Screening And Bioactivity Of Peptides That Could Specifically Bind To Sodium Pump α1Subunit M3-M4Extramembrane Domain

Sodium pump （Na⁺,K⁺-ATPase） is a transmembrane carrier proteinmediating ion transport in mammalian biological membranes. It consists ofthree different subunits （α, β, and γ） and and α-subunit has the catalyticactivity. α-subunit consists of α1, α2, α3, α4subtypes and the basic structureof each subtype is highly conserved, which is composed of10transmembranedomains （M1-M10）. The extramembrane domain of α-subunit contain multipleregulating factors binding sites, including the sites of sodium pumpinhibitors-endogenous ouabain. The second transmembrane region M3-M4of α-subunit is currently considered as an important specific binding site forendogenous ouabain. Ouabain can inhibit sodium pump activity by interactingwith it, and then promotes hypertension. In this study, the specific bindingpeptide of sodium pump α1subunit M3-M4extramembrane domain wasscreened from phage display random peptide libraries, which can blockouabain M3-M4binding site and inhibit the combination of ouabain andsodium pump competitively. And the bioactivity and influence of the specificbinding peptide on endothelial cells and SHR rats was also detected, which laythe foundation for the further exploring the possible interaction mechanism ofsodium pump with its inhibitory factor and the treatment of hypertension.1. Screening the binding peptide of the sodium pump α1subunit M3-M4extramembrane domainObjective: To screen the specific binding peptides from phage displayrandom peptide libraries with M3-M4extramembrane domain peptide as atarget, which can block the inhibition of ouabain to sodium pump, and todetect whether competitive inhibition between the specific binding peptide andouabain occurred. Methods:（1） Screen specific binding peptides using phage randompeptide library technology with Sodium pump α1subunit M3-M4extracellulardomain sequence （ILEYTWLEAGGGS） as a target. The phages that can bindwith the target protein were enriched after three turns "adsorption-elution-amplification" panning.（2） Double-sandwich ELISA identify positive phageclones: The sodium pump α1subunit M3-M4extramembrane domain peptide（ILEYTWLEAGGGS） was used to coated ELISA plate and the negativecontrol was set at the same time.30random amplified phage clones wereadded to the plate, and then HRP-Anti-M-13antibody was added and the ODvalues were measured at420nm.（3） The concentration-dependent experiments:The sodium pump α1subunit M3-M4extramembrane domain peptide（ILEYTWLEAGGGS） was coated as the target protein, and each clone thatto be identified was coated by eight holes. Phage clones are amplified andphage titer was determined. About10¹² pfu phages were added to plate andserially diluted phages by five-fold were added, too. And thenHRP-Anti-M-13antibody was added and OD values were measured. The plotof the absorbance value versus the dilution factor was generated.（4） Theextraction and sequence analysis of phage ssDNA: Phage ssDNA wasextracted by M13phage ssDNA rapid extraction reagent kit. After sequencing,the DNA sequence of the insert peptide was determined according to “randompeptide-gIII fusion protein N-terminal sequence”, and the correspondingamino acid sequence was deduced according to “genetic code table”.（5）Ouabain competitive inhibition test:96-well plates were coated by sodiumpump α1subunit M3-M4extramembrane domain peptide. The phagesupernatants were diluted by two-fold serially, and the mixed liquors of50μlphage supernatants and50μl100μg/ml ouabain were added into96-well plates.The negative control was set at the same time. And then HRP-Anti-M-13antibody was added and OD values were measured. The plot of the absorbancevalue versus the dilution factor was generated.Results:（1） Phage12peptide library screening results: The output/input ratio of the positive phage was gradually increased during three turns panning,which means that the target phages from peptide library were enrichedselectively.（2） Double-sandwich ELISA test: The OD ratio of21sample holes:control holes was≥2.1among30random selected phage clones. So the21phage clones were identified as positive clones.（3） Theconcentration-dependent test: The results showed that21positive clones wereall concentration-dependent and showed good affinity.（4） Extraction andsequence analysis of the phage ssDNA: The ssDNA of the21positive phageclones were extracted and1%agarose gel electrophoresis was performed. Theresults showed that the molecular weight of the bands of7500bp was in linewith the phage DNA. The sequencing results showed that the21positivephages contained three kinds of binding peptides. Peptide A:SWYQEGPSTQPE, consistent rate of47.6%（10/21）. Peptide B:SSNTAWQNMTSL, consistent rate of28.6%（6/21）. Peptide C:GENPWENVAVAM the consistent rate was23.8%（5/21）. Peptide A wassynthesized as sodium pump binding peptide.（5） Ouabain competitiveinhibition test: The results showed that the OD value of the same concentrationof phage supernatant was reduced than the negative control on the presence ofouabain. The binding of the binding peptide to sodium pump was decreasingwith the reduction of the concentration of the peptide in phage supernatant,which means that the binding peptide and ouabain were competitive inhibited,and the binding peptide was able to combine specifically with the epitoperecognition sites of sodium pump α1subunit M3-M4extramembrane domain.Conclusion: A specific binding peptide （SWYQEGPSTQPE） wassuccessfully obtained from phage random12peptide library with M3-M4extramembrane domain peptide （ILEYTWLEAGGGS） as a target, whichcan inhibit competitively the binding of ouabain to sodium pump. This laid thefoundation to further explore the action mechanism of sodium pump withouabain and the treatment of hypertension. 2. The biological activity analysis of sodium pump binding peptideObjective: To detect the influence of sodium pump binding peptide toouabain stimulating endothelial cell proliferation or apoptosis and the effect ofit to the blood pressure of SHR, and to explore the possible application valueof the binding peptide in the treatment of hypertension.Methods:（1） MTT assay was performed to detect the influence ofsodium pump binding peptides to the proliferation of endothelial cellEA.hy926stimulated by ouabain within physiological concentration ranges:96-well plates were coated by EA.hy926cells, and the mixtures of ouabainwithin physiological concentration ranges (0.3×10^-9,0.6×10^-9,0.9×10^-9mol/L)and10^-6mol/L sodium pump binding peptides were added. The groups withoutsodium pump binding peptides were set as the negative control. After24,48,and72h incubation,20μL MTT was added and the mixture was continuallycultured for4h. The supernatant was removed and DMSO was added, and theabsorption values at570nm were measured.（2） Flow cytometry was used todetect the influence of sodium pump binding peptides to the apoptosis ofvascular endothelial cell stimulated by ouabain: The cells adherent fully wereselected and ouabain with high concentration (10^-4,10^-6mol/L) and10^-4mol/Lsodium pump binding peptides were added separately. The groups withoutsodium pump binding peptides were the negative control. After24hincubation, the cells were collected by centrifugation and fixed by70%coldethanol, and then stored at4℃. After staining with propidium iodide （PI） for30min, the percentage of cell apoptosis of was detected by flow cytometry.（3）The influence of sodium pump binding peptide to the blood pressure of SHR:SHR for treatment group were injected with2mg/kg sodium pump bindingpeptide by tail vein and the blood pressure was measured respectively after15min,30min,45min,60min. The group of normal saline was used as thenegative control.Results:（1） MTT assay was performed to detect the influence of sodiumpump binding peptides to the proliferation of endothelial cell EA.hy926stimulated by ouabain within physiological concentration ranges:0.6×10^-9 mol/L ouabain had the greatest proliferative effect （P<0.05）. When coupledwith the sodium pump binding peptide, the cell proliferation rate wasdecreased compared to ouabain alone, by10.24%,18.35%,16.18%, for24h,48h,72h, respectively.（2） Flow cytometry was used to detect the influenceof sodium pump binding peptides to the apoptosis of vascular endothelial cellstimulated by ouabain: The results showed that ouabain with highconcentrations (10^-4mol/L,10^-6mol/L) can stimulate the apoptosis ofEA.hy926cell, with significant difference （P<0.01）. The apoptosis rate of10^-6mol/L ouabain treatment group was significantly decreased when10^-4mol/Lsodium pump binding peptide was added （P<0.01）.（3） The blood pressure ofSHR began to decrease after2mg/kg sodium pump binding peptides wereinjected by tail vein, and reached a minimum value of179.8±4.83mmHg at15min, compared to the original level of179.8±4.83mmHg, which reducedby11.5%. And then the blood pressure increased gradually, and was restoredto baseline value at about60min.Conclusion: The sodium pump binding peptides could antagonize thebinding of ouabain to sodium pump, and affect the proliferation or apoptosisof endothelial cell induced by ouabain. What’s more, it can reduce the bloodpressure of SHR, which implied that the sodium pump binding peptides couldbe used as a potential new drug against hypertension.