Snake venom metalloproteinase

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Overview

Snake venom metalloproteinase (SVMP) is a group of multi-domain enzymes that are responsible for many of the pathological symptoms that arise from a snake’s bite, primarily from snakes in the Viperidae and Crotalinae subfamilies. Symptoms induced by these enzymes include hemorrhaging, necrosis, proteolytic degradation of fibrin and fibrinogen, and inhibition of platelet aggregation. Strands of SVMP fall under the enzyme commission number 3.4.24, which defines metalloendopeptidases. These are protease enzymes (enzymes that catalyze the breakdown of proteins into smaller peptides) that require divalent metal ions in order to function. All SVMPs were derived from a common ancestral ADAM (a disintegrin and metalloproteinase) enzyme. This is to say that their catalytic zinc-dependent metalloprotease domain is structurally conserved, and they all perform proteolysis on extracellular matrix proteins. Still, these enzymes have since diversified greatly into a variety of different isoforms and classes.

Five representative SVMPs have been identified according to the IUBMB Enzyme Commission (EC):

• Bothropasin (EC 3.4.24.49)
  • Other names: Bothrops jararaca venom metalloproteinase

• Trimerelysin I (EC 3.4.24.52)
  • Other names: Trimeresurus metalloendopeptidase I; hemorrhagic proteinase HR1A; hemorrhagic metalloproteinase HR1A; metalloproteinase HR1A

• Trimerelysin II (EC 3.4.24.72)
  • Other names: Trimeresurus metalloendopeptidase II; proteinase H2; H2-proteinase

• Fibrolase (EC 3.4.24.53)
  • Other names: fibrinolytic proteinase; Agkistrodon contortrix contortrix metalloproteinase; Agkistrodon contortrix contortrix venom metalloproteinase

• Venombin A (EC 3.4.24.74)
  • Other names: α-fibrinogenase; habutobin; zinc metalloproteinase Cbfib1.1; zinc metalloproteinase Cbfib1.2; zinc metalloproteinase Cbfib2; ancrod

Classes

The reaction pathway for each SVMP is very similar with regards to the core proteolytic mechanism. SVMPs, however, have diversified amongst three distinct classes with different levels of virulence due to the number of domains they each exhibit. These classes are P-I (20-30 kDa), P-II (30-60 kDa), and P-III (60-100 kDa) SVMPs, and even within these classes, the biological effects of the enzymes vary depending on their specific structure and context within the particular snake.

P-I: This class contains the simplest forms of SVMP, containing a pro-domain (PD; non-matured proteins) and metalloproteinase (M) domain. The reaction pathway for these enzymes begins with the zinc²⁺ cation being coordinated by the conserved amino acid sequence (HEXXHXXGXXH) along with a catalytic water molecule. A glutamate residue in the active site then turns the water molecule into a nucleophile, which then attacks the carbonyl carbon of a peptide bond in the substrate (targeted substrate varies depending on the specific snake). An oxyanion tetrahedral intermediate is formed as a result, stabilized by the zinc ion and other residues nearby. As the peptide bond has been hydrolyzed, the polypeptide is cleaved.

P-II: These enzymes are more complex and larger than P-I SVMPs and they contain a pro-domain (PD), metalloproteinase (M) domain, and disintegrin (D) domain. These enzymes follow the same reaction pathway as P-I SVMPs, although with the addition of the disintegrin domain bonded to the protease. After the protease domain has been translated, the enzyme undergoes autocatalytic processing. The disintegrin domain is either cleaved from the protein or, in lesser known cases, can remain attached as part of the toxin. The domain will then bind integrins on platelets, thereby inhibiting platelet aggregation.

P-III: These enzymes are the largest out of the classes. They conserve the metalloproteinase domain and pro-domain, similar to P-I and P-II SVMPs, and they additionally have a disintegrin-like (D) domain and a cysteine-rich (C) domain. The disintegrin-like domain is not identical to the disintegrin domains present in P-II SVMPs, as in P-III, they are never cleaved from the enzyme, resulting in the binding of integrins with different specificity. The cysteine-rich domain presents additional binding regions on the enzyme to assist it in settling on larger ECM proteins. Other than these added domains, P-III SVMPs again follow the same catalytic pathway as P-I and P-II SVMPs.

Domains

The pro-domain contains a conserved sequence of PKMCGVT and it sits on the N-terminus of the metalloproteinase domain. The role of the pro-domain is to control the maturation of SVMP by assisting in protein folding and by acting as an autoinhibitor, blocking the catalytic zinc site so as to prevent the enzyme from digesting the snake’s own tissues. The metalloproteinase domain has an oblate ellipsoidal structure with a smaller lower region and larger upper region bearing the active site. The C-terminus of the lower region is in a helical structure preceded by an irregular folded domain. The domain contains a conserved zinc-binding sequence followed by a conserved “Methionine-turn” motif. The primary difference among structures of P-I SVMPs as they relate to hemorrhaging potency is the flexibility of the loop near the enzyme’s active site. Based on data from molecular dynamics simulations, non-hemorrhaging strands of P-I SVMP have a high level of flexibility in the loop region after the Met-turn whereas hemorrhaging strands have greater flexibility before the Met-turn. The disintegrin domain in P-II and P-III SVMPs is adjacent to the C-terminal of the metalloproteinase domain. P-II SVMPs contain 12 uniquely patterned cysteine residues (some members contain two additional cysteine residues) potentially responsible for the diversification of SVMP enzymes. In P-III SVMPs, the metalloproteinase domain, disintegrin-like domain, and cysteine-rich domain form a C-shaped structure which places the hyper-variable region (HVR) of the cysteine-rich domain adjacent to the catalytic site of the metalloproteinase domain. This formation aids in substrate recognition by creating flexibility between the catalytic site and exosite. The cysteine-rich domain structure is composed of two α-helices, four β-strands, and loops, of which the loop is stabilized by a disulfide bond. This domain can bind to free or membrane-bound proteins, aligning the metalloproteinase domain with the substrate for proteolytic attack.

Function

Snake venom metalloproteinases are secreted proteins, acting primarily on extracellular matrix (ECM) proteins and integrins. When an SVMP hydrolyzes an ECM protein (primarily nidogen, laminin, fibronectin, and type IV collagen), basement membranes degrade, resulting in hemorrhaging and vascular leakage. When P-II and P-III SVMPs bind integrins on endothelial cells, platelet aggregation becomes disrupted, thereby impairing hemostasis. Some SVMPs also interfere with coagulation by cleaving fibrinogen or activating clotting factors such as factor X (enzyme of coagulation cascade). These reactions cause reduced oxygen and nutrient delivery, prompting tissue cells to switch to anaerobic metabolism. Energy-demanding wound healing processes are also triggered such as collagen synthesis and ECM rebuilding to deal with cell death.

References

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