Structural and functional properties of the Kunitz-type and C-terminal domains of Amblyomin-X supporting its antitumor activity.

Amblyomin-X is a Kunitz-type FXa inhibitor identified through the transcriptome analysis of the salivary gland from Amblyomma sculptum tick. This protein consists of two domains of equivalent size, triggers apoptosis in different tumor cell lines, and promotes regression of tumor growth, and reduction of metastasis. To study the structural properties and functional roles of the N-terminal (N-ter) and C-terminal (C-ter) domains of Amblyomin-X, we synthesized them by solid-phase peptide synthesis, solved the X-Ray crystallographic structure of the N-ter domain, confirming its Kunitz-type signature, and studied their biological properties. We show here that the C-ter domain is responsible for the uptake of Amblyomin-X by tumor cells and highlight the ability of this domain to deliver intracellular cargo by the strong enhancement of the intracellular detection of molecules with low cellular-uptake efficiency (p15) after their coupling with the C-ter domain. In contrast, the N-ter Kunitz domain of Amblyomin-X is not capable of crossing through the cell membrane but is associated with tumor cell cytotoxicity when it is microinjected into the cells or fused to TAT cell-penetrating peptide. Additionally, we identify the minimum length C-terminal domain named F2C able to enter in the SK-MEL-28 cells and induces dynein chains gene expression modulation, a molecular motor that plays a role in the uptake and intracellular trafficking of Amblyomin-X.

Isolation and pharmacological characterization of AdTx1, a natural peptide displaying specific insurmountable antagonism of the alpha1A-adrenoceptor.

BACKGROUND AND PURPOSE: Venoms are a rich source of ligands for ion channels, but very little is known about their capacity to modulate G-protein coupled receptor (GPCR) activity. We developed a strategy to identify novel toxins targeting GPCRs. EXPERIMENTAL APPROACH: We studied the interactions of mamba venom fractions with alpha(1)-adrenoceptors in binding experiments with (3)H-prazosin. The active peptide (AdTx1) was sequenced by Edman degradation and mass spectrometry fragmentation. Its synthetic homologue was pharmacologically characterized by binding experiments using cloned receptors and by functional experiments on rabbit isolated prostatic smooth muscle. KEY RESULTS: AdTx1, a 65 amino-acid peptide stabilized by four disulphide bridges, belongs to the three-finger-fold peptide family. It has subnanomolar affinity (K(i)= 0.35 nM) and high specificity for the human alpha(1A)-adrenoceptor subtype. We showed high selectivity and affinity (K(d)= 0.6 nM) of radio-labelled AdTx1 in direct binding experiments and revealed a slow association constant (k(on)= 6 x 10(6).M(-1).min(-1)) with an unusually stable alpha(1A)-adrenoceptor/AdTx1 complex (t(1/2diss)= 3.6 h). AdTx1 displayed potent insurmountable antagonism of phenylephrine's actions in vitro (rabbit isolated prostatic muscle) at concentrations of 10 to 100 nM. CONCLUSIONS AND IMPLICATIONS: AdTx1 is the most specific and selective peptide inhibitor for the alpha(1A)-adrenoceptor identified to date. It displays insurmountable antagonism, acting as a potent relaxant of smooth muscle. Its peptidic nature can be exploited to develop new tools, as a radio-labelled-AdTx1 or a fluoro-labelled-AdTx1. Identification of AdTx1 thus offers new perspectives for developing new drugs for treating benign prostatic hyperplasia.

Structure-based secondary structure-independent approach to design protein ligands: Application to the design of Kv1.2 potassium channel blockers.

We have developed a structure-based approach to the design of protein ligands. This approach is based on the transfer of a functional binding motif of amino acids, often referred as to the "hot spot", on a host protein able to reproduce the functional topology of these residues. The scaffolds were identified by a systematic in silico search in the Protein Data Bank for proteins possessing a group of residues in a topology similar to that adopted by the functional motif in a reference ligand of known 3D structure. In contrast to previously reported studies, this search is independent of the particular secondary structure supporting the functional motif. To take into account the global properties of the host protein, two additional criteria were taken into account in the selection process: (1) Only those scaffolds sterically compatible with the positioning of the functional motif as observed in a reference complex model were retained. (2) Host proteins displaying electrostatic potentials, in the region of the transferred functional motif, similar to that of the reference ligand were selected. This approach was applied to the development of protein ligands of the Kv1.2 channel using BgK, a small protein isolated from the sea anemone Bunodosoma granulifera, as the reference ligand. Four proteins obtained by this approach were produced for experimental evaluation. The X-ray structure of one of these proteins was determined to check for similarity of the transferred functional motif with the structure it adopts in the reference ligand. Three of these protein ligands bind the Kv1.2 channel with inhibition constants of 0.5, 1.5, and 1.6 microM. Several mutants of these designed protein ligands gave binding results consistent with the presumed binding mode. These results show that protein ligands can be designed by transferring a binding motif on a protein host selected to reproduce the functional topology of this motif, irrespective to the secondary structure supporting the functional motif, if the host protein possesses steric and electrostatic properties compatible with the binding to the target. This result opens the way to the design of protein ligands by taking advantage of the considerable structural repertoire of the Protein Data Bank.

Relative spatial position of a snake neurotoxin and the reduced disulfide bond alpha (Cys192-Cys193) at the alpha gamma interface of the nicotinic acetylcholine receptor.

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.

Chemical engineering of a three-fingered toxin with anti-alpha7 neuronal acetylcholine receptor activity.

Though it possesses four disulfide bonds the three-fingered fold is amenable to chemical synthesis, using a Fmoc-based method. Thus, we synthesized a three-fingered curaremimetic toxin from snake with high yield and showed that the synthetic and native toxins have the same structural and biological properties. Both were characterized by the same 2D NMR spectra, identical high binding affinity (K(d) = 22 +/- 5 pM) for the muscular acetylcholine receptor (AChR) and identical low affinity (K(d) = 2.0 +/- 0.4 microM) for alpha7 neuronal AchR. Then, we engineered an additional loop cyclized by a fifth disulfide bond at the tip of the central finger. This loop is normally present in longer snake toxins that bind with high affinity (K(d) = 1-5 nM) to alpha7 neuronal AchR. Not only did the chimera toxin still bind with the same high affinity to the muscular AchR but also it displayed a 20-fold higher affinity (K(d) = 100 nM) for the neuronal alpha7 AchR, as compared with the parental short-chain toxin. This result demonstrates that the engineered loop contributes, at least in part, to the high affinity of long-chain toxins for alpha7 neuronal receptors. That three-fingered proteins with four or five disulfide bonds are amenable to chemical synthesis opens new perspectives for engineering new activities on this fold.

How do snake curaremimetic toxins discriminate between nicotinic acetylcholine receptor subtypes.

Curaremimetic toxins from snake venoms form a large family of small proteins that adopt a similar fold and which bind to Torpedo nicotinic acetylcholine receptors with high affinity. Notwithstanding its apparent homogeneity, the toxin family is subdivided into short-chain (60-62 residues and four disulfide bonds) and long-chain toxins (66-74 residues and five disulfide bonds). In agreement with this structurally-based distinction we recently showed that only long-chain toxins bind with high affinity to the neuronal nicotinic acetylcholine alpha7 receptor. We suggested that a small loop cyclized by a disulfide bond and uniquely present in long-chain toxins may act as a major discriminative element. To assess the validity of this proposal we prepared various derivatives of a long-chain toxin, using stepwise solid-phase synthesis. We found that replacement of both half cystines of the small loop by a serine caused a 35-fold affinity decrease for the neuronal receptor and only a 6-fold affinity decrease for Torpedo receptor. In addition, insertion of this loop at a homologous position of a short-chain toxin caused a 20-fold affinity increase for the neuronal receptor whereas it did not modify its affinity for the Torpedo receptor. Our findings, therefore, reveal that a small structural deviation from a toxin fold can generate exquisite discriminative recognition for some receptor subtypes.

On the immunogenic properties of retro-inverso peptides. Total retro-inversion of T-cell epitopes causes a loss of binding to MHC II molecules.

Retro-inversion is considered an attractive approach for drug and vaccine design since it provides the modified peptides with higher resistance to proteolytic degradation. We therefore investigated in detail the effect of retro-inversion on the immunological properties of synthetic peptides. We have synthesized retro-inverso analogues of MHC II restricted peptides that thus contained the correct orientation of the side chains but an inverse main chain. Retro-inversion made the peptides unable to compete in I E(d) or I A(d) binding tests, demonstrating a very low, if any, capacity to bind to MHC II molecules. These results confirm previous structural data that hydrogen bonds between residues of MHC II molecules and the main chain of antigenic peptides play a major interacting role. In vito experiments further showed that retro-inversion of a T-cell epitope causes its inability to either sustain in vitro T-cell stimulation or to prime specific T cells. Moreover, the retro-inverso peptide was not recognized by antibodies raised against the native peptide and did not elicit antibodies when injected into BALB/c mice. Retro-inverso peptides appear to be poor immunogens as a result of their weak capacity to bind to MHC II molecules. As an advantage, they are not expected to trigger undesirable humoral responses such as hypersensitivity or allergic disease. These results also provide a molecular explanation regarding the weak immunogenicity of D-amino acids containing polypeptides.

Fine chemical modifications at N- and C-termini enhance peptide presentation to T cells by increasing the lifespan of both free and MHC-complexed peptides.

We investigated the effect of modifying the N- and/or C-termini of the snake toxin peptide 24-36 on its presentation to T cells. Acetylation at the N-terminus as well as amidation at the C-terminus enhanced the capacity of the peptide to activate T cells. Simultaneous modifications further increased the stimulating activity, the peptide becoming approximately 100-fold more potent than the unmodified peptide. Clearly, the introduced modifications increased the lifetime of the peptide free in solution, by decreasing its proteolytic degradation, during the T cell stimulation assays. Paradoxically, however, at similar concentrations of free peptides, the modified ones, especially those having an acetylated N-terminus, were much more active than the unmodified peptide, irrespective of the experimental conditions. These observations suggested that components other than protection from proteolytic degradation should be associated with the higher stimulating activities of the modified peptides. Accordingly, chasing experiments with APC revealed that acetylation at N-terminus caused a higher persistence of the peptides at APC surface. Together, our data indicate that (i) the T cell stimulating capacity of a peptide is associated with its lifespans in the free and MHC II bound states; and (ii) these lifespans can be greatly enhanced by introducing fine chemical modifications at N- and C-termini. These data may have some implications in designing more potent peptidic immunomodulators.

Probing immunogenicity of a T cell epitope by L-alanine and D-amino acid scanning.

All residues of the I-Ed restricted fragment 24-36 of a snake toxin were individually changed into L-alanine and the corresponding D-enantiomer. Four analogs substituted with L-Ala at positions 25;30, 31 and 33, and nine analogs substituted with a D-residue along the stretch 25-33 lost most (position 28) or all their capacity to stimulate a toxin-specific T hybridoma. None of these analogs stimulated splenocytes from mice immunized with the peptide 24-36. Only the L-A31 and D-W29 modified analogs could prime a T cell response which, however, showed no cross-reactivity with the native peptide, demonstrating that T cell response selectivity can be deeply modified by mutation or configuration inversion of a single residue. Our data suggest that (i) the region 25-33 is the core of the T epitope that binds to I-Ed, and (ii) Y25 R30 and R33 contribute to the peptide binding by anchoring into pockets of I-Ed. In agreement with T cell priming observations, only the L-A31 and D-W29 modified analogs elicited strong antibody responses, just like the peptide 24-36, whereas nearly all other analogs were less immunogenic. All but the L-Ala30 and L-Ala33 modified analogs were recognized by a 24-36 specific antiserum as well as the native peptide. Altogether, our results show that substitution by D-amino acid in a peptide could be particularly well-suited for either minimizing the risk of hypersensitivity or designing peptidic vaccines.

Immunogenicity of T epitope-containing cyclic peptides. Increasing neutralizing antibody responses by introducing fine chemical changes.

We showed previously that the disulfide-containing T peptide 24-41 C from a highly structured snake toxin elicits, in a free state, Abs that neutralize the toxin, and only a turn structure commonly exists in 24-41 C and the corresponding toxin region. To tentatively increase the neutralizing capacity of antipeptide Abs, we 1) replaced Gly-40 by an aminoisobutyric moiety (24-41 Aib), 2) substituted the half cystines 24 and 41 by penicillamine moieties (24-41 Pen), and 3) introduced an amide bond between the epsilon NH2 of Lys-27 and the gamma-COOH of Glu-38 (24-41 K-E). A solution ELISA made with antitoxin Abs revealed that 24-41 Pen is more antigenic than 24-41 Aib and 24-41 C, which are more antigenic than 24-41 K-E, suggesting that the conformation of 24-41 Pen is most closely related to the corresponding region in the native toxin. The peptides 24-41 Pen, 24-41 Aib, and 24-41 C stimulate T cells from BALB/c mice, whereas 24-41 K-E has lost this property and thereby fails to elicit Abs. Finally, anti-24-41 Pen Abs are more potent at neutralizing the native toxin than anti-24-41 C Abs, which are more potent than anti-24-41 Aib Abs. The efficacy of anti-24-41 Pen Abs was similar to that of a toxin specific mAb. Therefore, introduction of appropriate constraints makes it possible to improve the neutralizing Ab response raised by a synthetic peptide. Such observations should be of interest for the design of efficient synthetic vaccines.

Immunogenicity of a disulphide-containing neurotoxin: presentation to T-cells requires a reduction step.

It is known that production in a host of antibodies against a protein is associated with various molecular events. These include the stimulation of specific T-lymphocytes, a step that implies the processing of the protein into peptides by various endosomal/lysosomal enzymes, such as cathepsins. Strikingly, however, we observed in vitro that cathepsins B and D have no degrading effect on toxin alpha from Naja nigricollis, a curaremimetic toxin of 61 amino acids and four disulphides. In sharp contrast, the enzymes exert an efficient cleavage of the toxin polypeptide chain once the toxin disulphides are reduced. We also found that the fully reduced toxin and the native toxin were presented with comparable efficiency to two different T-hybridomas by antigen-presenting cells (APC). Together, the data suggest that presentation of toxin fragments to T-cells requires a reduction step of toxin disulphides and, in agreement with previous findings, that this step may be achieved by APC. We wish to suggest that this phenomenon may commonly occur for any toxic proteins that contain disulphides.

Boc-Cys(Npys)-OH (BCNP): an appropriate reagent for the identification of T cell epitopes in cystine and/or cysteine-containing proteins.

Some T cell epitopes become inactive when their thiols are blocked with various irreversible reagents (Régnier-Vigouroux, 1988; Maillère, 1992; Maillère et al., 1993). Blocking protein and peptide thiols with BCNP (Boc-Cys(Npys)-OH) constitutes a most appropriate strategy when searching for thiol-containing T cell epitopes. Free cysteines can thus be readily transformed into disulphide-like moieties which not only resist undesirable oxidative reactions but which also remain susceptible to reduction by antigen presenting cells, a prerequisite for the activity of thiol-dependent T cell epitopes. We describe the use of this reagent in a study of the intact disulphide-rich protein, toxin alpha from Naja nigricollis, and also two disulphide-containing toxin fragments.

Interaction of protein ligands with receptor fragments. On the residues of curaremimetic toxins that recognize fragments 128-142 and 185-199 of the alpha-subunit of the nicotinic acetylcholine receptor.

Using a solid-phase assay, we found that 3H-labeled alpha Cobtx from Naja naja siamensis, a long-chain curaremimetic toxin, and 3H-labelled toxin alpha from Naja nigricollis, a short-chain toxin both bind specifically but with substantially different affinities (Kd = 4 x 10(-7) M and 50 x 10(-6) M) to fragment 185-199 (T alpha 185-199) of the alpha-subunit of the acetylcholine receptor (AcChoR) from Torpedo marmorata. Then we show that monoderivatizations of residues common to both long-chain and short-chain toxins (Tyr-25, Lys-27, Trp-29, and Lys-53) or to long-chain toxins only (Cys-30 and Cys-34) do not affect the binding of the toxins to T alpha 185-199, suggesting that none of these invariant residues in implicated in the recognition of this AcChoR region. alpha Cobtx and toxin alpha bind to the fragment 128-142 (T alpha 128-142) with more similar affinities (Kd = 3 x 10(-7) M and 1.4 x 10(-6) M) and their binding is dramatically affected by the single abolition of the positive charge of Lys-53, an invariant residue that contributes to AcChoR recognition. Therefore, the data indicate that Lys-53 more specifically recognizes the 128-142 region of AcChoR. Other monoderivatizations have no effect on toxin binding. The approach described in this paper may be of great help to identify toxin residues that establish direct contact with receptor fragments.

Role of thiols in the presentation of a snake toxin to murine T cells.

We isolated and characterized two T hybridomas specific for a highly stable snake toxic protein. One hybridoma, called T1C9, is I-E(d)-restricted and stimulated by both the native and reduced and carboxymethylated (RCM) toxins and by synthetic fragments containing the region 24-36. The other hybridoma, called T1B2, is I-A(d)-restricted and stimulated by the native toxin, only. Neither the RCM toxin nor any of the initial synthetic peptides used in our study could stimulate it. We show that this lack of effect is associated with the presence, in the epitope-containing fragment, of irreversible blocking groups on cysteine residues. Indeed, when the fragment 32-49 has its cysteines involved in either intra-(32-49SS) or mixed disulfides, a stimulation of T1B2 was observed. Fixed APC do not present native toxin to either hybridomas but present RCM toxin to T1C9. Strikingly, fixed APC present the peptide 32-49SS to T1B2; however, we show that this is possible only because the peptide disulfide is reduced. The thiol dependence of this epitope suggests that the native toxin can stimulate T1B2 only after disulfide reduction. This reaction may constitute a major step during the processing of the toxin and more generally of any disulfide-containing Ag.

Studies on immunoassays of peptide factors. II. Fluorescence enzyme immunoassay for human little-gastrin.

The fluorogenic chymotrypsin substrate N alpha-(4-carboxybutyryl)-L-phenylalanine (4-methyl-7-coumaryl)amide was converted to a thiol-containing compound via its condensation at the carboxyl function with cystamine followed by reduction of the resulting disulfide compound to a cysteamine derivative. By subsequent reaction of the thiol group with N alpha-maleoyl-beta-alanyl-human-little-gastrin-I-[2-17] a fluorogenic substrate-labeled gastrin, fully immunoreactive against antigastrin antisera, was obtained. This tracer was then applied for developing a fluorescence immunoassay based on separation of bound and free tracer followed by chymotryptic digestion of the fluorogenic substrate in the supernatant. The fluorescence intensity of the extracted fluorophore i.e., 7-amino-4-methyl-coumarin, was found to monitor gastrin concentrations in a reproducible manner. With the model peptide hormone human little-gastrin-I the sensitivity of this alternative immunoassay procedure was well documented.

Studies on immunoassays of peptide factors. IV. New synthesis of a gastrin/peroxidase conjugate.

We have shown that structurally well-defined homogeneous maleoyl-peptides are synthetically accessible. These anchor-modified peptide derivatives allow their selective covalent linkage to thiol-containing proteins via the maleimide-thiol procedure. Correspondingly mercaptosuccinylated horseradish peroxidase was reacted with N alpha-maleoyl-beta-alanyl-human-little gastrin-I-[2-17] to produce the gastrin/peroxidase conjugate in good yields at 1:1 stoichiometry. The conjugate exhibited full enzymatic activity and identical binding affinity to antigastrin antisera as the parent gastrin. This approach proved to be well suited for the preparation of enzyme labeled peptide factors as tracers for immunoassays.

Studies on immunoassays of peptide factors. V. Synthesis of cholecystokinin-58-[1-11]/iso-1-cytochrome c conjugate.

In previous studies on model compounds we have found that the maleimide function is sufficiently stable under the conditions of peptide synthesis to allow its incorporation at preselected positions of a peptide chain in earlier steps of the synthetic route. Taking advantage of this observation the N-terminal undecapeptide of canine cholecystokinin-58 containing at its N-terminus the maleimide group became accessible in high yields as chromatographically homogenous and analytically well characterized compound. Via the incorporated anchor group the undecapeptide was linked selectively at its N-terminus to the cysteine residue 107 of iso-1-cytochrome c to yield a well characterized conjugate of 1:1 stoichiometry for immunization experiments.

Studies on immunoassays of peptide factors. III. Gastrin/iso-1-cytochrome C as immunogen for raising anti-gastrin antisera.

Iso-1-cytochrome c contains in penultimate position of its sequence a cysteine residue which in analogy to the known tertiary structures of cytochromes c should be exposed on the surface of the protein. Its selective reaction with N alpha-maleoyl-beta-alanyl-human-little-gastrin-I-[2-17] led to a well characterized and homogeneous gastrin conjugate to be used as immunogen in rabbits. The antisera raised by this procedure exhibited a degree of specificity for the hormone gastrin parallel to that of the gastrin receptor. This is clearly documented by comparison of the immune crossreactivities of gastrin-peptides of increasing chain length and of fragments corresponding to various regions of the hormone molecule with their biological activity. The immune response provoked in the animals by the use of an homogeneous immunogen was found to be highly reproducible in terms of specificity of the antigastrin antibodies.

4-aminobutyric acid methyl ester hydrochloride, a precursor of 4-aminobutyric acid.

4-Aminobutyric methyl ester hydrochloride (GME) is able to cross the blood-brain barrier after intracardiac administration to the rat. GME has an LD50 of 1300 mg/kg in mice and 950 mg/kg in rats, exhibits an antiaggressive effect and is able to decrease isoniazid-induced convulsions in the rat. GME is hydrolyzed to 4-aminobutyric acid (GABA) by brain homogenates, acts as an inhibitor of GABA binding to crude synaptic plasma membranes, activates the release and inhibits the uptake of GABA by rat synaptosomes and acts as a competitive inhibitor of the so-called GABAse system in vitro.

Pharmacokinetics and in vitro effects of a 4-aminobutyric acid derivative with anticonvulsant action.

N-trimethyl-acetyl-4-aminobutyric acid, called PG2, is a new anticonvulsant acting as a 4-aminobutyric acid (GABA) pro-drug. The stimulatory effect of PG2 on synaptosomal 14C-GABA release and its inhibitory effect of synaptosomal 14C-GABA uptake were studied together with its pharmacokinetics after intracardiac and oral administration to rat. PG2 is a weak inhibitor of the GABAse system in vitro with an apparent Ki value of 0.83 mmol/l.