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.

Isotopic double-labeling of two honeybee odorant-binding proteins secreted by the methylotrophic yeast Pichia pastoris.

Odorant-binding proteins (OBPs) are soluble, low-molecular-weight proteins secreted in the sensillum lymph surrounding the dendrites of olfactory sensilla from a wide range of insect species. These proteins play a role in the solubilization, transport and/or deactivation of pheromones and odorants. In order to study the relationships between the molecular structure in solution and their ligand-binding properties, we have (13)C/(15)N-double-labeled two divergent honeybee OBPs, called ASP1 and ASP2, in sufficient quantities to permit a full determination of the structure and dynamics using heteronuclear NMR spectroscopy. The recombinant labeled proteins produced by the methylotrophic yeast Pichia pastoris have been secreted into a buffered minimal medium using native insect signal peptide. Mass spectrometry and Edman sequencing showed a native-like processing with a labeling efficiency of secreted proteins greater than 98%. After dialysis, the recombinant proteins were purified to homogeneity by one-step reversed-phase liquid chromatography. The final yield after 4-day shake-flask liquid culture was approximately 60 and 100 mg/L for ASP1 and ASP2, respectively. The inexpensive overproduction of labeled recombinant ASP1 and ASP2 should allow NMR studies of the structures and ligand-binding analysis in order to understand the relationships between structure and biological function of these proteins.

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.

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.

Valyl-tRNA synthetase from Escherichia coli MALDI-MS identification of the binding sites for L-valine or for noncognate amino acids upon qualitative comparative labeling with reactive amino-acid analogs.

Bromomethyl ketone derivatives of L-valine (VBMK), L-isoleucine (IBMK), L-norleucine (NleBMK) and L-phenylalanine (FBMK) were synthesized. These reagents were used for qualitative comparative labeling of Escherichia coli valyl-tRNA synthetase (ValRS), an enzyme with Val/Ile editing activity, in order to identify the binding sites for L-valine or noncognate amino acids. Labeling of E. coli ValRS with the substrate analog valyl-bromomethyl ketone (VBMK) resulted in a complete loss of valine-dependent isotopic [32P]PPi-ATP exchange activity. L-Valine protected the enzyme against inactivation. Noncognate amino acids analogs isoleucyl-, norleucyl- and phenylalanyl-bromomethyl ketones (IBMK, NleBMK and FBMK) were also capable of abolishing the activity of ValRS, FBMK being less efficient in inactivating the synthetase. Matrix-assisted laser desorption-ionization mass spectrometry designated cysteines 424 and 829 as the target residues of the substrate analog VBMK on E. coli ValRS, whereas, altogether, IBMK, NleBMK and FBMK labeled His266, Cys275, His282, His433 and Cys829, of which Cys275, His282 and His433 were labeled in common by all three noncognate amino-acid-derived bromomethyl ketones. With the exception of Cys829, which was most likely unspecifically labeled, the amino-acid residues labeled by the reagents derived from noncognate amino acids were distributed between two fragments 259-291 and 419-434 in the primary structure of E. coli ValRS. In fragment 419-434, Cys424 was specifically labeled by the substrate analog VBMK, while His433 was labeled in common by all the used bromomethyl ketone derivatives of noncognate amino acids, suggesting that the synthetic site where aminoacyl adenylate formation takes place on E. coli ValRS is built up of two subsites. One subsite containing Cys424 might represent the catalytic locus of the active center where specific L-valine activation takes place. The second subsite containing His433 might represent the binding site for noncognate amino acids. The fact that Cys275 and His282, fragment 259-291, were labeled by IBMK, NleBMK and FBMK, but not by the substrate analog VBMK, suggests that these residues might be located at or near the editing site of E. coli ValRS. Comparison of fragment 259-291 with all the available ValRS amino-acid sequences revealed that His282 is strictly conserved, with the exception of its replacement by a glycine in a subgroup corresponding to the archaebacteria. Because a nucleophile is needed in the editing site to achieve hydrolysis of an undesired product at the level of the carbonyl group thereof, it is proposed that the conserved His282 of E. coli ValRS is involved in editing.

Posttranslational modifications of the lutropin receptor: mass spectrometric analysis.

Our present knowledge of the lutropin (LH/hCG) receptor structure derives from deductions made from its amino acid sequence as established by studying the cDNA. To obtain direct experimental information, luteinizing hormone (LH) receptor expressed in L cells was immunopurified in sufficient amounts to warrant analysis by mass spectrometry and microsequencing. The mature receptor, complexed to human chorionic gonadotropin (hCG), was purified by using monoclonal antibodies recognizing the hormone, whereas the mannose-rich non-hormone-binding precursor was purified by use of antireceptor antibodies. Determination of the N-terminus showed that (2)/(3) of protein molecules started at Thr24 whereas (1)/(3) started at Ala28. All these molecules bound hCG, suggesting that the most N-terminal region of the receptor does not participate in hormone binding. Six N-glycosylation sites have been predicted from the amino acid sequence. One of them (Asn299) was found to be nonglycosylated in both the precursor and the mature protein. The most heavily glycosylated residue was Asn291, followed by Asn195 and Asn99. These three sites accounted for 82% and 97% of carbohydrate moieties in the mature receptor and in the mannose-rich precursor, respectively. The presence of some receptor molecules nonglycosylated at sites 99, 174, and 195 in hormone-receptor complexes dismisses a direct role of these glycosylation sites in hormone binding or in the correct folding of the protein. The mature carbohydrate chains were homogeneous at position 174, 195, and 313 (absence of Golgi mannosidase II activity at positions 174 and 313, absence of GlcNAc tranferases III and IV activity at position 195). Heterologous carbohydrates were present at sites 99 and 291. The latter, which is highly variable in carbohydrate chains, is unlikely to participate in a direct interaction with hormone. Site 313 thus remains as the main candidate for a role in hormone binding.

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.

Polyglutamylation of nucleosome assembly proteins.

Polyglutamylation is an original posttranslational modification, discovered on tubulin, consisting in side chains composed of several glutamyl units and leading to a very unusual protein structure. A monoclonal antibody directed against glutamylated tubulin (GT335) was found to react with other proteins present in HeLa cells. After immunopurification on a GT335 affinity column, two prominent proteins of approximately 50 kDa were observed. They were identified by microsequencing and mass spectrometry as NAP-1 and NAP-2, two members of the nucleosome assembly protein family that are implicated in the deposition of core histone complexes onto chromatin. Strikingly, NAP-1 and NAP-2 were found to be substrates of an ATP-dependent glutamylation enzyme co-purifying on the same column. We took advantage of this property to specifically label and purify the polyglutamylated peptides. NAP-1 and NAP-2 are modified in their C-terminal domain by the addition of up to 9 and 10 glutamyl units, respectively. Two putative glutamylation sites were localized for NAP-1 at Glu-356 and Glu-357 and, for NAP-2, at Glu-347 and Glu-348. These results demonstrate for the first time that proteins other than tubulin are polyglutamylated and open new perspectives for studying NAP function.

Enzyme-induced covalent modification of methionyl-tRNA synthetase from Bacillus stearothermophilus by methionyl-adenylate: identification of the labeled amino acid residues by matrix-assisted laser desorption-ionization mass spectrometry.

Methionyl-tRNA synthetase (MetRS) from Bacillus stearothermophilus was shown to undergo covalent methionylation by a donor methionyl-adenylate, the mixed carboxylic-phosphoric acid anhydride synthesized by the enzyme itself. Covalent reaction of methionyl-adenylate with the synthetase or other proteins proceeds through the formation of an isopeptide bond between the carboxylate of the amino acid and the epsilon-NH2 group of lysyl residues. The stoichiometries of labeling, as followed by TCA precipitation, were 2.2 +/- 0.1 and 4.3 +/- 0.1 mol of [14C]Met incorporated by 1 mol of the monomeric MS534 and the native dimeric species of B. stearo methionyl-tRNA synthetase, respectively. Matrix-assisted laser desorption-ionization mass spectrometry designated lysines-261, -295, -301 and -528 (or -534) of truncated methionyl-tRNA synthetase as the target residues for covalent binding of methionine. By analogy with the 3D structure of the monomeric M547 species of E. coli methionyl-tRNA synthetase, lysines-261, -295, and -301 would be located in the catalytic crevice of the thermostable enzyme where methionine activation and transfer take place. It is proposed that, once activated by ATP, most of the methionine molecules react with the closest reactive lysyl residues.

Cloning and expression of a queen pheromone-binding protein in the honeybee: an olfactory-specific, developmentally regulated protein.

Odorant-binding proteins (OBPs) are small abundant extracellular proteins thought to participate in perireceptor events of odor-pheromone detection by carrying, deactivating, and/or selecting odor stimuli. The honeybee queen pheromone is known to play a crucial role in colony organization, in addition to drone sex attraction. We identified, for the first time in a social insect, a binding protein called antennal-specific protein 1 (ASP1), which binds at least one of the major queen pheromone components. ASP1 was characterized by cDNA cloning, expression in Pichia pastoris, and pheromone binding. In situ hybridization showed that it is specifically expressed in the auxiliary cell layer of the antennal olfactory sensilla. The ASP1 sequence revealed it as a divergent member of the insect OBP family. The recombinant protein presented the exact characteristics of the native protein, as shown by mass spectrometry, and N-terminal sequencing and exclusion-diffusion chromatography showed that recombinant ASP1 is dimeric. ASP1 interacts with queen pheromone major components, opposite to another putative honeybee OBP, called ASP2. ASP1 biosynthetic accumulation, followed by nondenaturing electrophoresis during development, starts at day 1 before emergence, in concomitance with the functional maturation of olfactory neurons. The isobar ASP1b isoform appears simultaneously to ASP1a in workers, but only at approximately 2 weeks after emergence in drones. Comparison of in vivo and heterologous expressions suggests that the difference between ASP1 isoforms might be because of dimerization, which might play a physiological role in relation with mate attraction.

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.

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.

The 2.1 A structure of an elicitin-ergosterol complex: a recent addition to the Sterol Carrier Protein family.

Elicitins, produced by most of the phytopathogenic fungi of the genus Phytophthora, provoke in tobacco both remote leaf necrosis and the induction of a resistance against subsequent attack by various microorganisms. Despite the recent description of the three-dimensional crystal structure of cryptogein (CRY), the molecular basis of the interactions between Phytophthora and plants largely remains unknown. The X-ray crystal structure, refined at 2.1 A, of a ligand complexed, mutated CRY, K13H, is reported. Analysis of this structure reveals that CRY is able to encapsulate a ligand that induces only a minor conformational change in the protein structure. The ligand has been identified as an ergosterol by gas chromatographic analysis coupled with mass spectrometry analysis. This result is consistent with biochemical data that have shown that elicitins are a distinct class of Sterol Carrier Proteins (SCP). Data presented here provide the first structural description of the pertinent features of the elicitin sterol interaction and permit a reassessment of the importance of both the key residue 13 and the mobility of the omega loop for the accessibility of the sterol to the cavity. The biological implications thereof are discussed. This paper reports the first structure of a SCP/sterol complex.

Optimization of the production of a honeybee odorant-binding protein by Pichia pastoris.

A honeybee putative general odorant-binding protein ASP2 has been expressed in the methylotrophic yeast Pichia pastoris. It was secreted into the buffered minimal medium using either the alpha-factor preprosequence with and without the Glu-Ala-Glu-Ala spacer peptide of Saccharomyces cerevisiae or its native signal peptide. Whereas ASP2 secreted using the alpha-factor preprosequence with the spacer peptide showed N-terminal heterogeneity, the recombinant protein using the two other secretion peptides was correctly processed. Mass spectrometry showed that the protein secreted using the natural peptide sequence had a mass of 13,695.1 Da, in perfect agreement with the measured molecular mass of the native protein. These data showed a native-like processing and the three disulfide bridges formation confirmed by sulfhydryl titration analysis. After dialysis, the recombinant protein was purified by one-step anion-exchange chromatography in a highly pure form. The final expression yield after 7-day fermentation was approximately 150 mg/liter. To our knowledge, this is the first report of the use of a natural insect leader sequence for secretion with correct processing in P. pastoris. The overproduction of recombinant ASP2 should allow ligand binding and mutational analysis to understand the relationships between structure and biological function of the protein.

Sequential cleavage and excision of a segment of the thyrotropin receptor ectodomain.

The thyrotropin (TSH) receptor belongs to a subfamily of G protein-coupled receptors, which also includes luteinizing hormone and follicle-stimulating hormone receptors. The TSH receptor (TSHR) differs from the latter by the presence of an additional specific segment in the C-terminal part of its ectodomain. We show here that this insertion is excised in the majority of receptor molecules. Preparation of specific monoclonal antibodies to this region, microsequencing, enzyme-linked immunosorbent assay, and immunoblot studies have provided insight into the mechanisms of this excision. In the human thyroid gland, N termini of the transmembrane receptor beta subunit were found to be phenylalanine 366 and leucines 370 and 378. In transfected L cells a variety of other more proximal N termini were found, probably corresponding to incomplete excisions. The most extreme N terminus was observed to lie at Ser-314. These observations suggest that after initial cleavage at Ser-314 the inserted fragment of TSHR is progressively clipped out by a series of cleavage reactions progressing up to amino acids 366-378. The impossibility of recovering the excised fragment from purified receptor, cell membranes, or culture medium supports this interpretation. The cleavage enzyme has previously been shown to be inhibited by BB-2116, an inhibitor of matrix metalloproteases. However, we show here that it is unaffected by tissue inhibitors of metalloproteases. The cleavage enzyme is very similar to TACE (tumor necrosis factor alpha-converting enzyme) in both these characteristics. However, incubation of the TSH receptor with the purified recombinant catalytic domain of TACE, co-transfection of cells with TACE and TSHR expression vectors, and the use of mutated Chinese hamster ovary cells in which TACE is inactive suggested that the TSHR cleavage enzyme is different from TACE. TACE and TSHR cleavage enzyme may thus possibly be related but different members of the adamalysin family of metzincin metalloproteases.

Isolation, sequence determination, physical and physiological characterization of the neuroparsins and ovary maturing parsins of Schistocerca gregaria.

Neurosecretory products immunologically related to either neuroparsin (NP) or ovary maturing parsin (OMP) of Locusta migratoria (Lom) were purified from the nervous corpora cardiaca of Schistocerca gregaria (Scg). The determination of both their molecular masses by mass spectrometry and their sequences by automated Edman degradation established that they are members of the NP and OMP families respectively. NP molecules of Schistocerca (Scg NPs) consisted of two major forms having about the same molecular masses as NPA and NPB of Locusta and 88% primary structure similarity. They had also the same antidiuretic activity. OMP molecules of Schistocerca (Scg OMPs) were composed in young adults of four isoforms: two long isoforms corresponding to Lom OMP, and differing by a tripeptide insertion (Pro-Ala-Ala) at position 21 and two short isoforms deprived of the 13-residue N-terminal peptide of Lom OMP and differing by the same tripeptide insertion. The PAA isoforms were observed in low amounts as compared to the other isoforms. In mature adults, only the two short isoforms were present. The complete sequence of PAA Scg OMP presents a large degree of sequence homology with Lom OMP (83%). The mixed Scg OMPs had the same biological effects as Lom OMPs. They induced precocious occurrence of both ecdysteroids and vitellogenin in the haemolymph and stimulated oöcyte growth.

Identification and developmental profiles of hexamerins in antenna and hemolymph of the honeybee, Apis mellifera.

Four distinct hexamerin subunits (referred to as "hexamerins" in the following text) have been identified in the developing honeybee, Apis mellifera, by N-terminal protein sequencing. Hexamerins are abundant in the hemolymph of late larval and early pupal stages, and gradually decline during metamorphosis and adult development. Three hexamerins in the 70 kDa range have been found (Hex70a, Hex70b, Hex70c). In worker and drone, Hex70a is the only hexamerin present in large amount in later adult stages. Hex70b and c exhibit a similar developmental profile, disappearing in the drone just before adult emergence, and in the worker just after. Hex70b or Hex70c are still detectable in the adult queen. Hex80/110 likely exist in at least 3 different subunits, 1 of 110 kDa, and 2 of around 80 kDa, which all share a common N-terminus. They disappear during metamorphosis earlier than Hex70b and c. All these hexamerins have been found also in the antenna, suggesting their utilization in building up of antennal cuticle structures.