Ganglioside identification on human monocyte membrane with Clostridium perfringens delta-toxin.

Clostridium perfringens delta-toxin was first described as a hemolysin with a restricted lytic spectrum. A selective cytotoxicity of the delta-toxin was then found on rabbit leukocytes: peritoneal and alveolar macrophages were uniformly killed, whereas thymocytes were essentially resistant. The toxin was shown to be specific for GM2 ganglioside or a GM2-like structure. In the present study we report the interaction of delta-toxin with human monocytes. A specific, saturable, and irreversible binding of 125I-delta-toxin was demonstrated. Binding was inhibited by preincubation of the radiolabeled toxin with GM2 and with high amount of GM1 ganglioside. As judged by dye exclusion, no cytotoxicity was observed on freshly isolated monocytes, but when added at the beginning of a culture of human adherent cells, the cytotoxic effect was detected after 48 hours of culture. Taken together, these data indicate the presence of monosialoganglioside(s) at the surface of human monocytes, and suggest a possible reorganisation of such structure into the cell membrane when monocytes mature in vitro toward macrophage-like cells.

Characterization of an anti-Borrelia burgdorferi OspA conformational epitope by limited proteolysis of monoclonal antibody-bound antigen and mass spectrometric peptide mapping.

Lyme borreliosis is a multisystem disorder caused by the spirochete Borrelia burgdorferi that is transmitted to humans by the tick Ixodes dammini. The immune response against the 31 kDa OspA, which is one of the most abundant B. burgdorferi proteins, appears to be critical in preventing infection and tissue inflammation. Detailed knowledge of the immunological and molecular characteristics of the OspA protein is important for the development of reliable diagnostic assays. In this study, we characterized a new conformational epitope present within the middle part of B. burgdorferi OspA. Our approach used enzymatic proteolyses of the immune complex followed by mass spectrometric identification of the peptides bound to the antibody. It appears to be one of the first reports on the characterization of a discontinuous epitope using mass spectrometry.

Beta2 toxin, a novel toxin produced by Clostridium perfringens.

A novel toxin (Beta2) and its gene were characterized from a Clostridium perfringens strain isolated from a piglet with necrotic enteritis. At the amino-acid level, Beta2 toxin (27670 Da) has no significant homology with the previously identified Beta toxin (called Beta1) (34861 kDa) from C. perfringens type B NCTC8533 ( Hunter, S.E.C., Brown, J.E., Oyston, P.C.F., Sakurai, J., Titball, R.W., 1993. Molecular genetic analysis of beta-toxin of Clostridium perfringens reveals sequence homology with alpha-toxin, gamma-toxin, and leukocidin of Staphylococcus aureus. Infect. Immun. 61, 3958-3965). Both Beta1 and Beta2 toxins were lethal for mice and cytotoxic for the cell line 1407, inducing cell rounding and lysis without affecting the actin cytoskeleton. The genes encoding Beta1 and Beta2 toxins have been localized in unlinked loci in large plasmids of C. perfringens. In addition, Beta2 toxin-producing C. perfringens strains were found to be associated with animal diseases such as necrotic enteritis in piglets and enterocolitis in horses.

Interaction of Clostridium perfringens delta toxin with erythrocyte and liposome membranes and relation with the specific binding to the ganglioside GM2.

The specific interaction of the cytolytic Clostridium perfringens delta toxin with membrane GM2 was indicated by: (i) characterization of this glycolipid in the membrane of sheep and goat erythrocytes, which are lysed by the toxin, whereas GM2 was undetectable in insensitive rabbit erythrocytes, (ii) demonstration of 125I-toxin binding to GM2, by autoradiography, following incubation with thin-layer chromatograms containing separated neuroblastoma gangliosides, and (iii) toxin fixation by phospholipid-cholesterol unilamellar vesicles containing either sheep gangliosides or GM2. In order to investigate the intramembrane events leading to membrane disruption following toxin binding, the photoreactive probe 12(4-azido-2-nitrophenoxy)stearoyl 1-14C glucosamine, which inserts into the outer layer and labels integral membrane proteins, was used to establish whether delta toxin penetrates into target cell membrane. No toxin labeling was found, suggesting that toxin action takes place at the membrane surface. This contention is supported by the observation that despite toxin binding, GM2 liposomes did not release entrapped 14C-glucose. Treatment of toxin with carboxypeptidases, but not aminopeptidases, abolished both toxin binding capacity onto erythrocytes and its combination with antitoxin neutralizing antibodies, suggesting that the carboxy terminal end of the toxin is critical for binding to cell membrane.

Binding of Clostridium perfringens 125I-labeled delta-toxin to erythrocytes.

Hemolytically active, 125I-labeled delta-toxin from Clostridium perfringens was used to study the binding of this cytolysin to sheep, goat, human, rabbit, horse, mouse, and guinea pig erythrocytes. The extent of toxin binding was correlated with the known hemolytic specificity of the toxin. Detailed studies of the binding were carried out on sheep erythrocytes which showed the highest sensitivity to lysis by delta-toxin. Simultaneous determination of toxin binding and release of intracellular 86Rb+ and hemoglobin suggested that toxin binding and membrane damage were separate sequential events. Toxin binding was rapid (2-5 min) and temperature-dependent. The extent of binding was temperature-independent. Binding was saturable, specific, relatively tight (Ka = 4.4 X 10(8) M-1) and largely irreversible. A single type of binding site (7,000/sheep erythrocyte) was found. Cell-bound toxin was extractable by chaotropic ions. Preincubation of the toxin with N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide (GM2 ganglioside) inhibited both binding and hemolysis. Toxin binding was affected by pretreatment of sheep erythrocytes with pronase but not with trypsin or chymotrypsin. Cell treatment with neuraminidase prevented toxin binding by 30%. Preincubation of the toxin with specific immune sera blocked its binding on target cells. It is suggested that GM2 ganglioside, a more complex membrane component containing this glycolipid or a structurally related molecule is the binding site for delta-toxin on the surface of sensitive erythrocytes.

Purification and characterization of Clostridium perfringens delta-toxin.

Delta-toxin, an extracellular hemolysin released by Clostridium perfringens type C, was purified from culture supernatant fluid by sequential ammonium sulfate precipitation, thiol-Sepharose gel chromatography, isoelectric focusing, and Sephadex G-75 gel filtration. The purified preparation had a specific activity of 320,000 hemolytic units per mg of protein and was homogeneous, as determined by immunochemical and electrophoretic tests. This toxin was characterized as a single polypeptide chain composed of 391 amino acid residues, 30% of which were hydrophobic. The molecular weight was found to be 42,000, and the isoelectric point was pH 9.1. Delta-toxin appeared to be amphiphilic by charge shift electrophoresis in a three-detergent system. It was immunogenic in rabbits and lethal to mice at a dose of 0.12 micrograms. The lytic activity of delta-toxin was restricted to erythrocytes of even-toed ungulates (sheep, goats, and pigs). This activity was inhibited by GM2 ganglioside but not by other gangliosides, cholesterol, lecithin, or sphingomyelin.

Damaging effects of Clostridium perfringens delta toxin on blood platelets and their relevance to ganglioside GM2.

The lytic effect of Clostridium perfringens delta toxin was investigated on goat, human, rabbit, and guinea pig platelets. In contrast to erythrocytes from the latter three species, which are insensitive to the toxin, the platelets were equally lysed by the same amount of toxin. These results suggest the presence of GM2 or GM2-like ganglioside(s) as a specific recognition site of the toxin on platelet plasmic membrane as previously established for sensitive erythrocytes. Plasmic membrane damage of human platelets was evidenced by the release of entrapped alpha-[14C]aminoisobutyric acid used as a cytoplasmic marker. The specific binding of hemolytically active 125I-delta toxin by human and rabbit platelets was practically identical, dose dependent, and inhibitable by GM2. Labeled toxin was also bound by various subcellular organelles separated from rabbit platelets except the 5-hydroxytryptamine (5-HT)-containing dense bodies, suggesting the absence or inaccessibility of GM2 on the surface of the latter organelles. This result correlates with the low amounts of 5-[3H]HT liberated after platelet challenge with delta toxin whereas this mediator was massively liberated upon lysis by the sulfhydryl-activated toxin alveolysin. The levels of M and P forms of phenol sulfotransferase (PST), involved in 5-HT catabolism, were determined in human platelet lysates after challenge with delta toxin, alveolysin, and other disruptive treatments. The low PST-M activities detected after lysis by delta toxin suggest that this isoenzyme is very likely associated to dense bodies in contrast to PST-P which is cytoplasmic. Platelet lysis by the toxin allows easy separation of these organelles.

Photolabeling of staphylococcal alpha-toxin from within rabbit erythrocyte membranes.

Intrinsic membrane proteins of rabbit red blood cells were labeled with the photoreactive amphipatic reagent 12-(4-azido-2-nitrophenoxy) stearoyl (1-14C) glucosamine, which inserts into the hydrophobic membrane region and generates a reactive nitrene upon ultraviolet irradiation. Photolabeling of membrane-bound staphylococcal alpha-toxin after lysis of probe-treated rabbit red blood cells by this toxin implies its penetration into the hydrophobic region of the outer leaflet of the membrane. In contrast clostridial theta-toxin and staphylococcal delta-toxin were not labeled, but extraction of intrinsic membrane proteins by delta-toxin was evidenced.

Selective cytotoxicity of Clostridium perfringens delta toxin on rabbit leukocytes.

Clostridium perfringens delta toxin was selectively cytotoxic for various rabbit leukocyte populations. The sensitivity of these populations to the toxin varied, depending on the tissue from which they were derived, from 28% (appendix) to 41% (bone marrow) and 32% (spleen). Macrophages were uniformly killed by delta toxin, whereas thymocytes were essentially resistant. Selective cytotoxicity was correlated to the specific binding of the radiolabeled toxin by target cells. The relationship between sensitivity to the toxin and the presence of GM2 ganglioside in the cell membrane of rabbit leukocytes is discussed. Delta toxin might prove a useful new tool for separating leukocyte subpopulations based on their differential sensitivity to the cytolethal effect of this protein.

Fibroblast growth factor (FGF) and an FGF-like molecule in pituitary extracts stimulate thymic epithelial cell proliferation.

A bovine pituitary extract (BPE) induces thymidine incorporation into human thymic epithelial cells (TEC) in vitro. Previous data demonstrated that its effects resulted in epithelial cell replication and suggested that the active component of BPE was likely to be a growth factor, different from epidermal growth factor (EGF) and interleukin 1 (IL1). In this report we present evidence to identify the active component of BPE as a fibroblast growth factor (FGF) family member, based upon several criteria: a) Purified bovine and human recombinant FGF also enhanced TEC proliferation in vitro. b) Neutralization studies showed antigenic similarities between BPE and basic FGF. c) Biochemical analytical studies allowed purification of BPE by its affinity for heparin with elution at 3M NaCl, and gel filtration sizing yielded a 73 KD molecule with which basic FGF co-eluted. The isoelectric point was found to be between 6.3 and 6.6 and this is the only finding not consistent with the characterization of basic FGF as the factor responsible for the activity of BPE on TEC proliferation.

Targeting of GM2-bearing tumor cells with the cytolytic Clostridium perfringens delta toxin.

The cytolytic Clostridium perfringens delta toxin lyses selectively cells which express ganglioside GM2. In this study, we investigated whether delta toxin can be used to characterize GM2 on tumor cell membranes and as an antitumor agent. The sensitivity to lysis by delta toxin of various murine and human malignant cell lines and also normal tissues was quantified using a 51Cr-release assay. The cytotoxicity titers were correlated with the 125I-labeled toxin binding capacity of sensitive and insensitive cells. Seven of eight human melanomas tested were lysed by the toxin and, of these, four were very sensitive (cytotoxicity titers below 12 ng of toxin). All neuroblastomas, gliomas and the retinoblastoma tested were lysed with 3-18 ng of toxin. Three of six carcinomas and one of two sarcomas were also very sensitive (cytotoxicity titers 0.6-15 ng) whereas leukemias and lymphoma cells were insensitive. Normal human tissues were insensitive (erythrocytes, skin fibroblasts) or poorly sensitive (brain, lung, spleen). The in vivo antitumor activity of delta toxin was tested in tumor-bearing mice. Daily intra-tumor injections of 0.5-1 mg of toxin for 4-5 days in carcinoma Me180- and melanoma A375-bearing nude mice, and neuroblastoma C1300-bearing A/J mice significantly inhibited tumor growth for 12-36 days. Intravenous administration of 100 ng of toxin per day for 5 days in Me180-bearing nude mice and C1300-bearing A/J mice gave significant inhibition of tumor growth only during the treatment period, and 10 injections of the same dose of toxin had no significant effect on SK-MEL28, a tumor lacking GM2.

A new kinetic approach for studying phospholipase C (Clostridium perfringens alpha toxin) activity on phospholipid monolayers.

The enzymatic activity of purified phospholipase C (alpha toxin) from Clostridium perfringens was investigated with various phospholipid monolayers. A two-step reaction was used. Enzymatic hydrolysis of insoluble lecithin films by phospholipase C, generating 1,2-diacylglycerol and water-soluble phosphocholine, was coupled with the action of pancreatic lipase in order to give rise to fatty acid and 2-monoacylglycerol, which are rapidly desorbed from the interface. With this new procedure, it is possible to obtain continuous and accurate kinetic measurements of the phospholipase C catalyzed reaction with phospholipid monolayers as the substrate. It is thus possible to avoid the use of radiolabeled substrates as necessary in previous studies, and the difficulties caused by diacylglycerol accumulation in the lipid film are minimized. No hydrolysis was detected when either phosphatidylethanolamine, phosphatidylserine, or phosphatidylglycerol films were used as substrates. By means of a film transfer technique, Ca2+ and Zn2+ ions were found to play a specific and critical role. The present study demonstrates clearly for the first time that Ca2+ is essential for enzyme binding to lipid films, whereas Zn2+ is specifically involved in the catalytic hydrolysis of the substrate.

Murine immunoprotective activity of Klebsiella pneumoniae cell surface preparations: comparative study with ribosomal preparations.

Cell surface preparations and ribosomal preparations were extracted from Klebsiella pneumoniae. Agar gel diffusion with antisera to cell surface preparations or ribosomal preparations indicated common antigenic components among the preparations. Lipopolysaccharide and capsular polysaccharide were identified in the cell surface preparations. These results and the previous identification of lipopolysaccharide and capsular polysaccharide in ribosomal preparations suggest that these antigens are responsible for the immunochemical cross-reactivity observed among these two bacterial extracts. Active protection could be induced in mice by these two preparations. On a dry-weight basis, cell surface preparations provided better immunoprotective activity than did ribosomal preparations. However, the 50% protective dose of both preparations is practically the same on the basis of their capsular polysaccharide content. These results are consistent with the hypothesis that the immunoprotective moiety of ribosomal preparations is the contaminating cell surface antigens. Furthermore, the low level of nucleotidic components detected in purified cell surface preparations led us to infer that the immunoprotective activity of capsular polysaccharide may not be dependent on the adjuvant activity of ribonucleic acid. The involvement of capsular polysaccharide in the immunoprotective capacity of cell surface preparations is demonstrated either by using a degradation of this antigen by K. pneumoniae bacteriophage K2-associated glycanase or by using a preparation extracted from a noncapsulated mutant of K. pneumoniae. Nevertheless, the low protective ability of purified capsular polysaccharides is in contrast to its greater activity when induced in bacterial cell surface preparations. The protective activity of K. pneumoniae capsular polysaccharide may be dependent on its association with other surface antigenic components present in cell surface preparations or may be dependent on its native form in the bacterial cell surface.

Specificities of multiple sclerosis cerebrospinal fluid and serum antibodies against mimotopes.

In order to characterize antigenic epitopes specifically targeted by the immune response of patients with multiple sclerosis (MS), the antibody specificities of cerebrospinal fluids (CSF) and sera from the same MS patients have been analyzed using a random pentadecapeptide library displayed on phage. The 3 peptides (mimotopes) selected with MS sera were not disease-specific. In contrast, the combination of 4 MS CSF selected mimotopes, allowed the detection of specific antibodies in 21 of 60 MS CSF whereas only 2 of 27 CSF from patients with other neurological diseases equally recognized the 4 mimotopes. Some amino acid similarities were found between two MS CSF selected mimotopes and two envelope regions (319-329 and 433-443, respectively) of MSRV (multiple-sclerosis-associated retrovirus) and the related endogenous retrovirus HERV-W.

Analysis of prostate specific antigen and alpha1-antichymotrypsin interaction using antipeptide monoclonal antibodies.

PURPOSE: The synthetic peptides E30D and D10P that correspond to prostate specific antigen (PSA) sequences 60-91 and 78-89, respectively, and contain the kallikrein loop were used to immunize mice to obtain anti-PSA monoclonal antibodies (mAbs). MATERIALS AND METHODS: Antipeptide mAb characteristics were studied using biosensor technology and enzyme-linked immunosorbent assay, and analyzing the mAb effects on PSA-alpha1-antichymotrypsin (ACT) complex formation and PSA enzymatic activity. Epitope mapping of these mAbs was performed using overlapping peptide synthesis on nitrocellulose membrane. RESULTS: Anti-E30D mAbs bound PSA coated on the solid phase only, whereas anti-D10P mAbs recognized PSA in detection as well as in capture. However, these mAbs appeared to be anti-total PSA mAbs. Anti-E30D and anti-D10P mAbs were directed against linear epitopes corresponding to residues H74-Y77 and N84-R88, respectively, of the PSA sequence. Anti-D10P mAb recognition of PSA and PSA-ACT complex was equimolar, although an existing molecular model suggested that the sequence corresponding to anti-D10P mAb epitope was involved in the interaction site of PSA with ACT. Furthermore, we were unable to inhibit the enzymatic activity of PSA as well as PSA-ACT complex formation. Finally, the epitope N84-R88 overlapped the cleavage site R85-F86 of PSA. CONCLUSIONS: The linear anti-D10P mAb epitope is located outside of the PSA-ACT binding site. However, these mAbs may be of value for evaluating the presence of different molecular PSA forms in sera.

An immunoreactive peptide of the FSH involved in autoimmune infertility.

The purpose of this study was to identify autoantigens contained in human ovary extracts. Serum samples from 36 infertile women with anti-ovary antibodies as detected with an ELISA technique were tested in Western blot against human ovary extracts. A reactive protein with a molecular mass matching that of the FSH was detected in 34 cases. These serum samples also reacted strongly in Western blot and ELISA with purified FSH and, in immunofluorescence, with pituitary cells. Using the Pepscan approach, with overlapping peptides matching the amino acid sequence of the human FSH beta-chain, several immunoreactive regions were evidenced. The 78-93 amino acid sequence of the human FSH beta-chain appeared as one of the major epitopes. Synthetic peptides of this region were prepared and demonstrated to react with human serum samples from women with anti-ovary antibodies. These data demonstrate that FSH can be an autoantigen, recognized by autoantibodies associated with infertility.

HCV core immunodominant region analysis using mouse monoclonal antibodies and human sera: characterization of major epitopes useful for antigen detection.

Monoclonal antibodies (MAbs) were generated by immunizing mice with a truncated recombinant protein corresponding to the immunodominant region (residues 1-120) of hepatitis C virus (HCV) nucleocapsid protein. The specific recognition by either human sera or mouse monoclonal antibodies of overlapping peptides spanning the core region 1-120 as well as the comparison with epitopes described earlier allowed the fine mapping of HCV core. Within the region 1-120, the major antigenic domain could be restricted to the first 45 amino acids. Indeed, the peptide S42G (residues 2-45) allowed the detection of an anti-HCV core response by all anticore-positive human sera examined. According to their epitope localization, three groups of mouse MABs could be evidenced that were directed against different regions of core. Group II MAbs recognized a strictly linear epitope (QDVKF, residues 20-24), whereas group I MABs were directed against a conformational epitope mainly located at the amino acid residues (QIVGG, 29-33). The epitope of group III MABs was also conformational (PRGRRQPI, residues 58-65). These three epitopes appeared close but different from the three major human epitopes RKTKRNTN, VYLLPR, and GRTWAQPGYPWPLY (residues 7-17, 34-39, and 73-86, respectively). Group II MAB 7G12A8 and group I MAB 19D9D6 were used in a sandwich ELISA for the capture and the detection, respectively, of viral core antigen in sera of patients with chronic HCV infection. After treatment of sera with triton x 100 in acidic conditions, amounts of viral antigen as low as 20 pg/ml of sera could be detected.

Anti-free prostate-specific antigen monoclonal antibody epitopes defined by mimotopes and molecular modeling.

BACKGROUND: Prostate-specific antigen (PSA) is an important marker for the diagnosis and management of prostate cancer, and the free PSA/total PSA ratio has been shown to be efficient for distinguishing prostate cancer from benign prostatic hyperplasia. We report here the characterization of seven mouse monoclonal antibodies (mAbs) and the partial localization of two conformational epitopes identified by anti-free PSA mAbs. METHODS: The mAbs were studied by competition and sandwich assays, and the epitope localization of the two anti-free PSA mAbs (6C8D8 and 5D3D11) was performed using phage displayed peptide libraries and molecular modeling. RESULTS: The seven mAbs were classified into three groups according to their recognition specificities and their ability to inhibit the enzymatic activity of PSA and the formation of PSA-alpha1-antichymotrypsin (ACT) complex. Among the anti-free PSA mAb group, 6C8D8 recognized the phage displayed peptide RKLRPHWLHFHPVAV, two parts of which presented similarities with two regions distant on the PSA sequence but joined in the tridimensional structure. mAb 5D3D11 recognized the peptide DTPYPWGWLLDEGYD, which is similar to a PSA region located on the board of the groove containing the PSA enzymatic site. Both epitopes were located in the theoretical ACT binding site described previously. Moreover, these mAbs were able to inhibit the enzymatic activity of PSA. CONCLUSIONS: These epitope localizations are in agreement with the ability of both mAbs to inhibit enzymatic activity and ACT fixation. The results presented here could bring information for the generation of clinically relevant PSA assays.