Biomedical subjects

J C Pernollet

Publications and source records attributed to J C Pernollet.

At least 37 records · Page 2Linked to original sources

PubMed1998

Separation, characterization and sexual heterogeneity of multiple putative odorant-binding proteins in the honeybee Apis mellifera L. (Hymenoptera: Apidea).

According to precise molar mass determined by mass spectrometry and N-terminal sequence, some 25 odorant-binding-like proteins were characterized from the antennae and legs of worker and drone honeybees. Antennal specific proteins, composed of six different molecules, were classified into three subclasses according to N-terminal sequence homology. The major sexual difference was shown to lie in the relative abundance of these antennal specific proteins and in the occurrence of a drone-specific isoform. At least 19 other related proteins were found to occur in antennae and legs, forming another class showing homology with insect OBP. Genotype comparison of two honeybee races revealed a variability limited to this second class. Provided that these odorant-binding-like proteins are indeed able to bind odorants or pheromones, the question of whether their peculiar multiplicity contributes to the remarkable capacity of the honeybee to discriminate among a wide range of odor molecules is raised.

Amino Acid Sequence↗

PubMed1997

Use of immobilized metal ion affinity chromatography and dye-ligand chromatography for the separation and purification of rainbow trout pituitary gonadotropins, GTH I and GTH II.

A new procedure is described for the purification of gonadotropic hormones (GTHs) from the pituitary glands of vitellogenic rainbow trout. The procedure utilizes immobilized metal ion affinity chromatography (IMAC) on a column containing immobilized iminodiacetic acid (Toyopearl AF Chelate) charged with Cu2+ ions as a critical step for the efficient separation of GTH I and GTH II. Further purification of both GTH fractions on Cibacron Blue F3GA immobilized on Toyopearl was followed by HPLC size-exclusion for GTH II. The resulting electrophoretically homogeneous preparations possessed a characteristic range of biological activity in the stimulation of steroidogenesis in vitro. N-Terminal sequences of the GTH I and GTH II subunits purified using reversed-phase HPLC revealed a high level of homology with those of GTH subunits found in three other salmonid fish species. In contrast to salmon GTH II, two different alpha-subunits were observed in trout GTH II. The cross-reactivity between GTH I and GTH II was studied in radioimmunoassay using antibodies against Chinook salmon GTH II beta-subunit and rainbow trout GTH I dimer.

Amino Acid Sequence↗

PubMed1997

Biochemical characterization, molecular cloning and localization of a putative odorant-binding protein in the honey bee Apis mellifera L. (Hymenoptera : Apidea).

A honey bee antennal water-soluble protein, APS2, was purified and characterized as the first Hymenoptera putative odorant-binding protein. Comparison of its measured Mr (13695.2+/-1.6) to that of the corresponding cDNA clone shows it does not undergo any post-translational modification other than a 19-residue signal peptide cleavage and formation of three disulfide bridges. These biochemical features are close to those of Lepidoptera odorant-binding proteins. In situ hybridization experiments demonstrated its specific expression in olfactory areas. Based on its higher expression in the worker than in the drone, ASP2 might be more involved in general odorant than in sex pheromone detection.

Amino Acid Sequence↗

PubMed1997

Three-dimensional solution structure of beta cryptogein, a beta elicitin secreted by a phytopathogenic fungus Phytophthora cryptogea.

Cryptogein belongs to a new family of 10-kDa proteins called elicitins. Elicitins are necrotic and signaling proteins secreted by Phytophthora spp. responsible for the incompatible reaction and systemic hypersensitive-like necroses of diverse plant species leading to resistance against fungal or bacterial plant pathogens. The solution structure of beta cryptogein from Phytophthora cryptogea fungus was determined by using multidimensional heteronuclear nuclear magnetic resonance spectroscopy. A set of 18 structures was calculated using 1360 NOE-derived distance restraints and 40 dihedral angle restraints obtained from 3JHNH alpha couplings. The RMS deviation from the mean structure is 0.87 +/- 0.14 A for backbone atoms and 1.34 +/- 0.14 A for all the non-hydrogen atoms of residues 2 to 98. The structure of beta cryptogein reveals a novel protein fold, with five helices and a double-stranded beta-sheet facing an omega-loop. One edge of the beta-sheet and the adjacent face of the omega-loop form a hydrophobic cavity. This cavity made of highly conserved residues represents a plausible binding site. Residue 13, which has been identified from directed mutagenesis and natural sequence comparison studies as a key amino acid involved in the differential control of necrosis, is surface exposed and could contribute to the binding to a ligand or a receptor. The solution structure is close to the X-ray structure, with slight differences lightly due to the crystal packing.

Algal Proteins↗

PubMed1997

Mapping the elicitor and necrotic sites of Phytophthora elicitins with synthetic peptides and reporter genes controlled by tobacco defense gene promoters.

Elicitins are 10-kDa proteins secreted by Phytophthora and Pythium fungi that elicit a hypersensitive-like necrotic reaction, leading to resistance against fungal and bacterial plant pathogens. Induction of necrosis and resistance were previously shown to be borne by different sites of the molecule. Furthermore, sequence comparison indicated several potential residues necessary for necrosis. The role of one of these residues was previously evidenced with site-directed mutagenesis. In order to locate other necrosis-determining sites and reveal the defense-eliciting sites, we synthesized a series of synthetic peptides. Tests were performed on two types of transgenic tobacco plants, both transformed with a construction containing the beta-glucuronidase reporter gene, in one case controlled by the promoter of the multiple stimulus response gene str 246C and in the other by the promoter of the pathogenesis-related gene PR1a. We report that only certain peptides were found to be active. Whereas PR1a induction was consistently correlated with induction of necrosis, four peptides were observed to induce only str 246C expression without necrosis, which led to differentiate the defense-eliciting sites from the necrotic sites. From the structure-function relationship thus obtained, two different defense pathways were inferred to be independently induced by elicitins.

Algal Proteins↗

PubMed1996

Crystal structure of a fungal elicitor secreted by Phytophthora cryptogea, a member of a novel class of plant necrotic proteins.

BACKGROUND: Elicitins form a novel class of plant necrotic proteins which are secreted by Phytophthora and Pythium fungi, parasites of many economically important crops. These proteins induce leaf necrosis in infected plants and elicit an incompatible hypersensitive-like reaction, leading to the development of a systemic acquired resistance against a range of fungal and bacterial plant pathogens. No crystal structures of this class of protein are available. The crystal structure determination of beta-cryptogein (CRY), secreted by Phytophthora cryptogea, was undertaken to identify structural features important for the necrotic activity of elicitins. RESULTS: The structure of CRY was determined using the multiwavelength anomalous diffraction technique and refined to 2.2 A resolution. The overall structure has a novel fold consisting of six alpha helices and a beak-like motif, whose sequence is highly conserved within the family, composed of an antiparallel two-stranded beta sheet and an omega loop. This motif is assumed to be a major recognition site for a putative receptor and/or ligand. Two other distinct binding sites seem to be correlated to the level of necrotic activity of elicitins. CONCLUSIONS: The determination of the crystal structure of a member of the elicitin family may make it possible to separate the activity that causes leaf necrosis from that inducing systemic acquired resistance to pathogens, making it feasible to engineer a non-toxic elicitin that only elicits plant defences. Such studies should aid the development of non-toxic agricultural pest control.

Algal Proteins↗

PubMed1996

PCR cloning and expression analysis of a cDNA encoding a pectinacetylesterase from Vigna radiata L.

A cDNA clone encoding a pectinacetylesterase (PAE) was isolated from 3-day-old mung bean seedlings using PCR-based techniques. Degenerate oligonucleotide primers were designed according to the N-terminus and internal peptides from the purified PAE. The full-length clone of 1453 bp codes for a signal peptide of 24 amino acids and a mature protein of 375 amino acids. The Mr and the pI of the cDNA-deduced amino acid sequence agree with the values estimated for the purified enzyme. No significant sequence identity between the PAE and any known protein could be found in the databases. Northern analysis revealed developmentally regulated expression of the mRNA in mung been seedlings.

Amino Acid Sequence↗

PubMed1996

Overexpression in Pichia pastoris and crystallization of an elicitor protein secreted by the phytopathogenic fungus, Phytophthora cryptogea.

A synthetic gene encoding beta-cryptogein, a member of the elicitin family, has been cloned into a vector for expression by the methylotrophic yeast, Pichia pastoris. Having first optimized the gene construction for secretion, we have overexpressed a modified beta-cryptogein in a secreted form. A purification scheme suited to this expression system has been developed and highly pure, biologically active protein has been obtained. For structural analysis of this recombinant beta-cryptogein, and new mutated forms thereof, optimal conditions for the crystallization of this protein have been determined and crystals that diffract to 2.2 A have been obtained.

Algal Proteins↗

PubMed1996

Pectinmethylesterase isoforms from Vigna radiata hypocotyl cell walls: kinetic properties and molecular cloning of a cDNA encoding the most alkaline isoform.

Peptide maps and partial amino acid sequences of the 3 main pectinmethylesterases (PMEs) solubilized from mung bean hypocotyl cell walls demonstrated that these proteins were different isozymes originating from a small multigene family. A cDNA clone encoding the most alkaline PME (PE gamma) have been obtained by PCR using degenerate oligonucleotide primers. Combining the protein and nucleotide sequencing data, the complete amino acid sequence of PE gamma was determined. The nature protein is composed of 318 amino acids with a calculated Mtau of 34 677 and an estimated pI of 9.84 consistent with the values previously obtained by SDS-PAGE and IEF. It shares most of the conserved regions of previously known PMEs. Enzymatic activities of the three isoforms were differently affected by the presence of cations in the incubation medium but, in all cases, infra-optimal cation concentrations induced two opposite effects: a decrease in the Vmax and an increase in the affinity of the enzymes for their substrate. The presence of cations in the assay modulates both the number of enzyme molecules available to the demethylation reaction and the conformation of the pectin and, in turn, the affinity of the PMEs for their substrate.

Amino Acid Sequence↗

PubMed1996

Purification and characterization of multiple forms of odorant/pheromone binding proteins in the antennae of Mamestra brassicae (Noctuidae).

Proteins extracted form the antennae of Mamestra brassicae (L.) (Lepidoptera: Noctuidae) adults were biochemically characterized as pheromone-binding proteins (PBP) and general odorant-binding proteins (GOBP). PBP and GOBP were purified by two successive and different HPLC (high performance liquid chromatography) systems and native polyacrylamide gel electrophoresis (native-PAGE). Their N-terminal sequence was determined by Edman microsequencing. The combined results showed evidence for three different PBPs in males, and two different PBPs in females. In addition, one GOBP was characterized in both males than in females antennae. In the males, two isoforms of PBP have the same N-terminal sequence, but different apparent mobilities and hydrophobicities: they could be separated by electrophoresis and reverse phase-HPLC (RP-HPLC). The other PBP sequence (SQEIM) showed particularly high homology (88%) with the PBP of Heliothis virescens, another noctuid moth. The existence of several forms of PBP in the same animal strongly supports the hypothesis of the specificity of binding between the proteins and their odorant ligands, the pheromonal compounds. The observed microdiversity at the soluble proteins level could provide a good model for studying their involvement in the initial stages of odor discrimination.

Amino Acid Sequence↗

PubMed1995

Chemical synthesis, expression and mutagenesis of a gene encoding beta-cryptogein, an elicitin produced by Phytophthora cryptogea.

Elicitins are 10 kDa holoproteins secreted by Phytophthora fungi, that elicit an incompatible hypersensitive reaction, leading to resistance against fungal and bacterial plant pathogens. Comparison of primary sequences of alpha-elicitins and beta-elicitins indicated several potential necrotic activity-determining residues. All of the highly necrotic beta-elicitins have a hydrophilic residue (usually lysine) at position 13, whereas in the less necrotic alpha-elicitins this residue is replaced by a valine. Here, we report the synthesis and expression of a gene encoding a highly necrotic elicitin, beta-cryptogein, and we show that the substitution of Lys-13 of this recombinant protein by a valine leads to a drastic alteration to the necrotic activity of the recombinant protein.

Algal Proteins↗

PubMed1995

A novel protein from mung bean hypocotyl cell walls with acetyl esterase activity.

An acetyl esterase was purified from cell walls isolated from mung bean hypocotyls. The purified enzyme had an apparent Mr of 43,300 and an apparent pI > 9. It rapidly deesterified triacetin and p-nitrophenylacetate and slowly released acetate from beet and flax pectins, the deesterification rate being increased by previous demethylation of the pectins. No significant peptide sequence identity between the acetyl esterase and any known protein could be found in protein data bases.

Acetylesterase↗

PubMed1995

High-level expression of recombinant porcine LH receptor in baculovirus-infected insect cells or caterpillars.

Porcine LH receptor ectodomain was overexpressed in insect cells and lepidopteran larvae using the recombinant baculovirus expression system. A low multiplicity of infection yielded the largest active production, of approximately 10(7) receptors/cell or 3 micrograms active receptor/mg total protein in infected cells. The truncated ectodomain solubilized with Triton X-100 bound its ligand with a high affinity which was comparable with that of the native membrane receptor. Increasing the multiplicity of infection resulted in an optimum protein production of 0.6 mg receptor/mg total protein in infected cells. This receptor was largely inactive, probably trapped within aggregation pools. Active receptor could be recovered by dilution of the samples. No secretion of recombinant receptor was ever observed whatever the conditions of infection. Expression of the recombinant receptor in insect larvae was also tested. This low-cost system failed both to increase the amount of active receptor and to induce secretion into the haemolymph. Two methods remain for producing sizeable amounts of active receptor with this baculovirus/insect cell system. One relies on immunoaffinity purification of the active protein and requires large-scale production, and the other is based on the purification of overexpressed inactive receptor followed by renaturation.

Amino Acid Sequence↗

PubMed1994

1H and 15N resonance assignment and secondary structure of capsicein, an alpha-elicitin, determined by three-dimensional heteronuclear NMR.

The backbone 1H and 15N resonance assignments and solution secondary structure determination of capsicein, a protein of 98 residues with a molecular mass of 10161 Da, are presented. Capsicein belongs to the elicitin family, elicitor molecules having toxic and signaling properties that are secreted by Phytophthora fungi, responsible for the incompatible hypersensitive reaction of diverse plant species leading to resistance against fungal or bacterial plant pathogens. The protein was uniformly labeled with 15N to overcome spectral overlap of the proton resonances. A combination of 3D HOHAHA-HMQC and 3D NOESY-HMOC experiments allowed the identification of spin systems with through-bond correlations, which were in turn correlated by through-space connections. The sequential assignment was obtained for main- and side-chain resonances and led to the identification of all secondary structures. A 3D HMQC-NOESY-HMQC experiment was performed which characterized the NH(i)-NH(i+1) connections specific to alpha-helical structures. This proved particularly useful for the assignment of degenerate amide protons of successive residues in alpha-helical structures. The data show five alpha-helical regions comprising residues 5-18, 26-33, 44-58, 59-67, and 86-98 and a two-stranded antiparallel beta-sheet involving residues 70-75 and 80-85, packed around a hydrophobic core grouping all of the aromatic residues. The C-terminal secondary structure motifs of capsicein evoke phospholipase structural features, which suggests that elicitins might interact with the lipidic molecules of the plasma membrane.

Amino Acid Sequence↗

PubMed1994

Resonance assignment, cysteine-pairing elucidation and secondary-structure determination of capsicein, an alpha-elicitin, by three-dimensional 1H NMR.

Difficulties encountered in the interpretation of two-dimensional NMR spectra of proteins exceeding roughly 100 amino acids, including resonance overlap and line broadening due to longer correlation times and/or aggregation phenomena, can be overcome by using three-dimensional 1H-NMR experiments. The improvement of spectral resolution using these experiments allows the size of molecules amenable to structure determination by NMR spectroscopy to be extended. A three-dimensional non-selective homonuclear Hartmann-Hahn/nuclear Overhauser effect spectroscopy experiment was performed on capsicein, a 10161-Da elicitin secreted by the Phytophthora capsici fungus. Sequential assignment and secondary structure determination is illustrated for beta-sheet, alpha-helix and loop structures by analysis of planar cross sections perpendicular to the omega 2 or omega 3 axis at the amide proton resonance frequencies. Cysteine pairing was established in the course of the investigation. The secondary structure topology of the molecule is composed of five helices and an antiparallel beta-sheet. Four of the helices compose two pairs running antiparallel while the last one is parallel to the beta-sheet.

Amino Acid Sequence↗

PubMed1994

PopA1, a protein which induces a hypersensitivity-like response on specific Petunia genotypes, is secreted via the Hrp pathway of Pseudomonas solanacearum.

This paper describes the identification of a new class of extracellular bacterial proteins, typified by PopA1 and its derivative PopA3, which act as specific hypersensitive response (HR) elicitors. These two heat-stable proteins, with HR-like elicitor activities on tobacco (non-host plant) but without activity on tomato (host plant), have been characterized from the supernatant of the plant pathogenic bacterium Pseudomonas solanacearum strain GMI1000. These two proteins induced the same pattern of response on Petunia, as a function of the genotypes tested. popA, the structural gene for PopA1, maps outside of the hrp gene cluster but belongs to the hrp regulon. The amino acid sequence of PopA1 does not show homology to any characterized proteins. Its secretion is dependent on hrp genes and is followed by stepwise removal of the 93 amino-terminal amino acids, producing the protein PopA3. Petunia lines responsive to PopA3 and its precursors were resistant to infection by strain GMI1000, whereas non-responsive lines were sensitive, suggesting that popA could be an avirulence gene. A popA mutant remained fully pathogenic on sensitive plants, indicating that this gene is not essential for pathogenicity. While lacking PopA1, this mutant, which remained avirulent on tobacco and on resistant Petunia lines, still produced additional extracellular necrogenic compounds. On the basis of both their structural features and the biological properties of the popA mutant, PopA1 and PopA3 clearly differ from hairpins characterized in other plant pathogenic bacteria.

Amino Acid Sequence↗

PubMed1994

Local structural differences between alpha- and beta-elicitins shown by circular dichroism and ultraviolet difference spectroscopy.

In order to investigate differences in the conformation of elicitins exhibiting different levels of activity (toxicity to tobacco plants), the environment of the tyrosyl residues in four elicitins has been compared by different spectroscopic methods (difference absorption and circular dichroism). We compared two alpha-elicitins (capsicein and parasiticein) and two beta-elicitins (beta-cryptogein and beta-cinnamomin), that are 50-100 times more toxic than the alpha-ones. Thermal difference UV spectroscopy and titration experiments clearly showed the exposure of Tyr-85 by comparison of parasiticein lacking Tyr-85 and the accessibility of its hydroxyl group to the solvent. The adjacent Tyr-87 was also suggested to be located at the surface. In beta-cryptogein, beta-cinnamomin and capsicein the pK was measured at between 10.5 and 10.8, while in parasiticein it is higher (11.5) owing to a difference in the local environment. Thermal difference UV spectroscopy showed one more exposed tyrosine in beta-elicitins than in alpha-ones. This difference was attributed to Tyr-12, considering the more hydrophilic characteristic of the sequence around residue 13 in beta-elicitins and the role of this region in the toxicity. However, no difference in titration behaviour was noted among elicitins concerning Tyr-12. The other two tyrosines also presented an abnormal pK of titration (> 12). In all elicitins Tyr-47 was probably exposed, while Tyr-33 was probably buried and not titrated, except in beta-cinnamomin at very alkaline pH.

Algal Proteins↗

PubMed1993

Crystallization and preliminary X-ray diffraction studies of beta-cryptogein, a toxic elicitin secreted by the phytopathogenic fungus Phytophthora cryptogea.

Crystals of the basic elicitin secreted by Phytophthora cryptogea have been obtained by the hanging-drop method of vapor diffusion from sodium chloride solutions. The crystals belong to the tetragonal space group P4(1)22 (or enantiomorph P4(3)22), with unit cell dimensions a = b = 47 A, c = 137 A and probably contain two molecules per asymmetric unit. The crystals are very stable to X-rays and diffract to 2.2 A resolution on a synchrotron radiation source.

Algal Proteins↗