Directed evolution of an LBP/CD14 inhibitory peptide and its anti-endotoxin activity.

<h4>Background</h4>LPS-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 bin...

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Autores principales:	Li Fang, Zhi Xu, Guan-song Wang, Fu-yun Ji, Chun-xia Mei, Juan Liu, Guo-ming Wu
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2014
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/016a6a8b933444e785bbc9523e9e0ac7
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spelling	oai:doaj.org-article:016a6a8b933444e785bbc9523e9e0ac72021-11-25T06:08:24ZDirected evolution of an LBP/CD14 inhibitory peptide and its anti-endotoxin activity.1932-620310.1371/journal.pone.0101406https://doaj.org/article/016a6a8b933444e785bbc9523e9e0ac72014-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/25025695/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203<h4>Background</h4>LPS-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.<h4>Methods</h4>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. 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.<h4>Results</h4>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. 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).<h4>Conclusion</h4>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. 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.Li FangZhi XuGuan-song WangFu-yun JiChun-xia MeiJuan LiuGuo-ming WuPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 9, Iss 7, p e101406 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Li Fang Zhi Xu Guan-song Wang Fu-yun Ji Chun-xia Mei Juan Liu Guo-ming Wu Directed evolution of an LBP/CD14 inhibitory peptide and its anti-endotoxin activity.
description	<h4>Background</h4>LPS-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.<h4>Methods</h4>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. 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.<h4>Results</h4>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. 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).<h4>Conclusion</h4>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. 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.
format	article
author	Li Fang Zhi Xu Guan-song Wang Fu-yun Ji Chun-xia Mei Juan Liu Guo-ming Wu
author_facet	Li Fang Zhi Xu Guan-song Wang Fu-yun Ji Chun-xia Mei Juan Liu Guo-ming Wu
author_sort	Li Fang
title	Directed evolution of an LBP/CD14 inhibitory peptide and its anti-endotoxin activity.
title_short	Directed evolution of an LBP/CD14 inhibitory peptide and its anti-endotoxin activity.
title_full	Directed evolution of an LBP/CD14 inhibitory peptide and its anti-endotoxin activity.
title_fullStr	Directed evolution of an LBP/CD14 inhibitory peptide and its anti-endotoxin activity.
title_full_unstemmed	Directed evolution of an LBP/CD14 inhibitory peptide and its anti-endotoxin activity.
title_sort	directed evolution of an lbp/cd14 inhibitory peptide and its anti-endotoxin activity.
publisher	Public Library of Science (PLoS)
publishDate	2014
url	https://doaj.org/article/016a6a8b933444e785bbc9523e9e0ac7
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Directed evolution of an LBP/CD14 inhibitory peptide and its anti-endotoxin activity.

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