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Lysenin is a pore-forming toxin (PFT) in the coelomic fluid of the earthworm Eisenia fetida. Pore-forming toxins are a group of proteins that act as virulence factors of several pathogenic bacteria. Following the general mechanism of action of PFTs lysenin is segregated as a soluble monomer that binds specifically to a membrane receptor, sphingomyelin in the case of lysenin. After attaching to the membrane, the oligomerization begins, resulting in a nonamer on top of membrane, known as a prepore. After a conformational change, which could be triggered by a decrease of pH, the oligomer is inserted into the membrane in the so-called pore state.

Monomer

Lysenin is a protein produced in the coelomocyte-leucocytes of the earthworm Eisenia fetida. This protein was first isolated from the coelomic fluid in 1996 and named lysenin (from lysis and Eisenia). Lysenin is a relatively small molecule with a molecular weight of 33 kDa (Figure 1). Using X-ray crystallography , the structure of lysenin was classified as a member of the Aerolysin family, as it shares a similar structure and function. Structurally, lysenin monomers are formed by a receptor binding domain (right grey globular part, Figure 1) and a Pore Forming Module (rest of the molecule, Figure 1); domains shared by the rest of Aerolysin family. Lysenin monomers show three different sphingomyelin binding motifs in the receptor binding domain. The part of the monomer that forms part of the β-barrel (green and yellow regions in Figure 1) evolved till the pore structure was reached.

Membrane receptors

The natural membrane target of Lysenin is an animal plasma membrane lipid called sphingomyelin, involving at least three of its Phosphatidylcholines (PC) groups. Sphingomyelin is usually found associated with cholesterol in lipid rafts. Cholesterol, which enhances oligomerization, provides a stable platform with high lateral mobility where monomer-monomer encounters are more probable. PFTs have shown to be able to remodel the membrane structure, sometimes even mixing lipid phases. In lysenin, the detergent belt is 32 Å in height. The detergent belt is the part of the β-barrel occupied by detergent in Cryogenic Electron Microscopy (Cryo-EM) studies of Lysenin pore,therefore, is the part of the β-barrel inmersed in the hydrophobic region of the membrane,. On the other hand, sphingomyelin/Cholesterol bilayers are about 4.5 nm height. This difference in height between the detergent belt and the sphingomyelin/cholesterol bilayer implies a bend of the membrane in the region surrounding the pore, called negative mismatch. This bending results in a net attraction between pores that induce pores aggregation.

Binding, oligomerization and insertion

Membrane binding is a requisite to initiate PFT oligomerization. Lysenin monomers bind specifically to sphingomyelin via the receptor binding domain. The final Lysenin oligomer is constituted by nine monomers without quantified deviations. When Lysenin monomers bind to sphingomyelin-enriched domains, they provide a stable platform with a high lateral mobility, hence favouring the oligomerization. Like most proteins, Lysenin oligomerization occurs in a two-step process, as was recently imaged.

The process begins with monomers being adsorbed into the membrane by specific interactions, resulting in an increased concentration of monomers. This increase is promoted by the small area where the membrane receptor accumulates owing to the fact that the majority of PFT membrane receptors are associated with lipid rafts. Another side effect, aside from the increase of monomer concentration, is the monomer-monomer interaction. This interaction increases lysenin oligomerization. After a critical concentration is reached, several oligomers are formed simultaneously. Complete oligomers consist in nine monomers, although incomplete oligomers has been also observed. In contrast to PFT of cholesterol dependent cytolysin family, incomplete lysenin oligomer evolution to complete oligomers has not been observed.

A complete oligomerization results in the so-called prepore state, a structure on the membrane. Determining the prepore's structure by X-ray or Cryo-EM is a challenging process that so far has not produced any results. The only available information about the prepore structure was provided by Atomic Force Microscopy (AFM). The measured prepore height was 90 Å; and the width 118 Å, with an inner pore of 50 Å (Figure 2). A model of the prepore was built aligning the monomer structure (PDB ID 3ZXD) with the pore structure (PDB ID 5GAQ) by their receptor-binding domains (residues 160 to 297). A recent study in aerolysin suggests that the currently accepted model for the lysenin prepore should be revisited, according to the new available data on the aerolysin insertion.

A conformational change transforms the PFM to the transmembrane β-barrel, leading to the pore state. The trigger mechanism for the prepore to pore transition in lysenin depends on three glutamic acids (E92, E94 and E97), and is activated by a decrease in pH, from physiological conditions to the acidic conditions reached after endocytosis. These three glutamic acids are located in an α-helix that forms part of the PFM, and glutamic acids are found in aerolysin family members in its PFMs. Such a conformational change produces a decrease in the oligomer height of 2.5 nm according to AFM measurements. The main dimensions, using lysenin pore X-ray structure, are height 97 Å, width 115 Å and the inner pore of 30 Å (Figure 2). However, the complete oligomerization is not a requisite for the insertion, since incomplete oligomers in the pore state can be found. The prepore to pore transition can be blocked in crowded conditions, a mechanism that could be general to all β-PFTs. The first hint of crowding effect on prepore to pore transition was given by congestion effects in electrophysiology experiments. Cite error: A <ref> tag is missing the closing </ref> (see the help page).

Insertion consequences

The ultimate consequences caused by lysenin pore formation are not well documented; however, there are three plausible consequences:

• Punching the membrane breaks the sphingomyelin asymmetry between the two leaflets of the lipid bilayer, which cause apoptosis of the cell. PFTs are supposed to induce lipid flip-flop (reorientation of a lipid from one leaflet of a membrane bilayer to the other) that can also break the sphingomyelin asymmetry.
• Increasing the calcium concentration in the cytoplasm, drives to apoptosis.
• Decreasing the potassium concentration in the cytoplasm causes apoptosis.

Biological role

The biological role of lysenin remains unknown. It was suggested that lysenin may play a role as a defeat mechanism against attackers such as bacteria, fungi or small invertebrates. Nevertheless, sphingomyelin starts to appear in the plasma membrane of chordates. Therefore, lysenin, being dependent on sphingomyelin as a binding factor, cannot affect bacteria, fungi or in general invertebrates. Another hypothesis is that the earthworm, which is able to expel coelomic fluid under stress, generates an avoidance behaviour to its vertebrate predators (such as birds, hedgehogs or moles). If that is the case, the expelled lysenin might be more effective if the coelomic fluid reached the eye, where the concentration of sphingomyelin is ten times higher than in other body organs. A complementary hypothesis is that the coelomic fluid, exuded under stressful situations by the earthworm, which has a pungent smell -giving the earthworm its specific epithet foetida- was suggested to be an anti-predator adaptation. It remains unknown whether lysenin contributes to avoidance of Eisenia by predators.

Applications

Lysenin conductive properties have been studied for years. As most pore-forming toxins lysenin forms an unspecific channel also permeable to small peptides. 2 Behind all those studies along more than three decades underlie the interest of finding a sequencing system biocompatible and with tunable conductive properties by point mutation. Owing its binding affinity for sphingomyelin, lysenin or lysenin receptor binding domain has been used as a fluorescence marker to detect sphingomyelin domain in membranes. </ref> Reiko Ishitsuka and Toshihide Kobayashi, “Lysenin: A New Tool for Investigating Membrane Lipid Organization,” Anatomical Science International 79, no. 4 (2004): 184. </ref>

References

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External links

• https://www.theses.fr/2017AIXM0124

• Protein toxins