Screening And Identification For Peptides Selectively Targeting Pulmonary Vasculature Of Mice With Endotoxic Shock

Nowadays the mortality rate of septic shock and sequential multiple organ dysfunction still remains high,despite great improvements made in critical care medicine.Numerous researches have indicated that sepsis may represent a complicated clinical syndrome,which is systemicly developed and continuously changed in sepsis patients.Sepsis is considered as a multi-factor disease in which all risk factors are reciprocally influenced and mutually regulated.Therefore,the traditional linear research approaches are much too limited to unravel the complicated pathogenesis of sepsis even draw a final conclusion in sepsis research.The treatment of sepsis may be improved only if the interactions between the risk factors are clarified and the key factors of in these pro-inflammatory interactions are uncovered.It has been indicated that endothelium plays an important role in the pathogenesis of sepsis.Vascular endothelium is considered as a primary target of lipopolysaccharide,cytokines,pro-inflammatory mediators,adhesion molecules,free radicals,etc.during the onset and development of sepsis,and is closely associated with leukocyte adhesion,rolling,transmigration,microthrombus formation,decreased vascular reactivity and increased vascular permeability.Furthermore,vascular endothelium is characterized with its heterology and functions as monitor and transducer of molecular signals origining from serum,to actively feel,integrate and response to the signals from physicochemical environment changes in blood stream and blood constituents.Therefore,as one of the most vulnerable organs,lung,it is easily to be highlighted concerning the folowing questions:what heterology is presented in vascular endothelial cell surface molecules,if these vascular endothelial cell surface molecules are closely associated with clinical phenotype changes in sepsis,then what function these characteristic molecules may have,and,if these differential molecules have potential value in the dignosis and treatment of sepsis.To answer these questions,we applied in vivo phage-display technique in combination with high-through bioinformatic analysis,trying to map the special function spectrum of pulmonary vasculature under endotoxic shock state in a protein-to-protein interation level and from a vascular proteomics perspective.In this study,BALB/c mice were intraperitoneally injected with lipopolysaccharide（35 mg/kg body weight）.6 hours following the injection,mean arterial blood pressure was decreased under 40 mmHg and endotoxic shock model was successfully induced.Then the endotoxic shock model was injected with a CX₇C library(1.8×10¹¹pfu) via caudal vein.Phage selectively target to lung in endotoxic shock mice were recovered,amplified and re-injected for 4 consecutive rounds of screening.After that,normal mouse was applied for another round of differtial screening.The results showed that phage selectively target to pulmonary vasculature was significantly accumulated,and the final output of recovered phage is about 8.13 times as much as that of the 1^st round of screening,while phage selectively target to liver,brain and kidney is significantly decreased.After the normal subtraction screening in vivo,about 1%of the injected pool was recovered. Finally,92 phages selectively target to lung of endotoxic shock mice were sequenced. It was showed that the codons enconding the phage-displayed peptides are all in NNK format（N=G or A or T or C;K=G or T）,including 22 non-redundant heptapeptides.We also found a high-frequency motif,CSAWSNKFC,was about 35.16%of the total sequenced clones.We also intravenously injected phage-displayed CSAWSNKFC into endotoxic shock mice to confirm the selective binding activity to lung.Significant difference of accumulated amount in lung,liver,brain,kidney and heart was observed（F=96.027, P＜0.001＜0.05） using analysis of variance for paired comparison.The amount of CSAWSNKFC accumulated in lung was 6.62,4.99,16.6 and 10.48 times as much as that of liver,kidney,heart and brain respectively.Further more,analysis of variance of factorial design indicated that the binding activity of pulmonary vasculature is significantly affected by different phage-displayed peptides（F=193.063, P＜0.001＜0.05）.The binding amount of phage-displayed CSAWSNKFC in lung of endotoxic shock mice is 23 times as much as that of control.Beside that,the binding activity in endotoxic shock mice is significantly influenced under the state of endotoxic shock（F=76.580,P＜0.001＜0.05）.Phage-displayed CSAWSNKFC selectively binding to lung of endotoxic shock mice is 3.8 folds as much as that of normal lung.Immunohistochemical analysis indicated that alveolar capillaries were strongly stained,while little positive staining was observed in liver,brain,kidney and heart.At the same time we found weaker positive staining in lung of normal mice vs lung of endotoxic shock mice and little positive staining was observed in endotoxic shock mice when control phage was injected.To further confirm the binding activity of phage-displayed CSAWSNKFC targeting to pulmonary vasculature in endotoxic shock mice,we constructed His-EGFP-CSAWSNKFC fusion protein as a competitor of phage-displayed CSAWSNKFC for binding test in vivo.A dose-dependent inhibition of phage-displayed CSAWSNKFC homing to lung of endotoxic shock mice was observed when His-EGFP-CSAWSNKFC protein was co-injected with the phage-displayed CSAWSNKFC,and the binding activity of phage-displayed CSAWSNKFC can be significantly inhibited by 1,000μg His-EGFP -CSAWSNKFC protein,but not by 1,000μg His-EGFP protein.To identify the receptor for this lung-homing peptide,we employed His-EGFP-fusion proteins to pull down lung plasma membrane proteins.We purified lung plasma membrane through differential centrifugation and sucrose dense centrifugation.The purity of plasma membrane was observed by immunoblotting. Lung plasma membrane was lysed with 2%OG His-EGFP-CSAWSNKFC and His-EGFP were respectively used to pull down lung plasma membrane proteins of endotoxic shock mice.His-EGFP-CSAWSNKFC was also used to pull down lung plasma membrane proteins of normal mice.Solution containing 7M urea,2M thioure and 4%CHAPS was prepared to elute protein complex.Complex of His-EGFP and lung plasma membrane proteins of endotoxic shock mice was labeled with Cy2. Complex of His-EGFP-CSAWSNKFC and lung plasma membrane proteins of normal mice was labeled with Cy3.Complex of His-EGFP-CSAWSNKFC and lung plasma membrane proteins of endotoxic shock mice was labeled with Cy5.All protein complexs were combined and separated on the same 2-dimensional gel.Six new protein spots labeled with Cy5 were found in comparison with that labeled with Cy3,and the amount of the differential protein spots labeled with Cy5 was more than 1.5 folds as much as that of labeled with Cy3.After that,mass spectrum was applied to identify the differential protein spots,which were finally confirmed asβ-actin, ATP synthase subunit beta,aldehyde dehydrogenase and 78 kDa glucose-regulated protein.In order to obtain much more information about the peptides selectively homing to lung of endotoxic shock mice,the phage in two rounds of screening following the subtraction round was sequenced.At last,we obtained 1,375 heptapeptides.We then used the CD-HIT software to filter the redundant peptides from the 1,375 heptapeptides and finally obtained 808 non-redundant heptapeptides.The 808 heptapeptides were divided into a series of consecutive tripeptides from both positive and negative direction.Then all the tripeptides were analyzed by Poisson distribution test.We noticed that the frequence of 152 tripeptides was significant (P≤2.5X10^-5).Clustal W program was used to perform multiple sequence alignment in these 164 tripeptides.We finally obtained 48 mimetic peptides with various properties of amino acid taking into account.After that,the mimetic peptides were aligned in on-line mouse protein sequence library（SWISS-PROT） using BLASTP program,and mouse proteins containing these mimetic peptides were obtained. Signal peptide predicting program SignalP 3.0 and transmembrane helicespredicting program TMHMM 2.0 were used to identify secreted proteins and transmembrane proteins respectively.With the bioinformatics analysis mentioned above,we acquired 137 proteins.We also analyzed the proteins functions using gene ontology （GO）,protein domain and motif information from on-line protein database and gene database.We found that in some cases several different identified motifs matched different domains within one protein,while in other cases,some motifs were identified to match the same domain within different proteins.The identified proteins are involved in cell adhesion,immune response,blood coagulation and anticoagulation,inflammation,energy metabolism,cellular apoptosis and proliferation,permeability of blood vessel,etc.Our conclusion is as follows:1.Vascular endothelial cell surface molecules can present heterogeneity under endotoxic shock state. 2.The peptide CSAWSNKFC can selectively target to pulmonary vascular endothelial cell of endotoxic shock mice,providing essential information for subsequent research.3.The peptide CSAWSNKFC probably interacts with ATP synthase beta subunit, aldehyde dehydrogenase,78 kDa glucose-regulated protein andβ-actin in pulmonary vascular endothelium of endotoxic shock mice.4.The expression of ATP synthase subunit beta can be up-regulated in lung plasma membrane in mice with endotoxic shock.5.Using in vivo screening phage displayed random peptide library in combination with high-through bioinformatic analysis,48 mimetic peptides selectively homing to pulmonary vasculature of endotoxic shock mice were obtained.6.137 functional proteins homing to vascular receptors are potentially mimicked by this 48 selected targeting motifs.The identified 137 functional proteins were involved in cell adhesion,immune response,blood coagulation and anticoagulation,inflammation,energy metabolism,cellular apoptosis etc.The 48 mimetic peptides and the target functional proteins,provide the basis and guidance for the subsequent research.