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Delmas Agnès


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2016   Références trouvées : 4

Decourt C., Robert V., Anger K., Galibert M., Madinier J. B., Liu X., Dardente H., Lomet D., Delmas A. F., Caraty A., Herbison A. E., Anderson G. M., Aucagne V. and Beltramo M.  (2016)

A synthetic kisspeptin analog that triggers ovulation and advances puberty

Scientific Reports (2016) 6, 26908 - doi : 10.1038/srep26908
The neuropeptide kisspeptin and its receptor, KiSS1R, govern the reproductive timeline of mammals by triggering puberty onset and promoting ovulation by stimulating gonadotrophin-releasing hormone (GnRH) secretion. To overcome the drawback of kisspeptin short half-life we designed kisspeptin analogs combining original modifications, triazole peptidomimetic and albumin binding motif, to reduce proteolytic degradation and to slow down renal clearance, respectively. These analogs showed improved in vitro potency and dramatically enhanced pharmacodynamics. When injected intramuscularly into ewes (15 nmol/ewe) primed with a progestogen, the best analog (compound 6, C6) induced synchronized ovulations in both breeding and non-breeding seasons. Ovulations were fertile as demonstrated by the delivery of lambs at term. C6 was also fully active in both female and male mice but was completely inactive in KiSS1R KO mice. Electrophysiological recordings of GnRH neurons from brain slices of GnRH-GFP mice indicated that C6 exerted a direct excitatory action on GnRH neurons. Finally, in prepubertal female mice daily injections (0.3 nmol/mouse) for five days significantly advanced puberty. C6 ability to trigger ovulation and advance puberty demonstrates that kisspeptin analogs may find application in the management of livestock reproduction and opens new possibilities for the treatment of reproductive disorders in humans.

The neuropeptide kisspeptin and its receptor, KiSS1R, govern the reproductive timeline of mammals by triggering puberty onset and promoting ovulation by stimulating gonadotrophin-releasing hormone (GnRH) secretion. To overcome the drawback of kisspeptin short half-life we designed kisspeptin analogs combining original modifications, triazole peptidomimetic and albumin binding motif, to reduce proteolytic degradation and to slow down renal clearance, respectively. These analogs showed improved in vitro potency and dramatically enhanced pharmacodynamics. When injected intramuscularly into ewes (15 nmol/ewe) primed with a progestogen, the best analog (compound 6, C6) induced synchronized ovulations in both breeding and non-breeding seasons. Ovulations were fertile as demonstrated by the delivery of lambs at term. C6 was also fully active in both female and male mice but was completely inactive in KiSS1R KO mice. Electrophysiological recordings of GnRH neurons from brain slices of GnRH-GFP mice indicated that C6 exerted a direct excitatory action on GnRH neurons. Finally, in prepubertal female mice daily injections (0.3 nmol/mouse) for five days significantly advanced puberty. C6 ability to trigger ovulation and advance puberty demonstrates that kisspeptin analogs may find application in the management of livestock reproduction and opens new possibilities for the treatment of reproductive disorders in humans.

Terrier V. P., Adihou H., Arnould M., Delmas A. F. and Aucagne V.  (2016)

A straightforward method for automated Fmoc-based synthesis of bio-inspired peptide crypto-thioesters

Chemical Science (2016) 7 (1) 339-345 - doi : 10.1039/C5SC02630J
Despite recent advances, the direct Fmoc-based solid phase synthesis of peptide [small alpha]-thioesters for the convergent synthesis of proteins via native chemical ligation (NCL) remains a challenge in the field. We herein report a simple and general methodology, enabling access to peptide thioester surrogates. A novel C-terminal N-(2-hydroxybenzyl)cysteine thioesterification device based on an amide-to-thioester rearrangement was developed, and the resulting peptide crypto-thioesters can be directly used in NCL reactions with fast N [rightward arrow] S shift kinetics at neutral pH. These fast kinetics arise from our bio-inspired design, via intein-like intramolecular catalysis. Due to a well-positioned phenol moiety, an impressive >50 fold increase in the kinetic rate is observed compared to an O-methylated derivative. Importantly, the synthesis of this new device can be fully automated using inexpensive commercially available materials and does not require any post-synthetic steps prior to NCL. We successfully applied this new method to the synthesis of two long naturally-occurring cysteine-rich peptide sequences.

Despite recent advances, the direct Fmoc-based solid phase synthesis of peptide [small alpha]-thioesters for the convergent synthesis of proteins via native chemical ligation (NCL) remains a challenge in the field. We herein report a simple and general methodology, enabling access to peptide thioester surrogates. A novel C-terminal N-(2-hydroxybenzyl)cysteine thioesterification device based on an amide-to-thioester rearrangement was developed, and the resulting peptide crypto-thioesters can be directly used in NCL reactions with fast N [rightward arrow] S shift kinetics at neutral pH. These fast kinetics arise from our bio-inspired design, via intein-like intramolecular catalysis. Due to a well-positioned phenol moiety, an impressive >50 fold increase in the kinetic rate is observed compared to an O-methylated derivative. Importantly, the synthesis of this new device can be fully automated using inexpensive commercially available materials and does not require any post-synthetic steps prior to NCL. We successfully applied this new method to the synthesis of two long naturally-occurring cysteine-rich peptide sequences.

Loth K., Alami S. A. I., Habès C., Garrido S., Aucagne V., Delmas A.F., Moreau, T., Zani M.-L. and Landon C.  (2016)

Complete 1H, 15N and 13C assignment of trappin-2 and 1H assignment of its two domains, elafin and cementoin

Biomolecular NMR Assignments (2016) 10 (1) 223-226 - doi : 10.1007/s12104-016-9671-1
Trappin-2 is a serine protease inhibitor with a very narrow inhibitory spectrum and has significant anti-microbial activities. It is a 10 kDa cationic protein composed of two distinct domains. The N-terminal domain (38 residues) named cementoin is known to be intrinsically disordered when it is not linked to the elafin. The C-terminal domain (57 residues), corresponding to elafin, is a cysteine-rich domain stabilized by four disulfide bridges and is characterized by a flat core and a flexible N-terminal part. To our knowledge, there is no structural data available on trappin-2. We report here the complete 1H, 15N and 13C resonance assignment of the recombinant trappin-2 and the 1H assignments of cementoin and elafin, under the same experimental conditions. This is the first step towards the 3D structure determination of the trappin-2.

Trappin-2 is a serine protease inhibitor with a very narrow inhibitory spectrum and has significant anti-microbial activities. It is a 10 kDa cationic protein composed of two distinct domains. The N-terminal domain (38 residues) named cementoin is known to be intrinsically disordered when it is not linked to the elafin. The C-terminal domain (57 residues), corresponding to elafin, is a cysteine-rich domain stabilized by four disulfide bridges and is characterized by a flat core and a flexible N-terminal part. To our knowledge, there is no structural data available on trappin-2. We report here the complete 1H, 15N and 13C resonance assignment of the recombinant trappin-2 and the 1H assignments of cementoin and elafin, under the same experimental conditions. This is the first step towards the 3D structure determination of the trappin-2.

Lelievre D., Terrier V. P., Delmas A. F. and Aucagne V.  (2016)

Native Chemical Ligation Strategy to Overcome Side Reactions during Fmoc-Based Synthesis of C-Terminal Cysteine-Containing Peptides

Organic letters (2016 18 (5) 920-923 - doi : 10.1021/acs.orglett.5b03612
The Fmoc-based solid phase synthesis of C-terminal cysteine-containing peptides is problematic, due to side reactions provoked by the pronounced acidity of the Calpha proton of cysteine esters. We herein describe a general strategy consisting of the postsynthetic introduction of the C-terminal Cys through a key chemoselective native chemical ligation reaction with N-Hnb-Cys peptide crypto-thioesters. This method was successfully applied to the demanding peptide sequences of two natural products of biological interest, giving remarkably high overall yields compared to that of a state of the art strategy.

The Fmoc-based solid phase synthesis of C-terminal cysteine-containing peptides is problematic, due to side reactions provoked by the pronounced acidity of the Calpha proton of cysteine esters. We herein describe a general strategy consisting of the postsynthetic introduction of the C-terminal Cys through a key chemoselective native chemical ligation reaction with N-Hnb-Cys peptide crypto-thioesters. This method was successfully applied to the demanding peptide sequences of two natural products of biological interest, giving remarkably high overall yields compared to that of a state of the art strategy.


2015   Références trouvées : 2

Beltramo, M., Robert, V., Galibert, M., Madinier, J.-B., Marceau, P., Dardente, H., Decourt, C., De Roux, N., Lomet, D., Delmas, A. F., Caraty, A. and Aucagne, V.  (2015)

Rational Design of Triazololipopeptides Analogs of Kisspeptin Inducing a Long-Lasting Increase of Gonadotropins

Journal of Medicinal Chemistry (2015) 58 (8) 3459-3470 - doi : 10.1021/jm5019675
New potent and selective KISS1R agonists were designed using a combination of rational chemical modifications of the endogenous neuropeptide kisspeptin 10 (KP10). Improved resistance to degradation and presumably reduced renal clearance were obtained by introducing a 1,4-disubstituted 1,2,3-triazole as a proteolysis-resistant amide mimic and a serum albumin-binding motif, respectively. These triazololipopeptides are highly potent full agonists of KISS1R and are >100 selective over the closely related NPFF1R. When injected in ewes with a quiescent reproductive system, the best compound of our series induced a much prolonged increase of luteinizing hormone release compared to KP10 and increased follicle-stimulating hormone plasma concentration. Hence, this KISS1R agonist is a new valuable pharmacological tool to explore the potential of KP system in reproduction control. Furthermore, it represents the first step to develop drugs treating reproductive system disorders due to a reduced activity of the hypothalamo–pituitary–gonadal axis such as delayed puberty, hypothalamic amenorrhea, and hypogonadotropic hypogonadism.

New potent and selective KISS1R agonists were designed using a combination of rational chemical modifications of the endogenous neuropeptide kisspeptin 10 (KP10). Improved resistance to degradation and presumably reduced renal clearance were obtained by introducing a 1,4-disubstituted 1,2,3-triazole as a proteolysis-resistant amide mimic and a serum albumin-binding motif, respectively. These triazololipopeptides are highly potent full agonists of KISS1R and are >100 selective over the closely related NPFF1R. When injected in ewes with a quiescent reproductive system, the best compound of our series induced a much prolonged increase of luteinizing hormone release compared to KP10 and increased follicle-stimulating hormone plasma concentration. Hence, this KISS1R agonist is a new valuable pharmacological tool to explore the potential of KP system in reproduction control. Furthermore, it represents the first step to develop drugs treating reproductive system disorders due to a reduced activity of the hypothalamo–pituitary–gonadal axis such as delayed puberty, hypothalamic amenorrhea, and hypogonadotropic hypogonadism.

Galibert M., Piller V., Piller F., Aucagne V., Delmas A.F.  (2015)

Combining solid phase chemical ligations and enzymatic glycosylations for the synthesis of glycoproteins

Chemical Science (2015) 6 (6) 3617-3623 - doi : 10.1039/C5SC00773A
The solid-phase chemical assembly of a protein through iterative chemoselective ligation of unprotected peptide segments can be followed with chemical and/or enzymatic transformations of the resulting immobilized protein, the latter steps thus benefitting from the advantages provided by the solid support. We demonstrate here the usefulness of this strategy for the chemo-enzymatic synthesis of glycoprotein analogues. A linker was specifically designed for application to the synthesis of O-glycoproteins : this new linker is readily cleaved under mild aqueous conditions compatible with very sensitive glycosidic bonds, but is remarkably stable under a wide range of chemical and biochemical conditions. It was utilized for solid-supported N-to-C peptidomimetic triazole ligation followed by enzymatic glycosylation, ultimately leading to a very large MUC1-derived glycoprotein containing 160 amino acid residues, 24 α-GalNAc moieties linked to Ser and Thr, and 3 triazoles as peptide bond mimetics.

The solid-phase chemical assembly of a protein through iterative chemoselective ligation of unprotected peptide segments can be followed with chemical and/or enzymatic transformations of the resulting immobilized protein, the latter steps thus benefitting from the advantages provided by the solid support. We demonstrate here the usefulness of this strategy for the chemo-enzymatic synthesis of glycoprotein analogues. A linker was specifically designed for application to the synthesis of O-glycoproteins : this new linker is readily cleaved under mild aqueous conditions compatible with very sensitive glycosidic bonds, but is remarkably stable under a wide range of chemical and biochemical conditions. It was utilized for solid-supported N-to-C peptidomimetic triazole ligation followed by enzymatic glycosylation, ultimately leading to a very large MUC1-derived glycoprotein containing 160 amino acid residues, 24 α-GalNAc moieties linked to Ser and Thr, and 3 triazoles as peptide bond mimetics.


2014   Références trouvées : 4

Meindre, F., Lelièvre, D., Loth, K., Mith, O., Aucagne, V., Berthomieu, P., Marquès, L., Delmas, A. F., Landon, C. and Paquet, F.  (2014)

The Nuclear Magnetic Resonance Solution Structure of the Synthetic AhPDF1.1b Plant Defensin Evidences the Structural Feature within the γ-Motif

Biochemistry (2014) 53 (49) 7745-7754 - doi : 10.1021/bi501285k
Plant defensins (PDF) are cysteine-rich peptides that are major actors in the innate immunity in plants. Besides their antifungal activity, some PDF such as Arabidopsis halleri PDF1.1b confer zinc tolerance in plants. Here we present (i) an efficient protocol for the production of AhPDF1.1b by solid-phase peptide synthesis followed by controlled oxidative folding to obtain the highly pure native form of the defensin and (ii) the three-dimensional (3D) nuclear magnetic resonance structure of AhPDF1.1b, the first 3D structure of plant defensin obtained with a synthetic peptide. Its fold is organized around the typical cysteine-stabilized α-helix ?-sheet motif and contains the ?-core motif involved in the antifungal activity of all plant defensins. On the basis of our structural analysis of AhPDF1 defensins combined with previous biological data for antifungal and zinc tolerance activities, we established the essential role of cis-Pro41 within the ?-core. In fact, the four consecutive residues (Val39-Phe40-Pro41-Ala42) are strictly conserved for plant defensins able to tolerate zinc. We hypothesized that structural and/or dynamic features of this sequence are related to the ability of the defensin to chelate zinc.

Plant defensins (PDF) are cysteine-rich peptides that are major actors in the innate immunity in plants. Besides their antifungal activity, some PDF such as Arabidopsis halleri PDF1.1b confer zinc tolerance in plants. Here we present (i) an efficient protocol for the production of AhPDF1.1b by solid-phase peptide synthesis followed by controlled oxidative folding to obtain the highly pure native form of the defensin and (ii) the three-dimensional (3D) nuclear magnetic resonance structure of AhPDF1.1b, the first 3D structure of plant defensin obtained with a synthetic peptide. Its fold is organized around the typical cysteine-stabilized α-helix ?-sheet motif and contains the ?-core motif involved in the antifungal activity of all plant defensins. On the basis of our structural analysis of AhPDF1 defensins combined with previous biological data for antifungal and zinc tolerance activities, we established the essential role of cis-Pro41 within the ?-core. In fact, the four consecutive residues (Val39-Phe40-Pro41-Ala42) are strictly conserved for plant defensins able to tolerate zinc. We hypothesized that structural and/or dynamic features of this sequence are related to the ability of the defensin to chelate zinc.

Herve, V., Meudal, H., Labas, V., Rehault-Godbert, S., Gautron, J., Berges, M., Guyot, N., Delmas, A.F., Nys, Y. and Landon, C.  (2014)

Three-dimensional NMR Structure of Hen Egg Gallin (Chicken Ovodefensin) Reveals a New Variation of the beta-Defensin Fold

Journal of Biological Chemistry (2014) 289 (10) 7211-7220 - doi : 10.1074/jbc.M113.507046
Gallin is a 41-residue protein, first identified as a minor component of hen egg white and found to be antimicrobial against Escherichia coli. Gallin may participate in the protection of the embryo during its development in the egg. Its sequence is related to antimicrobial beta-defensin peptides. In the present study, gallin was chemically synthesized 1) to further investigate its antimicrobial spectrum and 2) to solve its three-dimensional NMR structure and thus gain insight into structure-function relationships, a prerequisite to understanding its mode(s) of action. Antibacterial assays confirmed that gallin was active against Escherichia coli, but no additional antibacterial activity was observed against the other Gram-positive or Gram-negative bacteria tested. The three-dimensional structure of gallin, which is the first ovodefensin structure to have been solved to date, displays a new five-stranded arrangement. The gallin three-dimensional fold contains the three-stranded antiparallel beta-sheet and the disulfide bridge array typical of vertebrate beta-defensins. Gallin can therefore be unambiguously classified as a beta-defensin. However, an additional short two-stranded beta-sheet reveals that gallin and presumably the other ovodefensins form a new structural subfamily of beta-defensins. Moreover, gallin and the other ovodefensins calculated by homology modeling exhibit atypical hydrophobic surface properties, compared with the already known vertebrate beta-defensins. These specific structural features of gallin might be related to its restricted activity against E. coli and/or to other yet unknown functions. This work provides initial understanding of a critical sequence-structure-function relationship for the ovodefensin family.

Gallin is a 41-residue protein, first identified as a minor component of hen egg white and found to be antimicrobial against Escherichia coli. Gallin may participate in the protection of the embryo during its development in the egg. Its sequence is related to antimicrobial beta-defensin peptides. In the present study, gallin was chemically synthesized 1) to further investigate its antimicrobial spectrum and 2) to solve its three-dimensional NMR structure and thus gain insight into structure-function relationships, a prerequisite to understanding its mode(s) of action. Antibacterial assays confirmed that gallin was active against Escherichia coli, but no additional antibacterial activity was observed against the other Gram-positive or Gram-negative bacteria tested. The three-dimensional structure of gallin, which is the first ovodefensin structure to have been solved to date, displays a new five-stranded arrangement. The gallin three-dimensional fold contains the three-stranded antiparallel beta-sheet and the disulfide bridge array typical of vertebrate beta-defensins. Gallin can therefore be unambiguously classified as a beta-defensin. However, an additional short two-stranded beta-sheet reveals that gallin and presumably the other ovodefensins form a new structural subfamily of beta-defensins. Moreover, gallin and the other ovodefensins calculated by homology modeling exhibit atypical hydrophobic surface properties, compared with the already known vertebrate beta-defensins. These specific structural features of gallin might be related to its restricted activity against E. coli and/or to other yet unknown functions. This work provides initial understanding of a critical sequence-structure-function relationship for the ovodefensin family.

Muench, S.P., Rawson, S., Eyraud, V., Delmas, A.F., Da Silva, P., Phillips, C., Trinick, J., Harrison, M.A., Gressent, F. and Huss, M.  (2014)

PA1b Inhibitor Binding to Subunits c and e of the Vacuolar ATPase Reveals Its Insecticidal Mechanism

The Journal of Biological Chemistry (2014) 289 (23) 16339-16408 - doi : 10.1074/jbc.M113.541250
The vacuolar ATPase (V-ATPase) is a 1MDa transmembrane proton pump that operates via a rotary mechanism fuelled by ATP. Essential for eukaryotic cell homeostasis, it plays central roles in bone remodeling and tumor invasiveness, making it a key therapeutic target. Its importance in arthropod physiology also makes it a promising pesticide target. The major challenge in designing lead compounds against the V-ATPase is its ubiquitous nature, such that any therapeutic must be capable of targeting particular isoforms. Here, we have characterized the binding site on the V-ATPase of pea albumin 1b (PA1b), a small cystine knot protein that shows exquisitely selective inhibition of insect V-ATPases. Electron microscopy shows that PA1b binding occurs across a range of equivalent sites on the c ring of the membrane domain. In the presence of Mg.ATP, PA1b localizes to a single site, distant from subunit a, which is predicted to be the interface for other inhibitors. Photoaffinity labeling studies show radiolabeling of subunits c and e. In addition, weevil resistance to PA1b is correlated with bafilomycin resistance, caused by mutation of subunit c. The data indicate a binding site to which both subunits c and e contribute and inhibition that involves locking the c ring rotor to a static subunit e and not subunit a. This has implications for understanding the V-ATPase mechanism and that of inhibitors with therapeutic or pesticidal potential. It also provides the first evidence for the position of subunit e within the complex.

The vacuolar ATPase (V-ATPase) is a 1MDa transmembrane proton pump that operates via a rotary mechanism fuelled by ATP. Essential for eukaryotic cell homeostasis, it plays central roles in bone remodeling and tumor invasiveness, making it a key therapeutic target. Its importance in arthropod physiology also makes it a promising pesticide target. The major challenge in designing lead compounds against the V-ATPase is its ubiquitous nature, such that any therapeutic must be capable of targeting particular isoforms. Here, we have characterized the binding site on the V-ATPase of pea albumin 1b (PA1b), a small cystine knot protein that shows exquisitely selective inhibition of insect V-ATPases. Electron microscopy shows that PA1b binding occurs across a range of equivalent sites on the c ring of the membrane domain. In the presence of Mg.ATP, PA1b localizes to a single site, distant from subunit a, which is predicted to be the interface for other inhibitors. Photoaffinity labeling studies show radiolabeling of subunits c and e. In addition, weevil resistance to PA1b is correlated with bafilomycin resistance, caused by mutation of subunit c. The data indicate a binding site to which both subunits c and e contribute and inhibition that involves locking the c ring rotor to a static subunit e and not subunit a. This has implications for understanding the V-ATPase mechanism and that of inhibitors with therapeutic or pesticidal potential. It also provides the first evidence for the position of subunit e within the complex.

Decostaire, I.E., Lelievre, D., Aucagne, V. and Delmas, A.F.  (2014)

Solid phase oxime ligations for the iterative synthesis of polypeptide conjugates

Organic & Biomolecular Chemistry (2014) 12 (29) 5536-5543 - doi : 10.1039/c4ob00760c
Peptide-based complex biomacromolecules are now optimally assembled by sequential ligation of unprotected peptide segments. However, this approach is still limited by the laborious chromatographic purification and handling steps needed for multiple successive chemoselective couplings, which leads to loss of material. An efficient alternative is solid phase chemical ligation (SPCL) initially developed for native chemical ligation. We report here an extension of this approach to iterative oxime ligation reactions, and describe a streamlined approach for the modular preparation of oxime-containing polypeptides. In particular, we determined optimal conditions to remove the Aloc group in the presence of aminooxy and oxime ether groups, and we extended the applicability of iterative C-to-N SPCL through simplification of the access to a C-terminally-grafted, unprotected peptide segment, using solid supported chemical transformations only. The high purity of the crude oxime-containing polypeptides highlights the efficiency of our approach.

Peptide-based complex biomacromolecules are now optimally assembled by sequential ligation of unprotected peptide segments. However, this approach is still limited by the laborious chromatographic purification and handling steps needed for multiple successive chemoselective couplings, which leads to loss of material. An efficient alternative is solid phase chemical ligation (SPCL) initially developed for native chemical ligation. We report here an extension of this approach to iterative oxime ligation reactions, and describe a streamlined approach for the modular preparation of oxime-containing polypeptides. In particular, we determined optimal conditions to remove the Aloc group in the presence of aminooxy and oxime ether groups, and we extended the applicability of iterative C-to-N SPCL through simplification of the access to a C-terminally-grafted, unprotected peptide segment, using solid supported chemical transformations only. The high purity of the crude oxime-containing polypeptides highlights the efficiency of our approach.


2013   Références trouvées : 2

Raibaut, L., Adihou, H., Desmet, R., Delmas, A.F., Aucagne and V. Melnyk, O.  (2013)

Highly efficient solid phase synthesis of large polypeptides by iterative ligations of bis(2-sulfanylethyl)amido (SEA) peptide segments

Chemical Science 4 (10) 4061-4066
Up to now, the advantages of solid phase protein synthesis have been largely under-utilized due to the difficulty of designing a simple and efficient elongation cycle enabling the concatenation of unprotected peptide segments. The combination of selective N-terminal anchoring (N3-Esoc linker) with the blocked thioester properties of the SEAoff group enabled the solid phase concatenation of unprotected peptide segments by N-to-C sequential formation of native peptide bonds. The strategy was applied to the synthesis of a 60 amino acid-long latent peptide thioester or to the assembly of five peptide segments to give a 15 kDa polypeptide.

Up to now, the advantages of solid phase protein synthesis have been largely under-utilized due to the difficulty of designing a simple and efficient elongation cycle enabling the concatenation of unprotected peptide segments. The combination of selective N-terminal anchoring (N3-Esoc linker) with the blocked thioester properties of the SEAoff group enabled the solid phase concatenation of unprotected peptide segments by N-to-C sequential formation of native peptide bonds. The strategy was applied to the synthesis of a 60 amino acid-long latent peptide thioester or to the assembly of five peptide segments to give a 15 kDa polypeptide.

Senille V., Lelievre D., Paquet F., Garnier N., Lamb N., Legrand A., Delmas A.F., Landon C.  (2013)

The Addressing Fragment of Mitogaligin : First Insights into Functional and Structural Properties

ChemBioChem 14 (6) 711-720
Mitogaligin is a mitochondrion-targeting protein involved in cell death. The sequence of the protein is unrelated to that of any known pro- or antiapoptotic protein. Mitochondrial targeting is controlled by an internal sequence from residues 31 to 53, and although this sequence is essential and sufficient to provoke cell death, the precise mechanism of action at the mitochondrial membrane remains to be elucidated. Here, by focusing on the [31–53] fragment, we first assessed and confirmed its cell cytotoxicity by microinjection. Subsequently, with the aid of membrane models, we evaluated the impact of the membrane environment on the 3D structure of the peptide and on how the peptide is embedded and oriented within membranes. The fragment is well organized, even though it does not contain a canonical secondary structure, and adopts an interfacial location. Structural comparison with other membrane-interacting Trp-rich peptides demonstrated similarities with the antimicrobial peptide tritrpcidin.

Mitogaligin is a mitochondrion-targeting protein involved in cell death. The sequence of the protein is unrelated to that of any known pro- or antiapoptotic protein. Mitochondrial targeting is controlled by an internal sequence from residues 31 to 53, and although this sequence is essential and sufficient to provoke cell death, the precise mechanism of action at the mitochondrial membrane remains to be elucidated. Here, by focusing on the [31–53] fragment, we first assessed and confirmed its cell cytotoxicity by microinjection. Subsequently, with the aid of membrane models, we evaluated the impact of the membrane environment on the 3D structure of the peptide and on how the peptide is embedded and oriented within membranes. The fragment is well organized, even though it does not contain a canonical secondary structure, and adopts an interfacial location. Structural comparison with other membrane-interacting Trp-rich peptides demonstrated similarities with the antimicrobial peptide tritrpcidin.


2012   Références trouvées : 6

Aucagne, V., Valverde, I.E., Marceau, P., Galibert, M., Dendane, N., Delmas, A.F.  (2012)

Towards the Simplification of Protein Synthesis : Iterative Solid-Supported Ligations with Concomitant Purifications

Angewandte Chemie International Edition, 51 (45) 11320-11324
Please release me : A new linker for the temporary tagging of peptides at their N-terminus after solid-phase elongation, and its potential for capture/release purification is demonstrated. This concept is extended to a remarkably efficient self-purifying N-to-C iterative triazole ligation strategy, which is applied to the synthesis of a polypeptide having 160 residues, in a high purity without the need for chromatographic purification (see picture ; orange blocks : peptide segments).

Please release me : A new linker for the temporary tagging of peptides at their N-terminus after solid-phase elongation, and its potential for capture/release purification is demonstrated. This concept is extended to a remarkably efficient self-purifying N-to-C iterative triazole ligation strategy, which is applied to the synthesis of a polypeptide having 160 residues, in a high purity without the need for chromatographic purification (see picture ; orange blocks : peptide segments).

Tavel, L., Jaquillard, L., Karsisiotis, A.I., Saab, F., Jouvensal, L., Brans, A., Delmas, A., Schoentgen, F., Cadene, M. and Damblon, C.  (2012)

Ligand binding study of human PEBP1/RKIP : interaction with nucleotides and Raf-1 peptides evidenced by NMR and Mass Spectrometry

PLoS ONE 7 (4) e36187
In the present study, we investigated human PEBP1 by NMR to determine the binding site of four different ligands : GTP, FMN, and one Raf-1 peptide in tri-phosphorylated and non-phosphorylated forms. The study was carried out by NMR in near physiological conditions, allowing for the identification of the binding site and the determination of the affinity constants KD for different ligands. Native mass spectrometry was used as an alternative method for measuring KD values. Our study demonstrates and/or confirms the binding of hPEBP1 to the four studied ligands. All of them bind to the same region centered on the conserved ligand-binding pocket of hPEBP1. Although the affinities for GTP and FMN decrease as pH, salt concentration and temperature increase from pH 6.5/NaCl 0 mM/20°C to pH 7.5/NaCl 100 mM/30°C, both ligands clearly do bind under conditions similar to what is found in cells regarding pH, salt concentration and temperature. In addition, our work confirms that residues in the vicinity of the pocket rather than those within the pocket seem to be required for interaction with Raf-1.

In the present study, we investigated human PEBP1 by NMR to determine the binding site of four different ligands : GTP, FMN, and one Raf-1 peptide in tri-phosphorylated and non-phosphorylated forms. The study was carried out by NMR in near physiological conditions, allowing for the identification of the binding site and the determination of the affinity constants KD for different ligands. Native mass spectrometry was used as an alternative method for measuring KD values. Our study demonstrates and/or confirms the binding of hPEBP1 to the four studied ligands. All of them bind to the same region centered on the conserved ligand-binding pocket of hPEBP1. Although the affinities for GTP and FMN decrease as pH, salt concentration and temperature increase from pH 6.5/NaCl 0 mM/20°C to pH 7.5/NaCl 100 mM/30°C, both ligands clearly do bind under conditions similar to what is found in cells regarding pH, salt concentration and temperature. In addition, our work confirms that residues in the vicinity of the pocket rather than those within the pocket seem to be required for interaction with Raf-1.

Gaulier, C., Hospital, A., Legeret, B., Delmas, A.F., Aucagne, V., Cisnetti, F. and Gautier, A.  (2012)

A Water Soluble CuI–NHC for CuAAC ligation of unprotected peptides under open air conditions

Chemical Communications 48 (33) 4005-4007
A reducing agent–free version of CuAAC able to operate

under open air conditions is reported. A readily–synthesizable, hydrophilic and highly stable CuI–NHC allows the clean ligations of unprotected peptides comprising sensitive side chains, at millimolar concentrations.

A reducing agent–free version of CuAAC able to operate
under open air conditions is reported. A readily–synthesizable, hydrophilic and highly stable CuI–NHC allows the clean ligations of unprotected peptides comprising sensitive side chains, at millimolar concentrations.

Buré, C., Marceau, P., Meudal, H. and Delmas, A.F.  (2012)

Synthesis and analytical investigation of C-terminally modified peptide aldehydes and ketone : application to oxime ligation

Journal of Peptide Science 18 (3) 147-154
C-terminally modified peptides aldehyde (glycinal and alpha-oxo aldehyde peptides) and ketone (pyruvic acid-containing

peptide) were synthesised to get new insights into the mechanism of acido-catalysed oxime ligation. Their tetrahedral

hydrated forms were investigated in solution and in the gas phase, using NMR and in-source collision-induced dissociation mass

spectrometry, respectively, and the kinetics of the oximation reactions followed using analytical HPLC. The results obtained confirmed

that the first step of the oximation reaction was the limiting step for the pyruvic acid-containing peptides because of the

steric effect and of the carbon angular strain of the ketone. The second step is the determining step for the aldehyde peptides

because the basicity of the oxygen of the hydroxyl function of the tetrahedral form is greater for glycinal than for alpha-oxo

aldehyde. These data strongly suggest that the hydrated form of the aldehyde partner has to be considered when oxime

reactions are performed in aqueous buffer.

C-terminally modified peptides aldehyde (glycinal and alpha-oxo aldehyde peptides) and ketone (pyruvic acid-containing
peptide) were synthesised to get new insights into the mechanism of acido-catalysed oxime ligation. Their tetrahedral
hydrated forms were investigated in solution and in the gas phase, using NMR and in-source collision-induced dissociation mass
spectrometry, respectively, and the kinetics of the oximation reactions followed using analytical HPLC. The results obtained confirmed
that the first step of the oximation reaction was the limiting step for the pyruvic acid-containing peptides because of the
steric effect and of the carbon angular strain of the ketone. The second step is the determining step for the aldehyde peptides
because the basicity of the oxygen of the hydroxyl function of the tetrahedral form is greater for glycinal than for alpha-oxo
aldehyde. These data strongly suggest that the hydrated form of the aldehyde partner has to be considered when oxime
reactions are performed in aqueous buffer.

Derache, C., Meudal, H., Aucagne, V., Mark, K.J., Cadène, M., Delmas, A.F., Lalmanach, A.C. and Landon, C.  (2012)

Initial insights into the structure-activity relationships of avian defensins

Journal of Biological Chemistry 287 (10) 7746-7755
Numerous β-defensins have been identified in birds and the potential use of these peptides as alternatives to antibiotics has been proposed, in particular to fight antibiotic-resistant and zoonotic bacterial species. Little is known about the mechanism of antibacterial activity of avian β-defensins (AvBDs), and the present work was carried out to obtain initial insights into the involvement of structural features or specific residues in the antimicrobial activity of chicken AvBD2. Chicken AvBD2 and its enantiomeric counterpart were chemically synthesized. Peptide elongation and oxidative folding were both optimized. The similar antimicrobial activity measured for both L- and D- proteins clearly indicates that there is no chiral partner. Therefore the bacterial membrane is in all likelihood the primary target. Moreover, this work evidences that the three-dimensional fold is required for an optimal antimicrobial activity, in particular for Gram-positive bacterial strains. The three-dimensional NMR structure of chicken AvBD2 defensin displays the structural 3-stranded antiparallel β-sheet characteristic of β-defensins. The surface of the molecule does not display any amphipathic character. In light of this new structure and of the king penguin AvBD103b defensin structure, the consensus sequence of avian β-defensin′s family was analyzed. Well conserved residues were highlighted and the potential strategic role of the lysine 31 residue of AvBD2 emphasized. The synthetic AvBD2-K31A variant displayed substantial N-terminal structural modifications and a dramatic decrease in activity. Taken together, these results demonstrate the structural as well as the functional role of the critical lysine 31 residue in antimicrobial activity.

Numerous β-defensins have been identified in birds and the potential use of these peptides as alternatives to antibiotics has been proposed, in particular to fight antibiotic-resistant and zoonotic bacterial species. Little is known about the mechanism of antibacterial activity of avian β-defensins (AvBDs), and the present work was carried out to obtain initial insights into the involvement of structural features or specific residues in the antimicrobial activity of chicken AvBD2. Chicken AvBD2 and its enantiomeric counterpart were chemically synthesized. Peptide elongation and oxidative folding were both optimized. The similar antimicrobial activity measured for both L- and D- proteins clearly indicates that there is no chiral partner. Therefore the bacterial membrane is in all likelihood the primary target. Moreover, this work evidences that the three-dimensional fold is required for an optimal antimicrobial activity, in particular for Gram-positive bacterial strains. The three-dimensional NMR structure of chicken AvBD2 defensin displays the structural 3-stranded antiparallel β-sheet characteristic of β-defensins. The surface of the molecule does not display any amphipathic character. In light of this new structure and of the king penguin AvBD103b defensin structure, the consensus sequence of avian β-defensin′s family was analyzed. Well conserved residues were highlighted and the potential strategic role of the lysine 31 residue of AvBD2 emphasized. The synthetic AvBD2-K31A variant displayed substantial N-terminal structural modifications and a dramatic decrease in activity. Taken together, these results demonstrate the structural as well as the functional role of the critical lysine 31 residue in antimicrobial activity.

Valverde, I., Lecaille, F., Lalmanach, G., Aucagne, V. & Delmas, A.F.  (2012)

Synthesis of a biologically active triazole-containing analogue of cystatin A through successive peptidomimetic alkyne–azide ligations

Angewandte Chemie International Edition 51 (3) 718-722
“Click” protein : CuI-catalyzed cycloaddition of azides and terminal alkynes has been applied to the successive ligations of three unprotected peptide fragments. Peptidomimetic triazole ligation (PTL, see scheme) as a new method for the chemical production of bioactive proteins is applied for the synthesis of a triazole-containing analogue of the 97 amino acid protein cystatin A.

“Click” protein : CuI-catalyzed cycloaddition of azides and terminal alkynes has been applied to the successive ligations of three unprotected peptide fragments. Peptidomimetic triazole ligation (PTL, see scheme) as a new method for the chemical production of bioactive proteins is applied for the synthesis of a triazole-containing analogue of the 97 amino acid protein cystatin A.


2011   Références trouvées : 1

Hardouin, J., Cremer, A. G. & Delmas, A. F.  (2011)

Investigation of in-source decay of oxime-linked peptide by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Rapid Communications in Mass Spectrometry 25 (14) 2106-2112


2010   Références trouvées : 1

Da Silva, P., Rahioui, I., Laugier, C., Jouvensal, L., Meudal, H., Chouabe, C., Delmas, A.F. & Gressent, F.  (2010)

Molecular requirements for the insecticidal activity of the plant peptide PA1b

J. Biol. Chem., 285, 32689-32694.


2009   Références trouvées : 4

Da Silva, P., Strzepa, A., Jouvensal, L., Rahioui, I., Gressent, F. & Delmas, A.F.  (2009)

A folded and functional synthetic PA1b, an interlocked entomotoxic miniprotein.

Biopolymers, 92, 436-444.

Valverde, I.E., Delmas, A.F. & Aucagne, V.  (2009)

Click a la carte : robust semi-orthogonal alkyne protecting groups for multiple successive azide/alkyne cycloadditions.

Tetrahedron 65, 7597-7602.

Derache, C., Labas, V., Aucagne, V., Meudal, H., Landon, C., Delmas, A.F., Magallon, T. & Lalmanach, A.C.  (2009)

Primary Structure and Antibacterial Activity of Chicken Bone Marrow-Derived beta-Defensins.

Antimicrob. Agents Chemother. 53, 4647-4655.

Bure, C., Maget, R., Delmas, A.F., Pichon, C. & Midoux, P.  (2009)

Histidine-rich peptide : evidence for a single zinc-binding site on H5WYG peptide that promotes membrane fusion at neutral pH.

J. Mass Spectrom. 44, 81-89.


2008   Références trouvées : 2

Lelièvre, D ; Barta, P ; Aucagne, V ; Delmas, AF  (2008)

Preparation of peptide thioesters using Fmoc strategy through hydroxyl side chain anchoring

Tetrahedron Letters 49 4016-4019

Bure, C ; Goffinont, S ; Delmas, AF ; Cadene, M ; Culard, F  (2008)

Oxidation-sensitive residues mediate the DNA bending abilities of the architectural MC1 protein

Journal of Molecular Biology 376 (1) 120-130
The Methanosarcina thermophila MC1 protein is a small basic protein that is able to bend DNA sharply. When this protein is submitted to oxidative stress through gamma irradiation, it loses its original DNA interaction properties. The protein can still bind DNA but its ability to bend DNA is decreased dramatically. Here, we used different approaches to determine the oxidations that are responsible for this inactivation. Through a combination of proteolysis and mass spectrometry we have identified the three residues that are oxidized preferentially.

The Methanosarcina thermophila MC1 protein is a small basic protein that is able to bend DNA sharply. When this protein is submitted to oxidative stress through gamma irradiation, it loses its original DNA interaction properties. The protein can still bind DNA but its ability to bend DNA is decreased dramatically. Here, we used different approaches to determine the oxidations that are responsible for this inactivation. Through a combination of proteolysis and mass spectrometry we have identified the three residues that are oxidized preferentially.


2007   Références trouvées : 1

Decostaire, IP ; Lelièvre, D ; Aucagne, V ; Delmas, AF  (2007)

Synergic effect of hydride and proton donors in the Pd(0)-mediated deprotection of N-a-Aloc proline derivatives

Tetrahedron Letters 48 6523-6526
In this study, the challenging Pd(O)-catalyzed N-Aloc removal from a proline residue using Me2NH-BH3 or PhSiH3 as allyl scavengers has been investigated. Standard conditions led to a large amount of an allylamine byproduct. A careful study of the reactions allowed us to design the optimal conditions for the quick and quantitative formation of the desired product, while taking advantage of a synergic effect between hydride and proton donors. (c) 2007 Elsevier Ltd. All rights reserved.

In this study, the challenging Pd(O)-catalyzed N-Aloc removal from a proline residue using Me2NH-BH3 or PhSiH3 as allyl scavengers has been investigated. Standard conditions led to a large amount of an allylamine byproduct. A careful study of the reactions allowed us to design the optimal conditions for the quick and quantitative formation of the desired product, while taking advantage of a synergic effect between hydride and proton donors. (c) 2007 Elsevier Ltd. All rights reserved.


2006   Références trouvées : 6

Cremer, GA ; Bureaud, N ; Lelievre, D ; Piller, V ; Kunz, H ; Piller, F ; Delmas, AF  (2006)

Modular synthesis and immunological evaluation of MUC-derived glycoproteine antigens

Journal of Peptide Science 12100-100 Suppl. S

Decostaire, IP ; Lelièvre, D ; Zhang, HH ; Delmas, AF  (2006)

Controlling the outcome of overacylation of N-protected aminooxyacetic acid during the synthesis of an aminooxy-peptide for chemical ligation

Tetrahedron Letters 47 (39) 7057-7060
An amino oxy-containing peptide, the nucleophile partner for oxime ligations, is usually grafted ona NH2-peptide resin by activating a protected aminooxyacetic acid as an active ester. Here, we have shown that its subsequent coupling to NH2-peptide resin competes with the overacylation of the -NH-O-nitrogen and that the overacylation level increases with the basicity of the reaction mixture. Moreover, we found that overacylation is prevented when the COOH of the Aoa-derivatives is engaged in an amide bond. (c) 2006 Elsevier Ltd. All rights reserved.

An amino oxy-containing peptide, the nucleophile partner for oxime ligations, is usually grafted ona NH2-peptide resin by activating a protected aminooxyacetic acid as an active ester. Here, we have shown that its subsequent coupling to NH2-peptide resin competes with the overacylation of the -NH-O-nitrogen and that the overacylation level increases with the basicity of the reaction mixture. Moreover, we found that overacylation is prevented when the COOH of the Aoa-derivatives is engaged in an amide bond. (c) 2006 Elsevier Ltd. All rights reserved.

Cremer, GA ; Bureaud, N ; Piller, V ; Kunz, H ; Piller, F ; Delmas, AF  (2006)

Synthesis and biological evaluation of a multiantigenic Tn/TF-containing glycopeptide mimic of the tumor-related MUC1 glycoprotein (Publication ayant donné lieu à la couverture du numéro)

Chemmedchem 1 (9) 965-968
The glycopeptide construct shown was synthesized using a convergent strategy based on oxime chemical ligation. It is composed of a universal T-helper and three tumor-related epitopes from the human mucin MUC1, an non-glycosylated repeat unit, and two units glycosylated with the Tn and TF epitopes, respectively. In association with a mild adjuvant suitable for human therapy, this construct elicited a strong specific immune response in mice, directed against natural tumor-associated structures.

The glycopeptide construct shown was synthesized using a convergent strategy based on oxime chemical ligation. It is composed of a universal T-helper and three tumor-related epitopes from the human mucin MUC1, an non-glycosylated repeat unit, and two units glycosylated with the Tn and TF epitopes, respectively. In association with a mild adjuvant suitable for human therapy, this construct elicited a strong specific immune response in mice, directed against natural tumor-associated structures.

Bure, C ; Castaing, B ; Lange, C ; Delmas, AF  (2006)

Location and base selectivity on fragmentation of brominated oligodeoxynucleotides

Journal of Mass Spectrometry 41 (1) 84-90
Bromine-modified oligodeoxynucleotides (ODNs) were fragmented in the electrospray source to study the influence of brominated bases on fragmentation. Several 13-mer ODNs containing a brominated pyrimidine base, BrdU (5-bromodeoxyuridine) or BrdC (5-bromodeoxycytidine), were analyzed. Low cone voltage fragmentation yields a loss of the brominated base with a preferential loss for the brominated base closer to the 5-end (2-position > 4-position > 12-position) as well as a preferential loss of BrdU over BrdC. Higher cone voltage produces backbone fragmentation with complementary a(n)-base and w(m) ions close to the brominated base. On the basis of these observations, we located the brominated base in the sequence for all of the ODNs studied. Copyright (c) 2005 John Wiley & Sons, Ltd.

Bromine-modified oligodeoxynucleotides (ODNs) were fragmented in the electrospray source to study the influence of brominated bases on fragmentation. Several 13-mer ODNs containing a brominated pyrimidine base, BrdU (5-bromodeoxyuridine) or BrdC (5-bromodeoxycytidine), were analyzed. Low cone voltage fragmentation yields a loss of the brominated base with a preferential loss for the brominated base closer to the 5-end (2-position > 4-position > 12-position) as well as a preferential loss of BrdU over BrdC. Higher cone voltage produces backbone fragmentation with complementary a(n)-base and w(m) ions close to the brominated base. On the basis of these observations, we located the brominated base in the sequence for all of the ODNs studied. Copyright (c) 2005 John Wiley & Sons, Ltd.

Hardouin, J ; Hubert-Roux, M ; Delmas, AF ; Lange, C  (2006)

Identification of isoenzymes using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Rapid Communications in Mass Spectrometry 20 (5) 725-732
The identification of isoforms is one of the great challenges in proteomics due to the large number of identical amino acids preventing their separations by two-dimensional electrophoresis. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) has become a rapid and sensitive tool in proteomics, notably with the new instrumental improvements. In this study, we used several acquisition modes of MALDI-TOFMS to identify isoforms of porcine glutathiones S-transferase. The use of multiple proteases coupled to the different acquisition modes of MALDI-TOFMS (linear, reflectron, post-source decay (PSD) and in-source decay, positive and negative modes) allowed the identification of two sequences. Moreover, a third sequence is pointed out from a PSD study of a tryptic ion revealing the modification of the amino acid tyrosine 146 to phenylalanine. Copyright (c) 2006 John Wiley & Sons, Ltd.

The identification of isoforms is one of the great challenges in proteomics due to the large number of identical amino acids preventing their separations by two-dimensional electrophoresis. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) has become a rapid and sensitive tool in proteomics, notably with the new instrumental improvements. In this study, we used several acquisition modes of MALDI-TOFMS to identify isoforms of porcine glutathiones S-transferase. The use of multiple proteases coupled to the different acquisition modes of MALDI-TOFMS (linear, reflectron, post-source decay (PSD) and in-source decay, positive and negative modes) allowed the identification of two sequences. Moreover, a third sequence is pointed out from a PSD study of a tryptic ion revealing the modification of the amino acid tyrosine 146 to phenylalanine. Copyright (c) 2006 John Wiley & Sons, Ltd.

Cremer, GA ; Tariq, H ; Delmas, AF  (2006)

Combining a polar resin and a pseudo-proline to optimize the solid-phase synthesis of a ’difficult sequence’

Journal of Peptide Science 12 (6) 437-442
This paper describes the optimization of a synthesis of a difficult sequence related to a 12-mer sequence of a Pan DR epitope (PADRE). Elongation was followed by on-line monitoring of the N-a-Fmoc removal adapted for the batch methodology. Studying the intrinsic factors related to the peptide-resin, such as substitution level, resin nature and backbone protecting group, has led to an increase in the elongation yield and purity of the crude peptide. Optimal elongation was obtained by combining a polar resin such as PEGA and a pseudo-proline as the backbone protecting group. Copyright (c) 2006 European Peptide Society and John Wiley & Sons, Ltd.

This paper describes the optimization of a synthesis of a difficult sequence related to a 12-mer sequence of a Pan DR epitope (PADRE). Elongation was followed by on-line monitoring of the N-a-Fmoc removal adapted for the batch methodology. Studying the intrinsic factors related to the peptide-resin, such as substitution level, resin nature and backbone protecting group, has led to an increase in the elongation yield and purity of the crude peptide. Optimal elongation was obtained by combining a polar resin such as PEGA and a pseudo-proline as the backbone protecting group. Copyright (c) 2006 European Peptide Society and John Wiley & Sons, Ltd.


2005   Références trouvées : 3

Marceau, P ; Bure, C ; Delmas, AF  (2005)

Efficient synthesis of C-terminal modified peptide ketones for chemical ligations

Bioorganic & Medicinal Chemistry Letters 15 (24) 5442-5445
Synthesis of a C-terminal modified peptide with an a-amido methylketone was efficiently carried out using a backbone-amide-type linker loading with a monofunctionalized diamine, provided that no base such as piperidine or diisopropylethylamine or a reducing agent such as triisopopylsilane was used for the synthetic pathway. The ketoxime-forming chemoselective ligation between a methylketone and an aminooxy was quantitative in 5 h at pH 2. (c) 2005 Elsevier Ltd. All rights reserved.

Synthesis of a C-terminal modified peptide with an a-amido methylketone was efficiently carried out using a backbone-amide-type linker loading with a monofunctionalized diamine, provided that no base such as piperidine or diisopropylethylamine or a reducing agent such as triisopopylsilane was used for the synthetic pathway. The ketoxime-forming chemoselective ligation between a methylketone and an aminooxy was quantitative in 5 h at pH 2. (c) 2005 Elsevier Ltd. All rights reserved.

Antonopoulos, A ; Hardouin, J ; Favetta, P ; Helbert, W ; Delmas, AF ; Lafosse, M  (2005)

Matrix-assisted laser desorption/ionisation mass spectrometry for the direct analysis of enzymatically digested kappa- iota- and hybrid iota/nu-carrageenans

Rapid Communications in Mass Spectrometry 19 (16) 2217-2226
Enzymatically digested oligosaccharides of kappa-, iota- and hybrid iota/nu-carrageenans were analysed using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry in the negative-ion mode. nor-Harmane was used as matrix. Depending on the stock concentration and the laser intensity applied, the oligosaccharides exhibited losses of sulphate units (neutralised by the Na+ ion, and thus non-stable), leaving the primary backbone structure in most cases with only the deprotonated sulphate groups (carrying the negative charge, stable).

Enzymatically digested oligosaccharides of kappa-, iota- and hybrid iota/nu-carrageenans were analysed using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry in the negative-ion mode. nor-Harmane was used as matrix. Depending on the stock concentration and the laser intensity applied, the oligosaccharides exhibited losses of sulphate units (neutralised by the Na+ ion, and thus non-stable), leaving the primary backbone structure in most cases with only the deprotonated sulphate groups (carrying the negative charge, stable).

Bure, C ; Boujard, O ; Bertrand, M ; Lange, C ; Delmas, AF  (2005)

Collision-induced dissociation of peptide thioesters : the influence of peptide length on fragmentation

European Journal of Mass Spectrometry 11 (1) 31-34
Five peptide thioesters of increasing length were fragmented using two processes, in-source and in-collision cell fragmentation, using an electrospray source coupled to a triple quadrupole mass spectrometer. Comparison of their fragmentations was made in regard to their length. The two fragmentation conditions show that the peptide length has no influence on structural information and that the fragmentation efficiency is higher for the smallest peptides than for the longest. The particularity of these peptide thioesters consists of the neutral loss of ethanethiol. The absence of the a(3) fragment ion and the presence of the (a(3)-17) ion on the collision-induced dissociation mass spectra are noted.

Five peptide thioesters of increasing length were fragmented using two processes, in-source and in-collision cell fragmentation, using an electrospray source coupled to a triple quadrupole mass spectrometer. Comparison of their fragmentations was made in regard to their length. The two fragmentation conditions show that the peptide length has no influence on structural information and that the fragmentation efficiency is higher for the smallest peptides than for the longest. The particularity of these peptide thioesters consists of the neutral loss of ethanethiol. The absence of the a(3) fragment ion and the presence of the (a(3)-17) ion on the collision-induced dissociation mass spectra are noted.


2004   Références trouvées : 3

Cremer, GA ; Bureaud, N ; Lelievre, D ; Piller, V ; Piller, F ; Delmas, A  (2004)

Synthesis of branched oxime-linked peptide mimetics of the MUC1 containing a universal T-helper epitope

Chemistry-A European Journal 10 (24) 6353-6360
Our goal was to develop mimics of MUC1, highly immunogenic to induce an efficient immune response against the tumor-associated form of MUC1, and sufficiently different from the natural antigen to bypass the tolerance barrier in humans. With the aim of obtaining a well-defined peptide construct as a means of evoking the precise immune responses required in immunotherapy, we synthesized artificial mimics of the MUC1 protein composed of two MUC1 repeat units of inverse orientation and a universal T-helper epitope. To synthesize these heteromeric peptide constructs, we followed a convergent approach using chemoselective ligation based on oxime chemistry. A stem peptide was first synthesized bearing two orthogonally masked aldehydes. After successive depfotection, two oxime bonds can be specifically generated. The proposed strategy proved to be concise and robust, and allowed the synthesis of the tri-branched protein in a very satisfactory yield. The different constructs were tested for their ability to generate antibodies able to recognize the MUC1 protein.

Our goal was to develop mimics of MUC1, highly immunogenic to induce an efficient immune response against the tumor-associated form of MUC1, and sufficiently different from the natural antigen to bypass the tolerance barrier in humans. With the aim of obtaining a well-defined peptide construct as a means of evoking the precise immune responses required in immunotherapy, we synthesized artificial mimics of the MUC1 protein composed of two MUC1 repeat units of inverse orientation and a universal T-helper epitope. To synthesize these heteromeric peptide constructs, we followed a convergent approach using chemoselective ligation based on oxime chemistry. A stem peptide was first synthesized bearing two orthogonally masked aldehydes. After successive depfotection, two oxime bonds can be specifically generated. The proposed strategy proved to be concise and robust, and allowed the synthesis of the tri-branched protein in a very satisfactory yield. The different constructs were tested for their ability to generate antibodies able to recognize the MUC1 protein.

Lelièvre, D ; Favard, C ; Antonny, B ; Chabre, M ; Delmas, A  (2004)

Design and synthesis of a peptide construct from p23 protein using oxime ligation

Biopolymers 71 (3) 404-404 Art No. P471

Bure, C ; Le Falher, G ; Lange, C ; Delmas, A  (2004)

Fragmentation study of peptide acetals and aldehydes using in-source collision-induced dissociation

Journal of Mass Spectrometry 39 (7) 817-823
The fragmentation of peptide acetals and peptide diols, corresponding to the hydrated form of the peptide aldehyde, is dominated by the successive losses of two molecules of MeOH and water, respectively. Using model peptides, the fragmentation mechanism, with respect to the loss of methanol and water, was elucidated. The first loss was certainly charge-directed whereas the second probably occurred via the nucleophilic attack of the nitrogen of an amine on the C-terminal carbon leading to a cyclic ion. Copyright (C) 2004 John Wiley Sons, Ltd.

The fragmentation of peptide acetals and peptide diols, corresponding to the hydrated form of the peptide aldehyde, is dominated by the successive losses of two molecules of MeOH and water, respectively. Using model peptides, the fragmentation mechanism, with respect to the loss of methanol and water, was elucidated. The first loss was certainly charge-directed whereas the second probably occurred via the nucleophilic attack of the nitrogen of an amine on the C-terminal carbon leading to a cyclic ion. Copyright (C) 2004 John Wiley Sons, Ltd.


2002   Références trouvées : 2

Lelièvre, D ; Turpin, O ; El Kazzouli, S ; Delmas, A  (2002)

Influence of polar support for the synthesis of large C-terminal peptide aldehyde : application to chemoselective ligation

Tetrahedron 58 (27) 5525-5533
Efficient conditions have been developed for the synthesis of large peptide aldehydes from solid support through nucleophilic displacement. Aminolysis of the ester bond between a deprotected peptide and the phenylacetamidomethyl linker with aminoacetaldehyde-dimethylacetal leads to a peptide aldehyde masked as an acetal. Besides the optimization of parameters such as solvents, workup procedure and temperature, the influence of the nature of the polymeric support was crucial. Among the solid supports tested, the poly(ethylene glycol)poly(acrylamide) resin proved to afford the best cleavage yield. This work underlines that the solid support has to be considered as a co-solvent rather than an inert carrier. Our methodology was further applied to the synthesis of a 33-mer with T-helper activity from the fusion protein of measles virus. The 33-mer peptide aldehyde was then chemoselectively ligated via an oxime bond to an (aminooxy) acetyl peptide with T-cytotoxic activity. (C) 2002 Elsevier Science Ltd. All rights reserved.

Efficient conditions have been developed for the synthesis of large peptide aldehydes from solid support through nucleophilic displacement. Aminolysis of the ester bond between a deprotected peptide and the phenylacetamidomethyl linker with aminoacetaldehyde-dimethylacetal leads to a peptide aldehyde masked as an acetal. Besides the optimization of parameters such as solvents, workup procedure and temperature, the influence of the nature of the polymeric support was crucial. Among the solid supports tested, the poly(ethylene glycol)poly(acrylamide) resin proved to afford the best cleavage yield. This work underlines that the solid support has to be considered as a co-solvent rather than an inert carrier. Our methodology was further applied to the synthesis of a 33-mer with T-helper activity from the fusion protein of measles virus. The 33-mer peptide aldehyde was then chemoselectively ligated via an oxime bond to an (aminooxy) acetyl peptide with T-cytotoxic activity. (C) 2002 Elsevier Science Ltd. All rights reserved.

Boillot, F ; Chabin, A ; Bure, C ; Venet, M ; Belsky, A ; Bertrand-Urbaniak, M ; Delmas, A ; Brack, A ; Barbier, B  (2002)

The Perseus Exobiology mission on MIR : Behaviour of amino acids and peptides in earth orbit

Origins of Life and Evolution of The Biosphere 32 (4) 359-385
Leucine, a-methyl leucine and two peptides were exposed to space conditions on board the MIR station during the Perseus-Exobiology mission. This long duration space mission was aimed at testing the delivery of prebiotic building blocks. During this mission, two amino acids (leucine and a-methyl leucine) and two peptides (leucine-diketopiperazine and trileucine thioethylester) were exposed in Earth orbit for three months. Basalt, clay and meteorite powder were also mixed with the samples in order to simulate the effects of potential meteorite protection.

Leucine, a-methyl leucine and two peptides were exposed to space conditions on board the MIR station during the Perseus-Exobiology mission. This long duration space mission was aimed at testing the delivery of prebiotic building blocks. During this mission, two amino acids (leucine and a-methyl leucine) and two peptides (leucine-diketopiperazine and trileucine thioethylester) were exposed in Earth orbit for three months. Basalt, clay and meteorite powder were also mixed with the samples in order to simulate the effects of potential meteorite protection.


2001   Références trouvées : 2

Lelièvre, D ; Bure, C ; Laot, F ; Delmas, A  (2001)

Synthesis of peptide di-aldehyde precursor for stepwise chemoselective ligations via oxime bonds

Tetrahedron Letters 42 (2) 235-238
To synthezise a triple-function branched peptide in a modular way, we present a new strategy based on orthogonal generation of two aldehyde functions from an acetal and a 2-amino alcohol. Successive unmaskings of aldehyde functions of the stem peptide affords stepwise chemoselective ligations of two (aminooxy)acetyl peptides via oxime bonds. (C) 2000 Elsevier Science Ltd. All rights reserved.

To synthezise a triple-function branched peptide in a modular way, we present a new strategy based on orthogonal generation of two aldehyde functions from an acetal and a 2-amino alcohol. Successive unmaskings of aldehyde functions of the stem peptide affords stepwise chemoselective ligations of two (aminooxy)acetyl peptides via oxime bonds. (C) 2000 Elsevier Science Ltd. All rights reserved.

Bure, C ; Gobert, W ; Lelièvre, D ; Delmas, A  (2001)

In-source fragmentation of peptide aldehydes and acetals : influence of peptide length and charge state

Journal of Mass Spectrometry 36 (10) 1149-1155
The use of in-source collision-induced dissociation (CID) was evaluated to generate structural information on peptide aldehydes, which represent an important class of compounds as inhibitors for serine and cysteine proteases and as key intermediates for protein engineering. By studying five peptide aldehydes of different lengths, and their peptide acetal counterparts, mass to charge (m/z) dependency of in-source fragmentation was established for peptides that differ only by their C-terminal functionalization. In-source fragmentation of peptide aldehydes and acetals leads to the same final ion, probably via a similar mechanism. Moreover, the gas-phase information obtained here reflects the equilibrium occurring in solution between the peptide aldehyde and its hydrated form, which was retained during the ionization process. The equilibrium constant was determined to be close to unity. Disturbance of this equilibrium should enable the stability of covalent hydration of a given series of aldehydes to be compared. Copyright (C) 2001 John Wiley & Sons, Ltd.

The use of in-source collision-induced dissociation (CID) was evaluated to generate structural information on peptide aldehydes, which represent an important class of compounds as inhibitors for serine and cysteine proteases and as key intermediates for protein engineering. By studying five peptide aldehydes of different lengths, and their peptide acetal counterparts, mass to charge (m/z) dependency of in-source fragmentation was established for peptides that differ only by their C-terminal functionalization. In-source fragmentation of peptide aldehydes and acetals leads to the same final ion, probably via a similar mechanism. Moreover, the gas-phase information obtained here reflects the equilibrium occurring in solution between the peptide aldehyde and its hydrated form, which was retained during the ionization process. The equilibrium constant was determined to be close to unity. Disturbance of this equilibrium should enable the stability of covalent hydration of a given series of aldehydes to be compared. Copyright (C) 2001 John Wiley & Sons, Ltd.


2000   Références trouvées : 2

Bure, C ; Lelièvre, D ; Delmas, A  (2000)

Identification of by-products from an orthogonal peptide ligation by oxime bonds using mass spectrometry and tandem mass spectrometry

Rapid Communications in Mass Spectrometry 14 (23) 2158-2164
Synthetic proteins with unusual architecture are obtained through chemoselective ligation, a method based on the condensation of unprotected peptides under mild aqueous conditions,. The last step of a new procedure leading to a tri-branched conjugate consists of the chemoselective ligation reaction between an (amioooxy)acetyl peptide and a peptide aldehyde resulting from a first ligation via an oxime bond. In order to optimize the reaction conditions, electrospray ionization mass spectrometry combined with Liquid chromatography and tandem mass spectrometry has been used.

Synthetic proteins with unusual architecture are obtained through chemoselective ligation, a method based on the condensation of unprotected peptides under mild aqueous conditions,. The last step of a new procedure leading to a tri-branched conjugate consists of the chemoselective ligation reaction between an (amioooxy)acetyl peptide and a peptide aldehyde resulting from a first ligation via an oxime bond. In order to optimize the reaction conditions, electrospray ionization mass spectrometry combined with Liquid chromatography and tandem mass spectrometry has been used.

Lugardon, K ; Raffner, R ; Goumon, Y ; Corti, A ; Delmas, A ; Bulet, P ; Aunis, D ; Metz-Boutigue, MH  (2000)

Antibacterial and antifungal activities of vasostatin-1, the N-terminal fragment of chromogranin A

Journal of Biological Chemistry 275 (15) 10745-10753
Vasostatin-1, the natural N-terminal 1-76 chromogranin A (CGA)-derived fragment in bovine sequence, has been purified from chromaffin secretory granules and identified by sequencing and matrix-assisted laser desorption time-of-flight mass spectrometry. This peptide, which displays antibacterial activity against Gram-positive bacteria at micromolar concentrations, is also able to kill a large variety of filamentous fungi and yeast cells in the 1-10 mu M range. We have found that the C-terminal moiety of vasostatin-1 is essential for the antifungal activity, and shorter active peptides have been synthesized.

Vasostatin-1, the natural N-terminal 1-76 chromogranin A (CGA)-derived fragment in bovine sequence, has been purified from chromaffin secretory granules and identified by sequencing and matrix-assisted laser desorption time-of-flight mass spectrometry. This peptide, which displays antibacterial activity against Gram-positive bacteria at micromolar concentrations, is also able to kill a large variety of filamentous fungi and yeast cells in the 1-10 mu M range. We have found that the C-terminal moiety of vasostatin-1 is essential for the antifungal activity, and shorter active peptides have been synthesized.


1999   Références trouvées : 2

Delmotte, C ; Delmas, A  (1999)

Synthesis and fluorescence properties of Oregon Green 514 labeled peptides

Bioorganic & Medicinal Chemistry Letters 9 (20) 2989-2994
Oregon Green 514, with high photostability and a pKa of 4.8, is suitable for imaging applications. Labeling of peptide-bound resin with the carboxylic acid form was optimized. Peptide conjugation resulted in altered fluorescence properties of the dye including a quenching of the intensity compared with that in aqueous buffers with a slight red-shift of the emission maximum. (C) 1999 Elsevier Science Ltd. All rights reserved.

Oregon Green 514, with high photostability and a pKa of 4.8, is suitable for imaging applications. Labeling of peptide-bound resin with the carboxylic acid form was optimized. Peptide conjugation resulted in altered fluorescence properties of the dye including a quenching of the intensity compared with that in aqueous buffers with a slight red-shift of the emission maximum. (C) 1999 Elsevier Science Ltd. All rights reserved.

Delmotte, C ; Le Guern, E ; Trudelle, Y ; Delmas, A  (1999)

Structural features of a chimeric peptide inducing cytotoxic T lymphocyte responses in saline

European Journal of Biochemistry 265 (1) 336-345
Little information is available correlating the structural properties of peptides with their immunogenicity in terms of responses via cytotoxic T lymphocytes (CTLs). The TT-NP6 chimeric peptide, consisting of two copies of a promiscuous T-helper epitope (T : residues 288-302 from the fusion protein of the measles virus) linked to the NP6 T-cytotoxic epitope (NP6 : residues 52-60 from the nucleoprotein of measles virus) was able to induce virus-specific CTL responses in the absence of any adjuvant and hydrophobic component. The present work was undertaken to gain insight into structural features of the TT-NP6 peptide that may be important in optimizing the CTL immunogenicity of the peptide. Circular dichroism data, obtained in a buffer of physiological ionic strength and pH, strongly suggest a self-associated state for the peptide, which was confirmed by a sedimentation velocity experiment.

Little information is available correlating the structural properties of peptides with their immunogenicity in terms of responses via cytotoxic T lymphocytes (CTLs). The TT-NP6 chimeric peptide, consisting of two copies of a promiscuous T-helper epitope (T : residues 288-302 from the fusion protein of the measles virus) linked to the NP6 T-cytotoxic epitope (NP6 : residues 52-60 from the nucleoprotein of measles virus) was able to induce virus-specific CTL responses in the absence of any adjuvant and hydrophobic component. The present work was undertaken to gain insight into structural features of the TT-NP6 peptide that may be important in optimizing the CTL immunogenicity of the peptide. Circular dichroism data, obtained in a buffer of physiological ionic strength and pH, strongly suggest a self-associated state for the peptide, which was confirmed by a sedimentation velocity experiment.


1998   Références trouvées : 1

Lelièvre, D ; Chabane, H ; Delmas, A  (1998)

Simple and efficient solid-phase synthesis of unprotected peptide aldehyde for peptide segment ligation

Tetrahedron Letters 39 (52) 9675-9678
We describe an efficient solid-phase synthesis of C-terminal peptide aldehyde. Making use of the stability of the PAM linker towards both acid and base conditions, a pentapeptide was synthesized starting from a PAM resin according to Fmoc/tBu chemistry. The side-chains were deprotected by TFA. The peptide was cleaved by aminolysis with aminoacetaldehyde-dimethylacetal leading to a C-terminal masked aldehyde. The unprotected peptide aldehyde was then coupled to amino-oxy derivatives by chemoselective ligation in aqueous solution. (C) 1998 Elsevier Science Ltd. All rights reserved.

We describe an efficient solid-phase synthesis of C-terminal peptide aldehyde. Making use of the stability of the PAM linker towards both acid and base conditions, a pentapeptide was synthesized starting from a PAM resin according to Fmoc/tBu chemistry. The side-chains were deprotected by TFA. The peptide was cleaved by aminolysis with aminoacetaldehyde-dimethylacetal leading to a C-terminal masked aldehyde. The unprotected peptide aldehyde was then coupled to amino-oxy derivatives by chemoselective ligation in aqueous solution. (C) 1998 Elsevier Science Ltd. All rights reserved.


1997   Références trouvées : 2

Lelièvre, D ; Hsu, SC ; Daubos, P ; Favard, C ; Vigny, P ; Trudelle, Y ; Steward, MW ; Delmas, A  (1997)

Structural properties of chimeric peptides containing a T-cell epitope linked to a fusion peptide and their importance for in vivo induction of cytotoxic T-cell responses

European Journal of Biochemistry 249 (3) 895-904
We have previously shown that when administered to mice without adjuvant, a chimeric peptide consisting of the fusion peptide F from measles virus protein linked at the C-terminus of a cytotoxic T-cell epitope from the M2 protein of respiratory syncytial virus efficiently primes for an major histocompatibility complex (MHC) class-I restricted cytotoxic T lymphocyte (CTL) response. In this report, we demonstrated by microspectrofluorometry that the fusion-peptide moiety bound to the plasma membrane of living cells.

We have previously shown that when administered to mice without adjuvant, a chimeric peptide consisting of the fusion peptide F from measles virus protein linked at the C-terminus of a cytotoxic T-cell epitope from the M2 protein of respiratory syncytial virus efficiently primes for an major histocompatibility complex (MHC) class-I restricted cytotoxic T lymphocyte (CTL) response. In this report, we demonstrated by microspectrofluorometry that the fusion-peptide moiety bound to the plasma membrane of living cells.

Partidos, CD ; Ripley, J ; Delmas, A ; Obeid, OE ; Denbury, A ; Steward, MW  (1997)

Fine specificity of the antibody response to a synthetic peptide from the fusion protein and protection against measles virus-induced encephalitis in a mouse model

Journal of General Virology 78 3227-3232 Part 12
A synthetic peptide representing residues 397-420 from the measles virus (MV) fusion (F) protein was tested for its structure, immunogenicity and protective capacity against intracerebral challenge with a neuroadapted strain of MV, Analysis of the peptide by mass spectrometry showed that it was linear, despite the presence of two cysteine residues in the sequence, Circular dichroism spectroscopy highlighted a weak preference for the peptide to adopt an a-helical conformation. The peptide was shown to be immunogenic in BALB/c and C57BL/6 mice after intraperitoneal immunization in Freund's adjuvant, and anti-peptide antibodies from both strains of mice reacted with the MV as a solid phase antigen on an ELISA plate.

A synthetic peptide representing residues 397-420 from the measles virus (MV) fusion (F) protein was tested for its structure, immunogenicity and protective capacity against intracerebral challenge with a neuroadapted strain of MV, Analysis of the peptide by mass spectrometry showed that it was linear, despite the presence of two cysteine residues in the sequence, Circular dichroism spectroscopy highlighted a weak preference for the peptide to adopt an a-helical conformation. The peptide was shown to be immunogenic in BALB/c and C57BL/6 mice after intraperitoneal immunization in Freund’s adjuvant, and anti-peptide antibodies from both strains of mice reacted with the MV as a solid phase antigen on an ELISA plate.


1996   Références trouvées : 5

Delmas, A ; Partidos, CD  (1996)

The binding of chimeric peptides to GM1 ganglioside enables induction of antibody responses after intranasal immunization

Vaccine 14 (11) 1077-1082
With a model peptide, the neutralizing epitope 50-75 of cholera toxin B subunit, two chimeric peptides were constructed. A T-cell epitope, the 174-187 peptide from the G protein of the respiratory syncytial virus, was co-linearly synthesized at the amino-(174-50) or carboxyl-terminus (50-174) of the 50-75 peptide. Although both chimeric peptides were equally immunogenic by the intraperitoneal route, the 50-174 peptide was more immunogenic than the 174-50 peptide by the intranasal (i.n.) route. Both chimeric peptides inhibited the binding of cholera toxin B subunit to GM1 ganglioside with the 50-174 peptide being more effective inhibitor than the 174-50 peptide. In addition, an effective priming of the immune system was achieved after the i.n. administration of immunogens. The observed unresponsiveness after the i.n. co-immunization with the 50-174 peptide and GM1 ganglioside emphasize the role of GM1 binding for the induction of an immune response after i.n. immunization. Copyright (C) 1996 Elsevier Science Ltd.

With a model peptide, the neutralizing epitope 50-75 of cholera toxin B subunit, two chimeric peptides were constructed. A T-cell epitope, the 174-187 peptide from the G protein of the respiratory syncytial virus, was co-linearly synthesized at the amino-(174-50) or carboxyl-terminus (50-174) of the 50-75 peptide. Although both chimeric peptides were equally immunogenic by the intraperitoneal route, the 50-174 peptide was more immunogenic than the 174-50 peptide by the intranasal (i.n.) route. Both chimeric peptides inhibited the binding of cholera toxin B subunit to GM1 ganglioside with the 50-174 peptide being more effective inhibitor than the 174-50 peptide. In addition, an effective priming of the immune system was achieved after the i.n. administration of immunogens. The observed unresponsiveness after the i.n. co-immunization with the 50-174 peptide and GM1 ganglioside emphasize the role of GM1 binding for the induction of an immune response after i.n. immunization. Copyright (C) 1996 Elsevier Science Ltd.

Partidos, CD ; Delmas, A ; Steward, MW  (1996)

Structural requirements for synthetic immunogens to induce measles virus specific CTL responses

Molecular Immunology 33 (16) 1223-1229
We have studied the immunogenicity of a synthetic peptide representing a cytotoxic T cell epitope (CTL) from the nucleoprotein of measles virus (MV). For the induction of peptide and MV-specific CTL responses after subcutaneous immunization, covalent linkage of the CTL epitope to a T-helper epitope was required. The presence of two copies of the T-helper epitope at the amino terminus of the CTL epitope (TT-CTL) resulted in the induction of strong CTL responses after administration in saline. In contrast, a chimeric peptide with one copy of the T-helper epitope at the amino terminus of the CTL epitope (T-CTL) was weakly immunogenic when given in saline. Analysis of the structure of the TT-CTL chimeric peptide by CD spectroscopy revealed an a-helical conformation, as compared to the random coil conformation favored by the T-CTL chimeric peptide.

We have studied the immunogenicity of a synthetic peptide representing a cytotoxic T cell epitope (CTL) from the nucleoprotein of measles virus (MV). For the induction of peptide and MV-specific CTL responses after subcutaneous immunization, covalent linkage of the CTL epitope to a T-helper epitope was required. The presence of two copies of the T-helper epitope at the amino terminus of the CTL epitope (TT-CTL) resulted in the induction of strong CTL responses after administration in saline. In contrast, a chimeric peptide with one copy of the T-helper epitope at the amino terminus of the CTL epitope (T-CTL) was weakly immunogenic when given in saline. Analysis of the structure of the TT-CTL chimeric peptide by CD spectroscopy revealed an a-helical conformation, as compared to the random coil conformation favored by the T-CTL chimeric peptide.

Hsu, SC ; Schadeck, EB ; Delmas, A ; Shaw, M ; Steward, MW  (1996)

Linkage of a fusion peptide to a CTL epitope from the nucleoprotein of measles virus enables incorporation into ISCOMs and induction of CTL responses following intranasal immunization

Vaccine 14 (12) 1159-1166
The introduction of soluble protein antigens into the endogenous processing pathway is a prerequisite for the efficient induction of MHC class-1 restricted cytotoxic T-lymphocytes (CTLs). Antigens incorporated into immunostimulating complexes (ISCOMs) containing lipids and Quil-A are able to induce CD8(+) CTL responses in vivo. Furthermore, lipopeptides have also been used to raise peptide-specific CTLs and bypass the requirement for the use of an adjuvant.

The introduction of soluble protein antigens into the endogenous processing pathway is a prerequisite for the efficient induction of MHC class-1 restricted cytotoxic T-lymphocytes (CTLs). Antigens incorporated into immunostimulating complexes (ISCOMs) containing lipids and Quil-A are able to induce CD8(+) CTL responses in vivo. Furthermore, lipopeptides have also been used to raise peptide-specific CTLs and bypass the requirement for the use of an adjuvant.

Lacassie, E ; Delmas, A ; Meunier, C ; Sy, D ; Trudelle, Y  (1996)

High thermal stability and cold-denaturation of an artificial polypeptide

International Journal of Peptide and Protein Research 48 (3) 249-258
An amphipathic polypeptide, P-n, with a tandemly repeated LKELPEKL sequence including a proline every eight residues, as well as a series of shorter peptides having the same sequence, P-2, P-3, P-4, P-5 and P,, were synthesized. Their conformation in aqueous solution was mainly studied by CD. At low temperature, these peptides and polypeptide are completely unordered and undergo a reversible transition leading to a partly a-helical structure upon heating. Such behavior has been demonstrated for a few proteins by other authors and has been called cold-denaturation. The transition temperature of the polypeptide is close to 20 degrees C. The conformational change does not depend on concentration, indicating a monomolecular process. The high-temperature structure seems to be compact as for globular proteins. A model of folded structure is proposed from experimental data and from molecular modelling studies. (C) Munksgaard 1996.

An amphipathic polypeptide, P-n, with a tandemly repeated LKELPEKL sequence including a proline every eight residues, as well as a series of shorter peptides having the same sequence, P-2, P-3, P-4, P-5 and P,, were synthesized. Their conformation in aqueous solution was mainly studied by CD. At low temperature, these peptides and polypeptide are completely unordered and undergo a reversible transition leading to a partly a-helical structure upon heating. Such behavior has been demonstrated for a few proteins by other authors and has been called cold-denaturation. The transition temperature of the polypeptide is close to 20 degrees C. The conformational change does not depend on concentration, indicating a monomolecular process. The high-temperature structure seems to be compact as for globular proteins. A model of folded structure is proposed from experimental data and from molecular modelling studies. (C) Munksgaard 1996.

Quesnel, A ; Hsu, SC ; Delmas, A ; Steward, MW ; Trudelle, Y ; Abastado, JP  (1996)

Efficient binding to the MHC class I K-D molecule of synthetic peptides in which the anchoring position 2 does not fit the consensus motif

Febs Letters 387 (1) 42-46
Peptides eluted from the MHC class I K-d molecule are generally nonamers that display a strong preference for Tyr in position 2 and Ile or Leu in position 9. We investigated the binding ability of several synthetic peptides which did not fit this consensus motif. In our peptides, Tyr(2) was substituted by other amino acids, i.e. Leu, Be or Met. These peptides were variants of the 252-260 K-d-restricted peptide SYIPSAEKI derived from the Plasmodium berghei circumsporozoite protein. They bound to purified K-d molecules in vitro with intermediate affinity. One of them was tested for in vivo stimulation of T cells and induced a cytotoxic response. These results demonstrate the importance of binding motif refinement to discover new binding characteristics and new ligands such as low-affinity peptides.

Peptides eluted from the MHC class I K-d molecule are generally nonamers that display a strong preference for Tyr in position 2 and Ile or Leu in position 9. We investigated the binding ability of several synthetic peptides which did not fit this consensus motif. In our peptides, Tyr(2) was substituted by other amino acids, i.e. Leu, Be or Met. These peptides were variants of the 252-260 K-d-restricted peptide SYIPSAEKI derived from the Plasmodium berghei circumsporozoite protein. They bound to purified K-d molecules in vitro with intermediate affinity. One of them was tested for in vivo stimulation of T cells and induced a cytotoxic response. These results demonstrate the importance of binding motif refinement to discover new binding characteristics and new ligands such as low-affinity peptides.


1995   Références trouvées : 1

Quesnel, A ; Delmas, A ; Trudelle, Y  (1995)

Purification of synthetic peptide libraries by affinity-chromatography using the avidin-biotin system

Analytical Biochemistry 231 (1) 182-187
The specific interaction between biotin and avidin was exploited in the affinity purification of solid-phase synthesized peptide libraries. During peptide library synthesis, by means of the single-resin method in which coupling on variable positions is carried out using an equimolar mixture of amino acids, biotin was used to cap the unreacted amino groups remaining after coupling of the equimolar amino acid mixture. The following synthesis and deprotection procedures were performed as usual in tert,-butyloxycarbonyl chemistry. The purification of the peptide mixture containing N-biotinylated sequences was performed by affinity chromatography on an avidin-agarose column.

The specific interaction between biotin and avidin was exploited in the affinity purification of solid-phase synthesized peptide libraries. During peptide library synthesis, by means of the single-resin method in which coupling on variable positions is carried out using an equimolar mixture of amino acids, biotin was used to cap the unreacted amino groups remaining after coupling of the equimolar amino acid mixture. The following synthesis and deprotection procedures were performed as usual in tert,-butyloxycarbonyl chemistry. The purification of the peptide mixture containing N-biotinylated sequences was performed by affinity chromatography on an avidin-agarose column.


1994   Références trouvées : 1

Lacassie, E ; Delmas, A ; Trudelle, Y  (1994)

Influence of the conformation of a macromolecule on the generation of T-cell proliferative response - a study with model polypeptides

Febs Letters 349 (3) 380-384
To study the influence of the conformation of polypeptidic macromolecules on the generation of T-cell epitopes, sequential polypeptides with an octamer repeat unit were designed and synthesized. They adopt mainly unordered and a-helical conformations. Among these polypeptides, those containing proline are fully or partly unordered, and are more effective at inducing T-cell proliferation than a proline-free very stable a-helical polypeptide. This extremely stable a-helical conformation, probably stabilized by aggregation, would enhance its stability against proteolytic processing.

To study the influence of the conformation of polypeptidic macromolecules on the generation of T-cell epitopes, sequential polypeptides with an octamer repeat unit were designed and synthesized. They adopt mainly unordered and a-helical conformations. Among these polypeptides, those containing proline are fully or partly unordered, and are more effective at inducing T-cell proliferation than a proline-free very stable a-helical polypeptide. This extremely stable a-helical conformation, probably stabilized by aggregation, would enhance its stability against proteolytic processing.


1992   Références trouvées : 1

Delmas, A ; Brack, A ; Trudelle, Y  (1992)

Studies of the influence of different cross-linking reagents on the immune-response against a B-epitope

Bioconjugate Chemistry 3 (1) 80-84
We have previously shown that the carrier polytuftsin obtained by polycondensation of tuftsin, a naturally occurring macrophage activator, increases significantly the antibody response against a linked B-epitope. In the present work, we have studied the influence of different cross-linking reagents on the quality of the conjugation and on the immune response, at both B-cell and T-cell levels. We observed that the cross-linking method used for coupling the B-epitope to the carrier influences the immune response. A hypothesis is put forward to explain the differences observed.

We have previously shown that the carrier polytuftsin obtained by polycondensation of tuftsin, a naturally occurring macrophage activator, increases significantly the antibody response against a linked B-epitope. In the present work, we have studied the influence of different cross-linking reagents on the quality of the conjugation and on the immune response, at both B-cell and T-cell levels. We observed that the cross-linking method used for coupling the B-epitope to the carrier influences the immune response. A hypothesis is put forward to explain the differences observed.


1989   Références trouvées : 2

Pedoussaut, S ; Delmas, A ; Milhaud, G ; Rivaille, P ; Gruaz-Guyon, A  (1989)

Oral immunization with a free peptide from cholera-toxin - local protection and IGA production

Molecular Immunology 26 (2) 115-119

Delmas, A ; Gruaz-Guyon, A ; Pedoussaut, S ; Pierre, P ; Rivaille, P ; Vaerman, JP  (1989)

Neutralization of cholera-toxin by rat IGA secretory antibodies induced by a free synthetic peptide

Biochemical and Biophysical Research Communications 159 (2) 707-712


1987   Références trouvées : 2

Trudelle, Y ; Brack, A ; Delmas, A ; Pedoussaut, S ; Rivaille, P  (1987)

Synthesis of a new carrier for immunization - polytuftsin - 2 examples of its use with peptides selected in the hepatitis-B surface-antigen

International Journal of Peptide and Protein Research 30 (1) 54-60

Guyon-Gruaz, A ; Pedoussaut, S ; Delmas, A ; Milhaud, G ; Rivaille, P  (1987)

A synthetic peptide selected in cholera-toxin sequence is immunogenic orally and induces local protection

Journal of Cellular Biochemistry 185-185 Suppl. 11b


1986   Références trouvées : 1

Guyon-Gruaz, A ; Delmas, A ; Pedoussaut, S ; Halimi, H ; Milhaud, G ; Raulais, D ; Rivaille, P  (1986)

Oral immunization with a synthetic peptide of cholera toxin-B subunit - obtention of neutralizing antibodies

European Journal of Biochemistry 159 (3) 525-528


1985   Références trouvées : 2

Delmas, A ; Guyon-Gruaz, A ; Halimi, H ; Raulais, D ; Rivaille, P  (1985)

Solid-phase synthesis of 2 cholera toxin-B subunit antigens

International Journal of Peptide and Protein Research 25 (4) 421-424

Delmas, A ; Milhaud, G ; Raulais, D ; Rivaille, P  (1985)

Epitopes of the 44-68 human parathyroid-hormone fragments - the importance of specific hydrophilic peptide sequences

Molecular Immunology 22 (6) 675-679


Mots-clés

Directeur de recherche , Responsable de groupe thématique , Protéines de synthèse et chimie bioorthogonale