Engineering of protein epitopes: a single deletion in a snake toxin generates full binding capacity to a previously unrecognized antibody

Structural features associated with the ability of a monoclonalantibody (mAb) to discriminate between protein variants areidentified and engineered. The variants are the curaremimetictoxin from Naja nigricollis and erabutoxin a or b from Laticaudasemifasciata which differ from each other by 16 substitutionsand one insertion. The neutralizing mAb M1 recognizes with highaffinity a topographical epitope on the surface of toxin , butfails to recognize the erabutoxins although they possess mostof the residues forming the presumed epitope. Examinations ofthe toxin and erabutoxin 3-D structures and molecular dynamicssimulations reveal several differences between the variants.In particular, the region involving the ß-turn 17–24is organized differently. Analysis of the differences foundin this region suggests that the insertion (or deletion) atposition 18 of the variant amino add sequences is particularlyimportant in determining the differential cross-reactivity.To test this proposal, residue 18 was deleted in one erabutoxinusing sitedirected mutagenesis, and the biological propertiesof the resulting mutant were examined. We found that full antigenicitywas restored in the previously unrecognized variant. The implicationsof this finding are discussed.     

Improving Escherichia coli alkaline phosphatase efficacy by additional mutations inside and outside the catalytic pocket

We describe a strategy that allowed us to confer on a bacterial (E. coli) alkaline phosphatase (AP) the high catalytic activity of the mammalian enzyme while maintaining its high thermostability. First, we identified mutations, at positions other than those occupied by essential catalytic residues, which inactivate the bacterial enzyme without destroying its overall conformation. We transferred concomitantly into the bacterial enzyme four residues of the mammalian enzyme, two being in the catalytic pocket and two being outside. Second, the gene encoding the inactive mutant was submitted to random mutagenesis. Enzyme activity was restored upon the single mutation D330N, at a position that is 12 A away from the center of the catalytic pocket. Third, this mutation was combined with other mutations previously reported to increase AP activity slightly in the presence of magnesium. As a result, at pH 10.0 the phosphatase activity of both mutants D330N/D153H and D330N/D153G was 17-fold higher than that of the wild-type AP. Strikingly, although the two individual mutations D153H and D153G destabilize the enzyme, the double mutant D330N/D153G remained highly stable (T(m)=87 degrees C). Moreover, when combining the phosphatase and transferase activities, the catalytic activity of the mutant D330N/D153G increased 40-fold (k(cat)=3200 s-1) relative to that of the wild-type enzyme (k(cat)=80 s-1). Due to the simultaneous increase in K(m), the resulting k(cat)/K(m) value was only increased by a factor of two. Therefore, a single mutation occurring outside a catalytic pocket can dramatically control not only the activity of an enzyme, but also its thermostability. Preliminary crystallographic data of a covalent D330N/D153G enzyme-phosphate complex show that the phosphate group has significantly moved away from the catalytic pocket, relative to its position in the structure of another mutant previously reported.     

Characterization of toxins within crude venoms by combined use of Fourier transform mass spectrometry and cloning

The standard analytical procedure for screening the proteomic profile of a venom often relies on an appropriate combination of sample extraction, electrophoresis, reversed-phase high-performance liquid chromatography, mass spectrometry, and Edman degradation. We present in this study a new approach for venom screening based on Fourier transform mass spectrometry (FTMS) analysis directly on the crude venom. The venom chosen is a unique sample from Atractaspis irregularis, a species never studied at the molecular level previously. This snake belongs to the Atractaspidae family that is known to produce highly toxic venoms containing endothelin-like peptides called sarafotoxins (SRTXs). Nanoelectrospray-FTMS spectrum of the crude venom allowed the identification of 60 distinct compounds with molecular masses from 600 to 14,000 Da, which would have been impossible without the resolution of this kind of instrument. De novo sequencing within the entire venom confirmed the sequences of two new families of sarafotoxins, whose precursors had been cloned, and allowed the characterization of a third one. One particularly interesting point was that the propolypeptides appeared processed not in one unique compound, but rather in different length molecules ranging from 15 for the shorter to 30 amino acids for the longer. Moreover, our results clearly establish that in the case of A. irregularis only one copy of mature sarafotoxin emerges from each precursor, which is a totally different organization in comparison of other precursors of SRTXs.     

Transformation of a non-enzymatic toxin into a toxoid by genetic engineering

Curaremimetic toxins are typical non-enzymatic toxins that bind to their target [the nicotinic acetylcholine receptor (AChR)] through multiple residues. Nevertheless, we show that the concomitant substitutions of only three of the ten functionally important residues of such a toxin sufficed to cause an affinity decrease of the toxin for AChR that is higher than four orders of magnitude. Despite these triple mutations, the overall conformation of the mutated protein remains similar to that of a related recombinant toxin, as judged from both circular dichroism analysis and investigation of antigenicity, using monoclonal and polyclonal antibodies. Furthermore, we show that the detoxified toxin is capable of eliciting antibodies that neutralize the binding of a wild-type toxin to AChR. Therefore, transformation of a non-enzymatic toxin into a toxoid can be achieved, like in the case of enzymatic toxins, by introducing a small number of mutations at positions identified to be critical for expression of toxicity.     

Direct expression in E.coli of a functionally active protein A-snake toxin fusion protein

We constructed a recombinant expression plasmid encoding a proteinA–neurotoxin fusion protein. The fused toxin is directlyexpressed in the periplasmic space of Escherichia coli and canbe purified in the milligram range by a single immuno-affinitystep. The LD₅₀ values of the fused toxin and native toxin are130 and 20 nmol/kg mouse respectively. The K_d values characterizingtheir binding to the nicotinic acetylcholine receptor (AcChoR)are respectively 4.8 ± 0.8 and 0.07 ± 0.03 nM.In contrast, the fused and native toxins are equally well recognizedby a toxin-specific monoclonal antibody which recognizes theAcChoR binding site. The lower toxicity of the fused toxin mightresult, therefore, from a steric hindrance, due to the presenceof the bulky protein A moiety (mol. wt = 31 kd) rather thanto a direct alteration of the ‘toxic’ site. Thefused toxin is more immunogenic than native toxin, since 1 nmolof hybrid toxin and 14 nmol of native toxin give rise to comparabletiters of antitoxin antibodies which, furthermore, are equallypotent at neutralizing neurotoxicity. The work described inthis paper shows that the use of fused toxins may be of paramountimportance for future development of serotherapy against envenomationby snake bites.     

Insertion of a disulfide-containing neurotoxin into E.coil alkaline phosphatase: the hybrid retains both biological activities

We have inserted a disulfide-containing snake neurotoxin intothe N-terminal end of Escherichia coli alkaline phosphatase,between residues +6 and +7 of the mature enzyme. For this purpose,we have designed a cloning and expression vector which allowsinsertion of foreign DNA between the corresponding codons, andvisual selection of the desired recombinant clones upon recoveryof phosphatase activity. The hybrid protein is exported to thebacterial periplasm, the alkaline phosphatase signal peptideis correctly processed, and both domains are functionally conformed.The phosphatase domain displays catalytic activity, and theinserted toxin is able to bind to its biological target, thenicotinic acetylcholine receptor. The hybrid molecule is remarkablystable and resistant to proteolysis. Crude periplasmic extractcontaining the hybrid can be used as a tracer-containing reagentin competitive enzymo-immuno and enzymo-receptor assays. Wepropose to use the system described in this paper for fast preparationof properly folded disulfide-containing enzymatic probes.     

Transformation of a non-enzymatic toxin into a toxoid by genetic engineering

Curaremimetic toxins are typical non-enzymatic toxins that bind to their target [the nicotinic acetylcholine receptor (AChR)] through multiple residues. Nevertheless, we show that the concomitant substitutions of only three of the ten functionally important residues of such a toxin sufficed to cause an affinity decrease of the toxin for AChR that is higher than four orders of magnitude. Despite these triple mutations, the overall conformation of the mutated protein remains similar to that of a related recombinant toxin, as judged from both circular dichroism analysis and investigation of antigenicity, using monoclonal and polyclonal antibodies. Furthermore, we show that the detoxified toxin is capable of eliciting antibodies that neutralize the binding of a wild-type toxin to AChR. Therefore, transformation of a non-enzymatic toxin into a toxoid can be achieved, like in the case of enzymatic toxins, by introducing a small number of mutations at positions identified to be critical for expression of toxicity.      

Cloning and sequence analysis of cDNAs encoding precursors of sarafotoxins. Evidence for an unusual "rosary-type" organization

Sarafotoxins (SRTXs) are 21-amino acid peptides structurally and functionally similar to endothelins (ETs). To understand how SRTXs are overproduced in venom glands of the snakes Atractaspis engaddensis and hence used as toxins, we cloned cDNAs encoding SRTXs and elucidated their nucleotide sequences. We predict that SRTX precursors are large prepropolypeptide chains with an unusual "rosary-type" structure made of 12 successive similar stretches of 40 residues (39 in the first stretch). Each stretch begins with a "spacer" of 19 invariant residues (18 in the first stretch) immediately followed by the sequence of one SRTX isoform. Six different isoforms are identified within a single precursor molecule. Maturation of the precursor may require endopeptidases that cleave the Leu-Cys bond and the Trp-Arg/Lys bond invariably found at the SRTX N and C termini, respectively.     

Precursors of vertebrate peptide antibiotics dermaseptin b and adenoregulin have extensive sequence identities with precursors of opioid peptides dermorphin, dermenkephalin, and deltorphins

The dermaseptins are a family of broad spectrum antimicrobial peptides, 27-34 amino acids long, involved in the defense of the naked skin of frogs against microbial invasion. They are the first vertebrate peptides to show lethal effects against the filamentous fungi responsible for severe opportunistic infections accompanying immunodeficiency syndrome and the use of immunosuppressive agents. A cDNA library was constructed from skin poly(A+) RNA of the arboreal frog Phyllomedusa bicolor and screened with an oligonucleotide probe complementary to the COOH terminus of dermaseptin b. Several clones contained a full-length DNA copy of a 443-nucleotide mRNA that encoded a 78-residue dermaseptin b precursor protein. The deduced precursor contained a putative signal sequence at the NH2 terminus, a 20-residue spacer sequence extremely rich (60%) in glutamic and aspartic acids, and a single copy of a dermaseptin b progenitor sequence at the COOH terminus. One clone contained a complete copy of adenoregulin, a 33-residue peptide reported to enhance the binding of agonists to the A1 adenosine receptor. The mRNAs encoding adenoregulin and dermaseptin b were very similar: 70 and 75% nucleotide identities between the 5'- and 3'-untranslated regions, respectively; 91% amino acid identity between the signal peptides; 82% identity between the acidic spacer sequences; and 38% identity between adenoregulin and dermaseptin b. Because adenoregulin and dermaseptin b have similar precursor designs and antimicrobial spectra, adenoregulin should be considered as a new member of the dermaseptin family and alternatively named dermaseptin b II. Preprodermaseptin b and preproadenoregulin have considerable sequence identities to the precursors encoding the opioid heptapeptides dermorphin, dermenkephalin, and deltorphins. This similarity extended into the 5'-untranslated regions of the mRNAs. These findings suggest that the genes encoding the four preproproteins are all members of the same family despite the fact that they encode end products having very different biological activities. These genes might contain a homologous export exon comprising the 5'-untranslated region, the 22-residue signal peptide, the 20-24-residue acidic spacer, and the basic pair Lys-Arg.     

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.