Does size matter? Proteomic comparison and fibrinogenolytic activity of Causus snake venoms from short and long venom glands.

F. C. P. COIMBRA1 , B. G. FRY1 1 Venom Evolution Lab, University of Queensland, St. Lucia QLD 4072 Australia.

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INTRODUCTION Causus lichtensteinii (Cl) METHODOLOGY

Causus (night adders) Venoms: • Six recognised species • No elongated venom gland • Pooled (multiple adults) lyophilised Causus lichtensteinii • Amphibian specialists system and Causus rhombeatus venom. • One of the smallest of its genus • Active pursuit predator Identification of venom proteins: • Fossorial to semi-fossorial • Feeds on small prey (gracile • 1D & 2D SDS-PAGE and tandem mass spectrometry • Morphologically unique frogs) sequencing. amongst vipers in having Fibrinogenolysis: Stago STA-R Max ® narrow heads • 1D SDS-PAGE of 6 time-point incubation series (0-60 min) of 10 venom concentrations (1.5625-800 μg/mL) • Some have elongated venom with purified human fibrinogen (1 mg/mL). glands • Time until clot measured via a Stago STA-R Max® Causus rhombeatus (Cr) coagulation analyser, incubating 17 venom concentrations (0.00935-2 mg/mL) with human fibrinogen • Elongated venom gland system (1.2 mg/mL), CaCl2, and phospholipid (37 ℃ for 60 min), with thrombin added post-incubation in a Claussian • Largest of its genus method (Clauss, 1957). • Feeds on disproportionaly large • Clot formation & strength measured via prey, up to the same weight as the thromboelastography (TEG®5000) with venom (0.2 snake mg/mL), human fibrinogen (2 mg/mL), CaCl2, and TEG ® 5000 phospholipid incubated at 37 ℃ for 30 min; thrombin added post-30 min and re-run for another 30 min.

RESULTS

Identification of venom proteins (fig.1) • Predominance of metalloproteases and kallikrein-like serine proteases in both venoms • Similar qualitative toxin composition between species • Typical viperid-venom protein classes

Fibrinogenolysis • Fibrinogenolytic activity similar for both venoms • Lysis of Aα and Bβ chains only, with Aα consistently quicker and before that of Bβ chain (fig.2) • Degradation of Bβ by Cr venom quicker than that of Cl venom (fig.3) • At a concentration of ≥ 0.4 mg/mL, Cr venom prevented clotting (fig.4) Figure 1. Proteomic comparison via 1D SDS-PAGE of C. Figure 2. Relative proteolytic action of venoms (1.525-800 μg/mL) upon human fibrinogen (1 • Clot strength (mm) reduced by half for both venoms (fig.5) lichtensteinii (Cl) and C. rhombeatus (Cr) venom. Red mg/mL) (Mean ± SD, N=3). boxes indicate protein bands identified via tandem • SAIMR snake polyvalent antivenom neutralised some of Cr venom mass spectrometry: 1) L-amino acid oxidases. 2-4) fibrinogenolytic activity; however, ineffective against Cl venom (data Snake-venom metalloproteases. 5,6) Kallikrein-like not shown) serine proteases. 7) Cysteine-rich secretory proteins.

8) Phospholipases A2

Figure 4. Anticoagulant effect on 1.2 mg/mL of human fibrinogen incubated with Cr venom for 60 min. (Mean ± Figure 3. Overall view of fibrinogenolytic action after 60 minutes of incubation with venoms SD, N=3. In some cases error bars are smaller than symbol). (Mean ± SD, N=3).

REFERENCES Figure 5. Thromboelastography tracings (N=3) of clot formation by post-30 min addition of Coimbra, F.C.P. et al. Does size matter? Venom proteomic and functional comparison between night adder species (Viperidae: Causus) thrombin to à priori venom (0.2 mg/mL) & fibrinogen (2.1 mg/mL) incubation, overlaid with with short and long venom glands. Comparative Biochemistry and Physiology, Part C (2018) 221: 7-14. negative control tracings (N=3).

CONCLUSIONS ACKNOWLEDGEMENTS

• Both venoms are dominated by snake-venom metalloproteases and kallikrein-like serine proteases, which in viper venoms have convergently evolved fibrinogenolytic activity and are thus likely responsible for the fibrinogenolysis observed in this study. • The cleavage of fibrinogen’s Aα and Bβ chains resulted in an anticoagulant effect and would likely result in hypofibrinogenemia in vivo. • Although toxin classes are conserved between both species, isoformic variability (2D PAGE not shown) may be responsible for differential SAIMR polyvalent antivenom effectiveness. • Given the venoms’ similarities, these results suggest a link between the elongated venom gland system and a predatory niche where venom yield is favoured over venom toxicity and/or diversification for predating upon proportionally larger prey. • However, this study does not represent the full suite of the venoms’ bioactivity, therefore future work on haemotoxic activity should include additional haemostatic components.