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The antihypertensive potential of a land snail by-product hydrolysate (SBH), obtained after an industrial treatment of the raw material, was studied in vitro and in vivo . The ACE inhibitory activity of SBH was characterised by an IC 50 value of 23 µg⋅mL −1 , which was not affected by in vitro digestion. SBH enhanced the Caco-2 intestinal cell metabolic activity and did not induce any toxicity in Wistar rats. The partial purification of SBH led to the obtainment of an active fraction characterised by an IC 50 of 0.007 µg⋅mL −1 . The sequences of the 17 most abundant peptides of the fraction were identified by LC/MS/MS analysis. Seven of them (YG, YA, VY, SF, FG, GF and VW) are known ACE inhibitory peptides. Finally, in vivo study on SHR rats showed that SBH significantly reduced systolic blood pressure. SBH represents therefore a new candidate as an ingredient for the design of functional foods against hypertension.

International audience; The Ku70-Ku80 (Ku) heterodimer binds rapidly and tightly to the ends of DNA double-strand breaks and recruits factors of the non-homologous end-joining (NHEJ) repair pathway through molecular interactions that remain unclear. We have determined crystal structures of the Ku-binding motifs (KBM) of the NHEJ proteins APLF (A-KBM) and XLF (X-KBM) bound to a Ku-DNA complex. The two KBM motifs bind remote sites of the Ku80 alpha/beta domain. The X-KBM occupies an internal pocket formed by an unprecedented large outward rotation of the Ku80 alpha/beta domain. We observe independent recruitment of the APLF-interacting protein XRCC4 and of XLF to laser-irradiated sites via binding of A- and X-KBMs, respectively, to Ku80. Finally, we show that mutation of the X-KBM and A-KBM binding sites in Ku80 compromises both the efficiency and accuracy of end joining and cellular radiosensitivity. A- and X-KBMs may represent two initial anchor points to build the intricate interaction network required for NHEJ.

International audience; In mammalian cells, classical non-homologous end joining (c-NHEJ) is critical for DNA double-strand break repair induced by ionizing radiation and during V(D)J recombination in developing B and T lymphocytes. Recently, PAXX was identified as a c-NHEJ core component. We report here that PAXX-deficient cells exhibit a cellular phenotype uncharacteristic of a deficiency in c-NHEJ core components. PAXX-deficient cells display normal sensitivity to radiomimetic drugs, are proficient in transient V(D)J recombination assays, and do not shift toward higher micro-homology usage in plasmid repair assays. Although PAXX-deficient cells lack c-NHEJ phenotypes, PAXX forms a stable ternary complex with Ku bound to DNA. Formation of this complex involves an interaction with Ku70 and requires a bare DNA extension for stability. Moreover, the relatively weak Ku-dependent stimulation of LIG4/XRCC4 activity by PAXX is unmasked by XLF ablation. Thus, PAXX plays an accessory role during c-NHEJ that is largely overlapped by XLF's function.

International audience; Non-homologous end joining is a ligation process repairing DNA double strand breaks in eukaryotes and many prokaryotes. The ring structured eukaryotic Ku binds DNA ends and recruits other factors which can access DNA ends through the threading of Ku inward the DNA, making this protein a key ingredient for the scaffolding of the NHEJ machinery. However, this threading ability seems unevenly conserved among bacterial Ku. As bacterial Ku differ mainly by their C-terminus, we evaluate the role of this region in the loading and the threading abilities of Bacillus subtilis Ku and the stimulation of the DNA ligase LigD. We identify two distinct sub-regions: a ubiquitous minimal C-terminal region and a frequent basic C-terminal extension. We show that truncation of one or both of these sub-regions in Bacillus subtilis Ku impairs the stimulation of the LigD end joining activity in vitro. We further demonstrate that the minimal C-terminus is required for the Ku-LigD interaction, whereas the basic extension controls the threading and DNA bridging abilities of Ku. We propose that the Ku basic C-terminal extension increases the concentration of Ku near DNA ends, favoring the recruitment of LigD at the break, thanks to the minimal C-terminal sub-region.

The three-dimensional structures of some components of snake venoms forming so-called ‘three-fingered protein’ domains (TFPDs) are similar to those of the ectodomains of activin, bone morphogenetic protein and transforming growth factor-β receptors, and to a variety of proteins encoded by the Ly6 and Plaur genes. The analysis of sequences of diverse snake toxins, various ectodomains of the receptors that bind activin and other cytokines, and numerous gene products encoded by the Ly6 and Plaur families of genes has revealed that they differ considerably from each other. The sequences of TFPDs may consist of up to six disulfide bonds, three of which have the same highly conserved topology. These three disulfide bridges and an asparagine residue in the C-terminal part of TFPDs are essential for the TFPD-like fold. Analyses of the three-dimensional structures of diverse TFPDs have revealed that the three highly conserved disulfides impose a major stabilizing contribution to the TFPD-like fold, in both TFPDs contained in some snake venoms and ectodomains of several cellular receptors, whereas the three remaining disulfide bonds impose specific geometrical constraints in the three fingers of some TFPDs.

BotXIV and LqhαIT are two structurally related long chain scorpion α-toxins that inhibit sodium current inactivation in excitable cells. However, while LqhαIT from Leiurus quinquestriatus hebraeus is classified as a true and strong insect α-toxin, BotXIV from Buthus occitanus tunetanus is characterized by moderate biological activities. To assess the possibility that structural differences between these two molecules could reflect the localization of particular functional topographies, we compared their sequences. Three structurally deviating segments located in three distinct and exposed loops were identified. They correspond to residues 8–10, 19–22, and 38–43. To evaluate their functional role, three BotXIV/LqhαIT chimeras were designed by transferring the corresponding LqhαIT sequences into BotXIV. Structural and antigenic characterizations of the resulting recombinant chimera show that BotXIV can accommodate the imposed modifications, confirming the structural flexibility of that particular α/β fold. Interestingly, substitution of residues 8–10 yields to a new electrophysiological profile of the corresponding variant, partially comparable to that one of α-like scorpion toxins. Taken together, these results suggest that even limited structural deviations can reflect functional diversity, and also that the structure–function relationships between insect α-toxins and α-like scorpion toxins are probably more complex than expected.

Toxin alpha from Naja nigricollis (61 amino acids, four disulfide bridges) belongs to the "three finger" fold family, which contains snake toxins with various biological activities and nontoxic proteins from different origins. In this paper, we report an extensive 1H and 15N NMR study of the dynamics of toxin alpha in solution. 15N relaxation, 1H off-resonance ROESY, and H-D exchange experiments allowed us to probe picosecond to hour motions in the protein. Analysis of these NMR measurements demonstrates that toxin alpha exhibits various time scale motions, i.e., particularly large amplitude picosecond to nanosecond motions at the tips of the loops, observable microsecond to millisecond motions around two disulfide bridges, second time scale motions around the C-N bonds of asparagine and glutamine side chains which are more or less rapid depending on their amino acid solvent accessibility, and minute to hour motions in the beta-sheet structure. The less well-defined regions of toxin alpha solution structures are subject to important picosecond to nanosecond motions. The toxic site is organized around residues belonging to the rigid core of the molecule but also comprises residues exhibiting dynamics on various time scales. The Malpha1 epitope is subject to large picosecond to millisecond motions, which are probably modified by the interaction with the antibody. This phenomenon could be linked to the neutralizing properties of the antibody.

In several instances, a monoclonal antibody raised against a receptor ligand has been claimed to mimic the ligand receptor. Thus, a specific monoclonal antibody (Malpha2-3) raised against a short-chain toxin from snake was proposed to mimic the nicotinic acetylcholine receptor (AChR) (). Further confirming this mimicry, we show that (i) like AChR, Malpha2-3 elicits anti-AChR antibodies, which in turn elicit anti-toxin antibodies; and (ii) the region 106-122 of the alpha-chain of AChR shares 66% primary structure identity with complementarity-determining regions of Malpha2-3. Also, a mutational analysis of erabutoxin a reveals that the epitope recognized by Malpha2-3 consists of 10 residues, distributed within the three toxin loops. Eight of these residues also belong to the 10-residue epitope recognized by AChR, a result that offers an explanation as to the functional similarities between the receptor and the antibody. Strikingly, however, most of the residues common to the two epitopes contribute differentially to the energetic formation of the antibody-toxin and the receptor-toxin complexes. Together, the data suggest that the mimicry between AChR and Malpha2-3 is partial only.

LMWP is the low molecular weight protein which copurifies with alpha-latrotoxin, the main neurotoxin from the black widow venom. It contains 70 residues and three disulfides. We found that its primary structure, including its 6 half-cystines, can be aligned with the amino acid sequences of crustacean hyperglycemic hormones (CHHs) which contain 72-73 residues and three disulfides. To further investigate this structural relationship, we produced a recombinant analog of LMWP in which the unique Met was changed in Leu (LMWPM35L). LMWPM35L was produced as a folded fusion protein in the periplasm of Escherichia coli and was generated in vitro by treating the fusion protein with cyanogen bromide. We showed that LMWPM35L and CHHs have an identical disulfide pairing pattern and possess some alpha-helical structure, as deduced from a comparison of their circular dichroism spectra. In addition, LMWPM35L and CHHs are consensually predicted to possess a helical structure within the region 13-17. Together, the data indicate that CHHs are structurally related to LMWPM35L and presumably also to LMWP. Finally, preliminary studies showed that LMWPM35L is not toxic to mice and does not form channels in lipid bilayers, two well-known properties of alpha-latrotoxin preparations.

A competitive enzyme immunoassay for rat prolactin using an immunogen and a tracer obtained by recombinant DNA technology is described. Polyclonal antibodies were raised in rabbits immunized with a purified chimeric protein consisting of rat prolactin fused with two synthetic immunoglobulin G binding domains derived from staphylococcal Protein A. The enzymatic tracer was obtained using an expression system which permits insertion of rPRL DNA sequence in the alkaline phophatase gene. Antibodies and tracer were used to develop a solid-phase competitive immunoassay for the measurement of rat prolactin in plasma with a minimal detectable concentration of 0.5 ng/ml. The mean intra- and inter-assay coefficients of variation were 7.8 and 13.2%, respectively. Rat plasma concentrations measured with this assay correlated well with those obtained with a conventional enzyme immunoassay ( r = 0.993, slope = 1.037, n = 24).

Cernunnos/XLF is a core protein of the nonhomologous DNA end-joining (NHEJ) pathway that processes the majority of DNA double-strand breaks in mammals. Cernunnos stimulates the final ligation step catalyzed by the complex between DNA ligase IV and Xrcc4 (X4). Here we present the crystal structure of the X 4 1–157 -Cernunnos 1–224 complex at 5.5-Å resolution and identify the relative positions of the two factors and their binding sites. The X-ray structure reveals a filament arrangement for X 4 1–157 and Cernunnos 1–224 homodimers mediated by repeated interactions through their N-terminal head domains. A filament arrangement of the X4–Cernunnos complex was confirmed by transmission electron microscopy analyses both with truncated and full-length proteins. We further modeled the interface and used structure-based site-directed mutagenesis and calorimetry to characterize the roles of various residues at the X4–Cernunnos interface. We identified four X4 residues (Glu 55 , Asp 58 , Met 61 , and Phe 106 ) essential for the interaction with Cernunnos. These findings provide new insights into the molecular bases for stimulatory and bridging roles of Cernunnos in the final DNA ligation step.

Two monoclonal antibodies, called M alpha 1 and M alpha 2,3, have been previously shown to neutralize the toxic activity of the curaremimetic toxin alpha from Naja nigricollis. In this paper, we report the mapping of the two corresponding epitopes, using affinity chromatography and proton 2D-NMR spectroscopy. The H-D exchange rates of labile amide hydrogens have been measured in toxin alpha bound to each antibody and in toxin alpha alone. Analysis of the exchange data revealed two regions containing amide hydrogens with decreased exchange rates in the bound toxin compared to the free toxin. These two regions correspond to the sites of interaction with M alpha 1 and M alpha 2,3, respectively. They are consistent with prior biochemical mapping studies, and they include several residues that were not previously identified. Thus, the two antigenic sites are found to be centered on two different loops of toxin alpha. Comparison of these antigenic sites with the active site of toxin alpha allows us to delineate the molecular events associated with the two neutralization processes.

We have constructed an expression vector to produce, in Escherichia coli, a fusion protein containing successively two IgG binding domains from staphyloccocal protein A, a nine-amino-acid linker peptide terminating in a methionine residue and the phospholipase A2 notechis 11'2L, an isoform of notexin of Notechis scutatus scutatus venom. Notechis 11'2L is a mutant of the naturally occurring notechis 11'2 [Bouchier, C., Boyot, P., Tesson, F., Tremeau, O., Bouet, F., Hodgson, D., Boulain, J. C. & Menez, A. (1991) Eur. J. Biochem. 202, 493-500] in which Met8 has been replaced by Leu. The fusion protein was recovered in the periplasmic extract with a yield of 0.25 mg/l culture. It was hydrolyzed with cyanogen bromide, yielding a protein having the molecular mass, amino acid composition and N-terminal sequence of notechis 11'2L. Notechis 11'2L and the wild notechis 11'2 displayed identical circular dichroic spectra and shared similar enzymatic, myotoxic and antigenic properties, suggesting that the recombinant notechis 11'2L was directly generated in a correctly folded form.

To study the site by which erabutoxin a (Ea) from Laticauda semifasciata binds to the nicotinic acetylcholine receptor, we mutated most residues that are shared with other curaremimetic toxins and studied the structural and biological consequences of introduced mutations. By site-directed mutagenesis, we changed Ser-8 into Gly (EaS8G), Lys-27 into Glu (EaK27E), Trp-29 into Phe (EaW29F) and His (EaW29H), Asp-31 into His (EaD31H), Phe-32 into Leu (EaF32L), Arg-33 into Lys (EaR33K) and Glu (EaR33E), Gly-34 into Ser (EaG34S), Glu-38 into Gln (EaE38Q) and Lys (EaE38K), Gly-49 into Val (EaG49V), and Leu-52 into Ala (EaL52A). All mutants were homogeneous as judged by various analytical procedures. EaE38Q, EaG49V, and EaL52A bound the nicotinic acetylcholine receptor with apparent Kd values close to 10(-10) M, virtually identical to wild Ea. Therefore, Glu-38, Gly-49, and Leu-52 are not important elements in the expression of curaremimetic function in Ea. Mutations of Phe-32 and Gly-34 provoked a 7-fold affinity decrease, suggesting that these residues moderately contribute to function. The 176-fold affinity decrease due to mutation of Ser-8 may reflect some structural change that operates in the polypeptide chain of the mutant, as detected by circular dichroism. Decreases in affinity by a factor of 175, 67, 46, and 318 were seen upon mutations of Lys-27 into Glu, Trp-29 into Phe, Asp-31 into His, and Arg-33 into Glu, with no concomitant change in secondary structure. These residues appear to be important elements of the curaremimetic function of Ea. Thus, a picture of the contribution of conserved residues to the function of a curaremimetic toxin is proposed on the basis of experimental evidence.

Much has been learned about the folding of proteins from comparative studies of the folding of proteins that are related in sequence and structure. Observation of the effects of mutations helps account for sequence-specific properties and large variations in folding rates observed in homologous proteins, which are not explained by structure-derived descriptions. The folding kinetics of variants of a β-stranded protein, toxin α from Naja nigricollis, depends on the length of their loop lk1. These proteins, named Tox60, Tox61, and Tox62, contain four disulfide bonds. We show that their oxidative refolding pathways are similar. Differences in these pathways are restricted to the last step of the reaction, that is, the closure of the last disulfide. At this step, two species of three-disulfide intermediates are observed: intermediate C lacking the B3 disulfide and intermediate D lacking the B2 disulfide. Surprisingly, D is the most productive intermediate for Tox61 despite the low accessibility of its free cysteines. However, in the case of Tox62, its conversion efficiency drops by 2 orders of magnitude and C becomes the most productive intermediate. NMR was used in order to study the structural dynamics of each of these intermediates. Both three-disulfide intermediates of Tox61 exist in two forms, exchanging on the 1- to 100-ms scale. One of these forms is structurally very close to the native Tox61, whereas the other is always significantly more flexible on a picosecond-to-nanosecond timescale. On the other hand, in the case of Tox62, the three-disulfide intermediates only show a native-like structure. The higher conformational heterogeneity of Tox61 intermediate D allows an increased accessibility of its free cysteines to oxidative agents, which explains its faster native disulfide formation. Thus, residue deletion in loop lk1 probably abrogates stabilizing intramolecular interactions, creates conformational heterogeneity, and increases the folding rate of Tox60 and Tox61 compared to Tox62.

International audience; In mammals, the majority of DNA double-strand breaks are processed by the Non Homologous End-Joining pathway (NHEJ), composed of seven factors: Ku70, Ku80, DNA-PKcs, Artemis, Xrcc4 (X4), DNA Ligase IV (L4) and Cernunnos/XLF. Cernunnos is part of the ligation complex, constituted by X4 and L4. In order to improve our knowledge on the structure and function of Cernunnos, we performed a systematic mutagenesis study on positions selected from an analysis of the recent 3D structures of this factor. Ten out of 27 screened mutants were non functional in several DNA repair assays. Outside amino acids critical for the expression and stability of Cernunnos, we identified three amino acids (Arg$^{64}$, Leu$^{65}$, and Leu$^{115}$) essential for the interaction with X4 and the proper function of Cernunnos. Docking the crystal structures of the two factors further validated this probable interaction surface of Cernunnos with X4.

The three-dimensional structures of some components of snake venoms forming so-called 'three-fingered protein' domains (TFPDs) are similar to those of the ectodomains of activin, bone morphogenetic protein and transforming growth factor-beta receptors, and to a variety of proteins encoded by the Ly6 and Plaur genes. The analysis of sequences of diverse snake toxins, various ectodomains of the receptors that bind activin and other cytokines, and numerous gene products encoded by the Ly6 and Plaur families of genes has revealed that they differ considerably from each other. The sequences of TFPDs may consist of up to six disulfide bonds, three of which have the same highly conserved topology. These three disulfide bridges and an asparagine residue in the C-terminal part of TFPDs are essential for the TFPD-like fold. Analyses of the three-dimensional structures of diverse TFPDs have revealed that the three highly conserved disulfides impose a major stabilizing contribution to the TFPD-like fold, in both TFPDs contained in some snake venoms and ectodomains of several cellular receptors, whereas the three remaining disulfide bonds impose specific geometrical constraints in the three fingers of some TFPDs.

SUMMARY: Proteolytic activities, active against casein and insulin are present in cell-free extracts of the psychrotrophic bacterium Arthrobacter globiformis S155. These activities were compared (i) following growth of the bacterium at different temperatures (10, 20 and 32°C) and (ii) after a temperature shift from 10 to 32°C. Both activities (measured at 20°C) were greater in cells grown at 32°C and after a temperature shift. Chloramphenicol prevented the increases in activities. The proteolysis of casein was stimulated by ATP; the stimulation was greater following a temperature shift. In addition, proteolysis of casein (measured at 20°C) was activated by pre-incubation at temperatures between 45 and 60°C; the proportion of proteolytic activity activated by such treatment also increased with growth temperature. Comparison by two-dimensional electrophoresis of the polypeptides synthesized at 10 and 32°C suggests that A. globiformis S155 produces proteins specific to both temperatures (13 species were either exclusive to or present in increased amounts during growth at 10°C, while at least 21 polypeptides were preferentially synthesized at 32°C). A temperature shift from 10 to 32°C promoted the synthesis of at least 16 polypeptides, many with M r values similar to those of heat shock proteins synthesized at higher temperatures in mesophiles. A temperature up-shift of A. globiformis S155 from 10 to 20°C did not provoke the synthesis of new proteins; heat shock proteins were produced only at 28°C and higher. The similarity in kinetics of the appearance of heat shock proteins and the increase in proteolytic activities suggest that in A. globiformis S155 some heat shock proteins might also possess protein catabolic function.

Proteins are poor immunogens that require an adjuvant to raise an immune response. Here we show that the human immunodeficiency virus, type 1 Tat protein possesses an autoadjuvant property, and we have identified the determinants and the molecular events that are associated with this unusual property. Using a series of chemically synthesized Tat101 derivatives, we show that the core region controls the autoadjuvant phenomenon independently of the B-cell recognition and T-cell stimulation that are associated with epitopes respectively located on the N-terminal region and the cysteine-rich region. We also show that cysteine-mediated oligomerization is a key molecular event of the adjuvant-free antibody response. In particular, a Tat dimer formed by the oxidation of two cysteine residues, at position 34 only, raises an adjuvant-free antibody response that is comparable with that observed with the wild-type protein. Unlike the parent protein, the Tat dimer has no transactivating activity and remains homogeneous for several weeks in solution. This construct might be of value for the design of an adjuvant-free Tat-based vaccine. Furthermore, we suggest that the specific autoadjuvanticity determinant of Tat could be used to provide other proteins with adjuvant-free immunogenicity.

International audience; Many studies that aim to characterize the proteome structurally or functionally require the production of pure protein in a high-throughput format. We have developed a fast and flexible integrated system for cloning, protein expression in Escherichia coli, solubility screening and purification that can be completely automated in a 96-well microplate format. We used recombination cloning in custom-designed vectors including (i) a (His)6 tag-encoding sequence, (ii) a variable solubilizing partner gene, (iii) the DNA sequence corresponding to the TEV protease cleavage site, (iv) the gene (or DNA fragment) of interest, (v) a suppressible amber stop codon, and (vi) an S·tag peptide-encoding sequence. First, conditions of bacterial culture in microplates (250 μL) were optimized to obtain expression and solubility patterns identical to those obtained in a 1-L flask (100-mL culture). Such conditions enabled the screening of various parameters in addition to the fusion partners (E. coli strains, temperature, inducer...). Second, expression of fusion proteins in amber suppressor strains allowed quantification of soluble and insoluble proteins by fluorescence through the detection of the S·tag. This technique is faster and more sensitive than other commonly used methods (dot blots, Western blots, SDS-PAGE). The presence of the amber suppressor tRNA was shown to affect neither the expression pattern nor the solubility of the target proteins. Third, production of the most interesting soluble fusion proteins, as detected by our screening method, could be performed in nonsuppressor strains. After cleavage with the TEV protease, the target proteins were obtained in a native form with a unique additional N-terminal glycine.

We propose a novel approach to the selection of Escherichia coli bacterial strains improved for the production of recombinant functional proteins. This approach is based on aggregation-induced toxicity of recombinant proteins. We show that selection of clones displaying a reduced toxicity is an efficient means of isolating bacteria producing recombinant protein with reduced aggregation in favour of correct folding. For an efficient selection, we found that time of toxicity induction must be precisely determined and recombinant protein must be expressed as a fusion with a protein whose activity is easily detectable on plates, thus allowing elimination of non-productive mutants. Choosing the expression to the periplasmic space of an scFv fragment fused to the N-terminus of alkaline phosphatase as a model, we selected chromosomal mutations that reduce aggregation-induced toxicity and showed that they concomitantly improve production of a functional recombinant hybrid. The effects of the mutations isolated could then be cumulated with those of other strategies used for recombinant scFv production. Thus, we could ensure a 6- to 16-fold increase in production of a functional scFv-PhoA hybrid. This is the first report demonstrating the possibility of directly selecting on agar plates E.coli strains improved for functional recombinant protein production from a large bacterial mutant library.

We determined the distances separating five functionally important residues (Gln(10), Lys(27), Trp(29), Arg(33), and Lys(47)) of a three-fingered snake neurotoxin from the reduced disulfide bond alpha(Cys(192)-Cys(193)) located at the alphagamma interface of the Torpedo nicotinic acetylcholine receptor. Each toxin position was substituted individually for a cysteine, which was then linked to a maleimido moiety through three different spacers, varying in length from 10 to 22 A. We estimated the coupling efficiency between the 15 toxin derivatives and the reduced cystine alpha(192-193) by gel densitometry of Coomassie Blue-stained gels. A nearly quantitative coupling was observed between alphaCys(192) and/or alphaCys(193) and all probes introduced at the tip of the first (position 10) and second (position 33) loops of Naja nigricollis alpha-neurotoxin. These data sufficed to locate the reactive thiolate in a "croissant-shaped" volume comprised between the first two loops of the toxin. The volume was further restrained by taking into account the absence or partial coupling of the other derivatives. Altogether, the data suggest that alphaCys(192) and/or alphaCys(193), at the alphagamma interface of a muscular-type acetylcholine receptor, is (are) located in a volume located between 11.5 and 15.5 A from the alpha-carbons at positions 10 and 33 of the toxin, under the tip of the toxin first loop and close to the second one.

Toxic proteins from animal venoms usually act on molecular targets that are critically involved in the function of a physiological system of a prey. With the ultimate view of designing new pharmacological tools as well as original drugs acting on these targets, animal toxins are the subject of extensive molecular analyses. In particular, the topographies by which toxins bind to their targets and hence affect the function of the associated physiological system clearly need to be identified.

We have investigated whether the nonpathogenic gram-positive bacteria Staphylococcus xylosus and S. carnosus can display a whole domain of a toxic protein on their surface and if such vectors are suitable for immunization of BALB/c mice. The nucleotide sequence encoding the receptor-binding domain (DTR; amino acids 382 to 535) of diphtheria toxin (DT) was inserted into plasmids pSE′mp18ABPXM and pSPPmABPXM, which were designed to display heterologous proteins on S. xylosus and S. carnosus cell surfaces, respectively. Western blot analysis of the resulting bacterial lysates indicates that DTR is produced by each expression system. However, analysis of rabbit anti-DTR antisera binding to the transformed live bacteria shows that DTR is not displayed on the surface of S. xylosus cells whereas it is efficiently exposed on S. carnosus . A significant anti-DT antibody response was raised in BALB/c mice immunized intraperitoneally with S. carnosus displaying DTR, and the antisera abolished DT cytotoxicity on Vero cells. Thus, only S. carnosus can display a whole domain of a toxic protein and represents a potential vector for humoral vaccination.

Curaremimetic toxins are typical non-enzymatic toxins thatbind to their target [the nicotinic acetylcholine receptor(AChR)] through multiple residues. Nevertheless, we showthat the concomitant substitutions of only three of the tenfunctionally important residues of such a toxin sufficed tocause an affinity decrease of the toxin for AChR that ishigher than four orders of magnitude. Despite these triplemutations, the overall conformation of the mutated proteinremains similar to that of a related recombinant toxin, asjudged from both circular dichroism analysis and investi-gation of antigenicity, using monoclonal and polyclonalantibodies. Furthermore, we show that the detoxified toxinis capable of eliciting antibodies that neutralize the bindingof a wild-type toxin to AChR. Therefore, transformationof a non-enzymatic toxin into a toxoid can be achieved,like in the case of enzymatic toxins, by introducing a smallnumber of mutations at positions identified to be criticalfor expression of toxicity.Keywords: snake toxin/site directed mutagenesis/toxoidIntroductionThe use of living vectors expressing selected recombinantantigens appears to be a promising approach for immunizinghumans or animals against various pathogens, including toxicproteins (Pozzi et al., 1992; Walker et al., 1992; N’Guyenet al., 1993; Stover et al., 1993; Gomez-Duarteet al., 1995).For evident safety reasons, it is of primary importance thatthe immunogen produced by a living organism is devoid oftoxic activity. In the case of toxic proteins, two situations canbe encountered. First, the toxin possesses enzymatic activityand its pathogenicity is directly associated with expression ofthis activity. In principle, therefore, abolition of the later shouldsuffice for detoxification and indeed, this strategy has beensuccessfully applied to bacterial enzymes such as pertussistoxin (Pizza et al., 1989; Loosmore et al., 1990; Rappuoliet al., 1992), cholera toxin (Hase et al., 1994; Fontana et al.,1995) or Shiga-like toxin (Gordon et al., 1992), in whichmutations of a few (one or two) crucial catalytic positionswere sufficient to fully inactivate the toxins. Second, the toxinsexert their function without expression of any enzymaticactivity but simply by binding to a biological target. This isthe case, for example, for some bacterial toxins, such asStaphylococcal toxins (Bonventre et al., 1995; Ulrich et al.,1995), cytolysins (Bhakdi et al., 1996) and of a large proportionof animal toxins, including most snake and scorpion toxins(Me´nez et al., 1991). Then, a related question is to what extent

Two recombinant fragments of diphtheria toxin (DT) were fused to an engineered tandem repeat of the immunoglobulin (Ig) binding domain of protein A, called ZZ. These fragments are (i) the receptor binding domain (DTR), which comprises amino acids 382 to 535 of DT, and (ii) a linear peptide (DT 168–220 ) which comprises residues 168 to 220 of the loop between fragment A and fragment B of DT. The fusion proteins were produced in Escherichia coli and purified by affinity chromatography. In vitro experiments showed that the DTR domain is responsible for the capacity of ZZ-DTR to bind to Vero cells and is capable of inhibiting the cytotoxicity of DT for these cells. These findings suggest that DTR binds to the cell surface receptors of DT and hence adopts a conformation that is similar to that of the receptor binding domain of DT. We compared the capacities of ZZ-DTR, ZZ-DT 168–220 , and a chemically detoxified form of DT currently used for vaccination to elicit antibodies in rabbits. The toxoid was more immunogenic than ZZ-DT 168–220 , which in turn was more immunogenic than ZZ-DTR. However, ZZ-DT 168–220 antiserum was poorly efficient at neutralizing DT cytotoxicity on Vero cells, whereas ZZ-DTR antiserum was only 15-fold less potent than anti-DT antisera.

Long chain and short chain curaremimetic toxins from snakes possess 66-74 residues with five disulfide bonds and 60-62 residues with four disulfide bonds, respectively. Despite their structural differences all of these toxins bind with high affinity to the peripheral nicotinic acetylcholine receptors (AChR). Binding experiments have now revealed that long chain toxins only, like the neuronal kappa-bungarotoxin, have a high affinity for a chimeric form of the neuronal alpha7 receptor, with Kd values ranging from about 1 to 12 nM. In contrast, all other toxins bind to the chimeric alpha7 receptor with a low affinity, with Kd values ranging between 3 and 22 microM. These results are supported by electrophysiological recordings on both the wild-type and chimeric alpha7 receptors. Amino acid sequence analyses have suggested that high affinities for the neuronal receptor are associated with the presence of the fifth disulfide at the tip of the toxin second loop. In agreement with this conclusion, we show that a long chain toxin whose fifth disulfide is reduced and then dithiopyridylated has a low affinity (Kd = 12 microM) for the neuronal alpha7 receptor, whereas it retains a high affinity (Kd = 0.35 nM) for the peripheral AChR. Thus, a long chain curaremimetic toxin having a reduced fifth disulfide bond behaves like a short chain toxin toward both the peripheral and neuronal AChR. Therefore, functional classification of toxins that bind to AChRs should probably be done by considering their activities on both peripheral and neuronal receptors.

The aim of this work was to produce and to label snake neurotoxins, disulfide-rich proteins. A mutant of a snake toxin, erabutoxin a, was used as a model. Its N-terminal part was fused to ZZ, a synthetic IgG-binding domain of protein A (B. Nilssonet al.,1987,Protein Eng.1, 107–113), thus preventing degradation in the bacterial cytoplasm and providing a simple affinity-purification method on IgG Sepharose. A soluble fusion protein was obtained with a yield of 60 mg/L, corresponding to 20 mg/L toxin. The toxin moiety was folded on the column while the hybrid was still bound. The oxidoreducing conditions for the refolding were optimized and were found to be oxidative but with a need for reducing molecules. The concentration of the hybrid bound to the column could be increased up to 3.3 mg/ml without significantly altering the folding process. CNBr cleavage of the fusion protein followed by a purification step yielded about 2 mg of biologically active toxin mutant per gram of dry cell weight. This procedure was applied to produce 55 mg of a toxin uniformly labeled with15N.

We have designed an expression vector permitting the production in the periplasm of Escherichia coli of a fusion protein comprising a dimer of bacterial alkaline phosphatase (PhoA) and two Fab or scFv fragments of a monoclonal antibody directed against human IgG. Each hybrid protein expressed both high specificity for the antigen and full PhoA activity. We show that crude periplasmic extracts containing these conjugates can be used as such in enzyme immunoassays for the detection of human IgG, as exemplified in the case of anti-hepatitis B immunoglobulin.

We constructed a synthetic gene encoding the published amino acid sequence of DTx from Dendroaspis angusticeps, a ligand of voltage-dependent postassium channels that facilitates neurotransmitter release. We expressed it in Escherichia coli as a fusion protein secreted in the culture medium. The recombinant DTx was generated in vitro by chemical treatment and recovered as two isoforms. One of them (rDTx), like the venom toxin, has an N-terminal pyroglutamate whereas the other (rQDTx) has a free N-terminal glutamine. Chromatographic differences between rDTx and natural DTx led us to re-examine the amino acid sequence of natural DTx. In contrast to what was previously published, position 12 was an Asp and not Asn. Despite this difference, rDTx and DTx had similar toxicity in mice and binding affinity to synaptosomes, suggesting that residue 12 is not important for DTx function. Nor is the N-terminal residue implicated in DTx function since rDTx and rQDTx also had similar biological activities. We also synthesized and expressed a mutant of the DTx gene in which the lysine triplet 28–30 was changed into Ala-Ala-Gly. The two resulting recombinant isoforms exhibited only small decreases in biological activity, excluding the possibility that the positively charged lysine triplet 28–30 of DTx is directly involved in the toxin functional site.

Recombinant erabutoxin a (Ea(r)) has been crystallized by vapour diffusion in hanging drops. The crystals belong to space group P2(1)2(1)2(1) with cell dimensions a = 55.8 A, b = 53.4 A, c = 40.8 A. Diffraction data have been recorded on a FAST detector up to 2.0 A. The atomic crystal structure of Ea(r) has been determined by initial refinement of the structure of the isotoxin erabutoxin b (Eb) the crystals of which were grown under identical conditions. The R-factor was 23% at 2.0 A resolution. The secondary and tertiary structures of Ea(r) are shown to be identical with that of wild-type Eb, within the experimental error.

The proteolytic activity in cell extracts of psychrotrophic bacterium Arthrobacter strain S1-55 has been compared after growth at 10°, 20° and 32°C. Extracts of cells grown at 32°C consistently possessed the greatest activity, and those from cells grown at 10°C the lowest when assayed at 10°, 20°, 32° and 37°C. The activity in extracts of 10°C-grown cells was significantly more heat-labile than that from the 20°C- and 32°C-grown cells. It is suggested that growth at the high (32°C) and low (10°C) temperatures yields qualitative and quantitative differences in proteolytic activity in this organism.

SUMMARY: Proteolysis of endogenous and exogenous substrates in cell-free extracts of the psychrotrophic bacterium Arthrobacter sp. S155 has been compared. Endogenous proteins were degraded only after treatment with cyanogen bromide. The hydrolysis of exogenous proteins of high Mr, (i.e. casein) was optimum at alkaline pH and was stimulated by Ca2+, Mg2+, Mn2+ and ATP. The serine protease inhibitor phenylmethylsulphonyl fluoride had no effect on ATP stimulation. Small peptides (i.e. insulin) were degraded at very high rates. This activity was optimum at slightly acidic pH and was stimulated by Ca2+, strongly inhibited by Mn2+, but not affected by ATP. Degradation of cyanogen bromide-treated cellular proteins displayed two pH optima which corresponded to the optimum pH for the degradation of insulin and casein. The characteristics of these acidic and alkaline activities were identical to those active against insulin and casein respectively. The proteases which degraded casein were much more heat resistant than those which degraded insulin.