Characterisation of metalloproteinases from Myanmar Russell's viper

Abstract

Russell’s viper bites are a major public health problem in tropical and subtropical regions. In Myanmar, a Russell’s viper (Daboia russellii siamensis) bite has a 60% morbidity rate and 8.2% fatality rate. Most victims encounter severe bleeding, renal failure and capillary leakage and the bite can possibly lead to death. Snake venom metalloproteinases (SVMPs) are the major components of the Viperidae venom and all mentioned lethal effects of the bites are attributed to these. The only available and partially effective agent for the treatment of the toxic effects is antivenom. Antivenom therapy is not always effective towards small toxins, however, and it can also provoke an anaphylactic response. The development of new therapeutic approaches is becoming increasingly important therefore. For the analysis of SVMP transcripts from Myanmar Russell’s viper, Next-Generation Sequencing (NGS) of mRNA from venom glands derived from 2 male snakes and 1 female snake was performed on an Illumina HiSeq2000 platform. De novo assembly of the reads was performed using Trinity software and the transcripts were annotated through Blastn against the collection of NCBI nucleotide sequences defined by the key-words (‘venom’ and ‘serpents’) search. Blastx hit results against the UniProtKB/Swiss-Prot (swissprot) database were also used for annotation of the transcripts. The abundance distribution (in term of FPKM value) of SVMPs toxin transcripts: disintegrin (75%), P-III SVMPs (25%) and P-II SVMPs (0.002 %), were the same for both male and female samples. No P-I SVMP transcripts were detected in the present analysis. A comparison of the contents of SVMP transcripts in adult male and female venom glands showed some gender-related differences. For example, a disintegrin transcript isoform (Dis 1b) was highly expressed only in the female venom gland, and some P-III SVMP isoforms (P-III 6, 7a, 7b) were only expressed at low level in the male venom glands. The P-II SVMP transcripts were expressed as different isoforms in male and female animals, which could reflect a sex-dimorphism of viper venom biological activities. This finding would support a requirement to use combined venoms of both sexes for preparation of antivenom. In addition to SVMP transcripts, mRNAs of novel tripeptide SVMP inhibitors (SVMPIs) were also discovered. These endogenous inhibitors have potential as a new treatment modality for neutralization of the effect of SVMP toxins. Two major snake SVMPs, RVV-X and daborhagin, were purified from Myanmar Russell’s viper venom using a new purification strategy. Moreover, the two novel endogenous tripeptides identified in transcript analysis, pERW and pEKW were identified and isolated from the crude venom. Both purified SVMPs showed caseinolytic activity. Additionally, RVV-X displayed specific proteolytic activity towards gelatin and Daborhagin showed potent fibrinogenolytic activity. These activities were inhibited by metal chelators. Notably, synthetic versions of the peptide inhibitors, pERW and pEKW, completely inhibited the gelatinolytic and fibrinogenolytic activities of the respective SVMPs when used at 5 mM concentration (estimated molar ratio of SVMP to tripeptide was 1:500). These complete inhibitory effects suggest that these tripeptides deserve further study as candidates for new therapeutic treatment against Russell’s viper envenomation.