Biomedical subjects

A Lecroisey

Publications and source records attributed to A Lecroisey.

At least 19 recordsLinked to original sources

PubMed1997

Conformational and functional properties of an undecapeptide epitope fused with the C-terminal end of the maltose binding protein.

Monoclonal antibody mAb164 is directed against the TrpB2 subunit of the Escherichia coli tryptophan synthase. It recognizes the synthetic peptide P11, constituted of residues 273-283 of TrpB, with high affinity. We constructed a hybrid protein in which the C-terminal end of protein MalE was linked with the N-terminal end of P11. Hybrid MalE-P11 was produced in E. coli from a plasmidic gene and purified in one step as MalE. MalE-P11 and the isolated P11 had identical conformational and functional properties according to the following criteria. The NMR spectra of MalE and MalE-P11 in TOCSY experiments showed that the P11 moiety of MalE-P11 moved independently from its MalE moiety. The chemical shifts of the protons for the P11 moiety of MalE-P11 and for the isolated P11 were very close and did not show significant deviations from random coil values. The equilibrium constant of dissociation (KD) from mAb164, measured by a competition ELISA, was identical for MalE-P11 and the isolated P11, around 6 nM. The change of the C-terminal residue of MalE-P11 from Lys into Ala increased 37-fold this dissociation constant. This increase showed that the P11 moiety of MalE-P11 was not degraded. The high molecular mass of MalE-P11 allowed us to follow its kinetics of interaction with immobilized mAb164 by surface plasmon resonance, using the BIAcore apparatus. The rates of association with mAb164 were similar for MalE-P11 and TrpB2, but the dissociation was faster for MalE-P11 than for TrpB2, as previously observed for the isolated P11 by a fluorometric method. Thus, the fusion of peptides with the C-terminal end of MalE could constitute an alternative to chemical synthesis for the study of their recognition by receptors, in vivo or in vitro.

ATP-Binding Cassette Transporters↗

PubMed1997

Purification and characterization of an extracellular heme-binding protein, HasA, involved in heme iron acquisition.

Many bacterial hemoproteins involved in heme acquisition have been isolated recently, comprising outer membrane receptors and extracellular heme-binding protein. The mechanisms by which these proteins extract heme have not been described up to now. One such protein, HasA, which can bind free heme as well as capture it from hemoglobin, is secreted by the Gram-negative bacteria Serratia marcescens under iron deficiency conditions. The fact that HasA does not present sequence similarities with other known hemoproteins suggests that it possesses a new type of heme binding site. This work describes the main physicochemical properties of HasA, essential for understanding its function. HasA is a monomer of 19 kDa that binds one b heme per molecule with high affinity. The electron paramagnetic resonance spectra indicate that the heme iron is in a low-spin ferric state and that the two iron axial ligands are His and His-. The low oxidation-reduction potential value (-550 mV vs standard hydrogen electrode) of the heme bound to HasA suggests that heme could be exposed to the solvent. According to circular dichroism data, the binding of heme does not seem to modify the conformation of HasA.

Amino Acid Sequence↗

PubMed1997

NMR studies on the flexibility of the poliovirus C3 linear epitope inserted into different sites of the maltose-binding protein.

A set of permissive positions that tolerate insertions/deletions without major deleterious consequences for the binding activity of the protein was previously identified in the maltose-binding protein. The C3 epitope from poliovirus VP1 protein (93DNPASTTNKDK103) was inserted into eight of these positions and two nonpermissive control sites. NMR studies were performed on the MalE protein, the insertion/deletion mutants, and the C3MalE hybrids to selectively determine the flexible regions in these proteins. Comparison of the C3 epitope mobility in the different hybrid proteins indicates that, whatever its insertion site and independently from the specific sequences of its linkers, the epitope is mostly flexible. The vector protein was shown to unfold partially only in the two C3MalE hybrids that correspond to nonpermissive positions. For one of them (insertion at site 339), both sides of the insert are flexible, and at most one side for all the other hybrids. This result correlates with the antigenicity data on the inserted epitope (Martineau, P., Leclerc, C., and Hofnung, M. (1997) Mol. Immunol, in press.

Amino Acid Sequence↗

PubMed1997

NMR studies on the flexibility of nucleoside diphosphate kinase.

Human NDP kinase B, product of the nm23-H2 gene, binds DNA. It has been suggested that a helix hairpin on the protein surface, part of the nucleotide substrate binding site, could accommodate DNA binding by swinging away. The presence of flexible regions was therefore investigated by 1H NMR dynamic filtering. Although TOCSY peaks could be assigned to five residues at the N terminus of Dictyostelium NDP kinase, no flexible region was detected in the human enzyme. These data favor the idea that the protein offers different binding sites to mono- and polynucleotides.

Amino Acid Sequence↗

PubMed1995

Phosphorylation mechanism of nucleoside diphosphate kinase: 31P-nuclear magnetic resonance studies.

The phosphorylation mechanism of Dictyostelium discoideum nucleoside diphosphate (NDP) kinase was investigated by NMR. 31P chemical shifts were measured on both native and denatured enzyme. In the enzymatically phosphorylated enzyme denatured by 9 M urea or 7 M guanidine hydrochloride, the NDP kinase phosphohistidine signal appeared between the signals of N delta and N epsilon free monophosphohistidines used as reference compounds and added to the sample. A signal with the same intermediate position was also observed in the pronase digest of the alkaline-denatured phosphorylated enzyme. However, when phosphohistidines of the phosphorylated synthetic peptide pGlu-His-Gly were taken as references, the NDP kinase and the N delta peptide phosphohistidine signals were shown to be identical, providing evidence that phosphorylation occurs on the N delta of the active site histidine residue. Moreover, the rate of hydrolysis of the histidine-bound phosphate is in agreement with a modification at the N delta position. Phosphorylation of the NDP kinase by phosphoramidate provided a result similar to that of the enzymatic phosphorylation. In both cases, phosphorylation could not be detected on any amino acid other than histidine. Particularly, no phosphoserine residue was observed.

Animals↗

PubMed1994

[Epitopes of the envelope of the hepatitis B virus: a structural approach].

Hepatitis B is a major public health problem. More than 300 million people are chronically infected by the virus. During infection very large quantities of complete virions and empty envelopes, consisting of spherical or filamentous lipoprotein particles, are present in the blood. DNA genome coding for envelopes is divided into three domains, preS1, preS2 and S. All available data suggest that the preS1 and preS2 products are exposed at the surface of the virions. These proteins are more immunogenic than S in terms of in vivo antibody response and the number of epitopes identified. The three dimensional mapping of antigenic sites of the HBV will provide important strategic information for vaccine development and identification of targets for immunorecognition or immunoregulation of the disease.

Epitopes↗

PubMed1991

Purification and characterization of an extracellular 29-kilodalton phospholipase C from Listeria monocytogenes.

We purified and characterized an extracellular phospholipase produced by Listeria monocytogenes. This enzyme was separated as a homogeneous protein of 29 kDa by chromatography on DEAE-52 cellulose and Bio-Gel P100 columns. It is a zinc-dependent phospholipase C (PLC) that is mainly active at pH 6 to 7 and expresses lecithinase activity and a weaker sphingomyelinase activity. The exoenzyme also hydrolyzed phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin but not phosphatidylinositol. It was distinct from the 36-kDa phosphatidylinositol PLC produced by L. monocytogenes and from the L. ivanovii sphingomyelinase. The pure protein expressed a weak, calcium-independent hemolytic activity and was not toxic in mice. Western immunoblot analysis using a rabbit immune serum raised against the enzyme showed that all virulent strains of L. monocytogenes tested produced in the culture supernatant a 29-kDa PLC. In contrast, no proteins antigenically related to the 29-kDa PLC were detected in supernatants of L. ivanovii, L. seeligeri, L. innocua, or L. welshimeri. The role in virulence of the 29-kDa PLC specifically produced by L. monocytogenes remains to be established.

Amino Acid Sequence↗

PubMed1991

Subgroup assignment of a human monoclonal anti-Rh(D) antibody.

Variable subgroups of both chains of a human monoclonal anti-RH(D) IgG1 (kappa), QA37C3G6, were determined from their N-terminal sequences. Sequence comparison with corresponding chains of other human antibodies indicated that the light chain belongs to the third subgroup of human kappa-light chains while the heavy chain belongs to the second subgroup of human heavy chains.

Amino Acid Sequence↗

PubMed1991

Partial amino acid sequence of the light chain of human anti-Rh(D) monoclonal antibody H2D5D2F5.

Antibody H2D5D2F5 is a human monoclonal anti-Rh(D) IgG1 (lambda) produced by Epstein-Barr virus-immortalized B lymphocytes from a healthy donor. The complete amino acid sequence of the light (L) chain, with the exception of positions 94-97, was determined by Edman degradation of the intact chain, containing 30 residues, and derived tryptic and thermolytic peptides. Sequences of the peptides were aligned by comparison with the sequences of previously reported L chains. H2D5D2F5 L chain belongs to the first variable subgroup of human chains. Its sequence does not reveal striking differences when compared to those of other human lambda chains issued from myeloma or hybridoma.

Amino Acid Sequence↗

PubMed1990

Modeling of protease I collagenolytic enzyme from the fiddler crab Uca pugilator.

Collagenolytic protease I from the fiddler crab Uca pugilator belongs to the serine proteases of the trypsin family. A graphic molecular model was built using information from sequences and X-ray structures of four homologous proteins which were superimposed to define structurally conserved regions. Protease I sequence was aligned, with sequences of the model proteins, without permitting any deletion or insertion in these regions. Elastase alpha-carbon chain was selected as a template molecule. For the structurally variable regions, fragments of the four homologous proteins which were 'closet' in sequence were selected. Intramolecular steric hindrance, that resulted from the substitution of the residues of the templates by protease I residues, was corrected by adjustment of the side-chain conformational angles. The model was then optimized by energy minimization. The primary specificity pocket in the model of collagenolytic protease I predict a substrate preference for both P1 hydrophobic and positively charged residues which is in agreement with the biochemical observations. As soybean trypsin inhibitor (STI) is known to inhibit collagenolytic protease I, a tentative model of the complex was constructed and possibilities of interaction examined.

Amino Acid Sequence↗

PubMed1990

Role of the gut proteinases from mosquito larvae in the mechanism of action and the specificity of the Bacillus sphaericus toxin.

Gut proteinases from larvae of mosquito species both susceptible and not susceptible to Bacillus sphaericus converted the 43-kDa toxin to a 40-kDa polypeptide exhibiting enhanced cytotoxicity to mosquito cell cultures. The toxin was also activated by gut proteinases from the nonsusceptible Lepidoptera Spodoptera littoralis in vitro and in vivo. Therefore, the specificity of Bacillus sphaericus toxin does not seem to be determined by gut proteinase action. However, susceptibility of mosquito cell cultures did not reflect the specificity of the toxin, which must now be investigated at the cellular level in the larvae.

Aedes↗

PubMed1990

[Model of protease I from the crab Uca pugilator].

Collagenolytic protease I from the fiddler crab Uca pugilator is one of the serine proteases of the trypsin family. A graphic molecular model was built on the basis of the sequences and crystalline structures of four homologous proteins which were superimposed in order to identify structurally conserved regions. The sequence of protease I was matched to sequences of the reference proteins, without allowing any deletions or insertions in these regions. For structurally variable regions, the most similar sequences of the four reference proteins were selected. Intramolecular steric clumping due to replacement of reference side-chains by protease I side-chains were corrected by adjusting side-chain conformations. The model was optimized by energy minimization. The conformation of the primary specificity pocket for protease I predicted by the model indicated a preference for P1 hydrophobic or positively charged substrates. This prediction is consistent with biochemical findings. Because soya bean trypsin inhibitor (STI) has been shown to inhibit protease I, a tentative model of the complex was constructed and possible protease I-STI interactions were analyzed.

Animals↗

PubMed1987

Complete amino acid sequence of the collagenase from the insect Hypoderma lineatum.

The primary structure of the Hypoderma lineatum collagenase was determined. Chymotrypsin digestion and thermolysin fragmentation of the chymotryptic core gave 30 and 5 peptides, respectively, accounting for all the residues of the protein. These peptides were aligned with overlapping peptides derived from tryptic and Staphylococcus aureus V8 proteinase digests. Hypoderma collagenase is a serine proteinase composed of 230 amino acids (Mr 25,223). It displays a high degree of sequential homology with the serine proteinases of the trypsin family, especially with another collagenolytic enzyme, the proteinase I of the crab Uca pugilator. The six half-cystinyl residues of Hypoderma collagenase correspond to 6 of the 10 half-cystinyl residues of chymotrypsin, and the residues forming the charge-relay system of the active site of chymotrypsin (His-57, Asp-102, and Ser-195) are found in corresponding regions. The prediction of the secondary structure of the collagenase is given.

Amino Acid Sequence↗

PubMed1985

Specificity of the collagenase from the insect Hypoderma lineatum.

Specificity of the collagenase from the larvae Hypoderma lineatum, a serine protease related to trypsin, has been investigated by using native collagen and non-collagenous substrates. At 25 degrees C and neutral pH the degradation of collagen by the larval enzyme in solution results in a 52% loss of specific viscosity, without loss of helicity. Electron microscopy of segment-long-spacing crystallites of the digest shows the occurrence of one cleavage region between bands 41 and 44 whereas Edman degradation indicates several cleavage loci in this region. Hypoderma collagenase differs from proteinases I and II from the crab Uca pugilator, which catalyse cleavages in multiple regions of the collagen molecule, and also from vertebrate collagenases, which cleave collagen only between residues 775 and 776. Apart of specific action on collagen, Hypoderma collagenase degrades the oxidized chain B of insulin; the major cleavage occurs at the Leu15-Tyr16 bond followed by two minor cleavages at the Arg22-Gly23 and Lys29-Ala30 bonds. The larval enzyme has no action on synthetic peptide substrates of trypsin or chymotrypsin.

Amino Acid Sequence↗

PubMed1984

Primary structure of alpha-clostripain light chain.

The primary structure of light chain of alpha-clostripain was determined by sequence analysis of peptides derived from tryptic digests purified by reverse-phase high-performance liquid chromatography. The 22 isolated tryptic peptides were aligned by peptides derived from chymotryptic and staphylococcal V8 proteinase digests. The light chain contains 133 amino acids residues and has a relative molecular mass of 15400. The prediction of its secondary structure is given.

Amino Acid Sequence↗

PubMed1983

Hypodermin B, a trypsin-related enzyme from the insect Hypoderma lineatum. Comparison with hypodermin A and Hypoderma collagenase, two serine proteinases from the same source.

Hypodermin B, a serine proteinase with a molecular weight of 23000, was purified to homogeneity from the larvae Hypoderma lineatum. It is stoichiometrically inhibited by diisopropylfluorophosphate and fully inactivated by N-tosyllysine chloromethyl ketone and soya bean and bovine pancreatic trypsin inhibitors. N-Tosylphenylalanine chloromethyl ketone and ovomucoid are without effect on its activity. Hypodermin B hydrolyses both amide and ester substrates of trypsin but does not display any chymotryptic activity on synthetic substrates. Its specificity on the B chain of insulin is slightly broader than that of bovine trypsin. Its amino acid composition and N-terminal sequence suggest structural homology with serine proteinases of the trypsin family and with two other serine proteinases, hypodermin A and Hypoderma collagenase, previously isolated from the same larvae. Hypodermins A and B are very similar with respect to their inhibition and specificity, they differ however strongly from Hypoderma collagenase.

Amino Acid Sequence↗

PubMed1983

Structural study on the active site of the collagenase from Hypoderma lineatum.

The collagenase from the larvae Hypoderma lineatum is a serine proteinase sequentially related to the trypsin family. The tryptic peptide containing the serine residue of the active site, labelled with [3H] diisopropylfluorophosphate was isolated and determined to be Ser-Pro-Cys-Phe-Gly-Asp-Ser-Gly-Gly-Pro-(Phe-Ser)-Lys. It is highly conservative with respect to the corresponding peptide in other serine proteinases related to trypsin.

Animals↗

PubMed1982

Specific antisera produced by direct immunization with slices of polyacrylamide gel containing small amounts of protein.

Rabbits were injected with slices of polyacrylamide gels containing entrapped insect proteins after separation by electrophoresis. Specific antibodies were produced independently of the nature of the gel (with or without sodium dodecyl sulphate) and of the staining technique (amido black or Coomassie Blue). The procedure appears to be a rapid and simple method for production of antibodies specific to proteins separated in minute quantities from a complex mixture.

Acrylic Resins↗