The CDR1 of the human lambdaVI light chains adopts a new canonical structure.

We performed a comparative analysis of the conformation of the CDR1 of the human lambdaVI variable domains JTO and WIL and the equivalent loop of the lambdaI light chains RHE and KOL, which are representative of the type I canonical structure for lambda light chains. On the basis of the differences found in the main chain conformation, as well as the identity of the residues at key positions, we showed that the L1 of some lambdaVI light chains adopts a conformation that represents a new type of canonical structure. The conformation of the L1 of those lambdaVI light chains, is primarily determined by the presence of an Arg residue at position 25. The analysis of the lambdaVI light chain sequences so far reported, showed that near 25% of those proteins have Gly at position 25 instead of Arg, which represents an allotypic variant of the lambdaVI variable locus. The presence of Gly at position 25 in the L1 of lambdaVI light chains would imply a different conformation for this loop. Additionally, the position 68 in lambdaVI light chains, which is at the top of the FR3 loop, showed such spatial orientation and variability that suggested its participation in the conformation of the antigen recognition surface in this subgroup of lambda chains.

Directed evolution, phage display and combination of evolved mutants: a strategy to recover the neutralization properties of the scFv version of BCF2 a neutralizing monoclonal antibody specific to scorpion toxin Cn2.

BCF2, a monoclonal antibody raised against scorpion toxin Cn2, is capable of neutralizing both, the toxin and the whole venom of the Mexican scorpion Centruroides noxius Hoffmann. The single chain antibody fragment (scFv) of BCF2 was constructed and expressed in Escherichia coli. Although its affinity for the Cn2 toxin was shown to be in the nanomolar range, it was non-neutralizing in vivo due to a low stability. In order to recover the neutralizing capacity, the scFv of BCF2 was evolved by error-prone PCR and the variants were panned by phage display. Seven improved mutants were isolated from three different libraries. One of these mutants, called G5 with one mutation at CDR1 and another at CDR2 of the light chain, showed an increased affinity to Cn2, as compared to the parental scFv. A second mutant, called B7 with a single change at framework 2 of heavy chain, also had a higher affinity. Mutants G5 and B7 were also improved in their stability but they were unable to neutralize the toxin. Finally, we constructed a variant containing the changes present in G5 and B7. The purpose of this construction was to combine the increments in affinity and stability borne by these mutants. The result was a triple mutant capable of neutralizing the Cn2 toxin. This variant showed the best affinity constant (KD=7.5x10(-11) M), as determined by surface plasmon resonance (BIAcore). The k(on) and k(off) were improved threefold and fivefold, respectively, leading to 15-fold affinity improvement. Functional stability determinations by ELISA in the presence of different concentrations of guanidinium hydrochloride (Gdn-HCl) revealed that the triple mutant is significantly more stable than the parental scFv. These results suggest that not only improving the affinity but also the stability of our scFv were important for recovering its neutralization capacity. These findings pave the way for the generation of recombinant neutralizing antisera against scorpion stings based on scFvs.

Selection of tumor-specific internalizing human antibodies from phage libraries.

Antibody internalization into the cell is required for many targeted therapeutics, such as immunotoxins, immunoliposomes, antibody-drug conjugates and for targeted delivery of genes or viral DNA into cells. To generate directly tumor-specific internalizing antibodies, a non-immune single chain Fv (scFv) phage antibody library was selected on the breast tumor cell line SKBR3. Internalized phage were recovered from within the cell and used for the next round of selection. After three rounds of selection, 40 % of clones analyzed bound SKBR3 and other tumor cells but did not bind normal human cells. Of the internalizing scFv identified, two (F5 and C1) were identified as binding to ErbB2, and one (H7) to the transferrin receptor. Both F5 and H7 scFv were efficiently endocytosed into SKBR3 cells, both as phage antibodies and as native monomeric scFv. Both antibodies were able to induce additional functional effects besides triggering endocytosis: F5 scFv induces downstream signaling through the ErbB2 receptor and H7 prevents transferrin binding to the transferrin receptor and inhibits cell growth. The results demonstrate the feasibility of selecting internalizing receptor-specific antibodies directly from phage libraries by panning on cells. Such antibodies can be used to target a variety of molecules into the cell to achieve a therapeutic effect. Furthermore, in some instances endocytosis serves as a surrogate marker for other therapeutic biologic effects, such as growth inhibition. Thus, a subset of selected antibodies will have a direct therapeutic effect.

Deletion of a repetitive extragenic palindromic (REP) sequence downstream from the structural gene of Escherichia coli glutamate dehydrogenase affects the stability of its mRNA.

A deletion that removes one repetitive extragenic palindromic sequence downstream of the structural gene of Escherichia coli glutamate dehydrogenase, reduces twofold the half-life of gdhA mRNA.

Identification of a functional promoter for the Escherichia coli gdhA gene and its regulation.

Glutamate dehydrogenase (GDH) catalyzes the synthesis of L-glutamate from 2-oxoglutarate and ammonia. The complete nucleotide sequence of the Escherichia coli gdhA gene, as well as its 5' and 3' flanking regions have been previously reported [Valle et al., Gene 23 (1983) 199-209; 27 (1984) 193-199]. In this paper we present data on the GDH specific activities using both excess and limiting concentrations of ammonia as nitrogen sources. Evidence is presented on the regulation of the mRNA levels for this enzyme by the ammonia concentration in the growth medium. We have identified a single and apparently invariant transcript for several metabolic growth conditions. We also report the identification of a functional promoter and the corresponding transcription start point under several growth conditions. Finally, possible regulatory sequences located at the 5' flanking region of the gdhA gene are discussed.

Repetitive extragenic palindromic (REP) sequences in the Escherichia coli gdhA gene.

Deletions of the 3' flanking DNA region of the glutamate dehydrogenase (GDH) structural gene from Escherichia coli K-12, have been produced on a plasmid that carries the complete gdhA gene. Those deletions include part of the repetitive extragenic palindromic (REP) sequences proposed by Stern et al. [Cell 37 (1984) 1015-1026], as a novel and major feature of the bacterial genome. The effect of these deletions on the final GDH level in the cell, has been determined. A broader compilation, analysis and alternative functions of the REP sequences, is also presented.

Cloning and characterization of cDNAs that code for Na(+)-channel-blocking toxins of the scorpion Centruroides noxius Hoffmann.

With the purpose of studying the organization and characteristics of the genes that code for toxins present in the venom of the Mexican scorpion, Centruroides noxius Hoffmann (CnH), we prepared a lambda gt11 cDNA library from the venom glands. Using specific oligodeoxyribonucleotides (oligos) designed according to known amino acid (aa) sequences of CnH toxins (STox), we detected several positive clones, determined their nucleotide (nt) sequences and deduced their aa sequences. A comparative analysis of these sequences with previously reported STox revealed that CnH cDNAs code for a family of very similar STox. The cDNA coding for a known STox, II-10, was cloned. Additionally, three other complete (new) nt sequences were obtained for cDNAs encoding peptides similar to STox 1 from CnH or variants 2 and 3 from Centruroides sculpturatus Ewing. Southern blot genomic DNA analysis showed a minimum size of approximately 600 bp as EcoRI fragments for elements of this family. PCR amplifications of CnH genomic DNA and hybridization of PCR products with specific probes indicated that the genomic structural regions that code for these genes do not contain introns, or at least not large introns.

Complete nucleotide sequence of the glutamate dehydrogenase gene from Escherichia coli K-12.

A 2.3-kb PstI- ClaI chromosomal DNA segment, carrying the complete coding region of the glutamate dehydrogenase (GDH) structural gene from Escherichia coli K-12, has been sequenced. The complete amino acid sequence (447 residues) of the GDH monomer has been deduced, and comparisons are made with reported amino acid sequences of GDH from other organisms.

Determination of the nucleotide sequence for the glutamate synthase structural genes of Escherichia coli K-12.

We have determined the complete nucleotide sequence of a 6.3-kb chromosomal HpaI-EcoRI fragment, that contains the structural genes for both the large and small subunits of the Escherichia coli K-12 glutamate synthase (GOGAT) enzyme, as well as the 5'- and 3'-flanking and intercistronic DNA regions. The Mrs of the two subunits, as deduced from the nucleotide (nt) sequence, were estimated as 166,208 and 52,246. Partial amino acid sequence of the GOGAT enzyme revealed that the large subunit starts with a cysteine residue that is probably generated by a proteolytic cleavage. Northern blotting experiments revealed a transcript of approximately 7300 nt, that at least contains the cistrons for both subunits. A transcriptional start point and a functional promoter were identified in the 5' DNA flanking region of the large subunit gene. The messenger RNA nontranslated leader region has 120 nt and shares identity with the leader regions of E. coli ribosomal operons, in particular around the so-called boxA sequence implicated in antitermination. Other possible regulatory sequences are described.

Phospholipin, a novel heterodimeric phospholipase A2 from Pandinus imperator scp6pion venom.

The primary structure of a phospholipase A2, with unique structural and functional characteristics, was determined. The large subunit has 108 amino acid residues, linked by a disulfide bridge to the small subunit, which contains 17 residues. Its gene was cloned from a cDNA library. The nucleotide sequence showed that the same RNA messenger encodes both subunits, separated only by a pentapeptide, that is processed during maturation.

Primary structure determination and cloning of the cDNA encoding toxin 4 of the scorpion Centruroides noxius Hoffmann.

A peptide (toxin II-10), shown to be a Na+ channel blocker, was purified from the venom of the scorpion Centruroides noxius Hoffmann and sequenced by Edman degradation. It has 66 amino acid residues with the C-terminal residue (asparagine) amidated, as demonstrated by mass spectrometry. In addition, we report the cloning and the nucleotide sequence of the cDNA (CngtV) that codes for this toxin. We discuss the mechanism for processing the precursor peptide to its final form and compare the primary structure to that of other Na+ channel toxins. Two distinct groups of toxins seem to emerge from this comparison, suggesting a structure-function relationship of these peptides towards the recognition of either mammalian or insect tissues.

Site directed mutants of Noxiustoxin reveal specific interactions with potassium channels.

Several site directed mutations were introduced into a synthetic Noxiustoxin (NTX) gene. Alanine scanning of the nonapeptide at the N-terminal segment of NTX (threonine 1 (T1) to serine 9 (S9)) was constructed and the recombinant products were obtained in pure form. Additionally, lysine 28 (K28) was changed to arginine (R) or glutamic acid (E), cysteine 29 was changed to alanine, and residues 37-39 (Tyr-Asn-Asn) of the carboxyl end were deleted. The recombinant mutants were tested for their ability to displace 125I-NTX from rat brain synaptosome membranes, as well as for their efficiency in blocking the activity of Kv1.1 K+ channels expressed in Xenopus laevis oocytes. The main results indicate that residues K6, T8 at the amino end, and K28 and the tripeptide YNN at the carboxyl end are involved in specific interactions of NTX with rat brain and/or Kv1.1 K+ channels.

The genomic region encoding toxin gamma from the scorpion Tityus serrulatus contains an intron.

The gene encoding toxin gamma from the scorpion, Tityus serrulatus, was amplified by PCR from genomic DNA employing synthetic oligonucleotides designed from the reported cDNA sequence. The nucleotide sequence of this gene reveals the presence of an intron of 475 base pairs (bp) which interrupts the region that encodes the signal peptide of the precursor toxin. A comparison of the intron boundary sequences of the gamma toxin gene with ones from other arachnid genes is also presented.

Peptides and genes coding for scorpion toxins that affect ion-channels.

Most scorpion toxins are ligand peptides that recognize and bind to integral membrane proteins known as ion-channels. To date there are at least 202 distinct sequences described, obtained from 30 different species of scorpions, 27 from the family Buthidae and three from the family Scorpionidae. Toxins that recognize potassium and chloride channels are usually from 29 to 41 amino acids long, stabilized by three or four disulfide bridges, whereas those that recognize sodium channels are longer, 60 to 76 amino acid residues, compacted by four disulfide bridges. Toxins specific for calcium channels are scarcely known and have variable amino acid lengths. The entire repertoire of toxins, independently of their specificity, was analyzed together by computational programs and a phylogenetic tree was built showing two separate branches. The K(+) and Cl(-) channel specific toxins are clustered into 14 subfamilies, whereas those of Na(+) and Ca(2+) specific toxins comprise at least 12 subfamilies. There are clear similarities among them, both in terms of primary sequence and the main three-dimensional folding pattern. A dense core formed by a short alpha helix segment and several antiparallel beta-sheet stretches, maintained by disulfide pairing, seems to be a common structural feature present in all toxins. The physiological function of these peptides is manifested by a blockage of ion passage through the channels or by a modification of the gating mechanism that controls opening and closing of the ion pore.

Toxins and genes isolated from scorpions of the genus Tityus.

Scorpion venoms contain a variety of low mol. wt peptides toxic to different organisms. These peptides have been intensively studied because they represent excellent models for investigating structure-function relationships and they are also fine probes for studying ionic channel functions. This review deals with the biological and chemical aspects of toxic peptides that affect Na+ or K+ channels and the cloning of the cDNAs and genes encoding the main alpha and beta neurotoxins present in the venom of the three most dangerous species of Brazilian scorpion, Tityus bahiensis, Tityus stigmurus and Tityus serrulatus, and the Venezuelan scorpion Tityus discrepans. At least 16 different peptides specific for Na+ channels and five affecting K+ channels were isolated and characterized from the venom of these scorpions. The isolation of cDNAs and genes encoding four distinct toxins has permitted the elucidation of their nucleotide sequences as well as their genomic organization. Venoms and isolated toxins from scorpions of the genus Tityus were shown to enhance the secretory activity of the pancreas. Antisera obtained against venom of T. serrulatus show cross-reactivity with other species of the Brazilian scorpions.

Fab fragments of the monoclonal antibody BCF2 are capable of neutralizing the whole soluble venom from the scorpion Centruroides noxius Hoffmann.

BCF2 is a murine hybridoma cell line that produces a neutralizing antibody against toxin 2 (Cn2) from the scorpion Centruroides noxius Hoffmann of Mexico. In this communication we report the preparation and use of the BCF2 antibody and its antigen binding fragments (Fab) in experiments aiming at obtaining protection of experimental albino mice (strain CD1) challenged with purified toxin Cn2, as well as, with whole soluble venom from C. noxius. The monoclonal antibody BCF2 in amounts of 1 mg neutralizes 28 LD50 of soluble venom of C. noxius, whereas the Fab fragments of BCF2 (1 mg) are capable of neutralizing 43 LD50 dose of the same venom. To reach the same level of neutralization, with the commercially available horse antiserum [F(ab')2], we need to use about ninefold more material.

Cloning and characterization of the genomic region encoding toxin IV-5 from the scorpion Tityus serrulatus Lutz and Mello.

By means of PCR and using synthetic oligonucleotides designed from the reported cDNA, we amplified the gene that codes for toxin IV-5 from the Brazilian scorpion Tityus serrulatus. The analysis of the nucleotide sequence shows that the amplified genomic region is composed of 659 base pairs (bp) comprising two exons (28 and 284 bp) and an intron of 347 bp interrupting the region that encodes the signal peptide of the precursor toxin. Based on these findings a model for the structural organization of scorpion toxin genes is proposed.

Synthesis and expression of the gene coding for noxiustoxin, a K+ channel-blocking peptide from the venom of the scorpion Centruroides noxius.

A set of six synthetic overlapping oligonucleotides coding for noxiustoxin were coupled into a continuous DNA fragment by means of recursive polymerase chain reaction. The polymerase chain reaction product was digested with SalI and HindIII, ligated into the E, coli vector pCSP 105 and expressed as a fusion protein. The fusion protein was purified and digested with trypsin and the hydrolysis products were separated by high-performance liquid chromatography. Approximately 1.3 mg of recombinant noxiustoxin per liter of culture was obtained. Amino acid analysis and N-terminal amino acid sequence of the recombinant noxiustoxin confirmed the nucleotide sequence of the cloned DNA. Binding experiments using rat brain synaptosomal membranes revealed that recombinant noxiustoxin displaced bound radioactive native NTX with a similar efficiency to cold native noxiustoxin.

Cloning and characterization of the cDNAs encoding Na+ channel-specific toxins 1 and 2 of the scorpion Centruroides noxius Hoffmann.

Using a cDNA library prepared from venomous glands of the Mexican scorpion Centruroides noxius Hoffmann the genes that encode toxins 1 and 2 were identified, cloned and sequenced. In view of the proposed mechanism for processing the mature peptides coded by these two genes, the corresponding peptide-toxins were sequenced de novo. Mass spectrometric and 1H-NMR analyses of the C-terminal peptide produced by enzymatic digestion of both toxins indicated that the last residue is serine-amide. Sequence comparison revealed that these two genes have a similarity of 56% and 80% at the amino acid and nucleotide levels, respectively. Small corrections to the published primary structures were introduced: Cn toxin 1 has an extra serine residue at position 65 and the residue in position 60 is a proline, while the amino acids at positions 34 and 35 of Cn 2 are, respectively, tyrosine and glycine. Sequence comparison of toxins from the genus Centruroides suggests the presence of at least three classes of distinct peptides in these venoms.

Isolation, characterization and comparison of a novel crustacean toxin with a mammalian toxin from the venom of the scorpion Centruroides noxius Hoffmann.

A novel crustacean-specific toxin, Cn5, containing 66 amino acid residues was isolated from the venom of the scorpion Centruroides noxius Hoffmann. It is stabilized by four disulfide bridges, formed between Cys12-Cys65, Cys16-Cys41, Cys25-Cys46 and Cys29-Cys48. Toxicity tests revealed that Cn5 is a toxin that affects arthropods but not mammals. However, at high concentrations, Cn5 does displace the mammal-specific toxin Cn2 from rat brain synaptosomes. The concentration of Cn5 that produces half-maximal inhibition (IC50) was estimated to be 100 microM. Sequence comparison of Cn5 with toxin Cn2, a mammal-specific toxin from the same scorpion, showed the presence of two sequence stretches, at positions 30 to 38 and 49 to 58, where the majority of the differences are concentrated. On the three-dimensional structure of Cn5 it is demonstrated that these two sequence stretches form a continuous surface region near the site thought to bind to the sodium channel. We assume that this region might be implicated in determining species specificity.