Determination of the primary and secondary structures of the dromedary (Camelus dromedarius) prolactin and comparison with prolactins from other species.

A non-glycosylated form of camel prolactin (camPRL), isolated from one-humped camel (Camelus dromedarius) pituitaries, was totally sequenced. A glycosylated form, separated by affinity chromatography on ConA-Sepharose, was partially sequenced. The comparison of the N-terminal amino acid sequences of the glycosylated and non-glycosylated forms showed that the only putative site of N-glycosylation (Asn-31) was indeed glycosylated. The far ultraviolet (UV) circular dichroism (CD) spectra of the two isohormones were identical, suggesting that the carbohydrate moiety had no effect on the global camPRL secondary structure. The far UV circular dichroism spectra of the two isohormones were analyzed in order to determine their relative proportions of periodic secondary structure, 60% of which was found to be in alpha-helix, as in prolactins of other species. The dromedary sequence was compared to those of other species and interpreted in term of evolutionary process. As already found for gonadotropins, the closest species to the dromedary was found to be the pig.

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.

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.

The relationships between the toxicity and the primary and secondary structures of elicitinlike protein elicitors secreted by the phytopathogenic fungus Pythium vexans.

Elicitins are toxic 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. Such proteins were observed in the culture filtrate of another species of the Oomycete genus, Pythium vexans. Two alpha elicitinlike proteins were purified and sequenced. One of these novel elicitins (Vex2) exhibited a 100-residue sequence instead of 98 while the other (Vex1) had an N-glycosylation site, effectively glycosylated (equivalent of 16 hexose residues). In addition to the point mutations already observed in Phytophthora species, we found several novel amino acid changes. Furthermore, circular dichroism revealed some differences in their structure in solution compared with the Phytophthora elicitins that were correlated with specific point mutations. These sequences permitted the establishment of a phylogenic tree, suggesting that Pythium vexans is a species close to the Phytophthora genus. The toxicity of the Pythium vexans elictins to tobacco leaves was investigated and correlated with the occurrence of the carbohydrate moiety of one of the two isoforms, observed for the first time in an elicitin.

Characterization of elicitin-like phospholipases isolated from Phytophthora capsici culture filtrate.

The phytopathogenic oomycete Phytophthora capsici secretes in culture a phospholipase activity. Two enzyme isoforms exhibiting a high phospholipase B activity were isolated by chromatography and electrophoresis. They differ in their apparent molar masses (22 and 32 kDa). Both proteins are glycosylated and share the same N-terminal amino acid sequence up to the 39th residue with a high homology with capsicein, the P. capsici elicitin. Although devoid of phospholipase activity, capsicein was shown by circular dichroism to specifically interact with negatively charged phospholipids, suggesting that the membrane lipids could be a potential target for elicitins.

Odorant and pheromone binding by aphrodisin, a hamster aphrodisiac protein.

Aphrodisin is a soluble glycoprotein of hamster vaginal discharges, which stimulates male copulatory behavior. Natural aphrodisin was purified and its post-translational modifications characterized by MALDI-MS peptide mapping. To evaluate its ability to bind small volatile ligands, the aphrodisiac protein was expressed in the yeast Pichia pastoris as two major isoforms differing in their glycosylation degree, but close in conformation to the natural protein. Dimeric recombinant aphrodisins were equally able to efficiently bind odors (2-isobutyl-3-methoxypyrazine and methyl thiobutyrate) and a pheromone (dimethyl disulfide), suggesting that they could act as pheromone carriers instead of, or in addition to, direct vomeronasal neuron receptor activators.

Large-scale preparation and characterization of recombinant ovine placental lactogen.

To clone ovine placental lactogen (oPL) cDNA, total RNA from sheep placental cotyledon was reverse transcribed and the single-stranded cDNA was PCR-amplified with 5' and 3' primers containing, respectively, NcoI and PstI sites. The oPL cDNA fragment amplified between these two primers extended from A(-1) to the natural stop codon. The PCR product was gel-purified and subcloned into a Puc vector and the insert was sequenced on both strands, revealing several differences relative to the published sequence: S19N, S69N, D129E and R165Q. We assume that these differences can be accounted for by the high level of individual polymorphism, which has been described in detail for PLs of different species. The insert was subcloned into NcoI/ PstI-digested pTrc99A procaryotic expression plasmid and protein expression was induced by isopropyl-1-thio-beta-D-galactopyranoside. Because of low expression, oPL's cDNA was further subcloned into pET8 procaryotic expression plasmid. Its expression in E. coli strain BL21 transformed with this vector yielded 30-40 mg/l. The expressed protein, found in the inclusion bodies, was refolded into a monomer and purified on a Q-Sepharose column to homogeneity. Structural analysis using circular dichroism revealed a spectrum similar to that of human GH (hGH) thereby indicating proper refolding. Gel filtration and binding experiments, including real-time kinetic measurements using the surface plasmon resonance method revealed that oPL forms transient homodimeric complexes with extracellular domains of prolactin receptors from rabbit, rat and bovine and with hGH receptor. The purified oPL was biologically active in an Nb2-11C cell proliferation bioassay, in its ability to stimulate beta-casein synthesis in explants of ovine and rabbit mammary gland and fat synthesis in explants of bovine mammary gland, and in a proliferation assay using FDC-P1 cells transfected with rabbit or hGH receptors.

Structures of elicitin isoforms secreted by Phytophthora drechsleri.

Most of the phytopathogenic fungi Phytophthora secrete holoproteins (elicitins) responsible for the incompatible reaction and systemic leaf necroses on tobacco. We found that Phytophthora drechsleri produces several elicitin isoforms of various toxicity on tobacco. The CD spectra showed that their secondary structure was largely conserved, exhibiting ca 50% alpha-helix and little or no beta-structure. These 98 residue proteins were sequenced and compared with other known elicitins. Only one point mutation correlated with the differences in necrotic activities. This residue could be either an active or a regulatory site, involved in the interaction with a receptor responsible for necrosis induction.

A novel elicitin necrotic site revealed by alpha-cinnamomin sequence and site-directed mutagenesis.

Elicitins are 10 kDa proteins secreted by Phytophthora fungi, that elicit resistance against certain plant pathogens. Various natural molecules, mutated recombinant elicitins and synthetic peptides were previously shown to differentially induce in tobacco leaf necrosis and defence genes, activities borne by several sites which were identified. We report a novel necrosis-determining residue at position 25, revealed by the comparison of the necrotic activity and sequence of alpha-cinnamomin with those of other known elicitins. Using a modified recombinant beta-cryptogein, expressed in Pichia pastoris, we show that the substitution of asparagine 25 by a serine leads to a significant enhancement of the necrotic activity.

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.

Structure-function relationships of α and ß elicitins, signal proteins involved in the plant-Phytophthora interaction.

Elicitins form a family of 10-kDa holoproteins secreted by various Phytophthora species. The large-scale purification of parasiticein, a novel elicitin secreted by P. parasitica, led to the determination of its sequence. We have compared the necrotic activities and the primary and secondary structures (determined through circular dichroism) of four elicitins. On tobacco plants, they could be classified into two classes: a, comprising capsicein and parasiticein (less necrotic), and ß, comprising cryptogein and cinnamomin (very toxic with a necrosis threshold of 0.1 µg per leaf). The features of elicitin structure which might be involved in the interaction of elicitins with the leaf target cells and that could explain the different necrosis-inducing properties of the two proteins are investigated. About 75% sequence identity was observed between the four elicitins: only two short terminal regions are heterologous, while the central core is mainly conserved. The circular-dichroism spectra showed that the secondary structure of the elicitins was largely conserved. All of them consisted of approx. 50% α-helix with little or no ß-structure. Comparisons of the complete sequences, amino-acid compositions, isoelectric points, hydropathy indices and the secondary-structure predictions correlated with the necrotic classification. Alpha elicitins corresponded to acidic molecules with a valine residue at position 13, while ß elicitins were basic with a lysine at this position, which appeared to be a putative active site responsible for necrosis induction.

Odorant binding and conformational changes of a rat odorant-binding protein.

Odorant-binding proteins (OBPs) are lipocalins secreted in the nasal mucus of vertebrates, which convey odorants to their neuronal receptors. We compared the binding properties of a recombinant rat OBP (OBP-1F) using a set of six odorants of various chemical structures. We examined the binding properties by both fluorescent probe competition and isothermal titration calorimetry. OBP-1F affinity constants, in the micromolar range, varied by more than one order of magnitude and were roughly correlated to the odorant size. The observed binding stoichiometry was found to be around one odorant per dimer. Using tyrosine differential spectroscopy, the binding of ligand was shown to induce local conformational changes. A three-dimensional structure of OBP-1F, modelled using the known structure of aphrodisin as template, allowed us to suggest the location of the observed structural changes outside of the binding pocket. These results are consistent with one binding site located in one of the two beta-barrels of the OBP-1F dimer and a subtle conformational change correlated with binding of an odorant molecule, which hampers uptake of a second odorant by the other hydrophobic pocket.

Ligand binding and physico-chemical properties of ASP2, a recombinant odorant-binding protein from honeybee (Apis mellifera L.).

In insects, the transport of airborne, hydrophobic odorants and pheromones through the sensillum lymph is generally thought to be accomplished by odorant-binding proteins (OBPs). We report the structural and functional properties of a honeybee OBP called ASP2, heterologously expressed by the yeast Pichia pastoris. ASP2 disulfide bonds were assigned after classic trypsinolysis followed by ion-spray mass spectrometry combined with microsequencing. The pairing [Cys(I)-Cys(III), Cys(II)-Cys(V), Cys(IV)-Cys(VI)] was found to be identical to that of Bombyx mori OBP, suggesting that this pattern occurs commonly throughout the highly divergent insect OBPs. CD measurements revealed that ASP2 is mainly constituted of alpha helices, like other insect OBPs, but different from lipocalin-like vertebrate OBPs. Gel filtration analysis showed that ASP2 is homodimeric at neutral pH, but monomerizes upon acidification or addition of a chaotropic agent. A general volatile-odorant binding assay allowed us to examine the uptake of some odorants and pheromones by ASP2. Recombinant ASP2 bound all tested molecules, except beta-ionone, which could not interact with it at all. The affinity constants of ASP2 for these ligands, determined at neutral pH by isothermal titration calorimetry, are in the micromolar range, as observed for vertebrate OBP. These results suggest that odorants occupy three binding sites per dimer, probably one in the core of each monomer and another whose location and biological role are questionable. At acidic pH, no binding was observed, in correlation with monomerization and a local conformational change supported by CD experiments.

Disulfide pairing and secondary structure of ASP1, an olfactory-binding protein from honeybee (Apis mellifera L).

In insects, the transport of airborne, hydrophobic odorants and pheromones through the sensillum lymph is accomplished by olfactory-binding proteins (CBPs). We report the structural characterization of a honeybee OBP called ASP1 found in workers and drones, previously observed to bind queen pheromone components. A novel method based on ion-spray mass spectrometry analysis of cyanylation-induced cleavage products of partially reduced protein with Tris(2-carboxyethyl)phosphine was needed to determine the recombinant ASP1 disulfide bond pairing. It was observed to be Cys(I)-Cys(III), Cys(II)-Cys(V), Cys(IV)-Cys(VI), similar to those already described for other OBPs from honeybee and Bombyx mori suggesting that this pattern occurs commonly throughout the diverse family of insect OBPs. Circular dichroism revealed that ASP1 is an all-alpha protein in accordance with NMR preliminary data, but unlike lipocalin-like vertebrate OBPs.

Ligand-binding properties and structural characterization of a novel rat odorant-binding protein variant.

After characterization of a novel odorant-binding protein (OBP) variant isolated from the rat nasal mucus, the corresponding cDNA was cloned by RT-PCR. Recombinant OBP-1F, the sequence of which is close to that of previously reported rat OBP-1, has been secreted by the yeast Pichia pastoris at a concentration of 80 mg.L-1 in a form identical to the natural protein as shown by MS, N-terminal sequencing and CD. We observed that, in contrast with porcine OBP-1, purified recombinant OBP-1F is a homodimer exhibiting two disulfide bonds (C44-C48 and C63-C155), a pairing close to that of hamster aphrodisin. OBP-1F interacts with fluorescent probe 1-aminoanthracene (1-AMA) with a dissociation constant of 0.6 +/- 0. 3 microM. Fluorescence experiments revealed that 1-AMA was displaced efficiently by molecules including usual solvents such as EtOH and dimethylsulfoxide. Owing to the large OBP-1F amounts expressed, we set up a novel biomimetic assay (volatile-odorant binding assay) to study the uptake of airborne odorants without radiolabelling and attempted to understand the odorant capture by OBP in the nasal mucus under natural conditions. The assay permitted observations on the binding of airborne odorants of different chemical structures and odors (2-isobutyl-3-methoxypyrazine, linalool, isoamyl acetate, 1-octanal, 1-octanol, dimethyl disulfide and methyl thiobutyrate). Uptake of airborne odorants in nearly physiological conditions strengthens the role of OBP as volatile hydrophobic odorant carriers in the mucus of the olfactory epithelium through the aqueous barrier towards the chemo-sensory cells.

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.

Physical characterization and sequence identification of the ovary maturating parsin. A new neurohormone purified from the nervous corpora cardiaca of the African locust (Locusta migratoria migratorioides.

A novel neurohormone, which anticipates ovarian maturation, was recently purified using liquid chromatography from the African locust nervous corpora cardiaca. Both its function and production by the pars intercerebralis of Locusta migratoria lead to its name, the ovary maturating parsin (Lom OMP). In this study, the Lom OMP was physically and chemically characterized. Its multiply charged ion spectrum was interpreted as two peaks of quite equal size having molecular masses of 6923.4 Da (major peak) and 6907.3 Da. The Lom OMP presented no periodic secondary structure according to the far ultraviolet circular dichroism spectrum obtained. It is composed of 65 amino acids and included a high concentration of alanine but is devoid of cysteine, isoleucine, methionine, lysine and threonine. The amino acid sequence indicated only one microheterogeneity, observed at position 26, consisted in the replacement of serine by alanine. The calculated Mr of the two acidic isoforms (calculated pHi = 4.87) were found to be in agreement with mass spectrometry measurements. When compared to the sequence libraries, the Lom OMP, the first insect gonadotropic neurohormone, was revealed as an unique protein.

Novel recombinant analogues of bovine placental lactogen. G133K and G133R provide a tool to understand the difference between the action of prolactin and growth hormone receptors.

Two new analogues of bovine placental lactogen (bPL), bPL(G133K) and bPL(G133R), were expressed in Escherichia coli, refolded, and purified to a native form. Binding experiments, which are likely to represent the binding to site 1 only, to intact FDC-P1 cells transfected with rabbit (rb) growth hormone receptor (GHR) or with human (h) GHR, to Nb2 rat lymphoma cells, or to rabbit mammary gland membranes prolactin receptor (PRLR), revealed only small or no reduction in binding capacity. The complex formation between these analogues and receptor extracellular domains (R-ECD) of various hormones was determined by gel filtration. Wild type bPL yielded 1:2 complex with hGHR-ECD, rat PRLR-ECD, and rbPRLR-ECD, whereas both analogues formed only 1:1 complexes with all R-ECDs tested. Real time kinetics experiments demonstrated that the ability of the analogues to form homodimeric complexes was compromised in both PRLR- and GHR-ECDs. The biological activity transduced through lactogenic receptors in in vitro bioassays in rabbit mammary gland acini culture and in Nb2 cells was almost fully retained, whereas the activity transduced through somatogenic receptors in FDC-P1 cells transfected with rbGHRs or with hGHRs was abolished. Both analogues exhibited antagonistic activity in the latter cells. To explain the discrepancy between the effect of the mutation on the signal transduced by PLR versus GHRs we suggest that: 1) the mutation impairs the ability of site 2 of bPL to form a stable homodimeric complex with both lactogenic and somatogenic receptors by a drastic shortening of the half-life of 2:1 complex; 2) the transient existence of the homodimeric complex is still sufficient to initiate the signal transduced through lactogenic receptors but not through somatogenic receptors; and 3) one possible reason for this difference is that JAK2, which serves as a mediator of both receptors, is already associated with lactogenic receptors prior to hormone binding-induced receptor dimerization, whereas in somatogenic receptors the JAK2 receptor association occurs subsequently to receptor dimerization.

Synthesis and CD studies of an 88-residue peptide containing the main receptor binding site of HTLV-I SU-glycoprotein.

Essential HTLV-1 biological functions, like host-cell receptor recognition, depend on the structural motives on the surface glycoprotein gp46. We defined a peptide of 88 amino acids [Arg147-Leu234] corresponding to the central part of the protein sequence, where major neutralizing epitopes are localized. After evaluating the feasibility of its chemical synthesis, the chosen sequence was realized using the stepwise solid-phase methodology. Multiple chromatographic purification steps were required to obtain a sample suitable for structural analysis. Correct folding was supported by strong binding of monooclonal antibodies, recognizing known exposed immunodominant regions. Circular dichroism studies confirmed a non-random conformation of at least 70-80% of the synthetic peptide. Investigation of the 3D-structure of the synthetic peptide will provide useful information for future vaccine and drug-design strategies.

Purification and structural analysis of a soluble human chorionogonadotropin hormone-receptor complex.

Receptors for the luteotropin/human chorionogonadotropin hormone belong to the G-protein-coupled receptor family by their membrane-anchoring domains. They also possess a large extracellular domain (ECD) responsible for most of the hormone-receptor interactions. Structure-function studies identified several contacts between hormone and receptor ECD, but the precise topology of the complex is still unknown because of the lack of suitable heterologous expression means. Receptor ECDs exhibit leucine repeats and have been modelized on the basis of the three-dimensional structure of the porcine ribonuclease inhibitor, the first structurally known leucine-rich repeats protein. Here we report overexpression (up to 20 mg per liter) and purification to homogeneity of a soluble human chorionogonadotropin-ECD receptor complex secreted by stably cotransfected Chinese hamster ovary cells. Biochemical analysis and surface plasmon resonance data were in favor of a unique dimer with a 1:1 ligand-receptor stoichiometry. Immunopurified complex was submitted to circular dichroism characterization; CD spectra deconvolution indicated more than 25% alpha helices contributed by the receptor, in agreement with the porcine ribonuclease inhibitor-based modelization.