Biomedical subjects

T Vernet

Publications and source records attributed to T Vernet.

At least 19 recordsLinked to original sources

PubMed2003

VL position 34 is a key determinant for the engineering of stable antibodies with fast dissociation rates.

Predictive engineering of antibodies exhibiting fast kinetic properties could provide reagents for biotechnological applications such as continuous monitoring of compounds or affinity chromatography. Based on covariance analysis of murine germline antibody variable domains, we selected position L34 (Kabat numbering) for mutational studies. This position is located at the VL/VH interface, at the base of the paratope but with limited antigen contacts, thus making it an attractive position for mild alterations of antigen binding properties. We introduced a serine at position L34 in two different antibodies: Fab (fragment antigen binding) 57P (Asn34Ser) and scFv (single chain fragment variable) 1F4 (Gln34Ser), that recognize peptides derived from the coat protein of tobacco mosaic virus and the oncoprotein E6, respectively. Both mutated antibodies exhibited similar properties: (i) expression levels of active fragments in Escherichia coli were markedly improved; (ii) thermostability was enhanced; and (iii) dissociation rate parameters (k(off)) were increased by 2- and at least 57-fold for scFv1F4 and Fab57P, respectively, while their association rate parameters (k(on)) remained unchanged. The L34 Ala and Thr mutants of both antibody fragments did not possess these properties. This first demontration of similar effects observed in two antibodies with different specificities may open the way to the predictive design of molecules with enhanced stability and fast dissociation rates.

Amino Acid Sequence↗

PubMed2001

BIACORE data processing: an evaluation of the global fitting procedure.

Data from real-time molecular interaction analysis using BIACORE are currently evaluated by numerical integration. We have investigated the ability of two software packages (BIAevaluation 3.0 and CLAMP99) to analyze complex interactions. Three experimental data sets of high quality obtained with BIACORE upgraded and 2000 instruments, representative of simple bimolecular, heterogeneous ligand, and mass-transport-limited interactions, were processed by the global fitting procedure. The two software, which differ mainly in the statistical assessment of the output values, were able to discriminate correctly between various interacting models and provided very close output parameters with satisfactory statistical tests.

Amino Acid Sequence↗

PubMed2001

Cysteine protease isoforms from Trypanosoma cruzi, cruzipain 2 and cruzain, present different substrate preference and susceptibility to inhibitors.

Cysteine-proteinases from parasitic protozoa have been recently characterized as factors of virulence and pathogenicity in several human and veterinary diseases. In Chagas' disease, the chronic infection caused by Trypanosoma cruzi, structure-functional studies on cysteine proteases were thus far limited to the parasite's major isoform, a cathepsin L-like lysosomal protease designated as cruzipain, cruzain or GP57/51. Encoded by a large gene family, cruzipain is efficiently targeted by synthetic inhibitors, which prevent parasite intracellular growth and differentiation. We have previously demonstrated that the multicopy cruzipain gene family includes polymorphic sequences, which could encode functionally different isoforms. We report here a comparative kinetic study between cruzain, the archetype of the cruzipain family, and an isoform, termed cruzipain 2, which is expressed preferentially by the mammalian stages of T. cruzi. Heterologous expression of the catalytic domain of cruzipain 2 in Saccharomyces cerevisae yielded an enzyme that differs markedly from cruzain with respect to pH stability, substrate specificity and sensitivity to inhibition by natural and synthetic inhibitors of cysteine proteases. We suggest that the structural-functional diversification imparted by genetic polymorphism of cruzipain genes may have contributed to T. cruzi adaptation to vertebrate hosts.

Amino Acid Sequence↗

PubMed2001

Molecular mechanisms of antibiotic resistance in gram-positive pathogens.

The widespread and uncontrolled use of antibiotics, both for human consumption and animal feed, has encouraged the development of drug resistance in a variety of pathogenic bacteria. Gram-positive species employ resistance mechanisms which include the modification of the antibiotic structure, mutagenesis of key amino acids in the macromolecular targets of specific chemotherapeutics, or drug efflux from the cell, among others. These three main mechanisms have been identified in resistance profiles for systems involved in protein biosynthesis, nucleic acid replication, and bacterial cell wall generation. This work will review how Gram-positive bacteria have manipulated all three classes of targets in the generation of resistance. Upcoming and recently approved antibacterials for human consumption will also be highlighted.

Anti-Bacterial Agents↗

PubMed2000

Self-assembly of the vascular endothelial cadherin ectodomain in a Ca2+-dependent hexameric structure.

Vascular endothelial cadherin (VE-cadherin) is a transmembrane protein essential for endothelial cell monolayer integrity (Gulino, D., Delachanal, E., Concord, E., Genoux, Y., Morand, B., Valiron, M. O., Sulpice, E., Scaife, R., Alemany, M., and Vernet, T. (1998) J. Biol. Chem. 273, 29786-29793). This molecule belongs to the cadherin family of cell-cell adhesion receptors, for which molecular details of homotypic interactions are still lacking. In this study, a recombinant fragment encompassing the four N-terminal modules of VE-cadherin (VE-EC1-4) was shown to associate, in solution, as a stable Ca(2+)-dependent oligomeric structure. Cross-linking experiments combined with mass spectrometry demonstrated that this oligomer is a hexamer. Gel filtration chromatography experiments and analytical ultracentrifugation analyses revealed the existence of an equilibrium between the hexameric and monomeric species of VE-EC1-4. The concentration at which 50% of VE-EC1-4 is in its hexameric form was estimated as 1 microm. The dimensions of the hexamer, measured by cryoelectron microscopy to be 233 +/- 10 x 77 +/- 7 A, are comparable to the thickness of adherens endothelial cell-cell junctions. Altogether, the results allow us to propose a novel homotypic interaction model for the class II VE-cadherin, in which six molecules of cadherin form a hexamer.

Animals↗

PubMed2000

Calcium-dependent conformational stability of modules 1 and 2 of human gelsolin.

Gelsolin modulates the actin cytoskeleton in the cytoplasm and clears the circulation of stray filaments. In vitro, gelsolin cleaves, nucleates and caps actin filaments, activities that are calcium-dependent. Both cellular and secreted forms share a sequence of 730 residues comprising six homologous modules termed G1-G6. A disulphide bond is formed in secreted G2, whereas in the cytoplasm it remains reduced. A point mutation in G2 causes an amyloidosis with neurological, ophthalmological and dermatological symptoms. This mutation does not affect the cytoplasmic form, while the secreted form is proteolysed. As a first step towards understanding how gelsolin folds and functions in different cellular compartments, we have characterized at equilibrium the urea-induced unfolding of G1 and G2, with or without calcium and/or disulphide bond. G1 and G2 both exhibit two-state unfolding behaviour and are stabilized by calcium. The disulphide bond also contributes to the stability of G2. In the absence of Ca(2+) and disulphide bond, G2 adopts a non-native conformation, suggesting that folding of G2 in the cytoplasm relies on the presence of surrounding modules or other molecular partners.

Calcium↗

PubMed2000

Deacylation kinetics analysis of Streptococcus pneumoniae penicillin-binding protein 2x mutants resistant to beta-lactam antibiotics using electrospray ionization- mass spectrometry.

Penicillin-binding proteins (PBPs) catalyze the transpeptidase reaction involved in peptidoglycan synthesis and are covalently inhibited by the beta-lactam antibiotics. In a previous work we have focused on acylation efficiency measurements of various Streptococcus pneumoniae PBP2x* mutants to study the molecular determinants of resistance to beta-lactams. In the present paper we have developed a method to improve an accurate determination of the deacylation rate constant using electrospray ionization-mass spectrometry. This method is adaptable to the analysis of deacylation of any beta-lactam. Compared to the fluorographic technique, the ESI-MS method is insensitive to variations in the concentration of functional proteins and is therefore more reliable. We have established that the resistance of PBPs to beta-lactams is mostly due to a decrease of the acylation efficiency with only marginal effects on the deacylation rates.

Acylation↗

PubMed2000

Expression, purification, crystallization and preliminary X-ray analysis of the iota-carrageenase from Alteromonas fortis.

This is the first crystallization report of a glycoside hydrolase which belongs to family 82. A recombinant form of His-tagged iota-carrageenase from Alteromonas fortis was expressed, purified and crystallized. Crystals were obtained by the vapour-diffusion method using polyethylene glycol (M(W) = 6000) as a precipitant. They belong to space group P2(1), with unit-cell parameters a = 56. 75, b = 91.04, c = 125.01 A, beta = 93.41 degrees. The unit cell contains two molecules in the asymmetric unit related by a non-crystallographic twofold axis. Crystals diffracted to 2.0 A resolution on a synchrotron beamline.

Alteromonas↗

PubMed1999

Schmid's metaphyseal chondrodysplasia mutations interfere with folding of the C-terminal domain of human collagen X expressed in Escherichia coli.

Human collagen X contains a highly conserved 161-amino acid C-terminal non-triple helical domain that is homologous to the C-terminal domain of collagen VIII and to the C1q module of the human C1 enzyme. We have expressed this domain (residues 545-680) in Escherichia coli as a glutathione S-transferase fusion protein. The purified fusion protein trimerizes spontaneously in vitro, and after thrombin cleavage, the purified C-terminal domain trimer (46.2 kDa) is extremely stable and trypsin-resistant. Mutations within the C-terminal domain have been observed in patients with Schmid's metaphyseal chondrodysplasia (SMCD). Some of these mutations (Y598D, G618V, W651X, or H669X; X is the stop codon) were constructed by site-directed mutagenesis. Each mutation had identical consequences regarding the fusion protein: 1) absence of trimeric formation, 2) copurification of the approximately 60-kDa GroEL chaperone protein, and 3) sensitivity of the monomeric fusion protein to trypsin digestion. These results show that the C-terminal domain of collagen X is sufficient to produce a very stable and compact trimer in the absence of collagen Gly-X-Y repeats. Moreover, mutations causing SMCD interfere in this system with the correct folding of the C-terminal domain. The existence of a similar mechanism in chondrocytes might explain the relative homogeneity of phenotypes in SMCD despite the diversity of mutations.

Chaperonin 60↗

PubMed1999

Mutations in the active site of penicillin-binding protein PBP2x from Streptococcus pneumoniae. Role in the specificity for beta-lactam antibiotics.

Penicillin-binding protein 2x (PBP2x) isolated from clinical beta-lactam-resistant strains of Streptococcus pneumoniae (R-PBP2x) have a reduced affinity for beta-lactam antibiotics. Their transpeptidase domain carries numerous substitutions compared with homologous sequences from beta-lactam-sensitive streptococci (S-PBP2x). Comparison of R-PBP2x sequences suggested that the mutation Gln552 --> Glu is important for resistance development. Mutants selected in the laboratory with cephalosporins frequently contain a mutation Thr550 --> Ala. The high resolution structure of a complex between S-PBP2x* and cefuroxime revealed that Gln552 and Thr550, which belong to strand beta3, are in direct contact with the cephalosporin. We have studied the effect of alterations at positions 552 and 550 in soluble S-PBP2x (S-PBP2x*) expressed in Escherichia coli. Mutation Q552E lowered the acylation efficiency for both penicillin G and cefotaxime when compared with S-PBP2x*. We propose that the introduction of a negative charge in strand beta3 conflicts with the negative charge of the beta-lactam. Mutation T550A lowered the acylation efficiency of the protein for cefotaxime but not for penicillin G. The in vitro data presented here are in agreement with the distinct resistance profiles mediated by these mutations in vivo and underline their role as powerful resistance determinants.

Acylation↗

PubMed1999

Kinetic analysis of the effect on Fab binding of identical substitutions in a peptide and its parent protein.

Monoclonal antibody 57P, which was raised against tobacco mosaic virus protein, cross-reacts with a peptide corresponding to residues 134-146 of this protein. Previous studies using peptide variants suggested that the peptide in the antibody combining site adopts a helical configuration that mimics the structure in the protein. In this study, we carried out a detailed comparison of Fab-peptide and Fab-protein interactions. The same five amino acid substitutions were introduced in the peptide (residues 134-151) and the parent protein, and the effect of these substitutions on antibody binding parameters have been measured with a Biacore instrument. Fabs that recognize epitopes located away from the site of mutations were used as indirect probes for the conformational integrity of protein antigens. Their interaction kinetics with all proteins were similar, suggesting that the substitutions had no drastic effect on their conformation. The five substitutions introduced in the peptide and the protein had minor effects on association rate constants (ka) and significant effects on dissociation rate constants (kd) of the antigen-Fab 57P interactions. In four out of five cases, the effect on binding affinity of the substitutions was identical when the epitope was presented in the form of a peptide or a protein antigen, indicating that antibody binding specifity was not affected by epitope presentation. However, ka values were about 10 times larger and kd values about 5 times larger for the peptide-Fab compared to the protein-Fab interaction, suggesting a different binding mechanism. Circular dichroism measurements performed for three of the peptides showed that they were mainly lacking structure in solution. Differences in conformational properties of the peptide and protein antigens in solution and/or in the paratope could explain differences in binding kinetics. Our results demonstrate that the peptides were able to mimic correctly some but not all properties of the protein-Fab 57P interaction and highlight the importance of quantitative analysis of both equilibrium and kinetic binding parameters in the design of synthetic vaccines and drugs.

Antibody Affinity↗

PubMed1999

Mapping of the interaction between the immunodominant loop of the ectodomain of HIV-1 gp41 and human complement protein C1q.

The human immunodeficiency virus type 1 transmembrane envelope glycoprotein gp41 has been previously shown to activate the C1 complex of human complement through direct interaction with its C1q subunit. The major interaction site has been located within the gp41 immunodominant region (residues 590-620), and a synthetic peptide overlapping residues 601-613 of gp41 (sequence GIWGCSGKLICTT) was shown to inhibit binding of gp41 to C1q in vitro (Thielens, N.M., Bally, I.M., Ebenbichler, C.F., Dierich, M.P. & Arlaud, G.J. (1993) J. Immunol. 151, 6583-6592). The ectodomain of gp41 (s-gp41) was secreted from the methylotrophic yeast Pichia pastoris and purified by immunoaffinity chromatography. Enzymatic deglycosylation of the recombinant s-gp41 was necessary to allow its in vitro interaction with C1q. A solid-phase competition assay was used to monitor the effect of mutant peptides derived from segment 601-613 of gp41 on the binding of deglycosylated s-gp41 to C1q. Whereas mutation of Ser606 had no effect, replacement of Ile602, Trp603, Lys608, Leu609 and Ile610 by Ala abolished the ability of the resulting peptides to inhibit binding of s-gp41 to C1q, suggesting that these residues participate in the interaction between gp41 and C1q. These findings are discussed in the light of a structural model of the immunodominant loop of gp41. It is proposed that the recognition of gp41 by C1q is driven by hydrophobic interactions, and that the sites of gp41 responsible for interaction with gp120 and C1q partly overlap.

Amino Acid Sequence↗

PubMed1999

Expression, purification, crystallization and preliminary x-ray analysis of the kappa-carrageenase from Pseudoalteromonas carrageenovora.

A recombinant form of His-tagged kappa-carrageenase from Pseudoalteromonas carrageenovora has been expressed, purified and crystallized. Crystals have been obtained by the vapour-diffusion method using polyethylene glycol (Mr = 4000) as a precipitant. These crystals belong to the space group P212121, with unit-cell parameters a = 58.2, b = 62.8, c = 77.9 A, and diffract to 2.2 A resolution on a rotating-anode X-ray source.

Bacterial Proteins↗

PubMed1999

Glycosyltransferase domain of penicillin-binding protein 2a from Streptococcus pneumoniae is membrane associated.

Penicillin-binding proteins (PBPs) are bacterial cytoplasmic membrane proteins that catalyze the final steps of the peptidoglycan synthesis. Resistance to beta-lactams in Streptococcus pneumoniae is caused by low-affinity PBPs. S. pneumoniae PBP 2a belongs to the class A high-molecular-mass PBPs having both glycosyltransferase (GT) and transpeptide (TP) activities. Structural and functional studies of both domains are required to unravel the mechanisms of resistance, a prerequisite for the development of novel antibiotics. The extracellular region of S. pneumoniae PBP 2a has been expressed (PBP 2a*) in Escherichia coli as a glutathione S-transferase fusion protein. The acylation kinetic parameters of PBP 2a* for beta-lactams were determined by stopped-flow fluorometry. The acylation efficiency toward benzylpenicillin was much lower than that toward cefotaxime, a result suggesting that PBP 2a participates in resistance to cefotaxime and other beta-lactams, but not in resistance to benzylpenicillin. The TP domain was purified following limited proteolysis. PBP 2a* required detergents for solubility and interacted with lipid vesicles, while the TP domain was water soluble. We propose that PBP 2a* interacts with the cytoplasmic membrane in a region distinct from its transmembrane anchor region, which is located between Lys 78 and Ser 156 of the GT domain.

Amino Acid Sequence↗

PubMed1998

Identification of a structural determinant for resistance to beta-lactam antibiotics in Gram-positive bacteria.

Streptococcus pneumoniae is the main causal agent of pathologies that are increasingly resistant to antibiotic treatment. Clinical resistance of S. pneumoniae to beta-lactam antibiotics is linked to multiple mutations of high molecular mass penicillin-binding proteins (H-PBPs), essential enzymes involved in the final steps of bacterial cell wall synthesis. H-PBPs from resistant bacteria have a reduced affinity for beta-lactam and a decreased hydrolytic activity on substrate analogues. In S. pneumoniae, the gene coding for one of these H-PBPs, PBP2x, is located in the cell division cluster (DCW). We present here structural evidence linking multiple beta-lactam resistance to amino acid substitutions in PBP2x within a buried cavity near the catalytic site that contains a structural water molecule. Site-directed mutation of amino acids in contact with this water molecule in the "sensitive" form of PBP2x produces mutants similar, in terms of beta-lactam affinity and substrate hydrolysis, to altered PBP2x produced in resistant clinical isolates. A reverse mutation in a PBP2x variant from a clinically important resistant clone increases the acylation efficiency for beta-lactams and substrate analogues. Furthermore, amino acid residues in contact with the structural water molecule are conserved in the equivalent H-PBPs of pathogenic Gram-positive cocci. We suggest that, probably via a local structural modification, the partial or complete loss of this water molecule reduces the acylation efficiency of PBP2x substrates to a point at which cell wall synthesis still occurs, but the sensitivity to therapeutic concentrations of beta-lactam antibiotics is lost.

Amino Acid Substitution↗

PubMed1998

Alteration of endothelial cell monolayer integrity triggers resynthesis of vascular endothelium cadherin.

Although cadherins appear to be necessary for proper cell-cell contacts, the physiological role of VE-cadherin (vascular endothelium cadherin) in adult tissue has not been clearly determined. To shed some light on this question, we have disturbed the adhesive function of VE-cadherin in human endothelial cell culture using a polyclonal anti-VE-cadherin antibody. This antibody disrupts confluent endothelial cell monolayers in vitro and transiently generates numerous gaps at cell-cell junctions. The formation of these gaps correlates with a reversible increase in the monolayer permeability. We present evidence that destruction of the homotypic interactions between the extracellular domains of VE-cadherin induces a rapid resynthesis of VE-cadherin, leading to restoration of endothelial cell-cell contacts. The expression of new molecules of VE-cadherin correlates with a modest but significant increase in VE-cadherin mRNA synthesis. Altogether, these results establish a critical role for VE-cadherin in the maintenance and restoration of endothelium integrity.

Amino Acid Sequence↗

PubMed1998

Identification, purification, and characterization of transpeptidase and glycosyltransferase domains of Streptococcus pneumoniae penicillin-binding protein 1a.

Resistance to beta-lactam antibiotics in Streptococcus pneumoniae is due to alteration of penicillin-binding proteins (PBPs). S. pneumoniae PBP 1a belongs to the class A high-molecular-mass PBPs, which harbor transpeptidase (TP) and glycosyltransferase (GT) activities. The GT active site represents a new potential target for the generation of novel nonpenicillin antibiotics. The 683-amino-acid extracellular region of PBP 1a (PBP 1a*) was expressed in Escherichia coli as a GST fusion protein. The GST-PBP 1a* soluble protein was purified, and its domain organization was revealed by limited proteolysis. A protease-resistant fragment spanning Ser 264 to Arg 653 exhibited a reactivity profile against both beta-lactams and substrate analogues similar to that of the parent protein. This protein fragment represents the TP domain. The GT domain (Ser 37 to Lys 263) was expressed as a recombinant GST fusion protein. Protection by moenomycin of the GT domain against trypsin degradation was interpreted as an interaction between the GT domain and the moenomycin.

Bacterial Proteins↗

PubMed1997

Role of the occluding loop in cathepsin B activity.

Within the lysosomal cysteine protease family, cathepsin B is unique due to its ability to act both as an endopeptidase and a peptidyldipeptidase. This latter capacity to remove C-terminal dipeptides has been attributed to the presence of a 20-residue insertion, termed the occluding loop, that blocks the primed terminus of the active site cleft. Variants of human procathepsin B, where all or part of this element was deleted, were expressed in the yeast Pichia pastoris. A mutant, where the 12 central residues of the occluding loop were deleted, autoprocessed, albeit more slowly than the wild type proenzyme, to yield a mature form of the enzyme with endopeptidase activity comparable with the wild-type cathepsin B, but totally lacking exopeptidase activity. This deletion mutant showed a 40-fold higher affinity for the inhibitor cystatin C, suggesting that the occluding loop normally restricts access of this inhibitor to the active site. In addition, the binding affinity of the cathepsin B propeptide, which is a potent inhibitor of this enzyme, was 50-fold increased, consistent with the finding that the loop reorients on activation of the proenzyme. These results suggest that the endopeptidase activity of cathepsin B is an evolutionary remnant since, as a consequence of its membership in the papain family, the propeptide must be able to bind unobstructed through the full length of the active site cleft.

Amino Acid Sequence↗