negative bacteria and is one of the major causes of acute respiratory distress syndrome (ARDS) [1]. 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 LBP and forms an LBP/LPS complex, that activates the LPS inflammatory signaling pathway [2,3,4]. During this process, the inflammatory effect of LPS is significantly amplified [5]. Blocking the LBP binding site for CD14 might interfere with the LPS-induced inflammatory signaling pathway without affecting the scavenging activity of LBP for LPS. We used in vitro directed evolution for this peptide analog to improve its in vivo and in vitro anti-endotoxin activity
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