中华蜜蜂性信息素结合蛋白ASP1的原核表达及配基结合特性分析

【目的】研究中华蜜蜂Apis cerana cerana信息素结合蛋白ASP1与蜜蜂信息素及某些植物挥发物分子的结合功能。【方法】构建中蜂ASP1的原核表达载体, 对其进行重组蛋白的诱导表达和分离纯化, 并得到具有生化活性的中蜂ASP1重组蛋白, 最后以1-NPN作为荧光报告探针, 通过荧光竞争结合实验研究中蜂重组ASP1蛋白与蜜蜂信息素及其他气味分子的结合功能。【结果】在22种潜在信息气味物质中, 有7种与中蜂ASP1有较强的结合能力, 能将1-NPN的相对荧光强度降至50%以下。其中发现蜂王信息素两种成分对 羟基苯甲酸甲酯和香草醇的竞争能力最强, 可分别引起1-NPN相对荧光值下降99.31%和95.50%, 解离常数K_D分别为13.39和98.44 μmol/L; 而与除蜂王信息素外的其他信息素如幼虫信息素和工蜂信息素等分子均不结合。此外中蜂ASP1对于水杨酸甲酯、 苯乙醛、 3, 4-二甲基苯甲醛4-烯丙基藜芦醚和β-紫罗兰酮等5种植物挥发物质能产生强度不一的结合。【结论】中蜂信息素结合蛋白ASP1对蜂王信息素具有非常强的特异性, 同时也能结合某些植物挥发性气味分子, 暗示中蜂ASP1是一种以蜂王信息素识别为主要功能、 植物挥发物识别为次要功能的多功能信息素结合蛋白。     

Probing a pheromone binding protein of the silkmoth Antheraea polyphemus by endogenous tryptophan fluorescence.

One subtype of the pheromone binding proteins of the silkmoth Antheraea polyphemus (ApolPBP1) has been analysed exploiting the two endogenous tryptophan residues as fluorescent probe. The intrinsic fluorescence exhibited a rather narrow spectrum with a maximum at 336 nm. Site-directed mutagenesis experiments revealed that one of the tryptophan residues (Trp37) is located in a hydrophobic environment whereas Trp127 is more solvent exposed, as was predicted modeling the ApolPBP1 sequence on the proposed structure of the Bombyx mori pheromone binding protein. Monitoring the interaction of ApolPBP1 as well as its Trp mutants with the three species-specific pheromone compounds by recording the endogenous fluorescence emission revealed profound differences; whereas (E6,Z11)-hexadecadienal induced a dose-dependent quenching of the fluorescence, both (E6,Z11)-hexadecadienyl-1-acetate and (E4,Z9)-tetradecadienyl-1-acetate elicited an augmentation of the endogenous fluorescence. These data indicate that although ApolPBP1 can bind all three pheromones, there are substantial differences concerning their interaction with the protein, which may have important functional implications.     

Cloning and characterization of a region of the Enterococcus faecalis conjugative plasmid, pCF10, encoding a sex pheromone-binding function.

In order to investigate the mechanism by which peptide sex pheromones induce expression of the conjugation functions of certain Enterococcus faecalis plasmids, a biological assay was developed to measure the ability of cells carrying the conjugative plasmid pCF10 to bind the sex pheromone cCF10. The data indicated that pCF10 endows its host E. faecalis cell with the ability to specifically remove (apparently by irreversible binding) cCF10 activity from culture medium. The pCF10 DNA encoding this ability was localized to a 3.4-kb segment within a region involved in negative control of expression of conjugal transfer functions. This segment also encoded ability to bind the pheromone inhibitor peptide iCF10. DNA sequencing revealed three open reading frames, which have been denoted prgW (pheromone responsive gene W), prgZ, and prgY. The deduced product of prgW resembled regulatory proteins from other bacteria and eucaryotes, with a very high degree of identity within a putative DNA-binding domain. The prgY gene actually extended into an adjacent region of pCF10 and could encode a protein with significant similarity to a protein called TraB, believed to be involved in shutdown of pheromone cAD1 production by cells carrying the pheromone-inducible hemolysin plasmid pAD1, according to F.Y. An and D.B. Clewell (Abstr. Gen. Meet. Am. Soc. Microbiol. 1992, H70, 1992). The prgZ gene product showed significant relatedness to binding proteins encoded by oligopeptide permease (opp) operons in gram-positive and gram-negative bacteria and is highly similar to a pAD1-encoded protein, TraC, which is believed to mediate sex pheromone cAD1 binding (K. Tanimoto, F. Y. An, and D. B. Clewell, submitted for publication). A Tn5 insertion into prgZ abolished cCF10 binding ability.     

NMR mapping of the recombinant mouse major urinary protein I binding site occupied by the pheromone 2-sec-butyl-4,5-dihydrothiazole.

The interactions between the mouse major urinary protein isoform MUP-I and the pheromone 2-sec-butyl-4,5-dihydrothiazole have been characterized in solution. (15)N-labeled and (15)N, (13)C-doubly-labeled recombinant MUP-I were produced in a bacterial expression system and purified to homogeneity. Racemic 2-sec-butyl-4, 5-dihydrothiazole was produced synthetically. An equilibrium diffusion assay and NMR titration revealed that both enantiomers of the pheromone bind to the recombinant protein with a stoichiometry of 1 equiv of protein to 1 equiv of racemic pheromone. A micromolar dissociation constant and slow-exchange regime dissociation kinetics were determined for the pheromone-protein complex. (1)H, (15)N, and (13)C chemical shifts of MUP-I were assigned using triple resonance and (15)N-correlated 3D NMR experiments. Changes in protein (1)H(N) and (15)N(H) chemical shifts upon addition of pheromone were used to identify the ligand binding site. Several amide signals, corresponding to residues on one side of the binding site, were split into two peaks in the saturated protein-ligand complex. Similarly, two overlapping ligand spin systems were present in isotope-filtered NMR spectra of labeled protein bound to unlabeled pheromone. The two sets of peaks were attributed to the two possible chiralities of the pheromone. Intermolecular NOEs indicated that the orientation of the pheromone in the MUP-I binding cavity is opposite to that modeled in a previous X-ray structure.     

Genetic fine-structural analysis of the Saccharomyces cerevisiae alpha-pheromone receptor.

The alpha-pheromone receptor encoded by the STE2 gene contains seven potential transmembrane domains. Its ability to transduce the pheromone signal is thought to require the action of a G protein. As an initial step toward defining the structural features of the receptor required for its activity, we examined the phenotypic consequences of linker insertion mutations (12 bp) at 10 different sites in the STE2 gene. Three mutant classes, which correspond to three different regions of the receptor protein, were observed. 1) The two mutants affecting the C-terminal region (C-terminal mutants) were essentially wild type for mating efficiency, pheromone binding, and pheromone sensitivity. 2) The three mutants in the N-terminus mated with reduced efficiency, showed reduced pheromone binding capacity, and were partially defective in pheromone induction of agglutinin production and cell division arrest. Increased gene dosage of these N-terminal alleles suppressed their mutant phenotypes, whereas the sst2-1 mutation, which blocks adaptation to pheromone, did not result in suppression. Thus, the N-terminal mutants were apparently limited by receptor production, but not by the adaptation function SST2. 3) The five mutants in the central region containing the seven transmembrane segments (central mutants) were completely defective for mating and did not respond to pheromone, but could be distinguished by their ability to bind pheromone. Inserts in or near transmembrane domains 2 and 4 blocked pheromone binding, whereas inserts into transmembrane domains 1, 5, and 6 retained partial pheromone binding activity even though they failed to transduce a signal. The central mutants were not suppressed by increased gene dosage, and one mutant (ste2-/101) was partially suppressed by sst2-1. Furthermore, the central core mutants were also distinguished from one another in that three of the five mutants were able to partially complement the temperature sensitivity of ste2-3.     

Do Pheromone Binding Proteins Converge in Amino Acid Sequence When Pheromones Converge?

Convergence in amino acid sequences between proteins can be strong evidence for selection. Here, I look for evidence of convergence in the amino acid sequences of pheromone binding protein (PBP) in response to convergence in pheromones. PBPs are involved in sex pheromone reception by the antennae of male moths. In this role PBPs may selectively bind pheromone components and experience convergent selection in response to convergence in pheromone components. However, examination of the PBPs of the taxa that have converged upon the use of (E)- or (Z)-11-tetradecenyl acetate as their major pheromone component reveals little evidence for convergence in the PBPs identified from these taxa. A few sites show a pattern consistent with convergence or parallelism; however, it cannot be ruled out that these sites share the ancestral state. Two of these sites fall within the proposed binding region of PBPs. These results suggest that PBPs either have not converged in sequence or have converged at very few sites in response to convergence on the same pheromone component. Received: 29 July 1999 / Accepted: 8 November 1999     

Pheromone induction of agglutination in Saccharomyces cerevisiae a cells.

a-Agglutinin, the cell surface sexual agglutinin of yeast a cells, was assayed by its ability to bind its complementary agglutinin, alpha-agglutinin. The specific binding of 125I-alpha-agglutinin to a cells treated with the sex pheromone alpha-factor was 2 to 2.5 times that of binding to a cells not treated with alpha-factor. Competition with unlabeled alpha-agglutinin revealed that the increased binding was due to increased cell surface expression of a-agglutinin, with no apparent change in the binding constant. The increase in site number was similar to the increase in cellular agglutinability. Increased expression of a-agglutinin followed the same kinetics as the increase in cellular agglutinability, with a 10-min lag followed by a 15- to 20-min response time. Induction kinetics were similar in cells in phases G1 and G2 of the cell cycle. Maximal expression levels were similar in cells treated with excess pheromone and in cells exposed to pheromone after destruction of constitutively expressed a-agglutinin.     

Pheromone discrimination by the pheromone-binding protein of Bombyx mori

Pheromone-binding proteins are postulated to contribute to the exquisite specificity of the insect's olfactory system, acting as a filter by preferentially binding only one of the components of the natural pheromone. Here, we investigated the possible discrimination of the two very similar components of the natural pheromone gland from the silk moth, Bombyx mori, bombykol and bombykal, by the only pheromone-binding protein (BmorPBP) known to be expressed in the pheromone-detecting sensilla. Free-energy calculations and virtual docking indicate that both bombykol and bombykal bind to BmorPBP with similar affinity. In addition, in vitro competitive binding assays showed that both bombykol and bombykal were bound by BmorPBP with nearly the same high affinity. While BmorPBP might filter out other physiologically irrelevant compounds hitting the sensillar lymph, discrimination between the natural pheromone compounds must be achieved by molecular interactions with their cognate receptors.     

Competition Among Homologous Polypeptide Pheromones of the Ciliate Euplotes raikovi for Binding to Each Other's Cell Receptors

ABSTRACT. Purified preparations of the polypeptide pheromones Er-1 and Er-2 were obtained from type I and II cells of Euplotes raikovi , respectively, radiolabeled, and used to study their ability to compete with each other in binding to the same type I and II cells and to type × cells secreting Er-10, another pheromone homologous to Er-1 and Er-2. It was shown that: 1) These radiolabeled pheromone preparations bind, with similar but not identical affinities, to cells from which they originate as well as to cells of the other types. 2) Every pheromone binding reaction can be completely inhibited by another homologous pheromone added in excess to cells. These results are discussed in relation to phenomena, common in ciliates, of instability of (homotypic) mating pairs formed between genotypically identical cells suspended with a foreign (nonself) pheromone.     

Cloning, E. coli overexpression, purification and binding properties of TraA and TraC, two proteins involved in the pheromone-dependent conjugation process in enterococci

The bacteriocin encoding plasmid pPD1 from Enterococcus faecalis is involved in a mating response to the sex pheromone cPD1 produced by recipient bacterial cells devoid of pPD1. Previous studies showed that cPD1 is internalized into donor cells in a process in which TraC plays the role of cell surface pheromone receptor. Inside the recipient cells, the pheromone binds to the plasmid-encoded cytoplasmic protein TraA, able to recognize specific DNA sequences and to modulate the conjugation process. To avoid self-induction of the conjugation process, donor cells produce the inhibitor iPD1, which competes with cPD1. This study was designed to produce recombinant TraA and TraC in a functionally active state and to evaluate their main functional properties. We have isolated the sequences encoding TraA and TraC from the plasmid pPD1 and cloned them in suitable expression vectors. The two recombinant proteins were successfully obtained in a soluble form using Escherichia coli as expression host and a T7 inducible expression system. TraC and TraA were purified to homogeneity by three or two chromatographic steps, respectively, leading to a final yield up to 4 mg/l of cell culture for TraC and up to 10 mg/l of cell culture for TraA. The ability of TraA and TraC to bind the specific pheromone and inhibitor peptides has been assessed by means of ESI-mass spectrometry. Moreover, the ability of recombinant TraA to bind DNA has been demonstrated by means of electrophoretic mobility shift assay. Overall these results are consistent with the heterologously expressed TraC and TraA being functionally active.     

Antagonistic and synergistic peptide analogues of the tridecapeptide mating pheromone of Saccharomyces cerevisiae.

Biologically inactive, truncated analogues of the Saccharomyces cerevisiae alpha-mating factor (WHWLQLKPGQPMY) either antagonized or synergized the activity of the native pheromone. An amino-terminal truncated pheromone [WLQLKPGQP(Nle)Y] had no activity by itself, but the analogue acted as an antagonist by competing with binding and activity of the mating factor. In contrast, a carboxyl-terminal truncated pheromone [WHWLQLKPGQP] was not active by itself nor did the peptide compete with alpha-factor for binding to the alpha-factor receptor, but it acted as a synergist by causing a marked increase in the activity of alpha-factor. The observation that residues near the amino terminus may be involved in signal transduction whereas those near the carboxyl terminus influence binding allows us to separate binding and signal transduction in the yeast pheromone response pathway. If found for other hormone-receptor systems, synergists may have potential as therapeutic compounds.     

Functional analysis of Plp1 and Plp2, two homologues of phosducin in yeast

Mammalian phosducins are known to bind G protein betagamma subunits in vitro, and are postulated to regulate their signaling function in vivo. Here we describe two homologues of phosducin in yeast, called PLP1 and PLP2. Both gene products were cloned, expressed, and purified as glutathione S-transferase fusions. Of the two isoforms, Plp1 bound most preferentially to Gbetagamma. Binding was enhanced by pheromone stimulation and by the addition of GTPgammaS, conditions that favor dissociation of Gbetagamma from Galpha. Gene disruption mutants and gene overexpression plasmids were prepared and analyzed for changes in signaling and nonsignaling phenotypes. Haploid spore products bearing the plp2Delta mutant failed to grow, suggesting that PLP2 is an essential gene. Cell viability was not restored by a mutation in STE7 that blocks signaling downstream of the G protein. Haploid products bearing the plp1Delta mutant were viable and exhibited a 6-7% increase in pheromone-mediated gene induction. Cells overexpressing PLP1 or PLP2 exhibited a 70-80% decrease in gene induction but no change in pheromone-mediated growth arrest. These data indicate that phosducin can selectively regulate early signaling events following pheromone stimulation and has an essential role in cell growth independent of its regulatory role in cell signaling.     

Localization and signaling of G(beta) subunit Ste4p are controlled by a-factor receptor and the a-specific protein Asg7p

Haploid yeast cells initiate pheromone signaling upon the binding of pheromone to its receptor and activation of the coupled G protein. A regulatory process termed receptor inhibition blocks pheromone signaling when the a-factor receptor is inappropriately expressed in MATa cells. Receptor inhibition blocks signaling by inhibiting the activity of the G protein beta subunit, Ste4p. To investigate how Ste4p activity is inhibited, its subcellular location was examined. In wild-type cells, alpha-factor treatment resulted in localization of Ste4p to the plasma membrane of mating projections. In cells expressing the a-factor receptor, alpha-factor treatment resulted in localization of Ste4p away from the plasma membrane to an internal compartment. An altered version of Ste4p that is largely insensitive to receptor inhibition retained its association with the membrane in cells expressing the a-factor receptor. The inhibitory function of the a-factor receptor required ASG7, an a-specific gene of previously unknown function. ASG7 RNA was induced by pheromone, consistent with increased inhibition as the pheromone response progresses. The a-factor receptor inhibited signaling in its liganded state, demonstrating that the receptor can block the signal that it initiates. ASG7 was required for the altered localization of Ste4p that occurs during receptor inhibition, and the subcellular location of Asg7p was consistent with its having a direct effect on Ste4p localization. These results demonstrate that Asg7p mediates a regulatory process that blocks signaling from a G protein beta subunit and causes its relocalization within the cell.     

Involvement of protein sulfhydryls in the trigger reaction of rhodotorucine A, a farnesyl peptide mating pheromone of Rhodosporidium toruloides.

The involvement of protein sulfhydryls for the signaling of rhodotorucine A, a mating pheromone produced by mating type A cells of Rhodosporidium toruloides, was investigated by the use of sulfhydryl compounds. The sulfhydryl-blocking reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB; Ellman's reagent) strongly inhibited both the biological effect of the pheromone on the recipient cell and the hydrolysis of the pheromone, which is catalyzed by the mating type-specific surface endopeptidase of the recipient cell. Conversely, the two reactions were markedly enhanced by the presence of the reducing reagent dithiothreitol. The inhibitory effect of DTNB on the pheromone response of the recipient cell was specific to an initial stage of the differentiation; once it had initiated, the reagent had no effect on its progression. The results suggested that dithiothreitol enhances and DTNB impairs the efficiency with which the pheromone triggers sexual d differentiation. The reaction of DTNB with cellular protein sulfhydryls was highly restricted to those at the exterior surface of the membrane due to the impermeability of the reagent through the membrane. Phosphorylation of endogenous proteins, which is modulated by the pheromone added to an in vitro phosphorylation system, was also blocked by DTNB. The results showed that sulfhydryl groups are involved in the pheromone hydrolysis by the surface endopeptidase of the recipient cell and that pheromone metabolism is indispensable for the signaling reaction. We suggest that the modulation of protein phosphorylation of membrane proteins by the pheromone is an initial transmembrane response coupled to pheromone metabolism.     

The identification of an age- and female-specific putative PBAN membrane-receptor protein in pheromone glands of Helicoverpa armigera: possible up-regulation by Juvenile Hormone

The present study was designed to determine the age and female specificity of a membrane protein that binds to a pheromone biosynthesis activating neuropeptide (PBAN) ligand and to elucidate the effect of Juvenile Hormone (JH) on binding as well as pheromone activation. The precise age at which developing adult females of Helicoverpa armigera begin to respond to PBAN was determined. PBAN activates in vitro pheromone biosynthesis as well as its intracellular second messenger, cAMP, only in intersegments of newly emerged adult female pheromone glands (i.e. 1-day-old females). An increase in response was observed in 2-day-old females. Intersegments of female pupae and the homologous tissues of adult males do not respond to PBAN. However, in the presence of Juvenile Hormone II (JH II) PBAN induced a response in females, 1 day before emergence (pharate females), but not in younger female pupae. This phenomenon was also observed after topical applications of the JH analog fenoxycarb (FX). In addition the response to PBAN by intersegments of FX-treated emerged adults increased significantly to the level of 2-day-old females. JH II also stimulated the level of incorporation of (35)S-labelled amino acids in female pupae into membrane proteins that are typical in adult intersegments. Using a photoaffinity-biotin labelled PBAN analog we demonstrate specific binding of a membrane protein (estimated MW: 50 kD) in adult females. This binding was not detected in female pupae 3 days before emergence. However, in such female pupae specific binding of the 50 kD protein by the photoaffinity-biotin labelled PBAN analog was induced after JH II or FX treatments thereby providing evidence that JH may up-regulate this putative receptor protein.