Mechanisms underpinning the permanent muscle damage induced by snake venom metalloprotease

Abstract

Snakebite is a major neglected tropical health issue that affects over 5 million people worldwide resulting in around 1.8 million envenomations and 100,000 deaths each year. Snakebite envenomation also causes innumerable morbidities, specifically loss of limbs as a result of excessive tissue/muscle damage. Snake venom metalloproteases (SVMPs) are a predominant component of viper venoms, and are involved in the degradation of basement membrane proteins (particularly collagen) surrounding the tissues around the bite site. Although their collagenolytic properties have been established, the molecular mechanisms through which SVMPs induce permanent muscle damage are poorly understood. Here, we demonstrate the purification and characterisation of an SVMP from a viper (Crotalus atrox) venom. Mass spectrometry analysis confirmed that this protein is most likely to be a group III metalloprotease (showing high similarity to VAP2A) and has been referred to as CAMP (Crotalus atrox metalloprotease). CAMP displays both collagenolytic and fibrinogenolytic activities and inhibits CRP-XL-induced platelet aggregation. To determine its effects on muscle damage, CAMP was administered into the tibialis anterior muscle of mice and its actions were compared with cardiotoxin I (a three-finger toxin) from an elapid snake (Naja pallida) venom. Extensive immunohistochemistry analyses revealed that CAMP significantly damages skeletal muscles by attacking the collagen scaffold and other important basement membrane proteins, and prevents their regeneration through disrupting the functions of satellite cells. In contrast, cardiotoxin I destroys skeletal muscle by damaging the plasma membrane, but does not impact regeneration due to its inability to affect the extracellular matrix. Overall, this study provides novel insights into the mechanisms through which SVMPs induce permanent muscle damage.PLOS Neglected Tropical Diseases | https://doi.Snakebite is a major neglected tropical disease that affects thousands of people in the rural areas of developing countries. As well as the deaths, snakebites result in a significant number of disabilities including permanent loss of limbs that alter the lifestyle of the victims. Snake venom is a mixture of different proteins with diverse functions; one of these major protein groups present in viper venoms are metalloproteases that primarily induce muscle damage. The mechanisms behind the development of snakebite (metalloprotease)induced permanent muscle damage are poorly studied. Here, we have purified a metalloprotease (CAMP) from the venom of the Western diamondback rattlesnake, and characterised its function in mice. To determine the actions of CAMP in the development of permanent muscle damage, it was injected into the muscle of mice in a parallel comparison with cardiotoxin I (from the venom of the Red spitting cobra). The effects of these proteins on muscle regeneration were analysed at 5 and 10 days after injection. The results demonstrate that through a combination of effects on the structural scaff...

Repurposing Cancer Drugs Batimastat and Marimastat to Inhibit the Activity of a Group I Metalloprotease from the Venom of the Western Diamondback Rattlesnake, Crotalus atrox

“…We have previously used the two-dimensional chromatography approach (e.g., a combination of ion exchange and gel filtration) as an effective method to purify various venom components, including a 50 kDa PIII SVMP CAMP from the venom of C. atrox [14,30]. Here, we have deployed a similar approach to purify a PI SVMP with a molecular weight of 23 kDa from the venom C. atrox.…”

“…A gel slice (from SDS-PAGE) containing CAMP-2 was subjected to tryptic digestion before undergoing mass spectrometry analysis at Alta Bioscience (Birmingham, UK), as we described previously [14]. Both MS and MS/MS scans were cross-referenced against the Uniprot protein database using the Sequest algorithm (Thermo fisher PD 1.4) in order to determine the identity of the purified protein.…”

“…SVMPs are zinc-dependent enzymes that vary in molecular mass from approximately 20 to 100 kDa and are responsible for the haemorrhagic effects and local tissue damage frequently seen upon viper envenomation [13,14]. SVMPs are classified into PI to PIV depending on the presence of additional domains [15]: PI-only a metalloprotease domain; PII-a metalloprotease and a disintegrin domain; PIII-a metalloprotease domain, a disintegrin-like and a cysteine-rich domain; PIV-two C-type lectin domains in addition to all the domains present in PIII.…”

The Urgent Need to Develop Novel Strategies for the Diagnosis and Treatment of Snakebites

“…Importantly, they also frequently induce haemorrhaging due to hydrolysis of the endothelial cell basement membrane components around blood capillaries [68]. These also affect muscle fibres impairing their regeneration [69]. These enzymes are in themselves a highly diverse family, and are separated into four groups depending on the domains present: P-I/Group I comprise just a metalloprotease domain, present in all groups.…”

“…These additional domains afford SVMPs a wide variety of different functions. For example, the disintegrin domains bind integrins blocking their functions in platelets and endothelial cells [72] and have the potential to bind the integrins in muscle cells, colocalising and exacerbating myotoxic effects [69]. Cysteine-rich domains have also been found to inhibit collagen induced platelet aggregation as well as to play a key role in the onset of inflammation [73].…”