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A pore-forming protein was detected and purified for the first time from a marine sponge (Tethya lyncurium). The purified protein has a polypeptide molecular mass of 21 kDa and a pI of 6.4. Tethya pore-forming protein (also called Tethya hemolysin) rapidly lysed erythrocytes from a variety of organisms. After binding to target membranes, the hemolysin resisted elution with EDTA, salt or solutions of low ionic strength and hence resembled an integral membrane protein. Erythrocytes could be protected from hemolysis induced by Tethya hemolysin by addition of 30 mM dextran 4 (4-6 kDa; equivalent hydrodynamic diffusion radius, 1.75-2.3 nm) to the extracellular medium, but not by addition of uncharged molecules of smaller size [sucrose, raffinose and poly(ethylene glycol) 1550; equivalent hydrodynamic diffusion radii, 0.46, 0.57 and 1.2 nm, respectively]. This result indicates that hemolysin is able to form stable transmembrane pores with an effective diameter of about 2-3 nm. Treatment of osmotically protected erythrocytes with Tethya hemolysin caused a rapid efflux of intracellular K+ and ATP, and a rapid influx of extracellularly added Ca2+ and sucrose. In negative-staining electron microscopy, target erythrocyte membranes exposed to purified Tethya hemolysin displayed ultrastructural lesions but without visible pores.

With the actual increasing menace of bacterial resistance, the antibiotic permeation mechanisms have been the centre of attention, in many research fields. OmpF is a pore forming protein, found in Gram-negative bacteria's outer membrane, known to have an important role in the uptake of nutrients and antibiotics (e.g. quinolones), towards the interior of the bacterial cell. Although several studies have been published of protein-drug interaction in the complex microbiological environment, an understanding of this mechanism at a molecular level is still lacking. Quinolones are one of the most prescribed classes of antibiotics, both in human and veterinary medicine, and this wide use seems to be the main cause for bacterial resistance. Fluoroquinolones were developed to increase quinolone antibacterial activity by changes in the quinolone structure. Enrofloxacin is a second generation fluoroquinolone being the first one to be introduced in Veterinary medicine. Its human analogue is the well known Ciprofloxacin. The main purpose of this work was to study Enrofloxacin permeation pathways. Permeation or interaction of antibiotics with lipid bilayers (liposomes) is of great importance in order to clarify the lipid component function's in membrane permeation. A first approach was the determination of partition coefficients Enrofloxacin/liposomes, which allowed the quantification of this interaction. After, OmpF was reconstituted in liposomes (proteoliposomes) by two different reconstitution methods (Gel exclusion chromatography and using detergent adsorbing beads) in order to compare size, homogeneity and protein orientation in proteoliposomes. Proteoliposomes quenching studies, using water soluble quencher KI and acrylamide, as well as ion conductance measurements were performed to establish the site of interaction/translocation of Enrofloxacin.

Both CD3- and CD3+ CD56+ effector cells can mediate non-MHC-restricted lysis in the absence of activation. Previous studies have shown that both of these subsets can be augmented with IL-2. In the present study, we have examined further the phenotypic markers expressed on these cells as well as the functional capacities of these subsets, including LAK activity, cytokine expression, and pore-forming protein (PFP) production. In addition, these populations were analyzed for clonality by Southern blot analysis of the T cell receptor beta chain gene constant region. The CD3-, CD56+ and CD3+, CD56+ lymphocytes were quite similar in their phenotypic markers, although the CD3+, CD56+ lymphocytes lacked high levels of IL-2 receptor beta chain and did not express CD16. The CD3+, CD56+ lymphocytes mediated non-MHC-restricted lysis, but failed to express LAK activity or be induced by IL-2 to secrete IFN gamma, a characteristic of the CD3-, CD56+ lymphocytes. The T cell receptor beta chain gene pattern of the CD3+, CD56+ lymphocytes was characteristic of a polyclonal cell population. Of interest, both populations of cells appeared morphologically to be large granular lymphocytes that contain PFP in their cytoplasmic granules. Therefore these CD56+ subsets provide a new model to study several questions related to non-MHC-restricted target cell lysis, including the identification of novel receptors involved in target cell recognition and/or triggering as well as the biochemical pathways implicated in cellular lysis.

We studied the 5' untranslated regions (UTRs) of the mouse lymphocyte pore-forming protein (PFP, perforin, and cytolysin). 5' UTRs were determined by primer extension analysis, sequencing PFP cDNA clone PFP-7, ribonuclease protection assays, and amplification of poly(A)+ RNA of cytolytic T lymphocyte using polymerase chain reaction (PCR). Two alternatively spliced 5' UTRs, designated type I and type II, of 222 and 115 bp, respectively, were found associated with PFP. Type II is identical to type I, except for being 107 bp shorter in the second exon. This deletion was generated by the use of alternative acceptor splice sites. The mouse PFP gene (Pfp) encodes three exons, is separated by two small introns, and spans a chromosomal region of approximately 7 kb. The first exon contains 79 bp of 5' UTR, the second exon contains 143 or 36 bp of 5' UTR (type I or type II UTR, respectively) plus the NH2-terminal region of the mouse PFP, and the third exon contains the rest of the COOH-terminal mouse PFP. The organization of the mouse Pfp is similar to that of the human gene. Moreover, the 5' flanking sequence of the mouse Pfp is highly homologous to that of the human Pfp. In contrast to the human sequence, the more immediate 5' flanking sequence of mouse Pfp contains two tandem "TATA" box-related elements and a GC box, but lacks a typical CAAT box-related sequence. Several other enhancer elements were found further upstream, including cAMP-, phorbol ester-, interferon-gamma-, and UV-responsive elements, and PU box-like and NFkB binding site-like elements. In addition, we found a nuclear inhibitory protein-like element, a transcriptional silencer, and a pair of purine-rich sequence motifs that were found in other T cell-specific genes, and three repeats of GGCCTG that may be a variation of a highly repetitious GCCCTG consensus sequence found in human Pfp.

Cytotoxic T lymphocytes (CTL) release from their granules a 70 kDa protein, called PFP, perforin or cytolysin, which inserts into the target cell plasma membrane in its monomeric form. Here it polymerizes into a macromolecular complex forming pores as large as 20 nm. Although purified PFP/perforin can effectively lyze all target cells tested. CTL are refractory to lysis. The mechanism underlying the resistance of CTL is currently unknown. This study represents a search for membrane structural properties that could confer resistance to CTL against PFP/perforin-mediated lysis. The fluorescent dye merocyanine 540 was used to measure the lipid head group packing of CTL and several target cells, and 1-[4-(trimethylamine)phenyl]-6-phenylhexa-1,3,5-triene was used to estimate the fluidity of the membrane hydrocarbon region. The resistance against PFP/perforin-mediated lysis was determined by the 51Cr release assay. A comparison of the membrane rigidity with cell resistance led to the conclusion that the membrane lipid structure cannot account for the unusually high resistance of CTL. In particular, the resistant CTL line CTLL-2 has a lipid head group packing that is looser than that of Yac-1, and the sensitive target cells Jy-25 and EL-4 have membrane acyl chains that are less fluid than those of the effector CTLL-R8.

The cDNAs encoding several proteins present in the granules of cytolytic effector lymphocytes have now been cloned. These included the cytolytic pore-forming protein (PFP) or perforin, and at least six serine esterases (SE), also called granzymes. The cDNA probes for PFP, SE-1, and SE-2 are used here to study the expression of these proteins in murine primary effector lymphocytes. Among the stimuli effective in inducing the expression of PFP, SE-1, and SE-2 were recombinant interleukin-2, the lectin concanavalin A in the presence of phorbol esters, and allogeneic cells in mixed lymphocyte cultures. Some correlation was seen between the levels of PFP and SE mRNAs and cytotoxicity measured in a standard 51Cr release assay. We also examined a panel of 13 cloned cytotoxic T Lymphocyte (CTL) lines and found that mRNAs for PFP and SE-2 were expressed in all CTL lines, including some that were previously considered not to produce PFP. Twelve of the 13 CTL lines also proved to possess the mRNA for SE-1. One thymoma cell line, TIMI.4, did not express mRNA for PFP, although it expressed mRNA for SE-1 and SE-2.

A pore-forming protein (PFP/perforin/cytolysin), stored in the cytoplasmic granules of cytolytic lymphocytes, lyses a variety of target cells but not the cytolytic lymphocytes. In the complement (C) system, a C8-binding protein (C8bp) or homologous restriction factor (HRF) has been described that protects cells against lysis mediated by homologous C. C8bp/HRF is known to bind to C8 and C9 and has also been suggested to protect lymphocytes against perforin-mediated lysis. Here, using an anti-idiotypic antibody approach, several polyclonal antisera were raised against IgGs that are specific for mouse perforin, and human C8 and C9. These anti-idiotypic antisera were shown to react against an overlapping epitope(s) on C8bp/HRF as indicated by the following evidence: (i) all three types of antisera reacted against partially purified C8bp/HRF and against a 65 kDa protein band in cell lysates; reactivity was only observed against disulfide-reduced antigens; (ii) the three antibodies react with a protein band in normal erythrocytes (E) but not with type III E of patients with paroxysmal nocturnal hemoglobinuria or with a mutant B lymphoblastoid cell line, both of which cell types are known to be deficient in C8bp/HRF; and (iii) the three antibodies compete with each other for binding to C8bp/HRF. Type III E and the C8bp/HRF-deficient mutant lymphoblastoid cell line, however, are as susceptible to perforin-mediated lysis as type I E and wild-type lymphoblastoid cell line, respectively, indicating that C8bp/HRF does not play a role in protecting cells against perforin-mediated lysis. These paradoxical findings suggest that perforin may share with C8 and C9 the same domain(s) that bind to C8bp/HRF and yet, unlike C8 and C9, perforin is not inactivated by this type of putative interaction. Since C8 and C9 are now readily available, the anti-idiotypic approach described here provides a convenient protocol for production of antisera specific for C8bp/HRF.

A cytolytic pore-forming protein (PFP, perforin) was purified from isolated granules of cloned NK-like cytolytic cells, which showed an apparent Mr of 70-75 kd (reduced) and 62-66 kd (nonreduced). Cytolysis produced by this protein occurred only in the presence of Ca2+ and was accompanied by the formation of membrane lesions of 160 A diameter. The purified protein depolarized cells and made lipid vesicles leaky to monovalent and divalent ions. This protein formed large, voltage insensitive and nonselective ion channels in planar bilayers that remained preferentially in the open state. The channels were heterogeneous in size distribution averaging 400 pS/U in 0.1 M NaCl. The membrane lesions formed by PFP were morphologically and functionally similar to those formed by intact NK-like cells and their granules. This PFP could be released from granules during cell killing, followed by its polymerization on target membranes to form large transmembrane pores.

We have constructed, by recombinant DNA techniques, one hybrid protein, colicin A-β-lactamase (P24), and two modified colicin As, one (P44) lacking a large central domain and the other (PX-345) with a different C-terminal region. The regulation of synthesis, the release into the medium and the properties of these proteins were studied. Only P44 was released into the medium. This suggests that both ends of the colicin A polypeptide chain might be required for colicin release. None of the three proteins was active on sensitive cells in an assay in vivo. However, P44 was able to form voltage-dependent channels in phospholipid planar bilayers. Its lack of activity in vivo is therefore probably caused by the inability to bind to the receptor in the outer membrane. PX-345 is a colicin in which the last 43 amino acids of colicin A have been replaced by 27 amino acids encoded by another reading frame in the same region of the colicin A structural gene; it was totally unable to form pores in planar bilayers at neutral pH but showed a very slight activity at acidic pH. These results confirm that the C-terminal domain of colicin A is involved in pore formation and indicate that at least the 43 C-terminal amino acid residues of this domain play a significant role in pore formation or pore function. Fifteen monoclonal antibodies directed against colicin A have been isolated by using conventional techniques. Five out of the 15 monoclonal antibodies could preferentially recognize wild-type colicin A. In addition, the altered forms of the colicin A polypeptide were used to map the epitopes of ten monoclonal antibodies reacting specifically with colicin A. Some of the antibodies did not bind to colicin A when it was pre-incubated at acidic pH suggesting that colicin A undergoes conformational change at about pH 4. The effects of monoclonal antibodies on activity in vivo of colicin A were investigated. The degree of inhibition observed was related to the location of the epitopes, with monoclonal antibodies reacting with the N terminus giving greater inhibition. The monoclonal antibodies directed against the C-terminal region promoted an apparent activation of colicin activity in vivo.

We have purified a major outer membrane protein from Aeromonas salmonicida. This 42-kilodalton protein shared several physical characteristics with enterobacterial porins in that it was noncovalently associated with the peptidoglycan, it was released from the peptidoglycan in the presence of 0.1 M NaCl and sodium dodecyl sulfate, and its mobility on sodium dodecyl sulfate-polyacrylamide gels was dependent on the solubilization temperature before electrophoresis. When added to the aqueous solution bathing a planar bilayer membrane it caused the conductance of the membrane to increase by several orders of magnitude. At lower protein concentrations, single channels with an average conductance of 1.6 nS in 1 M KCl were incorporated into the membrane in a stepwise fashion. Evidence that the protein formed a large, relatively nonselective, water-filled channel was obtained by performing single-channel experiments at different NaCl concentrations and in a variety of different salts. Current through the channel was a linear function of the applied voltage, and no evidence of voltage gating was observed. In addition, we obtained evidence for a 43-kilodalton channel-forming protein in the outer membrane of A. hydrophila with a similar single-channel conductance as the 42-kilodalton protein in 1 M NaCl.

Peroxisomes were purified from livers of clofibrate-treated rats. Permeability measurements on the isolated organelles revealed that peroxisomes are permeable to small solutes, including sucrose and the cofactors for fatty acid oxidation NAD+, CoA, ATP, and carnitine. The intraperoxisomal distribution volume was equal for all solutes. Peroxisomal solute uptake was rapid, not saturable and not visibly influenced by temperature. NAD+ and carnitine uptake in the solute accessible volume was not diminished by a variety of analogs and inhibitors. Subfractionation of peroxisomes and reconstitution of the subfractions into liposomes preloaded with solutes made the liposomes reconstituted with the integral membrane protein fraction, but not those reconstituted with the other subperoxisomal protein fractions, permeable to the same solutes that entered intact peroxisomes. Solute leakage from the preloaded liposomes was rapid and not visibly influenced by temperature. Leakage activity was destroyed by heat treatment of the integral membrane protein fraction and was not present in lipid extracts of the membrane. Separation of the integral membrane proteins on sucrose density gradients and reconstitution of the gradient fractions into liposomes indicated that the leakage activity was caused by a polypeptide of rather low molecular weight. The gradient distribution of leakage activity corresponded most closely to the presence of a 22- and a 28-kDa polypeptide. Our experiments indicate that the nonspecific permeability of the peroxisomal membrane to small solutes is based on the presence in the membrane of a nonselective pore-forming protein.

Treating oxidized cholesterol black lipid membranes with Aplysia hemocyanin induces the formation of channels with two conductivity states; at the fundamental level of conductance, the lifetime is several hours. Transitions from this state to a different conductivity state occur. Membranes with many of these channels have a voltage-dependent conductance and transitions between different conductivity values occurring in a few ms. Thus molluscan hemocyanins can be considered as a general class of pore-forming proteins.

Pore-forming protein from Entamoeba histolytica forms cation-selective channels in planar bilayers. With increasing potentials, the open-state probability of these channels decreases, and channel aggregates collapse (Young, J.D.-E. and Cohn, Z.A. (1985) J. Cell. Biochem. 29, 299-308). In this communication we report the following observations: (i) incorporation of the pore in black-lipid membranes was stimulated by membrane potential, (ii) pores were rectifying, (iii) breakdown of pores resulted in a continuous spectrum of subconductance states, (iv) the open-state probability increased strongly with pH. This pattern of behaviour is similar to that of the barrel-stave aggregates (alamethicin and related toxins). We therefore conclude that the amebal pores, like those of the barrel-stave class, may consist of complexes involving variable numbers of membrane-spanning subunits.

The cloned phoE genes from Enterobacter cloacae and Klebsiella pneumoniae are normally expressed and regulated in Escherichia coli K-12, and their products are correctly assembled into the outer membrane. Differences between the three PhoE proteins were found with binding of two out of ten monoclonal antibodies directed against the cell-surface-exposed part and in pore characteristics, but not in phage receptor function. The DNA sequences of the E. cloacae and K. pneumoniae phoE genes were determined and used to predict the primary structures of the encoded proteins. In the upstream non-coding regions, which showed more variations among the three genes than the coding regions, conserved sequences were identified which might be involved in regulation of phoE gene expression. Comparison of the predicted PhoE primary structures revealed a high degree of homology, with 81% of the amino acid residues being identical in all three proteins. Four small variable regions were found where differences are the most pronounced, corresponding to regions which were previously predicted to be exposed at the cell surface. Implications of the sequence comparison for structure-function relationships in PhoE protein are discussed.

We show here that human peripheral blood NK cells contain a pore-forming protein (PFP) with an Mr of 70,000-72,000 that assembles structural lesions (with an average internal diameter of 150-170 A) and forms functional channels. The PFP was isolated by affinity chromatography from human NK cells, using a specific anti-C9 antiserum as the immunoadsorbent. The NK cells were isolated from PBL by positive or negative selection by indirect rosetting using a panel of monoclonal antibodies directed against different NK and T cell surface antigens. PFP was identified in NK cells freshly isolated and isolated from cultured PBL, both stimulated with interleukin 2, but not in NK cell-depleted lymphocytes. In planar bilayers, the channels formed by the NK cell-derived PFP are highly voltage resistant, with most channels persisting in the open state once they have inserted into the bilayer. The unit conductances of these channels range 0.3-1 nS in 0.1 M NaCl. The channels show poor selectivity for monovalent and divalent ions. The PFP is also released from human NK cells stimulated with the calcium ionophore A23187, suggesting that this protein, like the one produced by murine CTL lines, may be similarly secreted during cell-mediated killing. Its identification in primary human NK cell cultures indicates that this protein may play an active role in NK cell-mediated killing.

The murine lymphocyte pore-forming protein (PFP) was purified to apparent homogeneity by successive steps of liquid chromatography. Monospecific antibodies were raised against purified PFP that detect only one protein band in murine CTL lines. 25% of the primary sequence of PFP (134 amino acids) was determined by amino terminal analysis of the purified protein and of some of its enzymatic cleavage products. These primary sequences were identical to sequences deduced by cDNA cloning. By isoelectric focusing, PFP was found to have a pI of 6.4. On the chromatofocusing column Mono P, however, PFP was found to elute at pH 4.7. This suggests a tertiary structure for monomeric PFP that is enriched in surface acidic amino acids.

Structural, functional and immunological similarities between the ninth component of complement (C9) and the lymphocyte pore-forming protein (PFP, perforin) have recently been described (8-10). PFP is shown here to be immunologically related to all other components of the membrane attack complex (MAC) of human complement, namely, C5b-6, C7, C8, and C9. Polyclonal antibodies raised against purified human C5b-6, C7, C8, or C9 react with other components of the MAC and with mouse lymphocyte PFP. The antigenic epitopes shared by human complement proteins and mouse lymphocyte PFP are limited to cysteine-rich domains. Only complement proteins that have been reduced and alkylated elicit the production of crossreactive antibodies when used as immunogens. The nonreduced forms of complement components or lymphocyte PFP neither react with these antibodies nor give rise to crossreactive antibodies. The homologous domains of complement proteins and lymphocyte PFP may play related functions in their attachment to lipid membranes and assembly of membrane lesions.

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.

Cytolytic granules from a T-cell line with specific cytolytic activity were isolated. Granules were solubilized and fractionated on a TSK 4000 gel filtration column. Lytic activity was eluted as a single retarded peak. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulfate indicated that the lytic fractions contained a single protein (perforin) with an apparent molecular weight of approximately 66 kDa. It separated well from the other proteins present in the granules. Isolated perforin polymerized and inserted into lipid bilayers in the presence of Ca2+, forming tubular structures with inner diameters varying from 6 to 16 nm. Lipid insertion of perforin was demonstrated using a membrane-restricted, photoactivatable probe. The lytic properties of perforin suggest an important role of this particular protein during cytolytic T-cell-mediated target cell lysis.

A membrane-bound cytolytic pore-forming protein (N-PFP) produced by the pathogenic ameboflagellate Naegleria fowleri was characterized. N-PFP was solubilized from ameba membranes by detergent and enriched 300-fold by gel filtration chromatography. When analyzed by gel electrophoresis, N-PFP migrates with a molecular mass of 66 kDa and 50-54 kDa, under reducing and non-reducing conditions, respectively. In addition to lysing erythrocytes, N-PFP is cytotoxic to several tumor cell lines tested. Its hemolytic activity is not dependent on the presence of divalent cations. N-PFP rapidly depolarizes the membrane potential of microelectrode-impaled chicken embryo myocytes, suggesting that functional channel formation may represent the mode of membrane damage. In planar bilayers, N-PFP forms ion channels with heterogeneous unit conductances ranging between 150 and 400 picosiemens in 0.1 M NaCl and that are relatively resistant to closing by high voltages. Upon heat treatment (75 degrees C, 30 min), N-PFP forms channels with unit conductances that are on average larger than those formed by untreated N-PFP. N-PFP channels are slightly more permeable to cations than to anions. Using a liposome swelling-shrinkage assay, the functional diameter of N-PFP channels is estimated to range between 3.6 and 5.2 nm. N-PFP is immunologically distinct from the PFP/perforin produced by lymphocytes, the terminal components of complement and a PFP from the ameba Entamoeba histolytica, all of which produce pores on target membranes. This protein may have a direct lytic role during target cell killing mediated by N. fowleri.

The permeability properties of the outer membrane of the chloroplast envelope were studied. The exclusion limit for the penetration of molecules through this membrane into the intermembrane space lies between M r values of about 7000–13 000. This is the largest exclusion limit measured so far for pores of the porin type. In addition, the pore-forming activity of the outer membrane of the chloroplast envelope was measured by reconstitution experiments. The pore has a single channel conductance of 0.72 nS, which is a linear function of the salt solution in the aqueous phase. The diameter of the chloroplast porin was calculated to be 2.5–3 nm, which is the largest diameter of all known porin pores, e.g., from mitochondria or Gram-negative bacteria.

Entamoeba histolytica is the human pathogen responsible for amebiasis. This infection is characterized by an invasive enteric illness that may spread to multiple organs. The parasite E histolytica may now be cultivated in vitro, with Diamond's axenic medium [1]. In culture, this protozoan remains cytolytic to a variety of cell types, including neutrophils and macrophages (reviewed in [2]). The mechanism(s) involved in the expression of this potent cytolysis remain(s) unclear. Previous studies from several laboratories have suggested that the cell killing mediated by amoeba is dependent on intimate contact of amoeba with the target cell membrane [3–9]. Following contact, the amoeba readily ingests the target cell (Fig. 1A). However, more recent cinemicroscopic and kinetic studies [8,9] (see also Fig. 1B) indicate that the cytolysis mediated by amoeba may occur prior to phagocytosis, raising the possibility of an extracellular cytolytic event triggered by surface contact. Previous work from this laboratory has described the properties of a highly enriched plasma membrane fraction and surface polypeptides from axenically grown E histolytica [10,11]. Studies from this laboratory on its extensive vacuolar apparatus and its role in pinocytosis and intracellular acidification have also been published [12,13]. Here, we review some recent evidence for the isolation and characterization of a pore-forming protein (PFP) extracted from this protozoan, which may be closely related to the killing of target cells mediated by pore-forming proteins of the immune system (the complement cascade as well as proteins extracted from granules of different immune cells [Young et al, in preparation]), which may thereby suggest a very similar and general mechanism of cell-mediated cytotoxicity.

Several studies have suggested that the granulated metrial gland (GMG) cells of the metrial gland (MG) may be natural killer (NK)-like cells. The cytotoxicity of NK cells involves secretion of a pore-forming protein termed perforin, which can polymerize on the target cell membrane to form transmembrane pores that are thought to be involved in target cell death. In the present study, we used an antiserum against perforin to determine whether this protein can be detected immunohistochemically in GMG cells. Mouse uteri were fixed by vascular perfusion with several fixatives on Day 14 of pregnancy, and tissue sections were labeled by an indirect immunofluorescence method. Specific perforin labeling was detected in GMG cells throughout the MG, in the decidua basalis, and in the labyrinthine placenta. The presence of perforin in GMG cells supports the suggestion that they may be NK-like cells.

A separation procedure based on Percoll gradient centrifugation is described here that allows enrichment for two main populations of granules, with average densities of 1.05 and 1.1 g/ml. The heavy-density granules, which comprise 5–20% of the total amount of membrane-lytic activity, are morphologically and biochemically more homogeneous than the light-density granules, which are contaminated with other subcellular organelles. A trypsin-like serine esterase activity, used in the past as a granule marker, was mainly found in the free cytosol and in the light-density fractions, while a small portion of this activity was associated with the heavy-density fractions. This enzyme marker is therefore not exclusively associated with granules and should not be used as the only granule marker.

Histocompatibility-restricted cytotoxic T lymphocytes produce circular lesions on target cell membranes. The pore-forming protein (PFP or perforin 1) that forms these membrane lesions has been purified from lymphocytes. At 37 degrees C, in the presence of Ca2+, this protein polymerizes into a supramolecular tubular complex of Mr greater than 10(6) that partially resists dissociation by SDS and reducing agents. It incorporates spontaneously into planar lipid bilayers during polymerization to form nonselective ion channels, showing heterogeneous size distribution, the smallest conductance per unit being identified as 400 pS in 0.1 M NaCl. PFP/P1 that had been assembled in lipid vesicles before incorporation into planar bilayer show much larger single channel conductance, ranging from 1 to 6 nS in 0.1 M NaCl, suggesting that PFP/P1 may assume multiple functional sizes in proportion to its state of polymerization. The reconstituted channels are relatively voltage-insensitive, with most channels persisting in the open state for seconds to minutes. Nucleated cells are rapidly depolarized by this protein. The purified protein lyses a variety of tumor cells. Polymerization and functional channel activity are absolutely Ca2+-dependent. The activity of this protein may play a direct role in T lymphocyte-mediated cytolysis.

The proteins of the outer membrane from rat liver mitochondria have been subfractionated by means of density gradient centrifugation. The different polypeptides of the membrane were incorporated into asolectin vesicles and black lipid membranes. It was observed that a polypeptide of Mr 32 000 renders asolectin vesicles permeable to ADP and forms pores in bilayer membrane. These pores showed the same properties as the channels which are formed in the lipid membrane after addition of Triton X-100 solubilized complete outer membrane. The properties of the pore are as follows: (1) The formation of pores depends on the type of phospholipid used for the preparation of the black membranes. (2) The pore is inserted asymmetrically into the membrane. (3) The pore is voltage gated but does not switch off completely at higher voltages. The pore seems to show different conductance states decreasing conductance being observed at increasing voltage. The implications of these findings for the regulation of transport processes across the outer membrane are discussed.

BACKGROUND: The permeabilization of cells with bacterial pore-forming proteins is an important technique in cell biology that allows the exchange of small reagents into the cytoplasm of a cell. Another notable technology is the use of caged molecules whose activities are blocked by addition of photoremovable protecting groups. This allows the photogeneration of reagents on or in cells with spatial and temporal control. Here, we combine these approaches to produce a caged pore-forming protein for the controlled permeabilization of cells. RESULTS: 2-Bromo-2-(2-nitrophenyl)acetic acid (BNPA), a water-soluble cysteine-directed reagent for caging peptides and proteins with the alpha-carboxy-2-nitrobenzyl (CNB) protecting group, was synthesized. Glutathione (gamma-Glu-Cys-Gly) was released in high yield from gamma-Glu-CysCNB-Gly by irradiation at 300 nm. Based on this finding, scanning mutagenesis was used to find a single-cysteine mutant of the pore-forming protein staphylococcal alpha-hemolysin (alpha HL) suitable for caging. When alpha HL-R104C was derivatized with BNPA, pore-forming activity toward rabbit erythrocytes was lost. Near UV irradiation led to regeneration of the cysteine sulfhydryl group and the restoration of pore-forming activity. CONCLUSIONS: Caged pore-forming proteins are potentially useful for permeabilizing one cell in a collection of cells or one region of the plasma membrane of a single cell. Therefore, alpha HL-R104C-CNB and other caged proteins designed to create pores of various diameters should be useful for many purposes. For example, the ability to introduce reagents into one cell of a network or into one region of a single cell could be used in studies of neuronal modulation. Further, BNPA should be generally useful for caging cysteine-containing peptides and single-cysteine mutant proteins to study, for example, cell signaling or structural changes in proteins.

Structural changes in staphylococcal alpha-hemolysin (alpha HL) that occur during oligomerization and pore formation on membranes have been examined by using a simple gel-shift assay to determine the rate of modification of key single-cysteine mutants with the hydrophilic sulfhydryl reagent, 4-acetamido-4'-((iodoacetyl)amino)stilbene-2,2'-disulfonate (IASD). The central glycine-rich loop of alpha HL lines the lumen of the transmembrane channel. A residue in the loop remains accessible to IASD after assembly, in keeping with the ability of the pore to pass molecules of approximately 1000 Da. By contrast, residues near the N-terminus, which are critical for pore function, become deeply buried during oligomerization, while a residue at the extreme C-terminus increases in reactivity after assembly, consistent with a location in the part of the pore that projects from the surface of the lipid bilayer.

Homogeneous maltoporin (lamB protein), an Escherichia coli outer membrane spanning protein, was incorporated in phospholipid planar bilayers. It generates aqueous channels distinct from those formed by the non-specific porin (OmpF) or by phosphoporin (phoE protein). The single conductance, 150 pS in 1 M NACl, is much smaller than that of the porins. The channels, which are poorly selective for cations and voltage independent, are specifically inhibited by maltose and maltodextrins. This inhibition, observed in the absence of maltose binding protein, demonstrates that the selectivity of maltoporin for maltose and maltodextrins is an intrinsic property of the protein.

The transmembrane protein TatA is the pore forming unit of the twin-arginine translocase (Tat), which has the unique ability of transporting folded proteins across the cell membrane. This ATP-independent protein export pathway is a recently discovered alternative to the general secretory (Sec) system of bacteria. To obtain insight in the translocation mechanism, the structure and alignment in the membrane of the well-folded segments 2–45 of TatAd from Bacillus subtilis was studied here. Using solid-state NMR in bicelles containing anionic lipids, the topology and orientation of TatAd was determined in an environment mimicking the bacterial membrane. A wheel-like pattern, characteristic for a tilted transmembrane helix, was observed in 15N chemical shift /15N–1H dipolar coupling correlation NMR spectra. Analysis of this PISA wheel revealed a 14–16 residue long N-terminal membrane-spanning helix which is tilted by 17° with respect to the membrane normal. In addition, comparison of uniformly and selectively 15N-labeled TatA2–45 samples allowed determination of the helix polarity angle.

The stalks (hyphae) of a prosthecate bacterium, directly sampled from the water surface of a hot pond, show extended regular patterns on their envelope in the electron microscope. Image processing revealed a structure of the crystalline complexes which is very similar to the gross morphology of the Escherichia coli porins OmpC and OmpF. The natural two-dimensional crystal of the outer membrane protein has p3 symmetry and a lattice constant of 7.95 nm. The three-dimensional structure of the stalk porin has been determined to an almost isotropic resolution of 1.7 nm. The reconstruction revealed a complex network of channels within the membrane matrix with a triplet of pores merging into a common outlet, similar to the structure of the E. coli porin OmpF in reconstituted membranes. In addition, a blindly ending pore exists which appears to be connected to the continuous pores via small channels. The significance of the regularly arrayed porin cylinders with respect to the shape and function of the stalks is discussed.

The great majority of trimeric porins of Gram-negative bacteria cannot be dissociated into monomers without disrupting their folded conformation. The porin of Campylobacter jejuni, however, displays two folded structures, a classical oligomer and a monomer resistant to detergent denaturation. We probed the transition of trimer to monomer using light scattering experiments and examined the secondary structures of these two molecular states by infra-red spectroscopy. The channel-forming properties of both trimer and monomer were studied after incorporation into artificial lipid bilayers. In these conditions, the trimer induced ion channels with a conductance value of 1200 pS in 1 M NaCl. The pores showed marked cationic selectivity and sensitivity to low voltage. Analysis of the isolated monomer showed nearly the same single-channel conductance and the same selectivity and sensitivity to voltage. These results indicate that the folded monomer form of C. jejuni MOMP displays essentially the same pore-forming properties as the native trimer.

A cytolytic protein (perforin) was rapidly purified from a cell line of mouse cytotoxic T-lymphocytes (CTL) by DEAE-cellulose, heparin-Sepharose, and phenyl-Sepharose chromatographies. The purified perforin was activated by heparin, the half maximal concentration being 3-10 ng/ml, depending on the calcium concentration. Other acid mucopolysaccharides, such as chondroitin sulfates A and C, keratan polysulfate, and heparin sulfate, also enhanced the lysis of erythrocytes by perforin, but the concentrations required for activation were more than 100-fold higher than that of heparin. Chondroitin, hyaluronic acid, and keratan sulfate, however, had no effect on the perforin activity. It was suggested that heparin potentiates the lytic activity of perforin and acid mucopolysaccharides may actually be involved in target cell lysis by CTL.

The cytolytic pore-forming protein (PFP, perforin) of lymphocyte granules has recently been isolated and characterized. The lytic activities expressed by both the isolated granules and the purified PFP require the presence of Ca2+. Here, we report on the extracellular release of PFP after stimulation of lymphocytes with the Ca2+ ionophore A23187, which degranulates the cells. The secreted protein associates with lipid to form structural and functional channels and supramolecular complexes that partially resist dissociation by sodium dodecyl sulfate and reducing agents. Immunoblots of the released material reveal positive identification with antibodies specific for mouse PFP and human complement component C9, indicating cross-reactivity between these two molecules. By using these specific antibodies as immunoadsorbents, the lymphocyte PFP has been affinity purified from the supernatant of stimulated cells. The extracellular release of PFP is associated with simultaneous formation of functional ion-nonselective channels with conductances of 550-600 pS in 0.15 M NaCl, as measured in planar model bilayers. In the absence of extracellular Ca2+, 15% of the maximal release activity is observed. Ca2+ appears to be required to elicit both secretion by lymphocytes and the assembly of the released PFP into tubular polymers. Similar secretion of PFP may occur during cell killing by lymphocytes, resulting in its assembly on target membranes to form tubular transmembrane lesions.

One of the major outer membrane proteins of yeast mitochondria was isolated and purified. It migrated as a single band with an apparent molecular weight of 30 kDa on a SDS-electrophoretogram. When reconstituted in lipid bilayer membranes the protein formed pores with a single channel conductance of 0.45 nS in 0.1 M KCl. The pores had the characteristics of general diffusion pores with an estimated diameter of 1.7 nm. The pore of mitochondrial outer membranes of yeast shared some similarities with the pores formed by mitochondrial and bacterial porins. The pores switched to substates at voltages higher than 20 mV. The possible role of this voltage-dependence in the metabolism of mitochondria is discussed.

Summary Purified murine lymphocyte pore-forming protein (PFP or perforin) was partially sequenced. Oligonucleotides synthesized on the basis of this sequence information were used to screen a murine cytotoxic T lymphocyte (CTL) cDNA library. Seven clones were obtained, two of which were sequenced, providing full-length sequence information on PFP. Murine PFP (534 a.a.) is 68% identical to human PFP. Hydropathic analysis revealed a predominantly hydrophilic protein with some hydrophobic domains, including a region (a.a. 191–251) that could contain putative membrane-spanning domains. PFP is approx. 20% identical to human C7, C8 and C9 within a region encompassing 270 a.a., confirming previous immunological cross-reactivity studies. Northern blot analysis showed that expression of PFP but not of a serine esterase transcript is enhanced in a CTL line by antigen receptor-stimulation. Southern blot analysis of mouse genomic DNA indicated that PFP is encoded as a single-copy gene with the coding region contained within 10 kilobases of genomic DNA.

The ninth component of complement (C9) and the pore-forming protein (PFP or perforin) from cytotoxic T lymphocytes polymerize to tubular lesions having an internal diameter of 100 A and 160 A, respectively, when bound to lipid bilayers. Polymerized C9, assembled by slow spontaneous or rapid Zn2+-induced polymerization, and polyperforin, which is assembled only in the presence of Ca2+, constitute large aqueous pores that are stable, nonselective for solutes, and insensitive to changes of membrane potential. Monospecific polyclonal antibodies to purified C9 and PFP show cross-reactivity, suggesting structural homology between the two molecules. The structural and functional homologies between these two killer molecules imply an active role for pore formation during cell lysis.

A pore-forming protein (PFP, perforin or cytolysin) has been found in the cytoplasmic granules of cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. Extraction of granules with high-salt buffers or by freezing-and-thawing results in the release of perforin, which occurs only when the buffer pH is above 7.0. While high-salt extraction and freezing-and-thawing of granules at low pH (below 7.0) do not result in perforin release, these treatments render granules susceptible to a subsequent incubation with low-salt buffers (pH 7–8) that then solubilizes perforin completely. Granules may thus have been made leaky by high-salt extraction or freezing-and-thawing that may occur regardless of the buffer pH, while dissociation of perforin from granules may be exquisitely pH-sensitive. Freezing-and-thawing intact CTL and NK cells in physiological buffers with pH in the range of 7–8 (but not below 7) also causes release of perforin activity to the cell supernatant, thus providing a simple procedure by which perforin activity can be quantitated in small cell samples. Our results suggest that during lymphocyte-mediated killing, the extracellularly released perforin may rapidly dissociate from granules as a result of pH change and, in the process, become cytolytically active.

We describe the partial purification and characterization of a pore-forming material (PEM) from Entamoeba histolytica. The formation of ion channels by PFM was examined in three systems. (a) PFM depolarizes J774 macrophages and mouse spleen lymphocytes as measured by [3H]TPP+ uptake. (b) PFM induces rapid monovalent cation flux across the membrane of phosphatidylcholine-cholesterol vesicles. (c) PFM confers a voltage-dependent conductance to artificial planar bilayers, which is resolved as a summation of opening of individually conducting steps of 67 pS in 0.1 M KCl. Monomers of PFM are functional; however, a preferential aggregation occurs in the planar bilayer. Activity is pronase, trypsin, and heat sensitive and is stable between pH 5-8. PFM is not secreted by unstimulated amoebae but after exposure to the calcium ionophore A23187, concanavalin A, and E. coli lipopolysaccharide, 5-10% of the total cell content of PFM is released into the medium within 5-10 min. High-performance gel filtration results in an approximately 1,000-fold purification of PFM and gives an Mr of 30,000. This protein may play a role in the cytotoxicity mediated by E. histolytica.

In rapidly growing, highly glycolytic hepatoma cells as much as 65% of the total cell hexokinase is bound to the outer mitochondrial membrane [Parry, D.M., & Pedersen, P.L. (1983) J. Biol. Chem. 258, 10904-10912]. In this paper, we describe the purification to apparent homogeneity of a mitochondrial pore-forming protein from the highly glycolytic AS-30D rat hepatoma cell line. The purified protein shows a single 35 000-dalton band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, an amino acid composition slightly more hydrophobic than that of the rat liver pore protein (also known as VDAC or mitochondrial porin), and a channel-forming activity of 136 channels min-1 (microgram of protein)-1. In addition to displaying the properties characteristic of VDAC (single-channel conductance, voltage dependence, and preference for anions), we observe that the AS-30D VDAC protein is one of only three mitochondrial proteins that bind [14C]dicyclohexylcarbodiimide (DCCD) at relatively low dosages (2 nmol of DCCD/mg of mitochondrial protein). Significantly, treatment of intact mitochondria isolated from either rat liver or the AS-30D hepatoma with DCCD results in an almost complete inhibition of their ability to binding hexokinase. Fifty percent inhibition of binding occurs at less than 2 nmol of DCCD/mg of mitochondrial protein. In contrast to DCCD, water-soluble carbodiimides are without effect on hexokinase binding. These results suggest that the pore-forming protein of tumor mitochondria forms at least part of the hexokinase receptor complex. In addition, they indicate that a carboxyl residue located within a hydrophobic region of the receptor complex may play a critical role in hexokinase binding.

Pore-forming protein (perforin, PFP) was isolated from a mouse large granular lymphocyte (LGL) [natural killer (NK-like)] cell line. Purified PFP lysed a variety of mouse tumor cell lines and helper T lymphocyte cell lines. However, LGL and cytotoxic T lymphocyte cell lines were resistant to PFP-mediated cell lysis. The presence of hemolytic activity in the granule was examined in these resistant cell lines. Four out of five of these resistant cell lines had hemolytically active granules. We determined whether NK cells freshly isolated from BALB/c nude mouse spleens were resistant to PFP-mediated cytolysis. Nylon column-passed spleen cells with an enriched content of NK cells exhibited more resistance than whole spleen cells. Moreover, when spleen cells were treated with PFP the remaining live cells showed enriched NK activity suggesting that normal peripheral cells with NK activity are resistant to PFP. These results indicate that cytolytic cells containing PFP have developed defense mechanisms to inhibit PEP-mediated cell lysis.

Lyme disease Borreliae are highly dependent on the uptake of nutrients provided by their hosts. Our study describes the identification of a 36 kDa protein that functions as putative dicarboxylate-specific porin in the outer membrane of Lyme disease Borrelia. The protein was purified by hydroxyapatite chromatography from Borrelia burgdorferi B31 and designated as DipA, for dicarboxylate-specific porin A. DipA was partially sequenced, and corresponding genes were identified in the genomes of B. burgdorferi B31, Borrelia garinii PBi and Borrelia afzelii PKo. DipA exhibits high homology to the Oms38 porins of relapsing fever Borreliae. B. burgdorferi DipA was characterized using the black lipid bilayer assay. The protein has a single-channel conductance of 50 pS in 1 M KCl, is slightly selective for anions with a permeability ratio for cations over anions of 0.57 in KCl and is not voltage-dependent. The channel could be partly blocked by different di- and tricarboxylic anions. Particular high stability constants up to about 28,000 l/mol (in 0.1 M KCl) were obtained among the 11 tested anions for oxaloacetate, 2-oxoglutarate and citrate. The results imply that DipA forms a porin specific for dicarboxylates which may play an important role for the uptake of specific nutrients in different Borrelia species.

The membrane attack complex (MAC) is an important innate immune effector of the complement terminal pathway that forms cytotoxic pores on the surface of microbes. Despite many years of research, MAC structure and mechanism of action have remained elusive, relying heavily on modelling and inference from biochemical experiments. Recent advances in structural biology, specifically cryo-electron microscopy, have provided new insights into the molecular mechanism of MAC assembly. Its unique ‘split-washer’ shape, coupled with an irregular giant β-barrel architecture, enable an atypical mechanism of hole punching and represent a novel system for which to study pore formation. This review will introduce the complement terminal pathway that leads to formation of the MAC. Moreover, it will discuss how structures of the pore and component proteins underpin a mechanism for MAC function, modulation and inhibition. This article is part of the themed issue ‘Membrane pores: from structure and assembly, to medicine and technology’.

Malaria remains one of the most serious health problems in developing countries. The causative agent of malaria, Plasmodium spp., have a complex life cycle involving multiple developmental stages as well as different morphological, biochemical and metabolic requirements. We recently found that γδ T cells control parasite growth using pore-forming proteins to deliver their cytotoxic proteases, the granzymes, into blood residing parasites. Here, we follow up on the molecular mechanisms of parasite growth inhibition by human pore-forming proteins. We confirm that Plasmodium falciparum infection efficiently depletes the red blood cells of cholesterol, which renders the parasite surrounding membranes susceptible to lysis by prokaryotic membrane disrupting proteins, such as lymphocytic granulysin or the human cathelicidin LL-37. Interestingly, not the cholesterol depletion but rather the simultaneous exposure of phosphatidylserine, a negatively charged phospholipid, triggers resistance of late stage parasitized red blood cells towards the eukaryotic pore forming protein perforin. Overall, by revealing the molecular events we establish here a pathogen-host interaction that involves host cell membrane remodeling that defines the susceptibility towards cytolytic molecules.

Sticholysin II (Stn II) is a cytolytic protein produced by the sea anemone Stichodactyla helianthus, its effect being related to pore formation. The conformation of the protein and its temperature-induced transitions, in the 1.5-12.0 pH range and in the 0-0.5 M NaCl concentration interval, have been studied by circular dichroism and fluorescence spectroscopy. At temperature35 degrees C, the protein maintains the same, high beta-structure content, folded conformation in the 1.5-11.0 pH range and ionic strength up to 0.5 M. In the 1.5-3.5 pH range and ionic strengthor = 0.1 M, Stn II shows a thermal transition, resulting in a partially folded state characterized by: (i) a native-like content of regular secondary structure, as detected by far-UV CD; (ii) a largely disordered tertiary structure, as detected by near-UV CD, with partially exposed tryptophan residues according to their fluorescence emission; and (iii) ability to bind the hydrophobic probe 2-anilinonaphthalene-6-sulfonic acid. In the pH range 4.0-10.5, thermally-induced protein aggregation occurs. The obtained results demonstrate the existence of partially folded state of Stn II, which may contribute to the pore formation ability of this cytolysin.