Functional and biological insights of rCollinein-1, a recombinant serine protease from Crotalus durissus collilineatus

Abstract

Background: The prevalent class of snake venom serine proteases (SVSP) in Viperidae venoms is the thrombin-like enzymes, which, similarly to human thrombin, convert fibrinogen into insoluble fibrin monomers. However, thrombin-like serine proteases differ from thrombin by being unable to activate factor XIII, thus leading to the formation of loose clots and fibrinogen consumption. We report the functional and biological characterization of a recombinant thrombin-like serine protease from Crotalus durissus collilineatus , named rCollinein-1. Methods: Heterologous expression of rCollinein-1 was performed in Pichia pastoris system according to a previously standardized protocol, with some modifications. rCollinein-1 was purified from the culture medium by a combination of three chromatographic steps. The recombinant toxin was tested in vitro for its thrombolytic activity and in mice for its edematogenicity, blood incoagulability and effect on plasma proteins. Results: When tested for the ability to induce mouse paw edema, rCollinein-1 demonstrated low edematogenic effect, indicating little involvement of this enzyme in the inflammatory processes resulting from ophidian accidents. The rCollinein-1 did not degrade blood clots in vitro , which suggests that this toxin lacks fibrinolytic activity and is not able to directly or indirectly activate the fibrinolytic system. The minimal dose of rCollinein-1 that turns the blood incoagulable in experimental mice is 7.5 mg/kg. The toxin also led to a significant increase in activated partial thromboplastin time at the dose of 1 mg/kg in the animals. Other parameters such as plasma fibrinogen concentration and prothrombin time were not significantly affected by treatment with rCollinein-1 at this dose. The toxin was also able to alter plasma proteins in mouse after 3 h of injection at a dose of 1 mg/kg, leading to a decrease in the intensity of beta zone and an increase in gamma zone in agarose gel electrophoresis Conclusion: These results suggest that the recombinant enzyme has no potential as a thrombolytic agent but can be applied in the prevention of thrombus formation in some pathological processes and as molecular tools in studies related to hemostasis.

State-of-the-art review - A review on snake venom-derived antithrombotics: Potential therapeutics for COVID-19-associated thrombosis?

“…Boldrini-França et al (2015) reported the heterologous expression of an SVSP named collinein-1 from the venom of Crotalus durissus collilineatus in Pichia pastoris (methyltropic yeast) that resulted in the production of 56 mg/L of recombinant collinein-1 (rCollinein-1), and importantly, retained its functional integrity to hydrolyze bovine fibrinogen. A subsequent study has shown that rCollinein-1 may have therapeutic potential in preventing thrombus formation [149] . An acute and repeated dose (28 days) toxicity study of a recombinant thrombin-like defibrinogenating enzyme, batroxobin expressed in Pichia pastoris , showed no adverse effects at a dose of 2.5 NIH u/kg in rats and 1 NIH u/kg in dogs, indicating clinical potential in the treatment of hemostatic disturbances [150] .…”

From Animal Poisons and Venoms to Medicines: Achievements, Challenges and Perspectives in Drug Discovery

“…Recently, collinein-1, a SVSP from Crotalus durissus collilineatus venom ( Boldrini-França et al., 2015 ) was recombinantly expressed in Pichia pastoris system ( Boldrini-Franca et al., 2019 ) and demonstrated to block, independently from its catalytic activity, the hEAG1 ion channel, which is overexpressed in several cell cancer lines. Collinein-1 reduced the viability of human breast cancer cell line MCF7, which displays high expression of hEAG1, but does not affect the HepG2 and MCF10A cell lines, which present low expression of this ion channel, demonstrating that the reduction of cell viability might be connected with hEAG1 inhibition by this protein ( Boldrini-França et al., 2020 ).…”

Beyond hemostasis: a snake venom serine protease with potassium channel blocking and potential antitumor activities

“…Collinein-1 is a 29.5 kDa thrombin-like serine protease isoform from C. d. collilineatus venom that cleaves preferentially the Aα chain of fibrinogen 23 . The recombinant form of collinein-1 (rCollinein-1) was previously produced with functional integrity, using the Pichia pastoris heterologous expression system 23,24 . The screening of collinein-1 activity on voltage-gated ion channels was performed with rCollinein-1 (5 μM) on different isoforms of voltage-gated potassium (Kv) and sodium (Nav) channels expressed in Xenopus laevis oocytes.…”

“…-1 (rCollinein-1). Recombinant collinein-1 was produced as described by Boldrini-França et al 24 . Pichia pastoris cells, transformed with the recombinant plasmid (containing collinein-1 coding sequence), was pre-inoculated with 10 mL of BMGY medium (1% yeast extract, 2% peptone, 1.34% YNB, 4 × 10 −5 M biotin, 1% glycerol, 100 mM phosphate potassium pH 6.0) and incubated at 30 °C under constant stirring of 200 rpm.…”