Colette Jolivet-Reynaud

Clostridium perfringens delta toxin is sequence related to beta toxin, NetB, and Staphylococcus pore-forming toxins, but shows functional differences.

Clostridium perfringens produces numerous toxins, which are responsible for severe diseases in man and animals. Delta toxin is one of the three hemolysins released by a number of C. perfringens type C and possibly type B strains. Delta toxin was characterized to be cytotoxic for cells expressing the ganglioside GM2 in their membrane. Here we report the genetic characterization of Delta toxin and its pore forming activity in lipid bilayers. Delta toxin consists of 318 amino acids, its 28 N-terminal amino acids corresponding to a signal peptide. The secreted Delta toxin (290 amino acids; 32619 Da) is a basic protein (pI 9.1) which shows a significant homology with C. perfringens Beta toxin (43% identity), with C. perfringens NetB (40% identity) and, to a lesser extent, with Staphylococcus aureus alpha toxin and leukotoxins. Recombinant Delta toxin showed a preference for binding to GM2, in contrast to Beta toxin, which did not bind to gangliosides. It is hemolytic for sheep red blood cells and cytotoxic for HeLa cells. In artificial diphytanoyl phosphatidylcholine membranes, Delta and Beta toxin formed channels. Conductance of the channels formed by Delta toxin, with a value of about 100 pS to more than 1 nS in 1 M KCl and a membrane potential of 20 mV, was higher than those formed by Beta toxin and their distribution was broader. The results of zero-current membrane potential measurements and single channel experiments suggest that Delta toxin forms slightly anion-selective channels, whereas the Beta toxin channels showed a preference for cations under the same conditions. C. perfringens Delta toxin shows a significant sequence homolgy with C. perfringens Beta and NetB toxins, as well as with S. aureus alpha hemolysin and leukotoxins, but exhibits different channel properties in lipid bilayers. In contrast to Beta toxin, Delta toxin recognizes GM2 as receptor and forms anion-selective channels.

Mapping of a conformational epitope shared between E1 and E2 on the serum-derived human hepatitis C virus envelope

Monoclonal antibody D32.10 produced by immunizing mice with a hepatitis C virus (HCV)-enriched pellet obtained from plasmapheresis of a chronically HCV1b-infected patient binds HCV particles derived from serum of different HCV1a- and HCV1b-infected patients. Moreover, this monoclonal has been shown to recognize both HCV envelope proteins E1 and E2. In an attempt to provide novel insight into the membrane topology of HCV envelope glycoproteins E1 and E2, we localized the epitope recognized by D32.10 on the E1 and/or E2 sequence using Ph.D.-12™ phage display peptide library technology. Mimotopes selected from the phage display dodecapeptide library by D32.10 shared partial similarities with 297RHWTTQGCNC306 of the HCV E1 glycoprotein and with both 613YRLWHYPCT621 and 480PDQRPYCWHYPPKPC494 of the HCV E2 glycoprotein. Immunoreactivity of D32.10 with overlapping peptides corresponding to these three HCV regions confirmed these localizations and suggested that the three regions identified are likely closely juxtaposed on the surface of serum-derived particles as predicted by the secondary model structure of HCV E2 derived from the tick-borne encephalitis virus E protein. This assertion was supported by the detection of specific antibodies directed against these three E1E2 regions in sera from HCV-infected patients.

Recombinant kallikrein expression: site-specific integration for hK6 production in human cells.

Abstract Kallikreins have been implicated in carcinogenesis and are promising biomarkers for the diagnosis and follow-up of various cancers. To evaluate the functions and clinical interest of kallikreins, it is important to be able to produce them as recombinant proteins. Here we summarize the various strategies used to produce kallikreins, emphasizing their advantages and limitations. We also describe an approach to achieve high-level production of kallikreins, such as hK6, with correct folding and activity, combining an expression system with targeted transgene integration and an efficient cultivation device to increase yield, the CELLine bioreactor. This novel method for recombinant kallikrein production will be useful to study their bio-pathological functions and to develop anti-bodies.

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.

Comparison of the crystallization and crystal packing of two Fab single-site mutant protein L complexes

Protein L from Peptostreptococcus magnus (PpL) is a multidomain protein composed of four or five immunoglobulin-binding domains that target the kappa light chain of a large repertoire of human and murine antibodies. Thus, a single domain of this protein can be used to aid the crystallization of Fab, free or complexed to their antigen when it is not possible to obtain crystals without it. Each wild-type PpL domain has two light-chain binding sites that target the same region of the light chain and can thus bring together two Fab-antigen complexes within the crystal lattice. In this context the small PpL domain is sandwiched between two Fab and cannot participate in crystal contacts, thus mutants are unlikely to increase the chances of crystallizing a particular complex. However, it is possible to design mutants that can bind at only one site by making use of the crystal structures obtained so far. Such mutants will have a free surface that can participate in crystal contacts and that can be modified to improve its crystal contact-forming properties. Here, a comparison of two single-site mutants that differ at three different positions is reported. In both mutants two different tryptophan residues participate in crystal-packing interactions, suggesting that this residue may be particularly interesting for enhancing crystal-contact formation.

ANALYSIS OF PROSTATE SPECIFIC ANTIGEN AND α1-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.

Different crystal packing in Fab-protein L semi-disordered peptide complex.

Proteins and peptides with variable degrees of disorder are a challenge for protein crystallization. These may be completely disordered or just contain regions with a high degree of mobility that may be represented by a multitude of discretely defined conformations. These difficulties are not insurmountable, but it may be unreasonable to expect a clean result from a structural point of view. The complex between a murine monoclonal antibody (19D9D6) and a synthetic peptide that encompasses the first 45 residues of the core protein of Hepatitis C virus that is poorly structured in solution has been crystallized. In order to make the crystallization possible, use was made of a single immunoglobulin-binding domain of protein L from Peptostreptococcus magnus (PpL), a bacterial protein that can bind the variable region (Fv) of a large population of antibodies through its light chain with no interference with antibody-antigen recognition. Crystals were obtained in different space groups where the size of the cavity that accommodates the peptide is different, although many of the crystal contacts and the overall lattice are preserved. The peptide can be considered to be semi-disordered and the larger cavity accommodates a better ordered peptide than the smaller one. The lattice is of interest for the design of a scaffold system for the crystallization of peptide-tagged proteins since a cavity that accommodates a disordered entity might be able to host ordered proteins of the same size and shape as the cavity. Here, the differences between the lattices formed by this trimolecular complex are described and it is discussed how such a system may be adapted to the crystallization of peptide-tagged proteins.

Development of monoclonal antibodies to human kallikrein-related peptidase 6 (KLK6) and their use in an immunofluorometric assay for free KLK6.

Abstract We have raised monoclonal antibodies against KLK6 and constructed a ‘sandwich’ type immunoassay using 8A8G3 as capture and 3H3E9 as tracer antibodies. 8A8G3 bound to one side of KLK6, whereas 3H3E9 probably bound near its catalytic site. The assay did not detect complexed forms of KLK6, indicating that it quantifies only free KLK6 (fKLK6). fKLK6 was higher in serum of patients with ovarian cancer (11.34 μg/l±1.37) than in controls (3.39 μg/l±0.42). The cerebrospinal fluid contained high concentrations of fKLK6 (257–965 μg/l). This assay could facilitate the evaluation of fKLK6 in human diseases.

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.