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Published on: November 3, 2014
Omics-guided discovery and functional profiling of Da-29, a bioactive serine protease from Deinagkistrodon acutus
Caojuan Chen1, Peng Ding2, Xinru Tao3
1Hunan University of Chinese Medicine, Changsha, Hunan, China; Institute of Innovative Traditional Chinese Medications, Hunan Academy of Chinese Medicine, Changsha, Hunan, China.
Background:
Snake venom serine proteases (SVSPs) are critical toxic components that interfere with the hemostatic balance and serve as a key candidate library for anticoagulant drug development. Despite the diversity of SVSPs in Deinagkistrodon acutus venom, systematic studies of low-abundance SVSPs with thrombin-like activity profiles are limited. Hence, this research aimed to isolate and characterize a bioactive SVSP candidate from D. acutus venom using integrated multi-omics and to assess its antithrombotic effects and bleeding risk.
Methods:
PacBio full-length transcriptome sequencing combined with label-free quantitative proteomics was used to screen SVSP candidates with matched transcription-translation associations. The target protein Da-29 was purified using gel filtration and reverse-phase chromatography, and its primary structure was confirmed using transcriptome sequencing and liquid chromatography-tandem mass spectrometry. In vitro enzymatic activity and fibrinogen hydrolysis specificity, as well as in vivo antithrombotic efficacy and bleeding risk, were evaluated using rodent thrombosis and bleeding models.
Results:
Da-29 was observed to be a 260-amino-acid SVSP containing a conserved catalytic triad and an S1 substrate-binding pocket. It exhibited significant amidase and fibrinogen-clotting activities and selectively hydrolyzed fibrinogen Aα and Bβ chains. Furthermore, Da-29 exerted dose-associated antithrombotic effects in vivo, extending the coagulation time, reducing the fibrinogen/TXB2 levels, and upregulating 6-keto-PGF1α, without causing considerable bleeding risks at effective doses.
Conclusion:
Da-29 exhibits antithrombotic activity associated with fibrinogen degradation and linked to concurrent alterations in TXB2 and 6-keto-PGF1α levels, establishing its potential as an antithrombotic experimental lead. This integrated omics strategy can effectively identify low- abundance bioactive molecules from snake venom, offering a reproducible paradigm for the discovery of functional proteins in natural products.
