title: Directed evolution of an LBP/CD14 inhibitory peptide and its anti-endotoxin activity.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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).
  
  
  CONCLUSIONS
  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.
authors.affiliation: Institute of Respiratory Disease, Xinqiao Hospital, Third Military Medical University, Chongqing, China.
authors.forename: Li
authors.initials: L
authors.lastname: Fang
authors.affiliation: Institute of Respiratory Disease, Xinqiao Hospital, Third Military Medical University, Chongqing, China.
authors.forename: Zhi
authors.initials: Z
authors.lastname: Xu
authors.affiliation: Institute of Respiratory Disease, Xinqiao Hospital, Third Military Medical University, Chongqing, China.
authors.forename: Guan-song
authors.initials: GS
authors.lastname: Wang
authors.affiliation: Institute of Respiratory Disease, Xinqiao Hospital, Third Military Medical University, Chongqing, China.
authors.forename: Fu-yun
authors.initials: FY
authors.lastname: Ji
authors.affiliation: Institute of Respiratory Disease, Xinqiao Hospital, Third Military Medical University, Chongqing, China.
authors.forename: Chun-xia
authors.initials: CX
authors.lastname: Mei
authors.affiliation: Institute of Respiratory Disease, Xinqiao Hospital, Third Military Medical University, Chongqing, China.
authors.forename: Juan
authors.initials: J
authors.lastname: Liu
authors.affiliation: Institute of Respiratory Disease, Xinqiao Hospital, Third Military Medical University, Chongqing, China.
authors.forename: Guo-ming
authors.initials: GM
authors.lastname: Wu
mesh_terms: D000209:Acute-Phase Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D061505:Cell Surface Display Techniques; D004731:Endotoxins / Q000037:antagonists & inhibitors*; D004789:Enzyme Activation / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000009:adverse effects / Q000494:pharmacology; D008168:Lung / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D016328:NF-kappa B / Q000378:metabolism; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D012128:Respiratory Distress Syndrome / Q000139:chemically induced / Q000188:drug therapy / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism; D020298:U937 Cells; D000099231:Lipopolysaccharide-Binding Protein
chemical_list: D000209:Acute-Phase Proteins; D002352:Carrier Proteins; D004731:Endotoxins; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D019151:Peptide Library; D010455:Peptides; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D000099231:Lipopolysaccharide-Binding Protein