Neurotoxin-specific immunoglobulins accelerate dissociation of the neurotoxin-acetylcholine receptor complex.

Toxin isolated from cobra venom and labeled with tritium was incubated with membranes rich in acetylcholine receptors. The amount of toxin bound to the receptors was determined and the kinetics of dissociation of the receptor-toxin complex was followed. Addition of an excess of horse antiserum to the venom resulted in a significant acceleration of the dissociation reaction. Similarly, a monoclonal antibody against the toxin accelerated dissociation of the receptor-toxin complex. The results indicate that specific antibody binding destabilizes the toxin-receptor complex.

Molecular evolution of snake toxins: is the functional diversity of snake toxins associated with a mechanism of accelerated evolution?

Recent studies revealed that animal toxins with unrelated biological functions often possess a similar architecture. To tentatively understand the evolutionary mechanisms that may govern this principle of functional prodigality associated with a structural economy, two complementary approaches were considered. One of them consisted of investigating the rates of mutations that occur in cDNAs and/or genes that encode a variety of toxins with the same fold. This approach was largely adopted with phospholipases A2 from Viperidae and to a lesser extent with three-fingered toxins from Elapidae and Hydrophiidae. Another approach consisted of investigating how a given fold can accommodate distinct functional topographies. Thus, a number of topologies by which three-fingered toxins exert distinct functions were investigated either by making chemical modifications and/or mutational analyses or by studying the three-dimensional structure of toxin-target complexes. This review shows that, although the two approaches are different, they commonly indicate that most if not all the surface of a snake toxin fold undergoes natural engineering, which may be associated with an accelerated rate of evolution. The biochemical process by which this phenomenon occurs remains unknown.

Analysis of cDNAs encoding the two subunits of crotoxin, a phospholipase A2 neurotoxin from rattlesnake venom: the acidic non enzymatic subunit derives from a phospholipase A2-like precursor.

We report the sequences of three cDNAs encoding the two subunits (CA and CB) of crotoxin, a neurotoxic phospholipase A2 from the venom of the South-American rattlesnake Crotalus durissus terrificus. CB is a basic and toxic phospholipase A2 and CA is an acidic, non toxic and non enzymatic three chain containing protein which enhances the lethal potency of CB. Two cDNAs encoding precursors of CB isoforms have been isolated from a cDNA library prepared from one venom gland. Both precursors are made of the same 16 residues signal peptide followed by a polypeptide of 122 amino acid residues. The two mature sequences differ from each other at eight positions and are in good agreement with the previous polypeptide sequence reported for CB. In the case of CA, the cDNA encodes a signal peptide identical to those found in CB precursors, followed by a polypeptide of 122 amino acids clearly homologous to phospholipases A2 and including three regions which correspond to the three chains of mature CA. This demonstrates that CA is generated from a phospholipase A2-like precursor, called pro-CA, by the removal of three peptides, leaving unchanged the molecule core cross-linked by disulfide bridges. The 5'-untranslated tracts of cDNAs encoding CA and CB are nearly identical and the 3'-untranslated tracts are very similar, suggesting that the mRNAs encoding the two crotoxin subunits may result from the alternative splicing of a single gene or from the existence of a recent gene conversion. Data have been analysed in light of recent results on other phospholipases A2 from different origins.

Complete amino-acid sequence of the beta-subunit of VTX from venom of the stonefish (Synanceia verrucosa) as identified from cDNA cloning experiments.

A cDNA encoding a subunit of the verrucotoxin (VTX) has been identified from a cDNA library derived from stonefish venom glands. It encodes a polypeptide of 708 amino-acid residues, followed by a 3'-untranslated region of 895 bp long. The ORF contains the complete mature sequence of the beta-subunit of the VTX, as inferred from both the presence of an identical N-terminus sequence and 96% homology among the 506 amino terminus residues found in the partial sequence of the beta-subunit of the stonustoxin from Synanceia horrida (Ghadessy, F.J., Jeyaseelan, K., Chung, M.C.M., Khoo, H.E., and Yuen, R. (1994) Toxicon 32, 1684-1688). Upstream the mature sequence, we noticed the presence of an incomplete peptide of a 13 amino acids, whose unusual primary structure supports the idea of the existence of a propeptide and/or of a new secretion signal.

Stability of a structural scaffold upon activity transfer: X-ray structure of a three fingers chimeric protein.

Fasciculin 2 and toxin alpha proteins belong to the same structural family of three-fingered snake toxins. They act on different targets, but in each case the binding region involves residues from loops I and II. The superimposition of the two structures suggests that these functional regions correspond to structurally distinct zones. Loop I, half of loop II and the C-terminal residue of fasciculin 2 were therefore transferred into the toxin alpha. The inhibition constant of the resulting chimera is only 15-fold lower than that of fasciculin 2, and as expected the potency of binding to the toxin alpha target has been lost. In order to understand the structure-function relationship between the chimera and its "parent" molecules, we solved its structure by X-ray crystallography. The protein crystallized in space group P3(1)21 with a=b=58.5 A, and c=62.3 A. The crystal structure was solved by molecular replacement and refined to 2.1 A resolution. The structure belongs to the three-fingered snake toxin family with a core of four disulphide bridges from which emerge the three loops I, II and III. Superimposition of the chimera on fasciculin 2 or toxin alpha revealed an overall fold intermediate between those of the two parent molecules. The regions corresponding to toxin alpha and to fasciculin 2 retained their respective geometries. In addition, the chimera protein displayed a structural behaviour similar to that of fasciculin 2, i.e. dimerization in the crystal structure of fasciculin 2, and the geometry of the region that binds to acetylcholinesterase. In conclusion, this structure shows that the chimera retains the general structural characteristics of three-fingered toxins, and the structural specificity of the transferred function.

Artificial evolution of an enzyme active site: structural studies of three highly active mutants of Escherichia coli alkaline phosphatase.

The crystal structure of three mutants of Escherichia coli alkaline phosphatase with catalytic activity (k(cat)) enhancement as compare to the wild-type enzyme is described in different states. The biological aspects of this study have been reported elsewhere. The structure of the first mutant, D330N, which is threefold more active than the wild-type enzyme, was determined with phosphate in the active site, or with aluminium fluoride, which mimics the transition state. These structures reveal, in particular, that this first mutation does not alter the active site. The second mutant, D153H-D330N, is 17-fold more active than the wild-type enzyme and activated by magnesium, but its activity drops after few days. The structure of this mutant was solved under four different conditions. The phosphate-free enzyme was studied in an inactivated form with zinc at site M3, or after activation by magnesium. The comparison of these two forms free of phosphate illustrates the mechanism of the magnesium activation of the catalytic serine residue. In the presence of magnesium, the structure was determined with phosphate, or aluminium fluoride. The drop in activity of the mutant D153H-D330N could be explained by the instability of the metal ion at M3. The analysis of this mutant helped in the design of the third mutant, D153G-D330N. This mutant is up to 40-fold more active than the wild-type enzyme, with a restored robustness of the enzyme stability. The structure is presented here with covalently bound phosphate in the active site, representing the first phosphoseryl intermediate of a highly active alkaline phosphatase. This study shows how structural analysis may help to progress in the improvement of an enzyme catalytic activity (k(cat)), and explains the structural events associated with this artificial evolution.

Further evidence showing that neurotoxin-acetylcholine receptor dissociation is accelerated by monoclonal neurotoxin-specific immunoglobulin.

We have demonstrated that the dissociation of Naja nigricollis alpha-toxin from the two acetylcholine receptor sites [Weber and Changeux, Molec. Pharmac. 10, 1-14 (1974); Rousselet et al., Eur. J. Biochem. 140, 31-37 (1984)], is markedly accelerated by a monoclonal neurotoxin-specific antibody. The dissociation of the toxin occurs in a biphasic manner in the presence of a 900 molar excess of immunoglobulin (with respect to toxin concn). The progress curves are characterized by first-order kinetics. Under these conditions the maximal dissociation rate is achieved as further rate enhancement cannot be induced by exposure to an increased immunoglobulin level. In contrast when a toxin-immunoglobulin complex is incubated with a large excess of receptor, the dissociation kinetics of the complex are not enhanced. The data fit a kinetic model which implicates the existence of a transient ternary complex involving the receptor, the toxin and the antibody.

A monoclonal antibody which recognized the functional site of snake neurotoxins and which neutralizes all short-chain variants.

We isolated a neurotoxin-specific monoclonal antibody (Mab) which is capable of recognizing and neutralizing all short-chain toxin variants that have been tested, including those with widely divergent sequences. The epitope incorporates the three invariant residues Lys-27, Trp-29 and Lys-47 which form part of the site by which the toxins bind to the nicotinic acetylcholine receptor. To our knowledge, this is the first Mab which possesses the universal capacity of neutralizing all natural variants of a toxic protein.

A recombinant snake neurotoxin generated by chemical cleavage of a hybrid protein recovers full biological properties.

We previously reported the production of a fused snake neurotoxin composed of protein A and erabutoxin a in E. coli. The hybrid had much lower toxicity and affinity for the acetylcholine nicotinic receptor than natural erabutoxin. By treating the hybrid with cyanogen bromide we generated a toxin which was purified in a single step by RP-HPLC. This compound, produced in a good yield, recovered all properties of native erabutoxin a, implying that the lower toxic activities of the hybrid were due to the bulky protein A and not to an incorrect folding of the toxin. This work serves as a basis for future studies of toxin-receptor interactions using engineered toxin mutants.

Three-dimensional crystal structure of recombinant erabutoxin a at 2.0 A resolution.

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.

Toxicity-based selection of Escherichia coli mutants for functional recombinant protein production: application to an antibody fragment.

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.

[Binding of a tritiated enkephalinergic analog (FK 33-824) to a mitochondrial fraction of rat brain]. / Liaison d'un analogue enképhalinergique [FK 33-824] tritié à une fraction mitochondriale de cerveau de rat.

FK-33-824 (Try-D-Ala-Gly-MePhe-Met(O)ol) is a potent enkephalin analog which has been tritium labelled with a high specific radioactivity (41 Ci/mmole). The labelled drug exhibits specific and saturable binding to rat brain crude mitochondrial fraction. Specific binding is inhibited by low concentrations of morphine, levallorphan and beta-endorphin, suggesting that FK 33-824 [3H] binds preferentially to mu opiate sites. Binding studies at equilibrium and kinetics of formation and dissociation of the labelled ligand-receptor complex indicate that FK 33-824 [3H] binds to two classes of specific sites. Their affinities are distinguishable at 0 degree (KD = 1.3 and 5.8 nM) and very close to each other at 37 degree (KD = 1.9 nM).

Recombinant single-chain Fv antibody fragment-alkaline phosphatase conjugate for one-step immunodetection in molecular hybridization.

Using phage-display technology, a recombinant single-chain Fv antibody fragment (scFv) was rapidly generated from the K16-16 hybridoma secreting mouse monoclonal antibody (MAb) that binds to acetylaminofluorene-labeled DNA (AAF-DNA). The selected A4 phage-scFv specifically bound to AAF-DNA. The anti-AAF scFv gene was then recloned into a fusion vector for the production of a hybrid protein comprising the antibody fragment fused to a potent bacterial alkaline phosphatase variant (PhoAv). The anti-AAF scFv-PhoAv hybrid protein was bifunctional and possessed both antigen binding capacity and PhoA activity. The recombinant conjugate was directly used, without further purification, for one-step immunodetection in dot-blot hybridization. The detection limit was identical and the test was quicker than the conventional two-step procedure with the purified anti-AAF MAb revealed with a secondary enzyme-labeled antibody. To assess the value of this new reagent for the immunodetection of genomic nucleic acids, genomic DNAs of Campylobacter jejuni and Campylobacter coli were then one-step immunodetected with non-purified recombinant scFv-PhoAv conjugate in a Southern-blot hybridization experiment. The present study shows that the genetic fusion with PhoAv provides a new tool for immunodetection which presents easier and quicker production and use with the same sensitivity and specificity as classical reagents. The recombinant anti-AAF scFv-PhoAv conjugate is a promising alternative reagent for applications involving the immunodetection of specific DNA or RNA sequences, such as the detection and characterization of microorganisms.

Recombinant technology in the preparation of immunogen and enzymatic tracer. Application to the development of an enzyme immunoassay for rat prolactin.

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 phosphatase 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).

Engineering of a recombinant colorimetric fusion protein for immunodiagnosis of insulin.

A synthetic DNA encoding human proinsulin was inserted in frame in the bacterial alkaline phosphatase gene. A homogeneous recombinant human proinsulin-alkaline phosphatase conjugate was obtained directly from the periplasm of Escherichia coli transformed with a plasmid carrying the hybrid gene. The recombinant conjugate was stable and could be produced in the bacteria. The immunological properties of the recombinant conjugate and those of the human insulin and human proinsulin were compared using a panel of six different human insulin-specific monoclonal antibodies. Three immunological groups were thus distinguished and one of them indiscriminately recognized all of the insulin-like molecules. One monoclonal antibody from this group was used in combination with the recombinant conjugate to develop successfully a competitive immunoenzymatic assay for detecting insulin.

Recombinant antibody-alkaline phosphatase conjugates for diagnosis of human IgGs: application to anti-HBsAg detection.

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.

Nucleotide sequence and structure analysis of cDNAs encoding short-chain neurotoxins from venom glands of a sea snake (Aipysurus laevis).

Two cDNAs from the sea snake Aipysurus laevis have been cloned and sequenced. They encode isoforms of short chain neurotoxins. One of them is toxin b, previously isolated from the venom of Aipysurus laevis and sequenced by Maeda and Tamiya (Biochem. J. 153, 79, 1976), whereas the other corresponds to an isoform which was not hitherto described. The two toxin sequences differ from each other by three amino-acid residues. Both cDNA structures were comparable with that previously determined in our laboratory for erabutoxin a from Laticauda semifasciata.

Amino acid sequence of a muscarinic toxin deduced from the cDNA nucleotide sequence.

We prepared a cDNA library from venom glands of the green mamba Dendroaspis angusticeps. A cDNA clone was isolated using an appropriate nucleotide probe. The nucleotide sequence codes for a 21 residue signal peptide followed by a 65 residue protein having the amino acid sequence of muscarinic toxin 2, as confirmed in the accompanying paper (Karlsson, E., Risinger, C., Jolkkonen, M., Wernstedt, C. and Adem, A.). The cDNA encoding the muscarinic toxin has been compared with those encoding other snake toxins. There are close similarities with short-chain curaremimetic neurotoxins.

Regional and accelerated molecular evolution in group I snake venom gland phospholipase A2 isozymes.

In accordance with detection of a few phospholipase A2 (PLA2) isozyme genes by Southern blot analysis, only two cDNAs, named NnkPLA-I , and NnkPLA-II, encoding group I PLA2s, NnkPLA-I and NnkPLA-II, respectively, were isolated from the venom gland cDNA library of Elapinae Naja naja kaouthia of Malaysia. NnkPLA-I and NnkPLA-II showed four amino acid substitutions, all of which were brought about by single nucleotide substitution. No existence of clones encoding CM-II and CM-III, PLA2 isozymes which had been isolated from the venom of N. naja kaouthia of Thailand, in Malaysian N. naja kaouthia venom gland cDNA library was verified by dot blot hybridization analysis with particular probes. NnkPLA-I and NnkPLA-II differed from CM-II and CM-III with four and two amino acid substitutions, respectively, suggesting that their molecular evolution is regional. The comparison of NnkPLA-I, NnkPLA-II and cDNAs encoding other group I snake venom gland PLA2s indicated that the 5'- and 3'-untranslated regions are more conserved than the mature protein-coding region and that the number of nucleotide substitutions per nonsynonymous site is almost equal to that per synonymous site in the protein-coding region, suggesting that accelerated evolution has occurred in group I venom gland PLA2s possibly to acquire new physiological functions.

Recombinant colorimetric antibodies: construction and characterization of a bifunctional F(ab)2/alkaline phosphatase conjugate produced in Escherichia coli.

We have designed a vector which allows the synthesis in Escherichia coli of bifunctional F(ab)2-alkaline phosphatase conjugates. The vector contains a di-cistronic operon encoding truncated heavy chain (Fd or VH-CH1) of an IgG inserted between residues +6 and +7 of bacterial alkaline phosphatase (PhoA), and the light chain of the same IgG. We demonstrate the utility of this approach with the heavy and light chain domains of a snake toxin-specific monoclonal antibody, M alpha 2-3. We show that the VH-CH1-PhoA hybrid and VL-CL are concomitantly expressed and exported to the periplasm of E. coli where they form a disulfide-linked chimeric protein. The hybrid has the same affinity as M alpha 2-3 for the snake toxin antigen and possesses PhoA enzymatic activity.