Screening And Identification Of Peptides Specifically Binding To Monocyte/macrophage In Sepsis

Sepsis is a systemic inflammatory response syndrome arising from infectioncaused by micro-organisms (usually bacteria) or their toxin invading the body. Sepsisis part of a spectrum of conditions ranging from the acute respiratory distresssyndrome(ARDS), systemic inflammatory response syndrome (SIRS) to septic shockand multiple organ dysfunction syndrome (MODS). Sepsis and its sequelae continueto be the main causes of morbidity and mortality in the intensive care unit (ICU). Themortality associated with these conditions ranges from around 26% in patients withSIRS to around 82% in septic shock, and shows no sign of decreasing despite optimalcurrent therapy. Sepsis therefore continues to have significant clinical and financialimplications and remains an area that attracts intense research interest.Lipopolysaccharide (LPS), or endotoxin, is the major component of the outersurface of Gram-negative bacteria. LPS is a potent activator of the cells of theimmune and inflammatory systems, including monocytes and macrophages, leads tothe release of inflammatory mediators and tissue factors, and contributes to thesystemic changes seen in gram-negative sepsis or septic shock. Activation by LPSconstitutes the first step in the cascade of events believed to lead to the manifestationof sepsis. Structure and function of monocytes and tissue macrophages change after being activated by LPS. Compared with untreated monocytes and maerophages, LPSstimulated monocytes and macrophages expose their active sites of cell surfacemolecules, which may be closely related with the development of sepsis and can actas therapeutic targets.As these changed molecules may act as binding sites on the cell surface for somecritical cytokines in inflammation cascade reaction, specific binding peptides of thesemolecules can competitively inhibit binding of cytokines with cells, and havepotential abilities to block biological effects of the cytokines. The biologically activepeptides that target to specific cells show properties of high performance with lowside effects. The committed step is to find peptides that specifically bind to LPSstimulated monocytes/macrophages.During the last decade, the development of phage display technique shines lighton finding specifically binding peptides. It is well known that interactions andrecognitions between proteins are mediated by several functional peptide residues.Also peptides containing critical residues can act as functional determinants of theproteins. The discovery of peptides that specifically bind to monocytes/macrophageswhich may block the biological effects of inflammatory mediators by phage displaytechnique will provides new concept and idea on the prevention and cure of sepsis.Phage-display technology is a powerful molecular tool that has been developedand widely used since the nineties of twenty century. It describes a selectiontechnique in which a peptide or protein is expressed as a fusion protein with a coatprotein of a bacteriophage, resulting in displaying of the fused protein on the surfaceof the virion, while the DNA encoding the fusion resides within the virion. Anessential feature of phage-display technology is that it links phenotype with genotype,such that, the expression of a particular phenotype on phage is physically linked to the genetic information contained within the phage particle. The expression of thedesired phenotype can be achieved by incorporating the relevant genetic material intothe phage genome. Phage display has been used to create a physical linkage betweena vast library of random peptide sequences to the DNA encoding each sequence,allowing rapid identification of peptide ligands for a variety of target molecules(antibodies, enzymes, cell-surface receptors, etc.) by an in vitro selection processcalled panning. In its simplest form, panning is carried out by incubating a library ofphage-displayed peptides with a plate (or bead) coated with the target, washing awaythe unbound phage, and eluting the specifically-bound phage. The eluted phage isthen amplified and taken through additional binding/amplification cycles to enrich thepool in favor of binding sequences. After 3～4 rounds, individual clones arecharacterized by DNA sequencing. Random peptide libraries displayed on phage havebeen used in a number of applications, including epitope mapping, mappingprotein-protein contacts, and identification of peptide mimics of non-peptide ligands.Bioactive peptides have been identified either by panning against immobilizedpurifed receptors or against intact cells.The Ph.D.-C7C Phage Display Peptide Library is based on a combinatoriallibrary of random peptide 7-mers fused to a minor coat protein (pⅢ) of M13 phage.The randomized sequence is flanked by a pair of cysteine residues. Undernonreducing conditions the cysteines will spontaneously form a disulfide cross-link,resulting in phage display of cyclized peptides, in contrast to the linear peptides.Disulfide-constrained peptide libraries have proven useful in identification ofstructural epitopes, mirror-image ligands for D-amino acid targets, and leads forpeptide-based therapeutics.Signaling molecules (membrane receptors) on the cell membrane reflectproperties and functional status of cells. The phage-display technique has some advantages in the study of protein-protein interaction. However, there are somedifficulties for membrane receptors to be purified and maintain the naturalconformation. It is better to use whole native cells or target receptor gene transfectedcells to select the ligands. As whole cells usually maintain the native conformation ofreceptor with normal posttranslaticnal modification, the ligands of receptors can beselected even without the information about the receptors. In this study, LPSstimulated THP-1 cells were used as target to select specifically binding peptidesfrom a phage peptide random library.In a very elegant study, Giordano and colleagues described a new approach forthe screening, selection and sorting of cell-surface binding peptides from phagelibraries. The method, termed bio-panning and rapid analysis of selective interactiveligands (BRASIL), allows separation of complexes formed by the cells and boundphage from the remaining unbound phage still in the suspension. This technique isbased on a differential centrifugation of the aqueous phage/cell suspension through anon-miscible organic lower phase. Centrifugation will drive the cells from ahydrophilic phase into a non-miscible (hydrophobic) organic phase. The passage ofcells from a hydrophilic to a hydrophobic setting will separate water-soluble materials,such as the unbound phage. The cell/phage pellet is then recovered from the bottomof the tube after immediate freezing in liquid nitrogen. Next, the cell pellet is thawedand bound phages are recovered by bacterial infection. As the method involves onecentrifugation and does not require repeated washes, it allows a simpler and moreconvenient phage recovery from cell membranes than other cell-panning techniques.In this study, LPS stimulated THP-1 cells were used as specifically binding cells anduntreated THP-1 cells as nonspecifically absorbing cell. After four rounds of subtractbiopanning, binding phages were enriched for about 155 times. All together 1,800phage clones were randomly selected and 1,033 of which were verified by cell-ELISA assay and DNA sequencing to be positive clones. The insert heptapeptidesequences were deduced according to the inserted DNA sequences.Some of the tripeptide fragments included in these heptapeptide sequences aremimotope of functional proteins, and may act as biochemical recognition units inprotein-protein interactions. In this study, shuffling algorithm and randomizedpermutation test were used to calculate the abundance and significance of thetripeptides. After calculation and statistics analysis, we got 3,085 distinct tripeptidesand eight of which the abundance exceeded 15 and four of which the appearance offrequency is significant. Then multiple sequence alignment of tripeptides withsignificance and most abundance was conducted with Clustal W program. Fromwhich and considered the properties of amino acid, we got some mimetic peptides.The mimetic peptides were aligned to human protein sequence library(SWISS-PROT)by BLASTP program and human proteins containing these mimetic peptides wereobtained. Signal peptide predicting program SignalP 3.0 and transmembrane helicespredicting program TMHMM 2.0 were used to identify secreted proteins andtransmembrane proteins respectively. With the bioinformatics analysis mentionedabove, we acquired human tumor necrosis factor-α(hTNF-α) and its mimetic peptideISPL.Immunofluorescence test identified that KSFISPL phage binds specifically to themembrane of LPS stimulated THP-1 cells. KSFISPL phage can also inhibit theproduction of IL-8 in THP-1 cells with the stimulation of hTNF-α. The bindingability relies on KSFISPL peptide instead of phage itself.On the whole, we screened LPS stimulated THP-1 cells and untreated THP-1cells for four rounds by BRASIL method and got some phage-displayed peptides thatbind specifically to LPS stimulated THP-1 cells. Functional proteins and their mimetic peptides were acquired by bioinformatics analysis and prediction andidentified to be biologic activity. Taken together, we draw the following conclusions:First, the new approach for screening, selection and sorting of cell-surfacebinding peptides from phage libraries, termed bio-panning and rapid analysis ofselective interactive ligands (BRASIL), allows separation of complexes formed bythe cells and bound phages from the remaining unbound phages still in the suspension.As it allows a simpler and more convenient phage recovery and effectively preventsthe loss of cells and ligands, BRASIL method is necessary to be popularized.Second, After four rounds of subtract biopanning, 1,033 phage clones whichbind specifically to LPS stimulated THP-1 cells were verified by celI-ELISA assayand DNA sequencing. By bioinformatics analysis, we got 3,085 distinct tripeptidesand eight of which the abundance exceeded 15 and four of which the appearance offrequency is significant.Third, multiple sequence alignment of tripeptides with significance and mostabundance was conducted with Clustal W program, from which and considered theproperties of amino acid, we got some mimetic peptides.Fourth, the mimetic peptides were aligned to human protein sequencelibrary(SWISS-PROT) by BLASTP program and human proteins containing thesemimetic peptides were obtained. Signal peptide predicting program SignalP 3.0 andtransmembrane helices predicting program TMHMM 2.0 were used to identifysecreted proteins and transmembrane proteins, and human tumor necrosis factor-α(hTNF-α) and its mimetic peptide ISPL were selected for further identification.Fifth, immunofluorescence test identified that KSFISPL phage binds specificallyto the membrane of LPS stimulated THP-1 cells. The binding ability relies onKSFISPL peptide instead of phage itself. Sixth, KSFISPL phage inhibits hTNF-αstimulated THP-1 cells from producingIL-8 and the ability relies on KSFISPL peptide instead of phage itself.In general, we have successfully established a high-performance method toscreen peptides that bind specifically to the membrane of cells. The method includesraw data acquiring through biology experiment, computational analysis of biologicaldata and biology experiment identification of results predicted by bioinformatics.