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==Monomer== ==Monomer==
⚫ ]
{{fig|1
|Lysenin monomer.tif
⚫ |Lysenin monomer structure. Lysenin water-soluble monomeric X-ray structure ({{PDB|3ZXD}}). The previously conceived as β-barrel forming region is coloured in green. X-ray data have shown that the yellow region, together with the green one, form the β-barrel of Lysenin pore.
}}


Lysenin is a ] produced in the ]-]s of the earthworm ''Eisenia fetida''.<ref>Yilmaz, N., Yamaji-Hasegawa, A., Hullin-Matsuda, F. & Kobayashi, T. Molecular mechanisms of action of sphingomyelin-specific pore-forming toxin, lysenin. in Seminars in cell & developmental biology (Elsevier, 2017).</ref> This protein was first isolated from the coelomic fluid in 1996 and named lysenin (from lysis and Eisenia).<ref>SEKIZAWA, Y., HAGIWARA, K., NAKAJIMA, T. & KOBAYASHI, H. A novel protein, lysenin, that causes contraction of the isolated rat aorta: its purification from the coelomic fluid of the earthworm, Eisenia foetida. Biomedical Research 17, 197–203 (1996).</ref> Lysenin is a relatively small molecule with a molecular weight of 33&nbsp;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.<ref name=Colibus>De Colibus, L. et al. Structures of lysenin reveal a shared evolutionary origin for pore-forming proteins and its mode of sphingomyelin recognition. Structure 20, 1498–1507 (2012).</ref> 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.<ref name=Colibus /> Lysenin monomers show three different ] 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. <ref name=Bokori>Bokori-Brown, M. et al. Cryo-EM structure of lysenin pore elucidates membrane insertion by an aerolysin family protein. Nat Commun 7: 11293–11296. (2016).</ref> Lysenin is a ] produced in the ]-]s of the earthworm ''Eisenia fetida''.<ref>Yilmaz, N., Yamaji-Hasegawa, A., Hullin-Matsuda, F. & Kobayashi, T. Molecular mechanisms of action of sphingomyelin-specific pore-forming toxin, lysenin. in Seminars in cell & developmental biology (Elsevier, 2017).</ref> This protein was first isolated from the coelomic fluid in 1996 and named lysenin (from lysis and Eisenia).<ref>SEKIZAWA, Y., HAGIWARA, K., NAKAJIMA, T. & KOBAYASHI, H. A novel protein, lysenin, that causes contraction of the isolated rat aorta: its purification from the coelomic fluid of the earthworm, Eisenia foetida. Biomedical Research 17, 197–203 (1996).</ref> Lysenin is a relatively small molecule with a molecular weight of 33&nbsp;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.<ref name=Colibus>De Colibus, L. et al. Structures of lysenin reveal a shared evolutionary origin for pore-forming proteins and its mode of sphingomyelin recognition. Structure 20, 1498–1507 (2012).</ref> 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.<ref name=Colibus /> Lysenin monomers show three different ] 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. <ref name=Bokori>Bokori-Brown, M. et al. Cryo-EM structure of lysenin pore elucidates membrane insertion by an aerolysin family protein. Nat Commun 7: 11293–11296. (2016).</ref>
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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 ] (AFM). The measured prepore height was 90 Å; and the width 118 Å, with an inner pore of 50 Å (Figure 2).<ref name=yfirst /> 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.<ref>Munguira, I. L. B., Barbas, A. & Casuso, ignacio. Mechanism of blocking and unblocking of the pore formation of the toxin lysenin regulated by local crowding. Under submission.</ref> 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 ] (AFM). The measured prepore height was 90 Å; and the width 118 Å, with an inner pore of 50 Å (Figure 2).<ref name=yfirst /> 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.<ref>Munguira, I. L. B., Barbas, A. & Casuso, ignacio. Mechanism of blocking and unblocking of the pore formation of the toxin lysenin regulated by local crowding. Under submission.</ref>



{{fig|2
⚫ ]
|Lysenin_action_mechanism.png
|align = right
|width = 500px
⚫ |Lysenin mechanism of action Scheme. '''a)''' Lysenin monomers are segregated as soluble proteins that bind specifically to sphingomyelin by its receptor binding domain. After binding, and reach a certain density, the oligomerization starts. '''b)''' After a complete oligomerization, the prepore is formed. The prepore model shown here was assembled from the monomer structure and aligned with the pore structure ({{PDB|5GAQ}}) by their receptor-binding domains (residues 160 to 297). The height of the prepore was set to agree with the Atomic Force Microscopy measurements. '''c)''' Membrane inserted Lysenin assembly ({{PDB|5GAQ}}). The height of the pore was measured from the detergent belt to the last residue, assuming that the detergent belt corresponds with the part of the pore surrounded by the membrane. The membrane was placed in the β-barrel of the pore to match with the detergent belt, that englobe all the hydrophobic residues of the β-barrel. The hydrophobic surface colour scale is according to the hydrophobicity scale of Kyte and Doolittle.
}}


A conformational change transforms the PFM to the transmembrane ], leading to the pore state.<ref name=Bokori /> 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,<ref>Munguira, I. L. B., Takahashi, H., Casuso, I. & Scheuring, S. Lysenin Toxin Membrane Insertion Is pH-Dependent but Independent of Neighboring Lysenins. Biophysical Journal 113, 2029–2036 (2017).</ref> 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&nbsp;nm according to AFM measurements.<ref name=yfirst /> The main dimensions, using lysenin pore X-ray structure, are height 97&nbsp;Å, width 115&nbsp;Å and the inner pore of 30&nbsp;Å (Figure 2).<ref name=Bokori /> However, the complete oligomerization is not a requisite for the insertion, since incomplete oligomers in the pore state can be found.<ref name=yfirst /> The prepore to pore transition can be blocked if the membrane reaches certain density of oligomers, a mechanism that could be general to all β-PFTs. <ref>Munguira, I. L. B., Barbas, A. & Casuso, ignacio. Mechanism of blocking and unblocking of the pore formation of the toxin lysenin regulated by local crowding. Under submission.</ref> A conformational change transforms the PFM to the transmembrane ], leading to the pore state.<ref name=Bokori /> 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,<ref>Munguira, I. L. B., Takahashi, H., Casuso, I. & Scheuring, S. Lysenin Toxin Membrane Insertion Is pH-Dependent but Independent of Neighboring Lysenins. Biophysical Journal 113, 2029–2036 (2017).</ref> 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&nbsp;nm according to AFM measurements.<ref name=yfirst /> The main dimensions, using lysenin pore X-ray structure, are height 97&nbsp;Å, width 115&nbsp;Å and the inner pore of 30&nbsp;Å (Figure 2).<ref name=Bokori /> However, the complete oligomerization is not a requisite for the insertion, since incomplete oligomers in the pore state can be found.<ref name=yfirst /> The prepore to pore transition can be blocked if the membrane reaches certain density of oligomers, a mechanism that could be general to all β-PFTs. <ref>Munguira, I. L. B., Barbas, A. & Casuso, ignacio. Mechanism of blocking and unblocking of the pore formation of the toxin lysenin regulated by local crowding. Under submission.</ref>

Revision as of 14:50, 2 May 2019

Pore-forming toxin found in the earthworm Eisenia fetida

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

Figure 1. Lysenin monomer structure. Lysenin water-soluble monomeric X-ray structure (PDB ID 3ZXD). The previously conceived as β-barrel forming region is coloured in green. X-ray data have shown that the yellow region, together with the green one, form the β-barrel of Lysenin pore.

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.


Figure 2. Lysenin mechanism of action Scheme. a) Lysenin monomers are segregated as soluble proteins that bind specifically to sphingomyelin by its receptor binding domain. After binding, and reach a certain density, the oligomerization starts. b) After a complete oligomerization, the prepore is formed. The prepore model shown here was assembled from the monomer structure and aligned with the pore structure (PDB ID 5GAQ) by their receptor-binding domains (residues 160 to 297). The height of the prepore was set to agree with the Atomic Force Microscopy measurements. c) Membrane inserted Lysenin assembly (PDB ID 5GAQ). The height of the pore was measured from the detergent belt to the last residue, assuming that the detergent belt corresponds with the part of the pore surrounded by the membrane. The membrane was placed in the β-barrel of the pore to match with the detergent belt, that englobe all the hydrophobic residues of the β-barrel. The hydrophobic surface colour scale is according to the hydrophobicity scale of Kyte and Doolittle.

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 if the membrane reaches certain density of oligomers, a mechanism that could be general to all β-PFTs.

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.

References

  1. Yilmaz, N., Yamaji-Hasegawa, A., Hullin-Matsuda, F. & Kobayashi, T. Molecular mechanisms of action of sphingomyelin-specific pore-forming toxin, lysenin. in Seminars in cell & developmental biology (Elsevier, 2017).
  2. SEKIZAWA, Y., HAGIWARA, K., NAKAJIMA, T. & KOBAYASHI, H. A novel protein, lysenin, that causes contraction of the isolated rat aorta: its purification from the coelomic fluid of the earthworm, Eisenia foetida. Biomedical Research 17, 197–203 (1996).
  3. ^ De Colibus, L. et al. Structures of lysenin reveal a shared evolutionary origin for pore-forming proteins and its mode of sphingomyelin recognition. Structure 20, 1498–1507 (2012).
  4. ^ Bokori-Brown, M. et al. Cryo-EM structure of lysenin pore elucidates membrane insertion by an aerolysin family protein. Nat Commun 7: 11293–11296. (2016).
  5. Ishitsuka, R. & Kobayashi, T. Cholesterol and lipid/protein ratio control the oligomerization of a sphingomyelin-specific toxin, lysenin. Biochemistry 46, 1495–1502 (2007).
  6. Simons, K. & Gerl, M. J. Revitalizing membrane rafts: new tools and insights. Nature reviews Molecular cell biology 11, nrm2977 (2010).
  7. Ishitsuka, R. & Kobayashi, T. Cholesterol and lipid/protein ratio control the oligomerization of a sphingomyelin-specific toxin, lysenin. Biochemistry 46, 1495–1502 (2007).
  8. ^ Ros, U. & García-Sáez, A. J. More than a pore: the interplay of pore-forming proteins and lipid membranes. The Journal of membrane biology 248, 545–561 (2015).
  9. Yilmaz, N. & Kobayashi, T. Visualization of lipid membrane reorganization induced by a pore-forming toxin using high-speed atomic force microscopy. ACS nano 9, 7960–7967 (2015).
  10. Quinn, P. J. Structure of sphingomyelin bilayers and complexes with cholesterol forming membrane rafts. Langmuir 29, 9447–9456 (2013).
  11. Guigas, G. & Weiss, M. Effects of protein crowding on membrane systems. Biochimica et Biophysica Acta (BBA)-Biomembranes 1858, 2441–2450 (2016).
  12. Munguira, I. L. B., Barbas, A. & Casuso, ignacio. Mechanism of blocking and unblocking of the pore formation of the toxin lysenin regulated by local crowdingThermometry at the nanoscale. Nanoscale 4, 4799–4829 (2018).
  13. Munguira, I. et al. Glasslike Membrane Protein Diffusion in a Crowded Membrane. ACS Nano 10, 2584–2590 (2016).
  14. Ishitsuka, R. & Kobayashi, T. Cholesterol and lipid/protein ratio control the oligomerization of a sphingomyelin-specific toxin, lysenin. Biochemistry 46, 1495–1502 (2007).
  15. Munguira, I. L. B., Barbas, A. & Casuso, ignacio. Mechanism of blocking and unblocking of the pore formation of the toxin lysenin regulated by local crowdingThermometry at the nanoscale. Nanoscale 4, 4799–4829 (2018).
  16. Lafont, F. & Van Der Goot, F. G. Bacterial invasion via lipid rafts. Cellular microbiology 7, 613–620 (2005).
  17. Munguira, I. L. B., Barbas, A. & Casuso, ignacio. Mechanism of blocking and unblocking of the pore formation of the toxin lysenin regulated by local crowding. Under submision.
  18. ^ Yilmaz, N. et al. Real-time visualization of assembling of a sphingomyelin-specific toxin on planar lipid membranes. Biophysical journal 105, 1397–1405 (2013).
  19. Mulvihill, E., van Pee, K., Mari, S. A., Müller, D. J. & Yildiz, Ö. Directly observing the lipid-dependent self-assembly and pore-forming mechanism of the cytolytic toxin listeriolysin O. Nano letters 15, 6965–6973 (2015).
  20. Munguira, I. L. B., Barbas, A. & Casuso, ignacio. Mechanism of blocking and unblocking of the pore formation of the toxin lysenin regulated by local crowding. Under submission.
  21. Munguira, I. L. B., Takahashi, H., Casuso, I. & Scheuring, S. Lysenin Toxin Membrane Insertion Is pH-Dependent but Independent of Neighboring Lysenins. Biophysical Journal 113, 2029–2036 (2017).
  22. Munguira, I. L. B., Barbas, A. & Casuso, ignacio. Mechanism of blocking and unblocking of the pore formation of the toxin lysenin regulated by local crowding. Under submission.
  23. Green, D. R. Apoptosis and sphingomyelin hydrolysis: the flip side. J Cell Biol 150, F5–F8 (2000).
  24. Orrenius, S., Zhivotovsky, B. & Nicotera, P. Calcium: Regulation of cell death: the calcium–apoptosis link. Nature reviews Molecular cell biology 4, 552 (2003).
  25. Yu, S. P. Regulation and critical role of potassium homeostasis in apoptosis. Progress in neurobiology 70, 363–386 (2003).
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  27. Kobayashi, H., Sekizawa, Y., Aizu, M. & Umeda, M. Lethal and non-lethal responses of spermatozoa from a wide variety of vertebrates and invertebrates to lysenin, a protein from the coelomic fluid of the earthworm Eisenia foetida. Journal of Experimental Zoology 286, 538–549 (2000).
  28. Sukumwang, N. & Umezawa, K. Earthworm-derived pore-forming toxin Lysenin and screening of its inhibitors. Toxins 5, 1392–1401 (2013).
  29. Kobayashi, H., Ohta, N. & Umeda, M. Biology of lysenin, a protein in the coelomic fluid of the earthworm Eisenia foetida. International Review of Cytology 236, 45–99 (2004).
  30. Swiderska, B. et al. Lysenin family proteins in earthworm coelomocytes–Comparative approach. Developmental & Comparative Immunology 67, 404–412 (2017).
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  32. Edwards, C. A. & Bohlen, P. J. Biology and Ecology of Earthworms. (Springer Science & Business Media, 1996).
  33. Bryant, S. et al. Insights into the Voltage Regulation Mechanism of the Pore-Forming Toxin Lysenin. Toxins 10, 334 (2018).
  34. Shrestha, N. et al. Stochastic sensing of angiotensin II with lysenin channels. Scientific reports 7, 2448 (2017).
  35. Deamer, D., Akeson, M. & Branton, D. Three decades of nanopore sequencing. Nature biotechnology 34, 518 (2016).

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