Lytic Pores Research Articles

Impaired binding of perforin on the surface of tumor cells is a cause of target cell resistance against cytotoxic effector cells

AbstractExocytosis of perforin, subsequent binding of perforin to the target cell membrane, and formation of lytic pores form an important pathway involved in the induction of tumor cell death by cytotoxic effector cells. Here we describe a novel escape mechanism employed by tumor cells to protect themselves from granule-mediated cell death: We were able to demonstrate that the resistance of the human leukemia cell line ML-2 to natural killer (NK)-cell–mediated killing is not caused by impaired NK-cell activation but by resistance against effector molecules contained in the granules of cytotoxic cells. No resistance was observed against other pore-forming agents like complement and streptolysin O. By using the NK-susceptible leukemia cell line K562, we could show that the induction of cell death by cytotoxic granules can be blocked completely by anti-perforin antibodies, indicating that perforin is essentially involved in this process. Flow cytometric data revealed that an impaired binding of perforin on the tumor cell membrane is mainly responsible for target cell resistance, because perforin turned out to bind well on K562 cells but is not able to attach to the surface of ML-2 cells. After impaired binding of perforin was identified as a potential mechanism of tumor cell resistance, leukemia cells from 6 patients with acute myeloid leukemia (AML) were examined. As predicted, AML cells that failed to bind perforin on their surface demonstrated complete resistance toward NK-cell–mediated cytotoxicity. Thus, perforin resistance could represent an important tumor escape mechanism that should be considered when cytotoxic effector cells are used for cellular immunotherapy.

Impaired binding of perforin on the surface of tumor cells is a cause of target cell resistance against cytotoxic effector cells

Exocytosis of perforin, subsequent binding of perforin to the target cell membrane, and formation of lytic pores form an important pathway involved in the induction of tumor cell death by cytotoxic effector cells. Here we describe a novel escape mechanism employed by tumor cells to protect themselves from granule-mediated cell death: We were able to demonstrate that the resistance of the human leukemia cell line ML-2 to natural killer (NK)-cell–mediated killing is not caused by impaired NK-cell activation but by resistance against effector molecules contained in the granules of cytotoxic cells. No resistance was observed against other pore-forming agents like complement and streptolysin O. By using the NK-susceptible leukemia cell line K562, we could show that the induction of cell death by cytotoxic granules can be blocked completely by anti-perforin antibodies, indicating that perforin is essentially involved in this process. Flow cytometric data revealed that an impaired binding of perforin on the tumor cell membrane is mainly responsible for target cell resistance, because perforin turned out to bind well on K562 cells but is not able to attach to the surface of ML-2 cells. After impaired binding of perforin was identified as a potential mechanism of tumor cell resistance, leukemia cells from 6 patients with acute myeloid leukemia (AML) were examined. As predicted, AML cells that failed to bind perforin on their surface demonstrated complete resistance toward NK-cell–mediated cytotoxicity. Thus, perforin resistance could represent an important tumor escape mechanism that should be considered when cytotoxic effector cells are used for cellular immunotherapy.

Complement activation by bacterial surface glycolipids: a study with planar bilayer membranes.

Planar asymmetric glycolipid/phospholipid bilayer membranes were used as a reconstitution model of the lipid matrix of the outer membrane of Gram-negative bacteria to study complement (C) activation by various bacterial surface glycolipids with the aim of defining the C activation pathway. As glycolipids the lipopolysaccharides of Salmonella enterica serovar Minnesota R mutant strains R595 (Re LPS) and R4 (Rd2 LPS), pentaacyl lipid A from the LPS of the Escherichia coli Re mutant F515, and glycosphingolipid GSL-1 of Sphingomonas paucimobilis IAM 12576 were used. Methylester and carboxyl-reduced derivatives of GSL-1 were used to elucidate the role of the carboxyl group as common functional group of LPS and GSL-1 for C activation. The formation of lytic pores was monitored via the measurement of changes in membrane current. For all glycolipids we observed a considerable increase in membrane current soon after addition of whole human serum due to the formation of lytic pores in the membranes. Pore formation was dependent on the presence of C9, indicating that the observed current changes were due to C activation. We found that in our reconstitution system of the outer membrane lipid A, Re LPS, and Rd2 LPS activated the classical pathway, the activation being independent of specific anti-LPS antibodies. In contrast, GSL-1 and the methylester derivative of GSL-1 activated the alternative pathway even at the low serum concentrations used in this study (about 0.2% v/v). Interestingly, the carboxyl reduced GSL-1 activated the classical pathway.

Self-assembled α-hemolysin pores in an S-layer-supported lipid bilayer

The effects of a supporting proteinaceous surface-layer (S-layer) from Bacillus coagulans E38-66 on a 1,2-diphytanoyl- sn-glycero-3-phosphatidylcholine (DPhPC) bilayer were investigated. Comparative voltage clamp studies on plain and S-layer supported DPhPC bilayers revealed no significant difference in the capacitance. The conductance of the composite membrane decreased slightly upon recrystallization of the S-layer. Thus, the attached S-layer lattice did not interpenetrate or rupture the DPhPC bilayer. The self-assembly of a pore-forming protein into the S-layer supported lipid bilayer was examined. Staphylococcal α-hemolysin formed lytic pores when added to the lipid-exposed side. The assembly was slow compared to unsupported membranes, perhaps due to an altered fluidity of the lipid bilayer. No assembly could be detected upon adding α-hemolysin monomers to the S-layer-faced side of the composite membrane. Therefore, the intrinsic molecular sieving properties of the S-layer lattice do not allow passage of α-hemolysin monomers through the S-layer pores to the lipid bilayer. In comparison to plain lipid bilayers, the S-layer supported lipid membrane had a decreased tendency to rupture in the presence of α-hemolysin.

The Role of the Amino Terminus in the Kinetics and Assembly of α-Hemolysin of Staphylococcus aureus

The nature of the involvement of an intact NH2 terminus in the assembly of alpha-hemolysin of Staphylococcus aureus was reinvestigated. For the first time, a deletion of the first four amino acids at the NH2 terminus of alpha-hemolysin yielded a novel mutant that undergoes all of the conformational changes to form a lytic pore. The experimental evidence shows unequivocally that the mutant toxin forms heat- and sodium dodecyl sulfate-stable heptameric oligomers. The concentration required to achieve 50% lysis of red blood cells is around 58-116 ng/ml, and the time taken to achieve lysis to the same extent as that of intact toxin is considerably longer. Transmission electron microscopic studies also suggest that the pores formed by this deletion mutant are similar to those by the full-length toxin. This is in contrast to the previously reported 2- and 11-amino acid deletions that failed to proceed further from a presumed prefinal nonlytic pore to a lytic pore. Studies on the kinetics of assembly indicate that this mutant can form heat- and sodium dodecyl sulfate-stable oligomers as fast as full-length alpha-hemolysin but that pore opening is slowed down. The data strongly suggest that these amino acids (Ala-Asp-Ser-Asp) are involved in the final stages of assembly of alpha-hemolysin in target membranes.

S-protein/vitronectin interaction with the C5b and the C8 of the complement membrane attack complex.

S-protein/vitronectin participates in the regulation of complement-mediated lysis by incorporating into the membrane attack complex C5b-9. Subsequently, soluble SC5b-7 and SC5b-9 macromolecules are not inserted into the membrane lipid bilayer nor induce lytic pore formation. Using a dot blot binding assay, it was determined that soluble S-protein/vitronectin interacted with immobilized C5b and C8 among the five separately immobilized components (C5b, C6, C7, C8, C9) of the membrane attack complex. Those interactions imply a new model of the formation of the complement membrane attack complex and its regulation by S-protein/vitronectin.

Structure of complement poly-C9 determined in projection by cryo-electron microscopy and single particle analysis

The ring-like complement ‘lesions’ found on membranes of complement lysed cells comprise a complex of components C5b through C9 that coalesce to form hollow cylinders which penetrate the membrane bilayer and create lytic pores. Walls of these C5b-9 membrane attack complex cylinders may consist primarily of the C9 component, since samples of purified, isolated C9 can polymerize into cylindrical structures which appear identical with the fully assembled C5b-9 complex. The structure of these poly-C9 molecules has been investigated using the techniques of cryo-electron microscopy and single particle analysis. Sets of single poly-C9 particles viewed as rings were selected from cryo-EM images, then particles were aligned and treated by correspondence analysis to identify the principle interparticle similarities and variations. The highest ranking variation found was the presence or absence of a dense inner ring of protein density. Other important variations were interpreted as different types of particle tilt. These results were used in selecting a subgroup of untilted particles for averaging and symmetry analysis. The rotational power spectrum of the initial average suggested 13-fold symmetry. The 13-fold symmetry was used to select and group particles for further analysis. Individual particles were 13-fold rotational averaged and those with enhanced peripheral features were placed into either a right-handed subgroup or into a left-handed subgroup based on orientation of the peripheral features. Particles within each group were aligned and averaged, and a poly-C9 structure was produced which shows important structural details and from which the C9 monomer structure can be deduced. The poly-C9 structure contains a dense inner ring of diameter between 113–181 Å and which is modulated into 13 discrete peaks with peak-to-peak separation of approx. 35 Å. The dense inner ring is surrounded by a less dense, concentric outer rim extending to 254 Å diameter. The outer rim contains projections that are contiguous with the inner peaks but are skewed relative to the ring radius to produce the appearance of a pin-wheel. These projections correspond with the peripheral features picked up in the rotationally averaged individual particles; the left- or right-handed orientation of projections may result from the up/down orientation of individual particles in ice. The C9 monomer structure within the cylinder is suggested by the density distribution. The monomer would be a rod with diameter of 35 Å, oriented parallel to the cylinder axis and would be roughly perpendicular to a membrane. A portion of the C9 monomer would form the projecting outer rim. In side views of the complex the outer rim density would be the torus structure seen distal to the membrane-inserting region. These results indicating an extended C9 structure are consistent with C9 unfolding in the transition from monomer to poly-C9 complex.

Functional complementation of staphylococcal alpha-hemolysin fragments. Overlaps, nicks, and gaps in the glycine-rich loop.

The final steps in assembly of the lytic pore formed by staphylococcal alpha-hemolysin (alpha HL) involve the formation of a nonlytic oligomeric pore precursor, followed by the formation of a transmembrane channel. In this study, truncation mutants of alpha HL encompassing the NH2-terminal or COOH-terminal half of the polypeptide chain and all, part, or none of the central glycine-rich loop were obtained by in vitro, coupled transcription and translation of mutant plasmid DNAs. These polypeptides were unable to oligomerize upon or cause lysis of rabbit erythrocytes (rRBCs). Twenty-one pairs of the same truncation mutants constituting discontinuous alpha HL chains with overlaps, nicks, and gaps in the central loop were obtained by cotranslation. When incubated with rRBCs, many of the pairs were able to form hetero-oligomers with wild-type alpha-hemolysin (s-alpha HL) and most of these formed homo-oligomers in the absence of s-alpha HL. However, only members of a subset of these pairs were able to lyse the cells. The lytic combinations contained overlaps, nicks, or gaps, but only two pairs, with nicks between amino acid residues 128 and 129 and between 131 and 132 had hemolytic activities approaching that of the wild-type polypeptide. Active combinations could also be obtained by separately translating NH2- and COOH-terminal truncation mutants and then combining them. These findings suggest that the integrity of the central loop is of little significance for oligomer formation but that it is more important for the final step in pore assembly or alternatively for determining the correct structure of the conductive channel. Our findings disagree with previous reports of NH2- and COOH-terminal fragments with hemolytic activity and of the prevention of hemolysis by proteolytic cleavage in the central loop. This discord is attributed to experimental and interpretative ambiguities in the earlier protein chemistry. For example, we show that loss of hemolytic activity after treatment with trypsin is not due to cleavage after Lys-131, as previously proposed, but to the removal of a small NH2-terminal peptide through cleavage after Lys-8.

Assembly of the oligomeric membrane pore formed by Staphylococcal alpha-hemolysin examined by truncation mutagenesis.

The alpha-hemolysin (alpha HL) from Staphylococcus aureus causes the lysis of susceptible cells such as rabbit erythrocytes (rRBCs). Lysis is associated with the formation of a hexameric pore in the plasma membrane. Here we show that truncation mutants of alpha HL missing 2 to 22 N-terminal amino acids form oligomers on the surfaces of rRBCs but fail to lyse the cells. By contrast, mutants missing 3 or 5 amino acids at the C terminus are very inefficient at oligomerization but do lyse rRBCs, albeit extremely slowly. The C-terminal truncation mutants, retarded as monomers on the cell surface, undergo a conformational change in which the protease-sensitive loop located near the midpoint of the polypeptide chain becomes occluded. Judged by this criterion, polypeptides truncated at the N terminus, frozen as nonlytic oligomers, are in a similar conformation. A second proteolytic site near the N terminus of the polypeptide becomes inaccessible in the lytic pore formed by the wild-type polypeptide, supporting the idea that a second conformational change occurs upon pore formation. These findings suggest a pathway for assembly of the lytic pore in which alpha HL first binds to target cells as a monomer, which is converted to a nonlytic oligomeric intermediate before formation of the pore. In keeping with this model, an N-terminal truncation mutant blocks the slow lysis induced by a C-terminal truncation mutant, presumably by diverting the weakly lytic subunits into inactive oligomers.

Colloid-osmotic lysis is a general feature of the mechanism of action of Bacillus thuringiensis δ-endotoxins with different insect specificity

The mechanism of action of Bacillus thuringiensis insecticidal δ-endotoxins has long been the subject of controversy. As our working hypothesis we propose a two-step model in which, after binding a specific plasma membrane receptor, the action of all the δ-endotoxins studied here is to generate small pores in the plasma membrane, either directly by inserting into the membrane, or indirectly by perturbing resident plasma membrane molecules. The creation of these pores will lead to colloid-osmotic lysis, i.e., an equilibration of ions through the pore resulting in a net inflow of ions, an accompanying influx of water, cell swelling and eventual lysis. Our observations that cell swelling precedes lysis, that small molecules leak out of the cell before large ones, that osmotic protectants inhibit or delay cytolysis, and that the toxin-induced pore of 0.5–1.0 nm radius will allow equilibration of ions but not leakage of cytoplasmic macromolecules, are in full agreement with the predictions of this hypothesis. To explain the specificity of the δ-endotoxin-induced lytic pore formation, we propose that prior interaction between the toxin and cell-specific plasma membrane recpetors is necessary before these toxins can insert into, or interact with, the membrane.

Dissociation of membrane binding and lytic activities of the lymphocyte pore-forming protein (perforin).

Granules isolated from CTL and NK cells contain a cytolytic pore-forming protein (PFP/perforin). At low temperatures (on ice), PFP binds to erythrocyte membranes without producing hemolysis. Hemolysis occurs when the PFP-bound erythrocytes are warmed up to 37 degrees C, which defines a temperature-dependent, lytic (pore-formation) step distinct from the membrane-binding event. Ca2+ and neutral pH are required for both membrane binding and pore formation by PFP. Serum, LDL, HDL, and heparin inhibit the hemolytic activity of PFP by blocking its binding to lipid membranes. Lysis by PFP that has bound to erythrocyte membranes is no longer susceptible to the effect of these inhibitors. The hemolytic activities associated with intact granules and solubilized PFP show different requirements for Ca2+ and pH, indicating that cytolysis produced by isolated granules may involve an additional step, possibly fusion of granules with membranes. It is suggested that three distinct Ca2+- and pH-dependent events may be involved during cell killing by CTL and NK cells: fusion of cytoplasmic granules of effector cells with their plasma membrane, releasing PFP from cells; binding of the released PFP to target membranes; and insertion of monomers and the subsequent formation of lytic pores in the target membrane. The serum-mediated inhibition of membrane binding by PFP could prevent the accidental injury of bystander cells by cell-released PFP, but would allow cytolysis to proceed to completion once PFP has bound to the target membrane.