Functional Mining of the Crotalus Spp. Venom Protease Repertoire Reveals Potential for Chronic Wound Therapeutics

Abstract

Chronic wounds are a major health problem that cause millions of dollars in expenses every year. Among all the treatments used, active wound treatments such as enzymatic treatments represent a cheaper and specific option with a fast growth category in the market. In particular, bacterial and plant proteases have been employed due to their homology to human proteases, which drive the normal wound healing process. However, the use of these proteases has demonstrated results with low reproducibility. Therefore, alternative sources of proteases such as snake venom have been proposed. Here, we performed a functional mining of proteases from rattlesnakes (Crotalus ornatus, C. molossus nigrescens, C. scutulatus, and C. atrox) due to their high protease predominance and similarity to native proteases. To characterize Crotalus spp. Proteases, we performed different protease assays to measure and confirm the presence of metalloproteases and serine proteases, such as the universal protease assay and zymography, using several substrates such as gelatin, casein, hemoglobin, L-TAME, fibrinogen, and fibrin. We found that all our venom extracts degraded casein, gelatin, L-TAME, fibrinogen, and fibrin, but not hemoglobin. Crotalus ornatus and C. m. nigrescens extracts were the most proteolytic venoms among the samples. Particularly, C. ornatus predominantly possessed low molecular weight proteases (P-I metalloproteases). Our results demonstrated the presence of metalloproteases capable of degrading gelatin (a collagen derivative) and fibrin clots, whereas serine proteases were capable of degrading fibrinogen-generating fibrin clots, mimicking thrombin activity. Moreover, we demonstrated that Crotalus spp. are a valuable source of proteases that can aid chronic wound-healing treatments.

A novel broad spectrum venom metalloproteinase autoinhibitor in the rattlesnakeCrotalus atroxevolved via a shift in paralog function

“…To identify and characterize proteins that confer resistance to metalloproteinase activity in the rattlesnake C. atrox , we used activity on a collagen-based substrate (Azocoll) as an initial assay for metalloproteinases and their inhibitors. Divalent cations such as Zn 2+ and Ca 2+ are critical for metalloproteinase function, and chelators such as EDTA inhibit their proteolytic activity ( 36 ). To test whether the activity of whole C. atrox venom on Azocoll was sensitive to EDTA, we added 1 millimolar (mM) EDTA and observed complete inhibition of the proteolytic activity, indicating that Azocoll was a suitable substrate for measuring the SVMP activity and its potential inhibition ( Fig.…”

Royal jelly from different floral sources possesses distinct wound-healing mechanisms and ingredient profiles

“…Polyubiquitin-A isoform X2 may not only stimulate cell growth, protein kinases and relevant signalling, but also possess antibacterial activity [74][75][76] . Venom serine carboxypeptidase isoform X1 may help clotting via its thrombin-like activity 77 . In contrast, the upregulation of esterase B1 present in BnRJ-Zj is related to cell apoptosis and is detrimental to wound healing 78 .…”

Crotalus Durissus Ruruima: Current Knowledge on Natural History, Medical Importance, and Clinical Toxinology

“…from North and Central Americas are more abundant and have been widely investigated. These studies have shown hemostatic effects such as fibrino(geno)lytic and inhibition of platelet aggregation activities as well as local hemorrhage caused by degradation of the capillary basement membrane and muscle damage with lower regeneration (91)(92)(93). As C. d. ruruima venom composition differs from other more prevalent C. durissus subspecies envenomings, an antivenom based on oligoclonal mixtures of antibodies could be a strategy to improve treatment.…”