The Binding Proteins And Immune Regulation Of Limulus Anti-lipopolysaccharide Factor Derived Peptide CLP-19in Mouse Macrophages RAW264.7

The looped peptide CLP-19is derived from a highly functional core region of theLimulus anti-LPS factor and exerts robust anti-LPS activity by directly interacting with LPSin the extracellular space.We previously showed that prophylactic administration of CLP-19even20h prior toLPS challenge might significantly increase the survival rate of a lethal endotoxin shockmouse model. Such effect may be associated with immune regulation of CLP-19.To investigate the underlying mechanisms, peptide affinity chromatography,immunofluorescence and Western blotting procedures were used to identify α-/β-tubulin asdirect and specific binding partners of CLP-19in the mouse macrophage cell line RAW264.7.Bioinformatic analysis using the AutoDock/Vina molecular docking and PyMOL moleculargraphics system predicted that CLP-19bond to the functional residues of both α-andβ-tubulin and located within the groove of microtubules. Tubulin polymerization assayrevealed that CLP-19might induce polymerization of microtubules and preventdepolymerization. The immune-regulatory effect of CLP-19involving microtubules wasinvestigated by flow cytometry, immunofluorescence and Western blotting, which showedthat CLP-19prophylactic treatment of RAW264.7cells significantly inhibited LPS-inducedsurface expression of Toll-like receptor4(TLR4). Taken together, these results suggest thatCLP-19binding to microtubules disrupts the dynamic equilibrium of microtubules, reducingthe efficacy of microtubules-dependent vesicular transport that would otherwise translocateTLR4from the endoplasmic reticulum (ER) to the cell surface.Methods1.1Preparation of peptidesThe head-to-tail looped peptide CLP-19(CRKPTFRRLKWKIKFKFKC; molecular weight2511.1Da) and the linear chain peptide LL-37(LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES; molecular weight4492.4Da)were synthesized by the Symphony Peptide Synthesizerusing a stepwise solid phase peptideassembly procedure starting with an Fmoc-Lys (Boc)-Wang resin. After drying, the peptideswere cleaved and purified by trifluoroacetic acid cocktail solution and HPLC. The resultantCLP-19and LL-37peptides and biotin were dissolved in DMF and mixed with resin. Thesolution was then reacted with HOBT, PYBOP and DIPEA in nitrogen gas for2h at roomtemperature before cleaving and purifying by trifluoroacetic acid cocktail solution and HPLC.The unbiotinylated CLP-19was also labeled with FITC at the last N-terminal cysteine sidechain. The resultant CLP-19peptide and FITC were reacted in a pyridine: DMF:dichloromethane (12:7:5v/v) solution in nitrogen gas for2h at room temperature, and thencleaved and purified by trifluoroacetic acid cocktail solution and HPLC.1.2Compare structural and biological properties of CLP-19, CLP-19B and CLP-19F1.2.1Secondary structures of CLP-19, CLP-19B and CLP-19F4μM of CLP-19, CLP-19B and CLP-19F were dissolved in Tris-HCl buffer (10mM,pH7.4). The lipid was prepared using POPC and POPG (1:1,1mM). CD measurements wereperformed at room temperature on a spectropolarimeter using a0.5cm path-length quartz cell.Spectra were recorded over a wavelength range of190~250nm with bandwidth1nm,scanning speed50nm/min, step size0.1nm and response time2s. Each CD spectrum was anaverage of10scans with the buffer background subtracted.1.2.2Effect of CLP-19, CLP-19B and CLP-19F on the release of TNF-αRAW264.7cells (1×10~5per well) were seeded in96-well plant and incubated overnight.Peptide was added and allowed to co-incubate at37°C. After five washes with PBS to removeunbound peptides,100ng/mL LPS was added and reacted for4h at37°C. The supernatantswere collected and measured the TNF-α level by using ELISA kits.1.2.3Effect of CLP-19, CLP-19B and CLP-19F on NF-κB signaling pathwayRAW264.7cells treated as in2.2. The samples were collected and measured thephosphorylated IκB-α (p-IκB-α) level by using ELISA kits.1.2.4Direct neutralizing LPS activity of CLP-19, CLP-19B and CLP-19FIncreasing concentrations of peptides were co-incubated with0.5EU/ml of LPS for30min at37°C.100μL of mixture was collected and reacted to an equal volume of the LAL reagent. The kinetic turbidity was measured using ATi-321tube reader.1.2.5Cytotoxicity of CLP-19, CLP-19B and CLP-19F toward RAW264.7cellsRAW264.7cells (1×10~4per well) were seeded in96-well plant and cultured for2h at37°C. Increasing concentrations of peptides were added and allowed to react for4h, followedby addition of20μL of MTTfor another4h. Afterward, the medium was removed and150μLDMSO was utilized to dissolve formazan. The OD540of each well was measured usingaspectrophotometer.2.1Identification of CLP-19peptide-binding proteins2.1.1Screen of the CLP-19-specific interacting proteins in RAW264.7cellsProteinswere co-incubated with CLP-19B for30min at37°C, then UltralinkImmobilized Neutr-Avidin resin was added and the incubation continued for another1h atroom temperature with mixing. The resin-bound complex was washed with binding bufferandboiled in SDS-PAGE sample buffer for10min to elute the bound proteins. The proteins wereseparated by12.5%SDS-PAGE and visualized by staining with Coomassie blue.2.1.2Identification of the CLP-19-specific interacting proteinsThe Coomassie-stained bands were manually excised from SDS-PAGE gels and washedwith50%acetonitrile until the gel slice became colorless. The slices were then freeze-driedand treated with dithiothreitol and iodoacetamide and incubated in sequencing gradetrypsinovernight at37°C. The digested proteins were then extracted using45%methylcyanides,5%formic acid, and50%deionized water and analyzed byHPLC-(micro)chip-MS/MS. The orthogonal nanoelectrospray source was operated at2000Vwith a PicoTip emitter. Protein identification was performed automatically by theaccompanying Spectrum Mill software.2.1.3Validation the of CLP-19-specific interacting proteinsThe proteins were elecrotransferred to polyvinylidene fluoride micropore membranesand blocked by incubating with skim milk (5%in TBST) for1h at37°C. After washing, themembranes were incubated with mouse monoclonal α-or β-tubulin antibodies (1:2000) orTLR4antibody (5μg/mL) overnight at4°C, followed by incubation with HRP-conjugatedgoat anti-mouse secondary antibody (1:5000) for1h at37°C. Immunoreactive proteins werevisualized by the DAB substrate. Cells were co-incubated with10μg/mL of CLP-19F for1hat37°C in the dark. After five washes with PBS, cells were fixed with ice-cold methanol for 10min and reacted with1%BSA for30min at room temperature. The mousemonoclonalβ-tubulin antibody (20μg/mL) was added and allowed to react for1h, followedby Alexa Fluor555-conjugated goat anti-mouse homologous IgG (1:500) reaction for1handDAPI (1:1000) reaction for5min. Coverslips were mounted and fluorescence was detectedwith anepifluorescent microscope.2.2Binding characteristics of CLP-19and microtubules2.2.1Direct interaction of CLP-19and microtubulesPurified bovine tubulinswere reconstituted in general tubulin bufferand polymerized tomicrotubules by incubating with cushion bufferfor exactly20min at35°C. The solution wasthen stabilized by diluting with200μL of general tubulin buffer plus taxolat roomtemperature. Co-incubations of diluted tubulins with0.8mg/mL CLP-19,0.3mg/mLmicrotubules-associated proteins (MTAPs, containing60%MTAP2at280kDa and40%tauproteins at40~70kDa), or0.2mg/mL BSA (containing100%BSA at68kDa) were carriedout for30min at room temperature. Each solution (including negative controls withoutmicrotubules) was then laid over respective cushion buffers containing taxol and centrifugedat100,000×g for40min at room temperature. The supernatants and pellets were collected,separated by SDS-PAGE, and visualized by staining with Coomassie blue.2.2.2Specificity of the CLP-19and microtubules interactionMicrotubules (1μM) were pre-incubated with increasing concentrations of CLP-19orLL-37(negative control) and then incubated with CLP-19B (250μM) and microtubules (1μM) or BSA (1mM) were incubated with increasing concentrations of CLP-19B. Pull-downproteins were resolved by SDS-PAGE and visualized by Coomassie blue stain.2.2.3Influence of ionic strength on CLP-19binding to microtubulesMicrotubules were interacted with CLP-19B in the presence of increasingconcentrations of NaCl. Pull-down proteins were resolved by SDS-PAGE and visualized byCoomassie blue stain.2.2.4Bioinformatic analysis of CLP-19and microtubules interactionThe three-dimensional structure of CLP-19was predicted by the Discovery Studiover2.55software. The heterodimer structure of α-/β-tubulin was extracted from the RCSBprotein data bank (No.1JFF). Computer-simulated docking studies were performed by theAutoDock/Vina molecular docking software, which allowed hydrogen atoms to be added to the grid set in the center of the active site region that involved all functional amino acidresidues. A set of possible binding models of CLP-19and α-/β-tubulin heterodimer wascreated according to the principle of minimum energy, and the functional residues of CLP-19and α-/β-tubulin heterodimer were further predicted by PyMOL software.3. Immunoregulatory effects of CLP-19and microtubules interaction3.1Effect of CLP-19on dynamic flux of microtubulesFree, purified bovine α-and β-tubulin subunitswere dissolved in chilled (4°C) tubulinpolymerization (TP) buffer and immediately mixed with pre-warmed (37°C) CLP-19solutionto achieve final concentrations of3mg/mL tubulins and20μM CLP-19. The polymerizationreaction was allowed to progress over60min at37°C and then switched the temperature to4°C for30min, during which time changes in the OD at340nm were recorded every5minutes using a spectrophotometer. Control reactions consisted of tubulins alone (to generatethe standard polymerization curve), with20μM LL-37, or with10μM paclitaxel.3.2Effects of CLP-19on cell surface expression of TLR4Adherent RAW264.7cells (5×10~6per treatment) were pre-treated with50μg/mLCLP-19at37°C. After five washes with PBS to remove unbound CLP-19,100ng/mLLPSwas added and allowed to react for4h at37°C. LPS-simulated cells, CLP-19-treated cellsand non-treated cells served as controls. After reaction, cells were collected, washed withice-cold PBS, and blocked with1%BSA for1h on ice. After an additional wash, the liveRAW264.7cells were incubated with mouse monoclonal TLR4antibody (1μg/10~6cells) orhomologous IgG control antibody (1μg/10~6cells) for20min on ice. After a final gentle wash,the cells were incubated with Alexa Fluor488-conjugated goat anti-mouse homologous IgG(1:500) for20min on ice and analyzed by flow cytometrywith gating for20,000events.3.3Effects of CLP-19on total TLR4RAW264.7cells treated as in3.2. Total proteins were resolved, blotted and probed withmouse monoclonal TLR4(5μg/mL) and α-tubulin antibodies (1:2000).3.4Relationship between prophylactic time and anti-LPS activity of CLP-19RAW264.7cells pre-treated with CLP-19(50μg/mL) in various time and subsequentlystimulated by LPS (100ng/mL). The surface TLR4and TNF-α in the supernatants wereanalyzed using flow cytometry and ELISA kit respectively. Results1. Preparation and comparation of peptide and its counterparts1.1Preparation of peptidesThe peptidesachieve a purity of98.4%for CLP-19,99.1%for LL-37,99.6%forCLP-19/biocytin (CLP-19B),95.2%for LL-37/biocytin (LL-37B) and98.8%forCLP-19/FITC (CLP-19F).1.2Similar structural and biological properties of CLP-19, CLP-19B and CLP-19F1.2.1Second structure of CLP-19, CLP-19B and CLP-19FCLP-19, CLP-19B and CLP-19F exhibited unstructured in free solution, but convertedto α-helical structure when in the presence of large unilamellar POPC-POPG (1:1) vesicles, ascharacterized by the maxima at195nm and minima at210and222nm.1.2.2Effect of CLP-19, CLP-19B and CLP-19F on the release of TNF-αAddition of peptides alone to the unstimulated cells had no effect on the amount ofTNF-α, compared to the completely untreated cells (p>0.05). Prophylactic peptides treatmentprior to LPS challenge was found to significantly attenuate the release of TNF-α in RAW264.7cells. The CLP-19B and CLP-19F displayed very similar effect (attenuation of TNF-αrelease) to CLP-19(p>0.05).1.2.3Effect of CLP-19, CLP-19B and CLP-19F on NF-κB signaling pathwayCLP-19and its counterparts were able to induce phosphorylation of IκB-α. Butprophylactic administration might inhibit the LPS-induced up-regulation of p-IκB-α. Threepeptides displayed similar activity to NF-κB signaling (p>0.05).1.2.4Direct neutralizing LPS activity of CLP-19, CLP-19B and CLP-19FThe LAL test showed there were no statistically significant differences inneutralizing-LPS activity of CLP-19B and CLP-19F, compared to CLP-19at concentrationsbetween10and100μM (p>0.05).1.2.5Cytotoxicity of CLP-19, CLP-19B and CLP-19F toward RAW264.7cellsCLP-19, CLP-19B and CLP-19F were not observed to be toxic to RAW264.7cells upto100μM concentration (p>0.05). Further increasing the concentration of peptides up to200μM, significant cytotoxicity exhibited. However, there were no significant differences ofcytotoxicity between CLP-19and its counterparts in this study (p>0.05). 2. Analysis of CLP-19peptide-binding proteins2.1Identification of tubulins as CLP-19peptide-binding proteins2.1.1Screening and identification of CLP-19peptide-binding proteinsThe majority of proteins found bound to CLP-19were of50kDa in size. MS analysisidentified the50kDa pull-down protein as α-/β-tubulin.2.1.2Validation the of CLP-19-specific interacting proteinsWestern blotting of CLP-19bound proteins showed immune-reactivity with themonoclonalα-and β-tubulin antibodies for the bands at55and50kDa, respectively.Immunofluorescence assay of live RAW264.7cells showed that CLP-19F co-localizedintracellularly with microtubules.2.2Binding characteristics of CLP-19and microtubules2.2.1Direct interaction of CLP-19and microtubulesThe CLP-19sample showed a band of2.5kDa (corresponding to CLP-19alone) in thesupernatant and not in the pellet fraction, suggesting that CLP-19could not pellet duringcentrifugation. The CLP-19+microtubules sample showed a2.5kDa band and a55kDa bandin the pellet and not in the supernatant fraction, suggesting that CLP-19was associated withmicrotubules and pelleted together during centrifugation.2.2.2Specificity of the CLP-19and microtubules interactionPre-incubation with unlabeled CLP-19led to a dose-dependent decrease in CLP-19Bbinding to microtubules. In contrast, pre-incubation with unlabeled LL-37control peptideproduced only a slight general decease in CLP-19B binding to microtubules. Moreover, asignificantly higher and dose-dependent binding percentage was observed with microtubules,compared to BSA.2.3Influence of ionic strength on CLP-19binding to microtubulesPull-down assay was performed in various concentrations of NaCl (0~400mM). Theamount of pulled-down microtubules changed slightly across the entire range, indicating thatthe ionic strength seldom interfered with the interaction of CLP-19and microtubules.2.4Bioinformatic analysis of CLP-19and microtubules interactionThe simulated docking models demonstrated that the looped CLP-19tended to bind inthe groove of the α-/β-tubulin heterodimer.3. Immunoregulatory effects of CLP-19interaction with microtubules 3.1Effect of CLP-19on dynamic flux of microtubulesAddition of CLP-19to the polymerization reaction not only increased the Vmax, buteliminated the nucleation phase and enhanced the overall polymer mass. Moreover, CLP-19was able to stabilize the microtubules against depolymerization by cold.3.2Effects of CLP-19on cell surface expression of TLR4Addition of CLP-19alone to the unstimulated cells had no effect on the amount ofevents (TLR4expression), compared to the completely untreated cells. However,pre-incubation with CLP-19prior to LPS stimulation resulted in significantly lower amountsof TLR4on the cell membranes.3.3Effects of CLP-19on total TLR4The amount of total TLR4was found nearly identical in the untreated cells andCLP-19-treated cells.3.4Relationship between prophylactic time and anti-LPS activity of CLP-19CLP-19had time-dependent inhibitory effect on the expression of surface TLR4andTNF-α in15h, indicating that the optimal prophylactic time of CLP-19was15h in vitro.Conclusions1. α-/β-tubulin as direct and specific binding partners of CLP-19in the mousemacrophage cell line RAW264.7.2. CLP-19interacting with microtubules perturbs the dynamic equilibrium ofmicrotubules, making microtubules-dependent vesicular transport of TLR4less effective.3. CLP-19alleviates the responses of macrophages associated with LPS viadown-regulating the expression of surface TLR4.