Study On Identification,biological Activity And Inhibitors Of The Enzymatic Constituents Of Jellyfish Venoms

In recent years,jellyfish outbreak in the world frequently and cause various environmental problems.As one of the consequences,the number of jellyfish envenomations worldwide is increasing,which also raises the public health concern.In China,the situation is getting worse with numerous people stung by jellyfish Nemopilema nomurai(N.nomurai)and Cyanea nozakii(C.nozakii)in the summer.Jellyfish venoms underlie the chemical basis for human envenomations,which are composed of various proteins and peptides and possess extensively biological activities.Proteins species in jellyfish venom could be grouped into enzymatically active proteins and enzymatically inactive proteins.So far,understanding of the enzymatic constituents and their biological functions are critically needed.In the present study,two common scyphozoan jellyfish species,N.nomurai and C.nozakii,were chosen as research subjects to study the enzymatic properties and biological activities of the venom extracted from their respective nematocysts.And as another goal of the present study,the inhibitory effects of secondary metabolites from marine-derived fungi against the enzymatic properties of jellyfish venom were assayed for developing natural antidotes of jellyfish stings.Resuts are given as follows:1.Based on the biochemical and kinetical profiling,the enzymatic properties of N.nomurai nematocyst venom(Nn NV)and C.nozakii nematocyst venom(Cn NV)were compared.The current data revealed that Nn NV and Cn NV exhibited various enzymatic activities,of which metalloproteinases activity and PLA2s-like activity were predominant.Under optimal conditions,the metalloproteinse activity of Nn NV was 1470.0 ± 63.5 U/mg,which was 7.1-fold higher than that of Cn NV.Moreover,the catalytic hydrolysis of azocasein by Nn NV was in accordance with Michaelis-Menten equation with KM and Vmax values of 17.0 ± 6.9 ?mol/L and 11.4 ± 1.2 U/mg/min.Results also indicated that Nn NV and Cn NV were found to degrade NOBA,a specific phospholipase A2 substrate,in a dose-dependent manner.The turnover numbers of the catalytic reactions of Nn NV PLA2s-like,calculated as kcat,was 7.5 ? 10-3 min-1,which was slightly higher than that of Cn NV PLA2s-like.Biochemical analysis revealed that the catalytic activities of metalloproteinases and PLA2s-like were dependent on different physiochemical conditions such as temperature,p H,divalent ions and inhibitors.The optimum p Hs of Nn NV metalloproteinase and PLA2s-like were 9.94 ± 0.04 and 8.0,respectively.In addition,Nn NV and Cn NV exhibited serine protease activity and fibrinogenolytic activity.2.The enzymatic components of Nn NV and Cn NV were functionally identified by combined in gel zymography and LC-MS/MS analysis.Various proteases were revealed by catalytic hydrolysis of gelatine,casein and fibrin in the zymograms of proteolytic enzymes,whose molecular weights ranged from 26 k Da to 200 k Da.The zymogram of PLA2 indicated lecithinolytic activity of Nn NV and Cn NV at 14.4 ~ 18.4 k Da.Moreover,marked hemolysis occurred at the location(14.4 ~ 18.4 k Da)where the lipase hydrolyzed the egg yolk substrate.Hyaluronic acid zymogram analysis further revealed two protein species exhibiting concentration-dependent lysis of hyaluronic acid,and their molecular weights were ~48 k Da and ~105 k Da,respectively.LC-MS/MS identification showed that the potential proteases responsible for the proteolytic activity observed in the zymography assays showed some homology to zinc metalloproteinase-disintegrin-like and astacin-like metalloproteinases.In addition,the LC-MS/MS identification also suggested that phospholipases(PLA1,PLA2,PLDs),hyaluronidases,serine proteases and L-amino acid oxidases were also potential components of jellyfish venom.Isolation of enzymatic components of Nn NV was conducted with Hi Prep 26/60 Sephacryl S-200 gel filtration column,fractions A ~ J were obtained from Nn NV.And peak peak D were found to possess potent metalloproteinase activity.Moreover,under non-reducing conditions,SDS-PAGE analysis revealed that peak D was composed of one single protein band at 60 k Da.And under reducing conditions this protein band split into two bands with lower moleclar weights,indicating that the protein at 60 k Da has two subunits bridged by disulfide bond.3.Biological functions of the enzymatic constituents of jellyfish venom were studied with specific inhibitors.Metalloproteinase inhibitors,EDTA,1,10-phenanthroline and batimastat were found to significantly inhibit the metalloproteinase activity of Nn NV.And among them,40 ?mol/L batimastat resulted in an approximate 62% reduction of jellyfish venom induced hemolysis compared to venom exposed sheep erythrocytes,suggesting that metalloproteinases contribute to hemolytic activity.At the same time,potent inhibition of sheep erythrocyte hemolysis was observed after venom pre-incubation with very low concentrations of varespladib(> 1.6 ?mol/L).This observation indicated the possible involvement of Nn NV PLA2-like in hemolytic activity to sheep erythrocytes.The potential roles of Nn NV metalloproteinases and PLA2 s in in vivo animal model of jellyfish envenomation were evaluated by pre-incubation Nn NV with varying doses of batimastat or varespladib.Results showed that Nn NV possessed lethal activity to juvenile grass carp Ctenopharyngodon idellus,could induce intensive paw edema within 30 min,elevated the extravasation of evans blue dye in dorsal or renal microvessel into outside tissues and led to thigh muscle tissue swelling of experimental animals.However,the extravasation of evans blue dye into local tissues was significantly reduced after pretreatement Nn NV with 200 ?mol/L of batimastat,indicating the important role of Nn NV metalloproteinases in changing vascular permeability.Type IV collogen and laminin are important structural constitutents of extracellular matrix(ECM)and vascular basement membranes,playing an important role in maintaining vascular integrity.Nn NV was found to degrade these two important structural constitutents in vitro within 12 h.This ECM-degrading capablity of Nn NV may constitute one of the important factors,which leads to changing the vascular permeability.In addition,preincubation of Nn NV with EDTA could extend the survival time of grass carp,but did not change the mortality of grass carp within 4 h.At the same time,it was also observed that preincubation of Nn NV with 50 ?mol/L,200 ?mol/L batimastat or varespladib did not significantly affect the acute swelling within 4 h,but preincubation of Nn NV with 200 ?mol/L batimastat or varespladib significantly reduced the degree of swelling after 12 h.These observations indicate that Nn NV metalloproteinases and phospholipase A2 are not acute inflammatory factors,but may fuction through their enzymatic hydrolysis of the local tissue.4.By means of the established enzymological model,marine-associated fungi from jellfish N.nomurai and unidentified sea anemone,were isolated and cultivated for obtaining their secondary metabolites.And then the Nn NV metalloproteinase inhibitory activity of these metabolites or pure compounds were tested.The current data suggested that co-cultures from jellyfish-derived fungi Aspergillus versicolor Sm T07 and Tilletiopsis sp.Sm U05 showed remarkable inhibitory activity against Nn NV metalloproteinase,and Et OAc,CHCl3 fractions from the cocultures exhibited significantly inhibitory potency.The current results also suggested that phenazine-1-carboxylic acid(PCA)isolated from sea anemone-associated fungus Emericella sp.SMA01 and jellyfish-associated fungi Aspergillus versicolor Sm T07 was found to inhibit the metalloproteinase activity of Nn NV for the first time,with the IC50 value of 708.26 ?g/m L.Moreover,phenazine-1-carboxylic acid was able to inhibit the fibrinogenolytic activity of Nn NV at the ratio of 1:0.6 ~ 1:10(w/w,Nn NV:PCA).