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• W2087514908 abstract "Intermedilysin (ILY) is an unusual member of the family of cholesterol-dependent cytolysins because it binds to human CD59 (hCD59) rather than directly to cholesterol-rich membranes. Binding of ILY to hCD59 initiates a series of conformational changes within the toxin that result in the conversion of the soluble monomer into an oligomeric membrane-embedded pore complex. In this study the association of ILY with its membrane receptor has been examined throughout the assembly and formation of the pore complex. Using ILY mutants trapped at various stages of pore assembly, we show ILY remains engaged with hCD59 throughout the assembly of the prepore oligomer, but it disengages from the receptor upon the conversion to the pore complex. We further show that the assembly intermediates increase the sensitivity of the host cell to lysis by its complement membrane attack complex, apparently by blocking the hCD59-binding site for complement proteins C8α and C9. Intermedilysin (ILY) is an unusual member of the family of cholesterol-dependent cytolysins because it binds to human CD59 (hCD59) rather than directly to cholesterol-rich membranes. Binding of ILY to hCD59 initiates a series of conformational changes within the toxin that result in the conversion of the soluble monomer into an oligomeric membrane-embedded pore complex. In this study the association of ILY with its membrane receptor has been examined throughout the assembly and formation of the pore complex. Using ILY mutants trapped at various stages of pore assembly, we show ILY remains engaged with hCD59 throughout the assembly of the prepore oligomer, but it disengages from the receptor upon the conversion to the pore complex. We further show that the assembly intermediates increase the sensitivity of the host cell to lysis by its complement membrane attack complex, apparently by blocking the hCD59-binding site for complement proteins C8α and C9. The cholesterol-dependent cytolysins (CDC) 2The abbreviations used are: CDC, cholesterol-dependent cytolysin; ILY, intermedilysin; MAC, membrane attack complex; hCD59, human CD59; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; HA, hemagglutinin; FRET, Förster resonance energy transfer; PFO, perfringolysin O; TRITC, tetramethylrhodamine isothiocyanate; mAb, monoclonal antibody; co-IP, co-immunoprecipitated; AGE, agarose gel electrophoresis. are a family of structurally related pore-forming toxins that are important virulence factors for a variety of Gram-positive pathogens (1Heuck A.P. Tweten R.K. Johnson A.E. Biochemistry. 2001; 40: 9065-9073Crossref PubMed Scopus (128) Google Scholar, 2Portnoy D. Jacks P.S. Hinrichs D. J. Exp. Med. 1988; 167: 1459-1471Crossref PubMed Scopus (655) Google Scholar, 3Bricker A.L. Cywes C. Ashbaugh C.D. Wessels M.R. Mol. Microbiol. 2002; 44: 257-269Crossref PubMed Scopus (102) Google Scholar, 4Boulnois G.J. J. Gen. Microbiol. 1992; 138: 249-259Crossref PubMed Scopus (86) Google Scholar). The CDCs are secreted by the bacterium as soluble monomers and then bind to cholesterol-rich eukaryotic cell membranes (5Waheed A.A. Shimada Y. Heijnen H.F. Nakamura M. Inomata M. Hayashi M. Iwashita S. Slot J.W. Ohno-Iwashita Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4926-4931Crossref PubMed Scopus (200) Google Scholar). Once bound, the monomers laterally diffuse and interact with one another to form a large oligomeric prepore structure comprised of 35–40 CDC monomers. One of the hallmarks of this family of toxins is the absolute requirement of their pore-forming mechanism on membrane cholesterol (1Heuck A.P. Tweten R.K. Johnson A.E. Biochemistry. 2001; 40: 9065-9073Crossref PubMed Scopus (128) Google Scholar). Membrane cholesterol serves to target the CDCs to the eukaryotic cell membrane and is necessary to convert the prepore oligomer to the inserted pore complex (6Soltani C.E. Hotze E.M. Johnson A.E. Tweten R.K. J. Biol. Chem. 2007; 282: 15709-15716Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Two classes of CDCs currently exist. The first class is typified by perfringolysin O (PFO) from Clostridium perfringens that appears to bind directly to cholesterol-rich membranes, an interaction mediated by three short loops in domain 4 (7Soltani C.E. Hotze E.M. Johnson A.E. Tweten R.K. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 20226-20231Crossref PubMed Scopus (111) Google Scholar). The second group includes intermedilysin (ILY) from Streptococcus intermedius and vaginolysin from Gardnerella vaginalis (8Gelber S.E. Aguilar J.L. Lewis K.L. Ratner A.J. J. Bacteriol. 2008; 190: 3896-3903Crossref PubMed Scopus (182) Google Scholar). These CDCs bind to the glycosylphosphatidylinositol-anchored protein human CD59 (hCD59). It has been shown for ILY that it first binds hCD59 and then inserts its domain 4 loops in a cholesterol-dependent fashion (7Soltani C.E. Hotze E.M. Johnson A.E. Tweten R.K. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 20226-20231Crossref PubMed Scopus (111) Google Scholar). Why the latter two CDCs have evolved to specifically bind hCD59 and whether they remain engaged with this receptor throughout the assembly of the pore complex remains unclear. S. intermedius is a pathogen frequently associated with abscesses of the oral cavity as well as with life-threatening abscesses of the head, neck, and liver (9Nagamune H. Whiley R.A. Goto T. Inai Y. Maeda T. Hardie J.M. Kourai H. J. Clin. Microbiol. 2000; 38: 220-226PubMed Google Scholar, 10Nagamune H. Ohnishi C. Katsuura A. Fushitani K. Whiley R.A. Tsuji A. Matsuda Y. Infect. Immun. 1996; 64: 3093-3100Crossref PubMed Google Scholar). ILY appears to be important in establishing these deep-seated abscesses as S. intermedius isolated from these sites produces levels of ILY 6–10 times greater than strains isolated from peripheral site infections or the oropharynx (9Nagamune H. Whiley R.A. Goto T. Inai Y. Maeda T. Hardie J.M. Kourai H. J. Clin. Microbiol. 2000; 38: 220-226PubMed Google Scholar). ILY binds only human cells, whereas other CDCs, such as PFO, bind to most cholesterol-rich eukaryotic membranes. The species selectivity of ILY is because of its specificity for human hCD59 and appears to be encoded in domain 4 of the toxin (11Giddings K.S. Zhao J. Sims P.J. Tweten R.K. Nat. Struct. Mol. Biol. 2004; 12: 1173-1178Crossref Scopus (190) Google Scholar, 12Giddings K.S. Johnson A.E. Tweten R.K. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 11315-11320Crossref PubMed Scopus (157) Google Scholar). CD59 is an 18–20-kDa surface-expressed glycoprotein tethered to the cell membrane via a glycosylphosphatidylinositol anchor. It is widely distributed on most human and nonhuman cell types. It is associated with a number of important cellular functions that include serving as an adaptor molecule for a candidate C1q receptor (C1qRO–2) (13Otabor I. Tyagi S. Beurskens F.J. Ghiran I. Schwab P. Nicholson-Weller A. Klickstein L.B. Mol. Immunol. 2004; 41: 185-190Crossref PubMed Scopus (14) Google Scholar, 14Ghiran I. Klickstein L.B. Nicholson-Weller A. J. Biol. Chem. 2003; 278: 21024-21031Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) and acting as a cell-signaling molecule (15Kimberley F.C. Sivasankar B. Morgan B.P. Mol. Immunol. 2007; 44: 73-81Crossref PubMed Scopus (113) Google Scholar). Its primary role, however, is regulating the terminal pathway of complement by inhibiting the formation of the membrane attack complex (MAC) on host cells by binding to C8α and C9, thus preventing the formation of the MAC pore (16Rollins S.A. Sims P.J. J. Immunol. 1990; 144: 3478-3483PubMed Google Scholar, 17Hamilton K.K. Ji Z. Rollins S. Stewart B.H. Sims P.J. Blood. 1990; 76: 2572-2577Crossref PubMed Google Scholar, 18Farkas I. Baranyi L. Ishikawa Y. Okada N. Bohata C. Budai D. Fukuda A. Imai M. Okada H. J. Physiol. (Lond.). 2002; 539: 537-545Crossref Scopus (92) Google Scholar). In various autoimmune diseases and inflammatory conditions, excessive complement activation can saturate the available CD59 resulting in MAC-mediated host cell injury (19Huang Y. Smith C.A. Song H. Morgan B.P. Abagyan R. Tomlinson S. J. Biol. Chem. 2005; 280: 34073-34079Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). CD59 exhibits species selectivity such that it most effectively inhibits only the homologous MAC (20Rollins S.A. Zhao J. Ninomiya H. Sims P.J. J. Immunol. 1991; 146: 2345-2351PubMed Google Scholar). ILY recognition of the same or similar structural differences in CD59 is the basis for its species selective activity (11Giddings K.S. Zhao J. Sims P.J. Tweten R.K. Nat. Struct. Mol. Biol. 2004; 12: 1173-1178Crossref Scopus (190) Google Scholar). ILY binding to hCD59 triggers a series of conformational changes in ILY leading to its membrane oligomerization into the prepore complex (6Soltani C.E. Hotze E.M. Johnson A.E. Tweten R.K. J. Biol. Chem. 2007; 282: 15709-15716Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). This is accompanied by the cholesterol-dependent insertion of three loops at the base of domain 4 and the insertion of the undecapeptide, events that are necessary for the conversion of the prepore to a pore complex (7Soltani C.E. Hotze E.M. Johnson A.E. Tweten R.K. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 20226-20231Crossref PubMed Scopus (111) Google Scholar). It is not known, however, whether ILY remains engaged with hCD59 throughout its assembly into the pore complex. Whether ILY remains engaged during and after the assembly of the pore complex may also impact the ability of the eukaryotic cell to protect itself from the host MAC because a previous study suggested the ILY-binding site on hCD59 overlaps that for complement proteins C8α and C9 (11Giddings K.S. Zhao J. Sims P.J. Tweten R.K. Nat. Struct. Mol. Biol. 2004; 12: 1173-1178Crossref Scopus (190) Google Scholar). To address these questions, we investigated the interaction of ILY with hCD59 during the assembly of the ILY pore complex. We further determined whether nonlytic assembly intermediates of ILY increase MAC-mediated damage to host cells by short circuiting the protective function of hCD59. These studies show ILY remains engaged during the assembly of its prepore complex and disengages from its receptor upon pore formation. In addition, we show that engagement of hCD59 by ILY prior to pore formation significantly increases the host cell sensitivity to the host MAC-mediated lysis. Bacterial Strains, Plasmids, and Chemicals—The gene for ILY was cloned into pTrcHisA (Invitrogen) as described previously (6Soltani C.E. Hotze E.M. Johnson A.E. Tweten R.K. J. Biol. Chem. 2007; 282: 15709-15716Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). ILYH242C (cysteine-containing derivative) is considered wild type toxin except where noted. All mutations were made in the native ILY background unless otherwise stated. All chemicals and enzymes were obtained from Sigma, VWR, and Research Organics except where noted. All fluorescent probes were obtained from Molecular Probes (Invitrogen). Anti-hCD59 H19-FITC conjugated and unconjugated was obtained from Pharmingen. Anti-hCD59 MEM43 and FITC-conjugated MEM43 were obtained from AbCAM. Anti-hCD59 10G10 expressing mouse B cell myeloma was a generous gift from Dr. Marilyn Telen (Duke University Medical Center). Purification of 10G10 antibody from tissue culture supernatants was performed using Affi-Gel Protein-A MAPS II antibody purification kit (Bio-Rad) as the manufacturer specified. Goat anti-mouse FITC-conjugated secondary antibody was obtained from AbCAM. All other secondary antibodies were obtained from Bio-Rad. Cells and Transfection—Chinese hamster ovary cells (CHO) transfected with a human CD59 (CHOhCD59)-expressing plasmid were a generous gift of Dr. Stephen Tomlinson (New York University Medical Center, New York) (19Huang Y. Smith C.A. Song H. Morgan B.P. Abagyan R. Tomlinson S. J. Biol. Chem. 2005; 280: 34073-34079Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The cells were maintained in F-12 Kaighn's medium (10% v/v fetal calf serum, 1% v/v penicillin/streptomycin) (Invitrogen). Two rounds of sorting by FLOW were used to obtain cell populations uniformly expressing high levels of hCD59. For sorting, CHO cells were detached with 5 mm EDTA in phosphate-buffered saline (PBS), washed once with PBS, and resuspended in F-12, 10% fetal calf serum. Eight rounds of QuikChange mutagenesis were performed on the hCD59 plasmid in order to replace the first nine amino acids of the malaria epitope tag (NANPNANPNALG located between residues 2 and 3 of the mature human CD59) with the HA epitope from influenza (YPYDVPDYA). CHO cells were then transfected at a 3:2 ratio using the FuGENE 6 reagent (Roche Applied Science) as per the manufacturer's instructions. Positive clones expressing the HA hCD59 (CHOHAhCD59) were selected using anti-HA-FITC antibody (Sigma) by cell sorting (Influx cell sorter, OUHSC Flow Cytometry Core Facility). CHOHAhCD59 Membrane Preparation—CHOHAhCD59 cells (10 × 106 cells) were resuspended in 1 ml of membrane buffer (1× PBS containing 1 complete protease inhibitor mixture (Roche Applied Science) and 0.5 mg/ml DNase (Roche Applied Science)). The cells were homogenized in a Dounce homogenizer for 20 strokes, and the mixture was then subjected to one freeze/thaw cycle with liquid nitrogen. This process was repeated a total of three times. The membranes were then centrifuged 13,000 × g for 15 min at 4 °C, and the pellet was resuspended in 100 μl of 1× PBS. Co-immunoprecipitation of ILY with Anti-HA Antibodies—CHOHAhCD59 membranes (2.5 μl) were incubated with ILYml, ILYpp, or ILYwt (171 nm) in a total volume of 100 μl of 1× PBS for 1 h at 37 °C. The samples were centrifuged 13,000 × g for 15 min to remove excess ILY. The pellet was then resuspended in 100 μl of CelLytic M Cell Lysis buffer (Sigma anti-HA immunoprecipitation kit) supplemented with 10 mm n-octyl β-d-glucopyranoside (Sigma) and allowed to incubate on ice for 10 min to solubilize the membranes. The samples were immunoprecipitated via the anti-HA immunoprecipitation kit according to the manufacturer's protocol (Sigma). Eluted samples were subjected to a 4–20% SDS-PAGE and then transferred to nitrocellulose for Western blotting analysis. Rabbit anti-ILY antibodies and mouse anti-CD59 (H19 from Pharmingen) were used for Western analysis. Antibodies and Serum—Rabbit antisera to CHO cell membranes were a kind gift from Dr. S. Tomlinson. Normal human serum was obtained from the blood of healthy volunteers. The serum was then incubated with 5 ml of Affi-gel protein A-agarose (Bio-Rad) as per manufacturer's instructions todeplete the serum of antibodies. The depleted serum was then quick frozen with liquid nitrogen and stored at –80 °C. Generation and Purification of ILY and Its Derivatives—The generation of amino acid substitutions in the gene for ILY was performed via PCR QuikChange mutagenesis (Stratagene). The Oklahoma Medical Research Foundation Core DNA sequencing facility performed DNA sequence analysis of each mutant toxin gene. The expression and purification of recombinant ILY and its derivatives from Escherichia coli were carried out as described previously (6Soltani C.E. Hotze E.M. Johnson A.E. Tweten R.K. J. Biol. Chem. 2007; 282: 15709-15716Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 21Shepard L.A. Heuck A.P. Hamman B.D. Rossjohn J. Parker M.W. Ryan K.R. Johnson A.E. Tweten R.K. Biochemistry. 1998; 37: 14563-14574Crossref PubMed Scopus (271) Google Scholar). Hemolytic Activity—The hemolytic activity of each ILY mutant on human erythrocytes was determined as described previously (21Shepard L.A. Heuck A.P. Hamman B.D. Rossjohn J. Parker M.W. Ryan K.R. Johnson A.E. Tweten R.K. Biochemistry. 1998; 37: 14563-14574Crossref PubMed Scopus (271) Google Scholar). The HD50 is defined as the concentration of toxin required to lyse 50% of the human erythrocytes under standard assay conditions. SDS-Agarose Gel Electrophoresis (SDS-AGE) and Immunoblot Analyses—SDS-AGE was carried out as described previously (6Soltani C.E. Hotze E.M. Johnson A.E. Tweten R.K. J. Biol. Chem. 2007; 282: 15709-15716Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 21Shepard L.A. Heuck A.P. Hamman B.D. Rossjohn J. Parker M.W. Ryan K.R. Johnson A.E. Tweten R.K. Biochemistry. 1998; 37: 14563-14574Crossref PubMed Scopus (271) Google Scholar, 22Hotze E. Tweten R.K. Ménez A. Perspectives in Molecular Toxicology. John Wiley & Sons Ltd., Chichester, UK2002: 23-37Google Scholar). Briefly, ILY (171 nm) was incubated in the presence or absence of human erythrocytes (1.5 × 106 cells) for 30 min at 37 °C. Samples were solubilized with SDS sample buffer at 37 °C for 2 min, and then the monomeric and oligomeric complexes were resolved on a 1.5% SDS-AGE gel and immunoblotted with rabbit anti-ILY antibody followed by anti-rabbit IgG horseradish peroxidase secondary antibody (Bio-Rad). Reactive species were visualized using a chemiluminescent substrate (ECL Western Blotting Detection Reagents, GE Healthcare) and autoradiography. Receptor Availability Assay—We have previously shown specific anti-hCD59 antibodies block ILY binding to hCD59 (11Giddings K.S. Zhao J. Sims P.J. Tweten R.K. Nat. Struct. Mol. Biol. 2004; 12: 1173-1178Crossref Scopus (190) Google Scholar). Therefore, we used these antibodies to probe the availability of the ILY-binding site on hCD59 at specific stages of its assembly process. Flow cytometry was used to monitor the ability of fluorescent derivatives of these antibodies (or fluorescently labeled secondary antibody to the primary mAb) to bind hCD59. If ILY disengaged from hCD59 at a specific stage of its assembly, then the binding sites for these antibodies would become accessible to the antibody. Therefore, the experimental approach was to use this panel of anti-CD59 monoclonal antibodies to probe the availability of hCD59 on erythrocytes preincubated with wild type ILY (ILYwt), monomer locked ILY (ILYml), or prepore locked ILY (ILYpp). PFO, a CDC that does not bind hCD59, was used as a negative control. Saturating levels of ILY and the ILY derivatives (ILYml (171 nm), ILYpp (85.5 nm), or ILYwt (85.5 nm)) were incubated with washed erythrocytes (1 × 106 cells) in PBS (reaction volume 100 μl) for 30 min at 4 °C. Samples were pelleted (14,000 × g for 5 min), and excess toxin was removed. The pellets were then resuspended in 100 μl of PBS containing 0.1% (w/v) BSA bovine serum albumin and kept on ice for 10 min to allow binding. The erythrocytes were washed twice with PBS and then resuspended in 100 μl of anti-hCD59 antibody (33.3 nm) and incubated on ice for 30 min. Samples were washed twice with PBS and then brought to a final volume of 500 μl of PBS and analyzed by a FACSCalibur flow cytometer (University of Oklahoma Health Sciences Center) and FLOWJO software (Tree Star). The emission wavelength was set to 530 nm, and the excitation was set at 488 nm with a bandpass of 30 nm. Samples assayed with 10G10 anti-hCD59 were resuspended in 100 μl of goat anti-mouse-FITC (33.3 nm) for 15 min on ice and washed twice with PBS. Samples assayed with either H19-FITC or MEM43-FITC anti-hCD59 were analyzed directly. Förster Resonance Energy Transfer (FRET) Analysis—Flow cytometry was used to monitor the interaction between ILY and its receptor during pore formation on the surface of erythrocytes by FRET. FRET between donor (FITC)-labeled anti-hCD59 MEM43 monoclonal antibody (AbCAM) and acceptor (TRITC)-labeled ILY or PFO was determined as described previously (6Soltani C.E. Hotze E.M. Johnson A.E. Tweten R.K. J. Biol. Chem. 2007; 282: 15709-15716Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 12Giddings K.S. Johnson A.E. Tweten R.K. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 11315-11320Crossref PubMed Scopus (157) Google Scholar) with the following modifications. Cell-associated hCD59 was indirectly labeled with a fluorescein-labeled monoclonal antibody that did not interfere with ILY binding or oligomerization. Briefly, human erythrocytes (1 × 106) were preincubated with anti-hCD59 MEM43-FITC-conjugated antibody (66.6 nm) for 1 h on ice in a reaction volume of 100 μl to label hCD59 with the donor fluorophore. Subsequently, either unlabeled or tetramethylrhodamine-labeled PFOpp, ILYpp, or ILYwt (42.7 nm) (acceptors) were added to the samples and incubated an additional 30 min on ice. PBS was added to the samples to a final volume of 500 μl, and each sample was analyzed by FACSCalibur flow cytometer (University of Oklahoma Health Sciences Center) and FLOWJO software (Treestar). Changes in the donor (FITC-labeled hCD59) fluorescence because of FRET with the acceptor (TRITC-labeled ILYpp) were determined by comparing donor emission intensity in the presence of unlabeled ILY to the donor emission intensity in the presence of acceptor-labeled ILY. Acceptor-labeled PFO was used as a negative control. Complement-mediated CHO Cell Lysis Assay—Assays were performed as described previously (19Huang Y. Smith C.A. Song H. Morgan B.P. Abagyan R. Tomlinson S. J. Biol. Chem. 2005; 280: 34073-34079Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Briefly, subconfluent CHO cells expressing human CD59 (CHOhCD59) were detached with 5 mm EDTA in PBS, washed once with PBS, and resuspended in F-12 Kaighn's media supplemented with 10% fetal calf serum (Invitrogen). 200 μl of cells (1 × 105 cells) were incubated in the presence or absence of the indicated amounts of monomer locked ILY (ILYml) or prepore locked ILY (ILYpp) on ice for 15 min. Cells were then sensitized with rabbit anti-CHO membrane serum (5% final concentration) for 15 min on ice. Normal human serum diluted in F-12 Kaighn's medium was then added to a final concentration of 10% (final volume of 400 μl). After 60 min at 37 °C, cell viability was determined by adding propidium iodide (5 μg/ml) and measuring the proportion of propidium iodide-stained (dead) cells to total cells by FACSCalibur flow cytometer (University of Oklahoma Health Sciences Center) and FLOWJO software (Treestar). These experiments were also performed with the monomer and prepore locked variants of PFO. Cells treated with 5 μg of PFO were used as 100% lysis controls. Cells treated without normal human serum were used as background controls. Characterization of Disulfide ILY Mutants Trapped in Monomeric or Prepore Stages—As described above, receptor binding by ILY initiates a series of ordered conformational changes that lead to oligomerization of membrane-bound monomers and the formation of the pore. Mutants of ILY trapped at distinct stages of the cytolytic mechanism were generated to determine whether it remained bound to the receptor during assembly of the pore. We previously reported two nonlytic mutants in the related CDC PFO that lock the toxin in the membrane-bound monomeric and prepore oligomeric stages of the pore-forming mechanism (23Ramachandran R. Tweten R.K. Johnson A.E. Nat. Struct. Mol. Biol. 2004; 11: 697-705Crossref PubMed Scopus (152) Google Scholar, 24Hotze E.M. Wilson-Kubalek E.M. Rossjohn J. Parker M.W. Johnson A.E. Tweten R.K. J. Biol. Chem. 2001; 276: 8261-8268Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Disulfide bridges were engineered into PFO to restrict critical structural transitions necessary for completion of the pore-forming mechanism. Inhibiting these structural changes trapped the toxin in either the membrane-bound monomer or prepore stage. The crystal structures of ILY and PFO are highly homologous so it was likely analogous disulfide bridges could be generated in ILY with similar results (25Rossjohn J. Feil S.C. McKinstry W.J. Tweten R.K. Parker M.W. Cell. 1997; 89: 685-692Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar, 26Polekhina G. Giddings K.S. Tweten R.K. Parker M.W. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 600-605Crossref PubMed Scopus (124) Google Scholar). The appropriate cysteine substitutions were generated in ILY to form the membrane-bound monomer locked mutants (Thr-346 to cysteine and Ile-361 to cysteine) and prepore locked mutants (Gly-83 to cysteine and Ser-217 to cysteine). ILYml and ILYpp were purified and assayed for cytolytic activity on both human erythrocytes and on Chinese hamster ovary cells expressing hCD59 (CHOhCD59). The oxidized forms of ILYml and ILYpp retained less than 0.1% of the cytolytic activity of native ILY on both human erythrocytes and the CHOhCD59 cells (Table 1). The addition of the reducing agent dithiothreitol prior to the assay reduced the disulfide bridge and restored activity to approximately wild type levels. These data show the cytolytic mechanism is reversibly inhibited by the introduction of the disulfide bridges.TABLE 1Hemolytic and cytolytic activity of disulfide-locked mutantsCDCDTTHemolytic activityCytolytic activity%%ILYwt-100100ILYml-<0.1<0.1ILYml+80.4NAILYpp-<0.1<0.1ILYpp+100NA Open table in a new tab SDS-AGE was used to verify the mutants were trapped at the specified stages of the pore-forming mechanism. As expected, in the presence of human erythrocytes the reduced forms of both ILYml (Fig. 1, lane 6) and ILYpp (lane 3) formed SDS-resistant oligomers similar to wild type ILY (lane 1). When the cysteines remained oxidized in a disulfide bridge ILYpp formed SDS-resistant oligomers (Fig. 1, lane 4). These oligomers are slightly less stable than those formed by the prereduced toxin, an observation consistent with what had been reported previously for PFOpp (24Hotze E.M. Wilson-Kubalek E.M. Rossjohn J. Parker M.W. Johnson A.E. Tweten R.K. J. Biol. Chem. 2001; 276: 8261-8268Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). As expected, the oxidized form of ILYml did not form SDS-resistant oligomers in the presence of erythrocytes, but it did form oligomers upon reduction of the disulfide bridge (Fig. 1, compare lanes 7 and 6). The results presented here are similar to those previously reported for the monomer locked PFO mutant (23Ramachandran R. Tweten R.K. Johnson A.E. Nat. Struct. Mol. Biol. 2004; 11: 697-705Crossref PubMed Scopus (152) Google Scholar). Monomer and Prepore Locked Mutants Block Antibody Binding to hCD59—We have previously shown that two anti-hCD59 monoclonal antibodies (10G10 and H19) could block binding of ILY to hCD59 (11Giddings K.S. Zhao J. Sims P.J. Tweten R.K. Nat. Struct. Mol. Biol. 2004; 12: 1173-1178Crossref Scopus (190) Google Scholar) whereas one did not (MEM43). 3R. K. Tweten unpublished data. ILY binding to hCD59 should inhibit the binding of mAbs 10G10 and H19 whether by occupying their binding sites or distorting it such that the mAb could not bind. Therefore, we can determine whether ILY disengages from hCD59 during pore formation by measuring the ability of these three different anti-hCD59 monoclonal antibodies to bind to hCD59 on the surface of erythrocytes preincubated with ILY or its monomer and prepore locked mutants. The anti-hCD59 antibodies were either directly conjugated with a fluorescent dye or were detected with a fluorescently labeled secondary antibody allowing receptor bound by antibody to be detected by flow cytometry. Therefore, when ILY is bound to hCD59 we should observe a significant decrease in the ability of these mAbs to bind to CD59, because their binding site will be occupied by ILY. All three monoclonal antibodies were used to probe the availability of hCD59 on erythrocytes preincubated with ILYml, ILYpp, or native ILY (Fig. 2, shaded peaks). As shown in Fig. 2, the 10G10 (Fig. 2A) and H19 (Fig. 2B) binding is significantly decreased when cells are prebound with saturating levels of either ILYml or ILYpp. Conversely, when cells are incubated with saturating amounts of ILYwt, both 10G10 and H19 bound hCD59 at levels similar to that observed in the absence of ILY (Fig. 2B, solid line) suggesting that hCD59 is no longer significantly engaged by ILY when it is converted to the pore complex. A low level of receptor occupancy by ILYwt was apparent, which suggested that some of the ILY does not disengage from receptor. This can be explained by the fact that the assembly of insertion-competent oligomers is a stochastic process, and so a fraction of ILYwt monomers will be incorporated into complexes that do not achieve an insertion-competent state and therefore will not disengage the receptor. In contrast to 10G10 and H19 mAbs, MEM43 is able to bind to hCD59 in the presence of ILYml (Fig. 2C). This result is in agreement with our previous findings that showed the application of MEM43 prior to ILY does not inhibit cytolytic activity. 4R. K. Tweten and K. S. Giddings, unpublished data. Interestingly, even though MEM43 does not interfere with pore formation, it appears the MEM43 epitope becomes occluded upon formation of the prepore complex (Fig. 2C). Once the prepore is converted to the pore, the site again becomes available, presumably because ILY dissociates from hCD59 upon the formation of the pore complex (Fig. 2C). Hence, these data suggest hCD59 is clustered around the ILY oligomer prior to formation of the pore complex. The decreased binding of 10G10 and H19 to the monomer- and prepore-engaged hCD59 could also result from conformational changes induced in the mAb epitopes by ILY binding. If true, it does not substantially change our interpretation because binding of ILY to CD59 would be responsible for deformation of the epitopes, and their structure is restored upon conversion of the prepore to the pore complex, suggesting that ILY disengages from the CD59. We replicated these studies with PFO, a CDC that does not bind to hCD59. As expected the prepore locked variant of PFO did not inhibit binding of the monoclonal antibodies to" @default.
• W2087514908 created "2016-06-24" @default.
• W2087514908 creator A5006542938 @default.
• W2087514908 creator A5009966637 @default.
• W2087514908 creator A5044088516 @default.
• W2087514908 date "2009-05-01" @default.
• W2087514908 modified "2023-10-01" @default.
• W2087514908 title "Intermedilysin-Receptor Interactions during Assembly of the Pore Complex" @default.
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