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.

Highly specific anti-estradiol antibodies: structural characterisation and binding diversity.

Subtle modulation of antibody-binding properties by protein engineering often lies with an accurate structural and energetic description of how an antigen is recognised. Thus, with the intent to increase the affinity and add a bias in favour of natural estradiol compared with its chemically modified immunogen, we have determined the crystal structure of two anti-estradiol monoclonal antibodies, 10G6D6 and 17E12E5. Although generated against the same estradiol derivative, these antibodies share little sequence identity, which is reflected in dissimilar binding pockets and in different positioning of the steroid. In both antibodies the characteristic 17-hydroxyl group is buried deeply at the bottom of hydrophobic pockets and stabilised by hydrogen bonds. Apart from this similarity, the steroid is oriented differently in the respective binding pockets. The high specificity of both antibodies has been mapped out, and even closely related steroids show low cross-reactivity. The structural studies of the complex formed between 10G6D6 and 6-CMO-estradiol have identified contacts between the 6-CMO coupling linker and an arginine residue from the heavy chain CDR2 segment. This segment is now being targeted by random mutagenesis to select mutants with a preference for natural estradiol compared to the branched hapten.

Slow folding of three-fingered toxins is associated with the accumulation of native disulfide-bonded intermediates.

Snake neurotoxins are short all-beta proteins that display a complex organization of the disulfide bonds: two bonds connect consecutive cysteine residues (C43-C54, C55-C60), and two bonds intersect when bridging (C3-C24, C17-C41) to form a particular structure classified as "disulfide beta-cross". We investigated the oxidative folding of a neurotoxin variant, named alpha62, to define the chemical nature of the three-disulfide intermediates that accumulate during the process in order to describe in detail its folding pathway. These folding intermediates were separated by reverse-phase HPLC, and their disulfide bonds were identified using a combination of tryptic hydrolysis, manual Edman degradation, and mass spectrometry. Two dominant intermediates containing three native disulfide bonds were identified, lacking the C43-C54 and C17-C41 pairing and therefore named des-[43-54] and des-[17-41], respectively. Both species were individually allowed to reoxidize under folding conditions, showing that des-[17-41] was a fast-forming nonproductive intermediate that had to interconvert into the des-[43-54] isomer before forming the native protein. Conversely, the des-[43-54] intermediate appeared to be the immediate precursor of the oxidized neurotoxin. A kinetic model for the folding of neurotoxin alpha62 which fits with the observed time-course accumulation of des-[17-41] and des-[43-54] is proposed. The effect of turn 2, located between residues 17 and 24, on the overall kinetics is discussed in view of this model.

Complementarity and redundancy of the binding specificity of HLA-DRB1, -DRB3, -DRB4 and -DRB5 molecules.

The second HLA-DR molecules, which are encoded by loci different from HLA-DRB1 are weakly polymorphic. Predominant alleles such as HLA-DRB3*0101, HLA-DRB4*0101 and HLA-DRB5*0101 are therefore interesting targets to define antigenic peptides with major impact for the entire population. Strikingly, they have been poorly investigated. Thus we have characterized peptides from the major bee venom allergen that bind efficiently to these molecules and compared them to peptides specific for preponderant HLA-DRB1 molecules. Interestingly, DRB5*0101 and DRB1*0701 molecules share four binding peptides and use some identical anchor residues. Similarities are also found between DRB3*0101 and its haplotype-associated molecules DRB1*0301 and DRB1*1301. In sharp contrast, DRB4*0101 exhibits a unique binding specificity, which results from particular structural features of its peptide binding site. Ybeta81 seems to alter the amino acid preferences of the P1 pocket, while Rbeta71, Ebeta74, Nbeta26 and Cbeta13 confer to the P4 pocket a unique topology. Our results show that the two HLA-DR molecules expressed in most haplotypes studied here have mostly complementary binding patterns. Only haplotype HLA-DR52 exhibits peptide binding redundancies. Finally our results document functional similarities among HLA-DR molecules and allow us to propose peptide sequences that might be useful for bee venom immunotherapy.

Functional characterization of two anti-estradiol antibodies as deduced from modelling and site-directed mutagenesis experiments.

Monoclonal antibodies are now widely used to measure the concentration of steroid hormones in human serum samples. The great development of molecular engineering techniques over the past 10 years has made possible the improvement of specificity and/or sensitivity of selected antibodies. We have obtained two monoclonal antibodies, 17E12E5 and 10G6D6, using estradiol-6-ethyl methoxy carbonyl (EMC)-bovine serum albumin (BSA) as immunogen. To tentatively improve their affinities for natural estradiol, we have initiated their structural and functional studies. For this purpose, we have cloned and sequenced the genes encoding the variable fragments of each antibody. Single chain variable fragments (scFv) were produced into the periplasmic space of E. coli using the pLIP6 expression vector. Mapping of the functional structures of both antibodies was obtained by combination of modelling and mutational analyses together with cross-reaction studies. The two binding pockets are described and models of estradiol complexed to 17E12E5 and 10G6D6 are proposed.

Crystal structure of alkaline phosphatase from human placenta at 1.8 A resolution. Implication for a substrate specificity.

Human placental alkaline phosphatase (PLAP) is one of three tissue-specific human APs extensively studied because of its ectopic expression in tumors. The crystal structure, determined at 1.8-A resolution, reveals that during evolution, only the overall features of the enzyme have been conserved with respect to Escherichia coli. The surface is deeply mutated with 8% residues in common, and in the active site, only residues strictly necessary to perform the catalysis have been preserved. Additional structural elements aid an understanding of the allosteric property that is specific for the mammalian enzyme (Hoylaerts, M. F., Manes, T., and Millán, J. L. (1997) J. Biol. Chem. 272, 22781-22787). Allostery is probably favored by the quality of the dimer interface, by a long N-terminal alpha-helix from one monomer that embraces the other one, and similarly by the exchange of a residue from one monomer in the active site of the other. In the neighborhood of the catalytic serine, the orientation of Glu-429, a residue unique to PLAP, and the presence of a hydrophobic pocket close to the phosphate product, account for the specific uncompetitive inhibition of PLAP by l-amino acids, consistent with the acquisition of substrate specificity. The location of the active site at the bottom of a large valley flanked by an interfacial crown-shaped domain and a domain containing an extra metal ion on the other side suggest that the substrate of PLAP could be a specific phosphorylated protein.

Isolation of a tarantula toxin specific for a class of proton-gated Na+ channels.

Acid sensing is associated with nociception, taste transduction, and perception of extracellular pH fluctuations in the brain. Acid sensing is carried out by the simplest class of ligand-gated channels, the family of H(+)-gated Na(+) channels. These channels have recently been cloned and belong to the acid-sensitive ion channel (ASIC) family. Toxins from animal venoms have been essential for studies of voltage-sensitive and ligand-gated ion channels. This paper describes a novel 40-amino acid toxin from tarantula venom, which potently blocks (IC(50) = 0.9 nm) a particular subclass of ASIC channels that are highly expressed in both central nervous system neurons and sensory neurons from dorsal root ganglia. This channel type has properties identical to those described for the homomultimeric assembly of ASIC1a. Homomultimeric assemblies of other members of the ASIC family and heteromultimeric assemblies of ASIC1a with other ASIC subunits are insensitive to the toxin. The new toxin is the first high affinity and highly selective pharmacological agent for this novel class of ionic channels. It will be important for future studies of their physiological and physio-pathological roles.

Design of a Gag pentapeptide analogue that binds human cyclophilin A more efficiently than the entire capsid protein: new insights for the development of novel anti-HIV-1 drugs.

Cyclophilin A (hCyp-18), a ubiquitous cytoplasmic peptidyl-prolyl cis/trans isomerase (PPIase), orchestrates HIV-1 core packaging. hCyp-18, incorporated into the virion, enables core uncoating and RNA release and consequently plays a critical role in the viral replication process. hCyp-18 specifically interacts with a single exposed loop of the Gag polyprotein capsid domain via a network of nine hydrogen bonds which mainly implicates a 7-mer fragment of the loop. As previously reported, the corresponding linear heptapeptide Ac-Val-His-Ala-Gly-Pro-Ile-Ala-NH(2) (2) binds to hCyp-18 with a low affinity (IC(50) = 850 +/- 220 microM) but a potentially useful selectivity for hCyp-18 relative to hFKBP-12, another abundant PPIase. On the basis of X-ray structures of Gag fragments:hCyp-18 complexes, we generated a series of modified peptides in order to probe the determinants of the interaction and hence to select a peptidic ligand displaying a higher affinity than the capsid domain of Gag. We synthesized a series of heptapeptides to test the energetic contribution of amino acids besides the Gly-Pro moiety. In particular the importance of the histidine residue for the interaction was underscored. We also investigated the influence of N- and C-terminal modifications. Hexapeptides containing either deaminovaline (Dav) in place of the N-terminal valine or substitution of the C-terminal alanine amide with a benzylamide group displayed increased affinities. Combination of both modifications gave the most potent competitor Dav-His-Ala-Gly-Pro-Ile-NHBn (28) which has a higher affinity for hCyp-18 (K(d) = 3 +/- 0.5 microM) than the entire capsid protein (K(d) = 16 +/- 4 microM) and a very low affinity for hFKBP-12. Some of our results strongly suggest that the title compound is not a substrate of hCyp-18 and interacts preferentially in the trans conformation.

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.

On the diversity and heterogeneity of H-2(d)-restricted determinants and T cell epitopes from the major bee venom allergen.

One of the main limitations of using synthetic peptides for immunotherapy in allergic patients is the difficulty to delineate the immunodominant T cell epitopes which are necessarily dependent on HLA molecules. We have thus addressed the question of the role of MHC II molecules in immunodominant epitopes selection in the particular case of the major bee venom allergen (API m1). To exhaustively and easily explore it, we used BALB/c mice whose H-2 haplotype is associated with high IgE and IgG responses to API m1. By means of extensive sets of synthetic peptides, we investigated the specificity of polyclonal T cells and monoclonal hybridomas from mice immunized with API m1 and delineated four immunodominant regions, restricted to either the I-E(d) or the I-A(d) molecule. All the peptides were also tested for their capacity to bind to immunopurified MHC II molecules. Eight determinants of high affinity were identified. They clustered into three distinct regions and were largely overlapping. They included all the immunodominant epitopes, but half of them were not capable of stimulating T cells. Strikingly, interacting surfaces with either the TCR or MHC II molecule greatly differed from one determinant to another. In one case, we observed that flanking regions exerted a particular action on T cell stimulation which prevented the fine epitope localization. Our results underline the diversity and complexity of MHC II-restricted determinants and T cell epitopes from the major bee venom allergen, even in a single haplotype. These data also participate in the development of alternative approaches to conventional immunotherapy.

Biochemical characterization and nuclear magnetic resonance structure of novel alpha-conotoxins isolated from the venom of Conus consors.

Two novel alpha-conotoxins were purified and characterized from the venom of the fish-hunting cone snail Conus consors. These peptides were identified by screening HPLC fractions of the crude venom and by binding experiments with Torpedo nicotinic acetylcholine receptor. The toxins named alpha-CnIA and alpha-CnIB exhibited sequences of 14 and 12 amino acids, respectively. The alpha-CnIA represents the main alpha-conotoxin contained in the venom, whereas alpha-CnIB is present in a relatively small amount. Chemical synthesis of alpha-CnIA was carried out using the Fmoc methodology by selective disulfide bond formation. The biological activity of the toxin was assessed in fish and mice. The alpha-CnIA inhibited the fixation of iodinated alpha-bungarotoxin to Torpedo nicotinic acetylcholine receptors with an IC50 of 0.19 microM which can be compared to the IC50 of 0.31 microM found for the previously characterized alpha-MI isolated from the piscivorous Conus magus. The synthetic alpha-CnIA blocked spontaneous and evoked synaptic potentials in frog and mouse isolated neuromuscular preparations at sub-micromolar concentrations. Solution NMR of this toxin indicated a conformational heterogeneity with the existence of different conformers in solution, at slow and intermediate exchange rates relative to the NMR chemical shift time scale, similar to that reported for alpha-GI and alpha-MI. NMR structures were calculated for the major NMR signals representing more than 80% of the population at 5 degrees C.

Conformational and functional variability supported by the BPTI fold: solution structure of the Ca2+ channel blocker calcicludine.

Calcicludine, a 60-amino acid protein isolated from the green mamba venom, has been recently identified as blocking a large set (i.e., L-, N- and P-type) of Ca2+ channels. The three-dimensional structure of calcicludine has been determined by NMR and molecular modeling using a data set of 723 unambiguous and 265 ambiguous distance restraints, as 33 phi and 13 chi1 dihedral angle restraints. Analysis of the 15 final structures (backbone root-mean-square deviation = 0.6 A) shows that calcicludine adopts the Kunitz-type protease inhibitor fold. Its three-dimensional structure is similar to that of snake K+ channel blockers dendrotoxins. Conformational differences with protease inhibitors and dendrotoxins are localized in the 3(10) helix and loop 1 (segments 1-7 and 10-19), the extremity of the beta-hairpin (segment 27-30), and loop 2 (segment 39-44). These regions correspond to the functional sites of bovine pancreatic trypsin inhibitor (BPTI) and dendrotoxins. The positioning of the N-terminal segment 1-7 relative to the rest of the protein is characteristic of calcicludine. The involvement of this segment and the positively charged K31 at the tip of the beta-hairpin in the biological activity of calcicludine is discussed.

Functional determinants by which snake and cone snail toxins block the alpha 7 neuronal nicotinic acetylcholine receptors.

Snakes and cone snails produce toxins which block muscular and/or neuronal nicotinic acetylcholine receptors (AChRs). This paper mostly focuses on the determinants by which a snake long chain curaremimetic toxin and the cone snail toxin ImI bind specifically to the alpha 7 neuronal receptor. In both cases, the site involves a small turn-like structure constrained by two half-cystines.

Increasing immunogenicity of antigens fused to Ig-binding proteins by cell surface targeting.

Fusion of antigenic proteins to Ig-binding proteins such as protein A from Staphylococcus aureus and its derived ZZ fragment is known to increase immunogenicity of the fused Ag in vivo. To shed light on the origin of this effect, we used snake toxins as Ags and observed that 1) fusion of toxins to ZZ enhanced their presentation to a toxin-specific T cell hybridoma (T1B2), using A20 B lymphoma cells, splenocytes, or peritoneal exudate cells as APCs; 2) this enhancement further increased when the number of fused Ig-binding domains varied from two with ZZ to five with protein A; and 3) the phenomenon vanished when the fusion protein was preincubated with an excess of free ZZ or when P388D1 monocytes cells were used as APCs. Therefore, ZZ-fused toxins are likely to be targeted to surface Igs of APCs by their ZZ moiety. Furthermore, ZZ-alpha and toxin alpha stimulated similar profiles of toxin-specific T cells in BALB/c mice, suggesting a comparable processing and presentation in vivo for both toxin forms. To improve the targeting efficiency, ZZ-alpha was noncovalently complexed to various Igs directed to different cell surface components of APCs. The resulting complexes were up to 10(3)-fold more potent than the free toxin at stimulating T1B2. Also, they elicited both a T cell and an Ab response in BALB/c mice, without the need of any adjuvant. This simple approach may find practical applications by increasing the immunogenicity of recombinant proteins without the use of adjuvant.

Pseudopeptide ligands for MHC II-restricted T cells.

The potential therapeutic use of peptides to activate or anergize specific T cells is seriously limited by their susceptibility to proteolytic degradation. Classically, peptides are stabilized by incorporation of non-natural modifications including main chain modifications. In the case of MHC II-restricted peptides, the peptide backbone actively participates to the interaction with the MHC molecule and hence may preclude the peptidomimetic approach. We thus investigated whether a single amide bond modification influenced the peptide capacity to bind to a MHC II molecule and to stimulate specific T cells. Twenty pseudopeptide analogs of the I-Ed binder 24-36 peptide, whose sequence was derived from a snake neurotoxin, were obtained by replacing each amide bond of the peptide central part, by either a reduced psi[CH2-NH] or N-methylated psi[CO-NMe] peptide bond. In agreement with the major interacting role played by the peptide backbone, several peptides displayed a low, if any, capacity to bind to the MHC II molecule and did not lead to T cell stimulation. However, one-third of the peptides were almost as active as the 24-36 peptide in I-Ed binding assays and one-fifth in T cell stimulation assays. Among them, two pseudopeptides displayed native-like activity. Good binders were not necessarily good at stimulating T cells, demonstrating that main chain modification also affected T cell recognition. We thus showed that a peptidomimetic approach could create a new type of MHC II ligand to control T cell responses.

Consequence of the removal of evolutionary conserved disulfide bridges on the structure and function of charybdotoxin and evidence that particular cysteine spacings govern specific disulfide bond formation.

Scorpion toxins are miniglobular proteins containing a common structural motif formed by an alpha-helix on one face, an antiparallel beta-sheet on the opposite face, and three disulfide bonds making up most of its internal volume. We have investigated the role of these evolutionary conserved bonds by replacing each couple of bridged cysteine residues of the scorpion charybdotoxin by a pair of nonbridging L-alpha-aminobutyric acid (Aba) residues. Three analogues were obtained by solid-phase synthesis, Chab I, Chab II, and Chab III, containing the Aba residues in positions 7 and 28, 13 and 33, 17 and 35, respectively. Circular dichroism analysis showed that the purified Chab II acquired a conformation similar to that of charybdotoxin, while the Chab I and Chab III possess decreased nativelike characteristics. All analogues block single high-conductance Ca(2+)-activated K+ channels from rat skeletal muscle inserted into planar lipid bilayers, but with different potencies. Chab II is the most active analogue (KD = 8.0 x 10(-8) M), with a 9-fold lower affinity as compared to native charybdotoxin. Chab I and Chab III have, respectively, 180- and 580-fold lower affinity. Therefore, the removal of evolutionary conserved disulfide bridges does not prevent the toxin to adopt a functional and presumably nativelike structure. However, removal of one disulfide bond affects the yields of formation of correct pairing between the remaining cysteine residues, and only Chab I preserves the ability to form the native disulfide pairings with high efficiency. This is the only analogue to preserve particular spacings of three and one residue between the cysteines, which have been described to thermodynamically disfavor disulfide bond formation between the cysteines [Zhang R., and Snyder, G. H. (1989) J. Biol. Chem. 264, 18472-18479]. Therefore, we conclude that the position of the cysteine residues in the sequence of charybdotoxin, by disfavoring specific pairings and favoring others, may govern selective formation of specific disulfide bonds, thus, explaining the efficient folding properties of Chab I and of native charybdotoxin. The structural properties of the Chab analogues and the discovered role of the cysteine spacings have interesting implications in protein design and engineering.

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.

NMR solution structure of a two-disulfide derivative of charybdotoxin: structural evidence for conservation of scorpion toxin alpha/beta motif and its hydrophobic side chain packing.

The alpha/beta scorpion fold consisting of a short alpha-helix and beta-sheet is a structural motif common to scorpion toxins, insect defensins, and plant gamma-thionins that invariably contains three disulfides. CHABII is a two-disulfide derivative of the scorpion toxin charybdotoxin (ChTX), chemically synthesized by inserting two L-alpha-aminobutyric acids in place of the two half-cystine residues involved in the disulfide 13-33. This disulfide is one of the two disulfides which connect the alpha-helix to the beta-sheet. The solution structure of CHABII was determined at pH 6.3 and 5 degrees C using 2D NMR and simulated annealing from 513 distance and 46 dihedral angle constraints. The NMR structure of CHABII is well-defined as judged from the low value of the averaged backbone rms deviation between the 30 lowest energy structures and the energy-minimized mean structure ((rmsd) = 0.65 A for the entire sequence and 0.48 A for the segment 3-36). Analysis and comparison of the solution structures of CHABII and ChTX lead to the following conclusions: (i) the fold of CHABII is similar to that of ChTX as indicated by the low value of the averaged backbone atomic rms deviation between the 10 lowest energy solution structures of the two proteins (1.44 A); (ii) the packing of the hydrophobic core is well-preserved, underlying the critical structural role of the hydrophobic interactions even for such a small and cysteine-rich protein as ChTX.

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.