| PartⅠMulti-omics comparative analysis of venoms of two dominant sea snake species from ChinaSea snakes are a subfamily of Elapidae.There are more than 70 species of sea snakes around the world and a total of 17 species in China.The dominant species are Hydrophis curtus（H.curtus）and Hydrophis cyanocinctus（H.cyanocinctus）,which are mostly distributed in Beibu Gulf and Hainan,Fujian coastal waters.Sea snake venoms are an extremely lethal mixture of proteins,peptides and other small molecules,mainly composed of neurotoxins and various enzymes.Due to the differences in sea snake species,their geographical distributions and prey objects,the protein compositions and proportions of sea snake venoms are also distinct.In order to better investigate the compositions of H.curtus venom（Hcu V）and H.cyanocinctus venom（Hcy V）in China,in partⅠ,we firstly constructed the venom gland transcriptome databases,and then identified the protein compositions of the two sea snake venoms using liquid chromatography-mass spectrometry.Based on the transcriptome and proteome databases of sea snake venoms,we further analyzed the biological properties of sea snake venoms.The main results were as follows:1.Firstly,the Illumina Hi Seq?platform was used to sequence the venom gland transcriptomes of two sea snakes in China,and high-quality venom gland transcriptome databases were successfully constructed.There were 38,710 and 35,716 unigenes obtained from venom glands of H.curtus and H.cyanocinctus,respectively.We have annotated the unigenes via a set of public databases including NR,KEGG,GO,KOG and Swiss Prot database.In the NR database,the annotation sequences of sea snake venom glands are mostly from Notechis scutatus,Pseudonaja textilis,and Ophiophagus Hannah.For the KEGG annotation,10,949 and 10,683 unigenes were annotated in the venom glands of H.curtus and H.cyanocinctus,mainly focusing on the pathways involved in signal transductions and disease occurrences.With regard to the simple sequence repeat（SSR）detection,15,171 and 12,908 SSR sites were found in unigenes of the two sea snake venom glands,respectively.Among them,the single nucleotide repeat units accounted for the highest proportion with over 50%.We further performed the Blast analysis between venom gland transcriptomes and Uni Prot animal toxin and venom database.A total of 17 kinds of snake venom proteins were screened out,fully mining the gene resources of functional protein in sea snake venom glands.2.Secondly,the original peptides of Hcu V and Hcy V were obtained by LC-MS/MS,and matched with toxins in the Uni Prot animal toxin database.A total of 47 and 38 putative toxins were identified in Hcu V and Hcy V,respectively,which could be grouped into 15functional categories,mainly including proteinases,phospholipases,three-finger toxins（3FTxs）,lectins,protease inhibitors,ion channel inhibitors,cysteine-rich venom proteins（CRVPs）and snake venom metalloproteases（SVMPs）.These toxins are mostly found in snakes but with a few found in other venomous organisms,including spiders（Lycosa singoriensis）,scorpions（Lychas mucronatus）,and snails（Conus textile）.3.After comparative analysis of venom proteomes,we found that the proportions of each toxin category varied between Hcu V and Hcy V,with 3FTx（54%in Hcu V/69%in Hcy V）and PLA2（38%in Hcu V/22%in Hcy V）ranking the top two most abundant proteins.The biological activity detection of sea snake venom showed that both Hcu V and Hcy V exhibited relative high lethality(LD50 in mice,0.34 mg/kg and 0.24 mg/kg,respectively),specific neurotoxicity and PLA2 activity,and mild indirect hemolytic toxicity.No obvious procoagulant and anticoagulant activities were identified in Hcu V and Hcy V.These results were consistent with those of transcriptomic and proteomic analysis.In conclusion,after multi-omics analysis of Hcu V and Hcy V,combined with biological activity detection of the toxins,this part revealed the molecular diversity of sea snake venoms from the two dominant species in China.It also indicated a possible relationship between the difference in the proportion of toxin proteins and the symptoms and lethal effects of sea-snake bites,providing an important basis for subsequent development of antivenom against sea snake bites in Chinese waters.PartⅡPreparation and Evaluation of Sea Snake AntivenomThe main component of sea snake venom is neurotoxin,which can block nerve synaptic transmission,resulting in a series of poisoning symptoms.As an extremely toxic venom,sea snake venom can induce severe respiratory failure and high mortality.The most effective first aid method for sea snake bites is to inject antivenom as soon as possible.However,in China,there were only several terrestrial snake antivenoms,whose antitoxic effects are not yet confirmed for treatment of sea snake bites.The results of partⅠindicated that there were differences among sea snake venoms from different areas and species.Therefore,it is necessary to develop an antivenom that was exclusive for the dominant species of sea snakes in Chinese waters.In partⅡ,we chose Hcu V as the antigen and a high-titer antivenom was prepared after a series of procedures including antigen processing,horse immunization and antibody purification.Furthermore,we evaluated the efficacy of sea snake antivenom in vivo and in vitro,and determined the effective dosage and timing of use,providing evidence for future clinical practice.The main results were as follows:1.To maximize the immunogenicity of toxin antigen,we used 0.6%formaldehyde to inactivate the Hcu V and added Freund incomplete adjuvant（FIA）for emulsification.The horse serum was collected 50 days after antigen-immunization with the highest titer.Through a series of purification processes such as pepsin digestion,ammonium sulfate precipitation,ultrafiltration and column chromatography,the purity of F（ab’）2 in the sea snake antivenom reached 91.6%,and it retained a definite toxin neutralization capacity.2.The assessment of antivenom activity in vitro showed that H.curtus antivenom（Hcu AV）had a cross-neutralizing effect towards Hcu V and Hcy V,and a variety of other sea snake venoms as well.In addition,it was showed that 1 m L Hcu AV could neutralize1.2 mg of Hcu V or 0.9 mg of Hcy V,providing a basis for the dosage of antivenom in vivo studies.3.The assessment of antivenom activity in vivo elucidated that an injection of Hcu AV after being poisoned could significantly improve the survival rate of experimental animals.To achieve a better treatment effect,it was necessary to inject adequate antivenom as soon as possible.Notably,preventive and delayed medication could not reduce the mortality rate.In addition,Hcu AV could also effectively alleviate the impairment of vital organs caused by sea snake venom.In this part,we prepared a high-titer,high-purity sea snake antivenom by choosing Hcu V as toxin antigen.Hcu AV could effectively neutralize Hcu V and Hcy V,and a variety of other sea snake venoms in Chinese waters.Adequate injection of antivenom as soon as possible after sea-snake bites could significantly reduce the mortality rate and prevent damage to vital organs,providing experimental evidence for the clinical practice of sea snake antivenom in the future.PartⅢPanning and Effect Analysis of Active Components in Sea Snake Venom GlandsSea snake venoms are composed of a variety of enzymes,non-enzymatic proteins,and small molecular peptides.As an important marine biological toxin,in addition to severe toxicity,sea snake venoms also contained a variety of active substances with specific structures and novel functions,which demonstrated various pharmacological effects such as:analgesia,anti-inflammatory,anti-tumor,anti-thrombosis and etc.The research on active substances in sea snake venoms is one of the hot topics in development and utilization of marine biological resources.In this part,one of the two dominant sea snake species in South China Sea,H.curtus,was chosen to be investigated.We constructed the M13 phage display library derived from H.curtus venom gland.Using LPS as the target molecule,high-affinity phage clones were screened out after multiple rounds of panning.After gene sequencing,amino acid sequence prediction and analysis,a functional peptide was confirmed and finally obtained by chemical synthesis.Subsequently,its antagonistic effect towards LPS was evaluated in vivo and in vitro.The main results of this section were as follows:1.We constructed a high-quality M13 phage display library derived from H.curtus venom gland,with the library volume up to 2.2×109,and the accuracy of foreign inserts was verified using random gene sequencing.C-myc tag detection proved that the inserts were efficiently displayed on the surface of phage shell.2.With LPS as a screening target of the M13 phage display library derived from H.curtus venom gland,a 21-peptide was obtained through 4 rounds of solid-phase panning,gene sequencing of positive clones,sequence prediction of amino acids and other processes.Property analysis of the 21-peptide showed that it was rich in basic amino acids,with a theoretical molecular weight and isoelectric point being 2523.15 and 10.76,respectively.In addition,with aα-helix secondary structure,the 21-peptide carried positive charges,presented an amphipathic structure,and could specifically bind to negatively charged hydrophobic LPS.3.The 21-peptide was successfully obtained by chemical synthesis with a purity of over 95%.The actual molecular weight determined by mass spectrometry was consistent with the theoretical molecular weight predicted by software,proving that the synthesized peptide was correct and could be used for further experiments.The binding ability of the21-peptide with LPS was measured using Bio-Layer Interferometry（BLI）,and the results showed that the peptide possessed a strong binding ability towards LPS,with a binding mode of fast binding and slow dissociation.Furthermore,endotoxin neutralization experiment showed that the 21-peptide derived from H.curtus venom gland had a distinct endotoxin neutralizing effect,and thus it was named H.curtus Endotoxin-neutralizing Peptide,or Hc-ENP for short.4.The experiments in vitro demonstrated that Hc-ENP had a definite LPS antagonistic effect and it could effectively inhibit the binding of LPS to RAW264.7 cells at a concentration of 5-10μg/m L.Furthermore,through blocking activations of MAPK and NF-κB pathway,Hc-ENP could inhibit a series of inflammatory responses generated by LPS-stimulated cells,including inflammatory factor release,increased expressions of inflammation-related enzymes and increase in ROS production,indicating a significant anti-inflammatory effect.5.The experiments in vivo clarified that Hc-ENP could also inhibit the systemic inflammatory responses caused by LPS stimulation,including inflammatory-cell infiltration,up-regulation of serum inflammatory factors,expression increase of inflammation-related enzymes in organs,histopathological changes and etc.In this part,we constructed a high-quality H.curtus venom gland M13 phage display library for panning,so as to screen out specific active molecules from sea snake venom glands.Finally,we obtained an endotoxin neutralizing peptide Hc-ENP derived from H.curtus venom gland,and it had a definite LPS antagonistic effect both in vivo and vitro.This study provides a new way for further development and application of sea snake resources. |
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