Faculty Opinions recommendation of CryoEM reveals how the complement membrane attack complex ruptures lipid bilayers.

Proteolysis of the fluid phase complement complex SC5b-9 transforms it into an arnphiphilic molecule which resembles the membrane attack complex of complement and reconstitutes into lipid vesicles. Complement-containing vesicles prepared in this way can be made to fuse with planar lipid bilayers transferring their protein content to the host membrane. Massive conductance increases can thus be observed, which are due to the insertion of a large number of ionic channels into the membrane. Using low concentrations of vesicles, single channels can be studied.

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Membrane attack complex formation on a supported lipid bilayer: initial steps towards a CARPA predictor nanodevice

European Journal of Nanomedicine ◽

10.1515/ejnm-2015-0016 ◽

2015 ◽

Vol 7 (3) ◽

Cited By ~ 8

Author(s):

Saziye Yorulmaz ◽

Seyed R. Tabaei ◽

Myunghee Kim ◽

Jeongeun Seo ◽

Walter Hunziker ◽

...

Keyword(s):

Real Time ◽

Lipid Bilayer ◽

Lipid Bilayers ◽

Ambient Medium ◽

Membrane Attack Complex ◽

Common Adverse Effect ◽

Individual Risk ◽

Supported Lipid Bilayer ◽

Activation Assay

AbstractThe rapid advance of nanomedicines and biologicals in pharmacotherapy gives increasing importance to a common adverse effect of these modern therapeutics: complement (C) activation-related pseudoallergy (CARPA). CARPA is a relatively frequent and potentially lethal acute immune toxicity of many intravenous drugs that contain nanoparticles or proteins, whose prediction by laboratory or in vivo testing has not yet been solved. Preliminary studies suggest that proneness of the drug to cause C activation in the blood of patients may predict the individual risk of CARPA, thus, a sensitive and rapid bedside assay for individualized assessment of a drug’s C activating potential could alleviate the CARPA problem. The goal of the present study was to lay down the foundations of a novel approach for real-time sensing of C activation on a supported lipid bilayer platform. We utilized the quartz crystal microbalance with dissipation (QCM-D) monitoring technique to measure the self-assembly of C terminal complex (or membrane attack complex [MAC]) on supported lipid bilayers rapidly assembled by the solvent-assisted lipid bilayer (SALB) formation method, as an immediate measure of C activation. By measuring the changes in frequency and energy dissipation of deposited protein, the technique allows extremely sensitive real-time quantification of the sequential assembly of MAC from its molecular components (C5b-6, C7, C8 and C9) and hence, measure C activation in the ambient medium. The present paper delineates the technique and our initial evidence with purified C proteins that the approach enables sensitive and rapid (real-time) quantification of MAC formation on a silicon-supported planar (phospho) lipid bilayer, which can be used as an endpoint in a clinically useful bedside C activation assay.

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CryoEM reveals how the complement membrane attack complex ruptures lipid bilayers

Nature Communications ◽

10.1038/s41467-018-07653-5 ◽

2018 ◽

Vol 9 (1) ◽

Cited By ~ 28

Author(s):

Anaïs Menny ◽

Marina Serna ◽

Courtney M. Boyd ◽

Scott Gardner ◽

Agnel Praveen Joseph ◽

...

Keyword(s):

Lipid Bilayers ◽

Membrane Attack Complex

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Exaptation of two ancient immune proteins into a new dimeric pore-forming toxin in snails

10.1101/2019.12.23.880021 ◽

2019 ◽

Cited By ~ 1

Author(s):

M.L. Giglio ◽

S. Ituarte ◽

V. Milesi ◽

M.S. Dreon ◽

T.R. Brola ◽

...

Keyword(s):

Lipid Bilayers ◽

Solution Structure ◽

Three Dimensional ◽

Membrane Attack Complex ◽

Membrane Conductance ◽

Dimensional Structure ◽

Apple Snail ◽

Negative Stain ◽

Negative Stain Electron Microscopy ◽

Macpf Domain

AbstractThe Membrane Attack Complex-Perforin (MACPF) family is ubiquitously found in all kingdoms. They have diverse cellular roles but MACPF but pore-forming toxic function are very rare in animals. Here we present the structure of PmPV2, a MACPF toxin from the poisonous apple snail eggs, that can affect the digestive and nervous systems of potential predators. We report the three-dimensional structure of PmPV2, at 15 Å resolution determined by negative stain electron microscopy (NS-EM) and its solution structure by small angle X-ray scattering (SAXS). We found that PV2s differ from nearly all MACPFs in two respects: it is a dimer in solution and protomers combine two immune proteins into an AB toxin. MACPF chain is linked by a single disulfide bond to a tachylectin chain, and two heterodimers are arranged head-to-tail by non-covalent forces in the native protein. MACPF domain is fused with a putative new Ct-accessory domain exclusive to invertebrates. Tachylectin is a six-bladed β-propeller, similar to animal tectonins. We experimentally validated the predicted functions of both subunits and demonstrated for the first time that PV2s are true pore-forming toxins. The tachylectin ..B.. delivery subunit would bind to target membranes, and then its MACPF ..A.. toxic subunit disrupt lipid bilayers forming large pores altering the plasma membrane conductance. These results indicate that PV2s toxicity evolved by linking two immune proteins where their combined preexisting functions give rise to a new toxic entity with a novel role in defense against predation. This structure is an unparalleled example of protein exaptation.

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CryoEM reveals how the complement membrane attack complex ruptures lipid bilayers

10.1101/392563 ◽

2018 ◽

Cited By ~ 1

Author(s):

Anaïs Menny ◽

Marina Serna ◽

Courtney M. Boyd ◽

Scott Gardner ◽

Agnel Praveen Joseph ◽

...

Keyword(s):

Lipid Bilayers ◽

Membrane Attack Complex ◽

Structural Basis ◽

Complement Proteins ◽

Cell Functions ◽

Outer Leaflet ◽

Membrane Barrier ◽

Membrane Rigidity ◽

Membrane Bending ◽

Pore Forming Proteins

AbstractThe membrane attack complex (MAC) is one of the immune system’s first responders. Complement proteins assemble on target membranes to form pores that lyse pathogens and impact tissue homeostasis of self-cells. How MAC disrupts the membrane barrier remains unclear. Here we use electron cryo-microscopy and flicker spectroscopy to show that MAC interacts with lipid bilayers in two distinct ways. Whereas C6 and C7 associate with the outer leaflet and reduce the energy for membrane bending, C8 and C9 traverse the bilayer increasing membrane rigidity. CryoEM reconstructions reveal plasticity of the MAC pore and demonstrate how C5b6 acts as a platform, directing assembly of a giant β-barrel whose structure is supported by a glycan scaffold. Our work provides a structural basis for understanding how β-pore forming proteins breach the membrane and reveals a mechanism for how MAC kills pathogens and regulates cell functions.

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Breaching the Bacterial Envelope: The Pivotal Role of Perforin-2 (MPEG1) Within Phagocytes

Frontiers in Immunology ◽

10.3389/fimmu.2021.597951 ◽

2021 ◽

Vol 12 ◽

Author(s):

Leidy C. Merselis ◽

Zachary P. Rivas ◽

George P. Munson

Keyword(s):

Lipid Bilayers ◽

Knockout Mice ◽

Clinical Manifestations ◽

Membrane Attack Complex ◽

Pivotal Role ◽

Missense Mutations ◽

Gram Negative ◽

Initial Description ◽

Membrane Spanning

The membrane attack complex (MAC) of the complement system and Perforin-1 are well characterized innate immune effectors. MAC is composed of C9 and other complement proteins that target the envelope of gram-negative bacteria. Perforin-1 is deployed when killer lymphocytes degranulate to destroy virally infected or cancerous cells. These molecules polymerize with MAC-perforin/cholesterol-dependent cytolysin (MACPF/CDC) domains of each monomer deploying amphipathic β-strands to form pores through target lipid bilayers. In this review we discuss one of the most recently discovered members of this family; Perforin-2, the product of the Mpeg1 gene. Since their initial description more than 100 years ago, innumerable studies have made macrophages and other phagocytes some of the best understood cells of the immune system. Yet remarkably it was only recently revealed that Perforin-2 underpins a pivotal function of phagocytes; the destruction of phagocytosed microbes. Several studies have established that phagocytosed bacteria persist and in some cases flourish within phagocytes that lack Perforin-2. When challenged with either gram-negative or gram-positive pathogens Mpeg1 knockout mice succumb to infectious doses that the majority of wild-type mice survive. As expected by their immunocompromised phenotype, bacterial pathogens replicate and disseminate to deeper tissues of Mpeg1 knockout mice. Thus, this evolutionarily ancient gene endows phagocytes with potent bactericidal capability across taxa spanning sponges to humans. The recently elucidated structures of mammalian Perforin-2 reveal it to be a homopolymer that depends upon low pH, such as within phagosomes, to transition to its membrane-spanning pore conformation. Clinical manifestations of Mpeg1 missense mutations further highlight the pivotal role of Perforin-2 within phagocytes. Controversies and gaps within the field of Perforin-2 research are also discussed as well as animal models that may be used to resolve the outstanding issues. Our review concludes with a discussion of bacterial counter measures against Perforin-2.

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Observation of Concanavalin-A receptors on hydrated planar erythrocyte membrane film by in situ electron microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100076640 ◽

1983 ◽

Vol 41 ◽

pp. 608-609

Author(s):

Neng-Bo He ◽

S.W. Hui

Keyword(s):

Lipid Bilayers ◽

Erythrocyte Membrane ◽

Lipid Membranes ◽

Surface Film ◽

Biological Membranes ◽

Electron Microscopic Observation ◽

Electron Microscopic ◽

Black Lipid Membranes ◽

Fine Mesh ◽

Planar Films

Monolayers and planar "black" lipid membranes have been widely used as models for studying the structure and properties of biological membranes. Because of the lack of a suitable method to prepare these membranes for electron microscopic observation, their ultrastructure is so far not well understood. A method of forming molecular bilayers over the holes of fine mesh grids was developed by Hui et al. to study hydrated and unsupported lipid bilayers by electron diffraction, and to image phase separated domains by diffraction contrast. We now adapted the method of Pattus et al. of spreading biological membranes vesicles on the air-water interfaces to reconstitute biological membranes into unsupported planar films for electron microscopic study. hemoglobin-free human erythrocyte membrane stroma was prepared by hemolysis. The membranes were spreaded at 20°C on balanced salt solution in a Langmuir trough until a surface pressure of 20 dyne/cm was reached. The surface film was repeatedly washed by passing to adjacent troughs over shallow partitions (fig. 1).

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Effect of Integral Proteins on Frozen Membrane Fracture

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s1431927600003287 ◽

1980 ◽

Vol 38 ◽

pp. 756-759 ◽

Cited By ~ 1

Author(s):

S. Kirchanski ◽

D. Branton

Keyword(s):

Lipid Bilayers ◽

Freeze Fracture ◽

Covalent Bond ◽

Erythrocyte Membranes ◽

Glycophorin A ◽

Experimental Protocol ◽

Transmembrane Glycoprotein ◽

Bond Breakage ◽

Human Erythrocyte Membranes ◽

Integral Proteins

We have investigated the effect of integral membrane proteins upon the fracturing of frozen lipid bilayers. This investigation has been part of an effort to develop freeze fracture labeling techniques and to assess the possible breakage of covalent protein bonds during the freeze fracture process. We have developed an experimental protocol utilizing lectin affinity columns which should detect small amounts of covalent bond breakage during the fracture of liposomes containing purified (1) glycophorin (a transmembrane glycoprotein of human erythrocyte membranes). To fracture liposomes in bulk, frozen liposomes are ground repeatedly under liquid nitrogen. Failure to detect any significant covalent bond breakage (contrary to (2)) led us to question the effectiveness of our grinding procedure in fracturing and splitting lipid bilayers.

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