Purification And Bioactivity Of The Hemolytic Protein And Polypeptide Growth Factors From Jellyfish Cyanea Capillata

INTRODUCTIONJellyfish belong to the phylum Cnidaria, which is one kind of lower invertebratemarine zooplankton. They are rich in species numbers, abundant and distribute widely, andplay an important role in the marine ecosystem. There are a large number of nematocystsdistributing in the tentacles and oral arms, and the bioactive compoundsof nematocysts include various enzymes, histamine, bioactive peptides and other accessorysubstances with the exception of toxins. Purification and identification of bioactivesubstances in the nematocysts is one of the most important prerequisites to take advantageof jellyfish venom. In present study, jellyfish Cyanea capillata, which has often bloomedin southeast Chinese coastal waters in recent years, was chosen as the research object forextraction, purification, identification and bioactivity of the jellyfish venom.METHODSChapter One: Hemolytic activity and purification of the hemolysin from thejellyfish Cyanea capillataSection I Tentacle extract (TE) is a potential alternative of the jellyfish venom, whichwas chosen as the research object, monitoring the protein concentration and hemolyticactivity of TE as well as SDS-PAGE analysis. Effects of physical and chemical factors andprocessing methods on protein stability and hemolytic activity of TE were observed. Anionexchange chromatography and gel filtration chromatography were utilized for thepurification of the hemolysin in TE, which was pretreated with40℃water bath for1h.Section II Oral arms extract (OAE) is another potential alternative of the jellyfishvenom, which was prepared through severe mechanical vibration and centrifugation. Amicroscopic observation of the emission of nematocysts in the oral arms was taken duringthe process of preparation. Effects of desalting, freeze-dring and ultrafiltration on thehemolytic activity of OAE were observed, and then anion exchange chromatography andgel filtration chromatography were utilized for separation of the hemolysin in OAE,respectively.Chapter2: Purification, identification and bioactivity of the polypeptide growthfactors from the jellyfish Cyanea capillataFirst, preparing two kinds of venom sample: rapid preparation of tentacle extract (rpTE) and crude nematocyst venom (cNV), and determining their hemolytic activity andother parameters. A microscopic observation of the emission and fragmentation ofnematocysts in the tentacles was taken during the process of preparation. Secondly, gelfiltration chromatography and HPLC, combining with MALDI-TOF mass spectrometryanalysis, were utilized for the purification of rpTE and cNV, respectively. Thirdly,determining the N-terminal sequence of the purified polypeptides and identifying theamino acid sequence of the5782.9Da polypeptide for bioinformatics analysis, combiningwith the enzymatic and reducing derivative reactions and MALDI-TOF mass spectrometry.Lastly, Applying the methods of filter paper diffusion, spectrophotometry, and CCK-8tostudy the antimicrobial and antithrombin activities and cell proliferation of the polypeptidegrowth factor Cc-GRN-1(5782.9Da peptides).RESULTSChapter One: Hemolytic activity and purification of the hemolysin from thejellyfish venomSection I TE caused a significant and dose-dependent hemolytic effect, and the HU50of TE against0.5%erythrocyte suspension from SD rat was226μg/mL. A40℃waterbath for1h could effectively remove inactive proteins contaminating in TE. TE retainedhemolytic activity at4℃for28days but it was unstable when kept at25℃over3days.TE was active in the pH range from6.0to11and the optimum pH was8.0. TE wasconcentrated1.3-fold and1.5-fold by100kDa and10kDa ultrafiltration tubes,respectively. Various buffer solutions had significantly different effects on the stability andhemolytic activity of TE, and a good salting-out effect was observed on the hemolyticprotein of TE while the concentration of ammonium sulfate solutions was greater than26%.After anion exchange chromatography, the first elution peak was hemolytic component,which was further separated by gel filtration chromatography and resulted in two elutionpeaks with no hemolysis. However, SDS-PAGE analysis showed the lane of the secondelution peak contained four protein bands, where the31kDa band was the most significantand the20kDa band was followed.Section II The HU50of OAE against0.5%erythrocyte suspension from SD rat was95μg/mL. There were three types of nematocysts in the oral arms, including spindle,ellipse and sphericity, which diameter were26-30μm,14μm and4μm, respectively. Thespheroid nematocysts were the most in quantity, followed by ellipsoidal nematocysts, fusiform nematocysts were the least. OAE retained hemolytic activity after the directtreatment of freeze-drying and reconsitution, but missed the hemolytic activity if it wasdesalted firstly, and then freeze-dried and reconstituted. The concentrated OAE afterultrafiltration with a membrane (cut off100kDa), but not the filtrate, was hemolytic. Allfractions had no hemolytic activity after anion exchange chromatography and gel filtrationchromatography.The precipitation emerging during ultrafiltration with a membrane (cutoff3kDa) was hemolytic.Chapter2: Purification, identification and bioactivity of the polypeptide growthfactors from the jellyfish Cyanea capillataFirst, there were three types of nematocysts in the tentacles, including spindle, ellipseand sphericity, the diameter of which were26-30μm,14μm and4μm, respectively. Thefusiform nematocysts were most in quantity, followed by spheroid nematocysts, whileellipsoidal nematocysts were the least. rpTE caused a significant and dose-dependenthemolytic effect. The HU50of rpTE against0.5%erythrocyte suspension from SD rat was200μg/mL, and the HU50of cNV was40μg/mL. Secondly, four polypeptides werepurified from the sample rpTE,5805.3Da,5782.9Da,5668.3Da and5747.4Da,respetively. There were five polypeptides purified from the sample cNV, such as5805.3Da,5782.9Da,5691.0Da,5861.3Da and5747.4Da. The5805.3Da,5782.9Da and5747.4Da polypeptides can be purified from both rpTE and cNV. Thirdly, the amino acidsequence of the polypeptide5782.9Da wasNVICPDGTSFCASGQTCCKLSSGSYGCCPLPNAVCCSDGVHCCPSGTTCDVSQGTCL(I)R, cantaining58amino acids and12cysteines. The N-terminal sequence of thepolypeptides5805.3Da,5747.4Da and5668.3Da areNVICPDGTSYCATGQTCCKLSSGGYGCCPL, VICPDGTSYCATGQT andVICPDGTSFCASGQTCCVLSSGQY, respectively, and their sequences have highsimilarity. The polypeptide5782.9Da belongs to the granulin family of growth factor(named as polypeptide growth factor Cc-GRN-1) with a theoretical isoelectric point5.29.There are six disulfide bonds in the molecule, so it was structural stability and stronghydrophilicity. Cc-GRN-1is secreted polypeptide comprising eight glycosylation sites andfive phosphorylation sites. Multiple sequence alignment suggested that the polypeptidegrowth factor Cc-GRN-1might have antibacterial and antithrombin activity. Functionalannotation and metabolic pathway analysis showed that Cc-GRN-1might play a major roleon protein binding and growth factor activity, involving in a variety of biological processes, such as response to stress, proteolysis, signal transduction, blastocyst hatching, embryoimplantation, positive regulation of epithelial cell proliferation, neuropeptide signalingpathway and defense response to bacterium. Lastly, there was no obvirous bacteriostaticring appearing around filter paper containing g/mL Cc-GRN-1solution, and wasalso not inhibitory to antithrombin activity at the concentration of1g/mL,2g/mL,4g/mL,8g/mL,12g/mL,25g/mL,50g/mL and100g/mL. The proliferation rateof A549, HUVEC, HaCaT, A9and L-929cells significantly increased with the increase ofCc-GRN-1concentration, while the NRK-52E cells showed no significant changes.CONCLUSION1. A40℃water bath for1h could effectively remove inactive proteins in TE andreduce its viscosity.4℃and pH8.0are the optimum conditions to maintain the proteinstability and hemolytic activity of TE. The hemolytic protein was enriched by thesalting-out effect when the concentration of ammonium sulfate solutions was greater than26%. Lyophilization did not affect the hemolytic activity of OAE, while the salt solutionwas conducive to maintaining its hemolytic activity. The molecular weight of hemolyticprotein from the jellyfish Cyanea capillata was greater than100kDa, and might becomposed of multi-subunit with structural instability, weak solubility and susceptibility tovarious physical and chemical conditions and processing methods.2. Hemolytic activity of the crude venom samples was cNV> OAE> rpTE> TE,respectively. Severe mechanical oscillations could effectively stimulate nematocystsemission and venom release. The preparation method of OAE and rpTE would efficientlyreduce the mix and interference from tissues or non-toxin proteins. The method in whichthe nematocysts were isolated first and then broken for extracting venom was conducive topurity and quality of the venom sample, but was difficult to get adequate amounts ofvenom.3. Six polypeptides5805.3Da,5782.9Da,5668.3Da,5747.4Da,5691.0Da and5861.3Da, respectively, were purified from rpTE and cNV, and their sequences showedhigh similarity. The5782.9Da polypeptide (Cc-GRN-1) belonged to the granulins,prompting that we found a group of similar molecular weight, highly homologouspolypeptide growth factors in the tentacle (especially nematocysts) of jellyfish Cyaneacapillata. 4. The polypeptide growth factor Cc-GRN-1showed no antibacterial andantithrombin activity, but could significantly promote the proliferation of A549, HUVEC,HaCaT, A9, and L-929cells.