Purification, characterization, and amino acid sequence of the mating pheromone Er-10 of the ciliate Euplotes raikovi

The mating pheromone Er-10 from mat-10 homozygous Euplotes raikovi was purified by a three-step purification procedure with an overall yield of 62%. It was identified as a protein of molecular weight 8000 having an isoelectic point of 3.9. Its complete primary structure was determined by automated Edman degradation of the whole protein after performic acid oxidation and of peptides generated by cyanogen bromide and Staphylococcus aureus V8 protease. The proposed sequence is Asp1-Leu-Cys-Glu-Gln-Ser-Ala-Leu-Gln-Cys10-Asn-Glu-Gln-Gly-Cys-His -Asn-Phe-Cys- Ser20-Pro-Glu-Asp-Lys-Pro-Gly-Cys-Leu-Gly-Met30-Val-Trp-Asn- Pro-Glu-Leu-Cys- Pro38. The calculated molecular weight of 4191.7, which is in good agreement with the value of m/z 4190.7 obtained by fission fragment ionization mass spectrometry, suggests that the native structure is a dimer with three intrachain disulfide bonds in each subunit. The amino acid sequence is 43% identical with that of the E. raikovi mating pheromone Er-1, with the identities concentrated in the amino-terminal half. The half-cystine locations are conserved, but Er-10 is two residues shorter than Er-1. Prediction of the secondary structure suggests that Er-10 may also contain a helical structure at the amino terminus. These results indicate that the mating pheromones of E. raikovi form a homologous family.     

Identification and initial characterization of an autocrine pheromone receptor in the protozoan ciliate Euplotes raikovi

The polypeptide pheromone Er-1, purified from the ciliate Euplotes raikovi of mating type I and genotype mat-1/mat-1, was iodinated with 125I-Bolton-Hunter reagent to a sp act of 0.45-0.73 mu Ci/microgram of protein. This preparation of 125I-Er-1 bound specifically to high affinity binding sites on the same cells of mating type I. Binding of 125I-Er-1 occurred with an apparent Kd of 4.63 +/- 0.12 X 10(-9) M in cells in early stationary phase. It was estimated that these cells carry a total number of approximately 5 X 10(7) sites/cell, with a site density that falls in the range of 1,600-1,700/microns 2 of cell surface. Unlabeled Er-1, other homologous pheromones such as Er-2 and Er-10, antibodies specific for Er-1, and human IL-2 were shown to act as effective inhibitors of specific binding of 125I-Er-1 to mating type I cells. The "autocrine" nature of the identified specific high affinity binding sites for Er-1 was further substantiated by cross-linking experiments. These experiments revealed that mating type-I cell membranes contain one protein entity of Mr = 28,000 that is capable of reacting specifically with the homodimeric native form of Er-1.     

Specific and common epitopes in mating pheromones of Euplotes raikovi revealed by monoclonal antibodies.

Polypeptide mating pheromones Er-1 and Er-2, purified from the supernatant of Euplotes raikovi cultures of mating type I and mating type II, respectively, were used to immunize mice and obtain monoclonal antibodies. Five hybridoma clones producing antibodies specific to the mating pheromones were selected. They were analyzed for immunospecificity by immunoperoxidase assay, immunoblotting, and for their efficacy in inhibition of mating pheromone activity. Monoclonal antibodies from two hybridoma clones recognized only the mating pheromone used as antigen: those from the other three clones reacted, to comparable extents, with both mating pheromones. On the basis of these results it was assumed that two immunogenic sites exist in Er-1 and Er-2, one specific and the other common to both mating pheromones.     

Purification to apparent homogeneity of the mating pheromone of mat-1 homozygous Euplotes raikovi

Mating type-specific mating pheromones autonomously released into the environment control cell-cell union in conjugation of the marine ciliate Euplotes raikovi. The mating pheromone, termed euplomone r-1, of cells of the homozygous mating type determined by the mat-1/mat-1 genotype was purified by means of a three-stage purification procedure which provides a yield of 79%. Starting from 10 liters of supernatant, 3.3 mg of euplomone r-1 were regularly recovered. Euplomone r-1 was identified with a protein showing a molecular weight of 12,000, an isoelectric point of 3.7, and unusually high contents of cysteine (15%) and tyrosine (5.8%). It appeared active at a concentration of 3.4 +/- 0.5 X 10(-12) M. Carbohydrate and a small colored compound, not yet identified, occurred in association with partially purified euplomone r-1 samples, whereas they were not detected in samples of the final product of purification. An acidic shift was apparently capable of destroying the carbohydrate/euplomone r-1 association, suggesting that it does not involve covalent bonds.     

Developmental analysis of the cell recognition mechanism in the ciliate Euplotes raikovi.

Euplotes raikovi, like other ciliates, passes through a postconjugal immaturity, operatively identified by an apparent cell inability to form mating pairs under experimental conditions that are the same as those used for inducing mating at maturity. In cells homozygous for the gene mat-2, which controls the pheromone Er-2, Er-2 mRNA synthesis and mature Er-2 secretion were shown to start from the very beginning of the life cycle and continue throughout immaturity, although to extents estimated to be 5- to 10-fold lower than at maturity. In addition, experiments of 125I-Er-2 binding and crosslinking provided evidence that autocrine pheromone-binding sites, showing values of the dissociation constant of the order of 10(-9) M, are on the surface of immature cells. The number of these sites per cell was estimated to increase from less than 10(6) per cell of 5-7 fissions of age, to about 16 x 10(6) at maturity. These results were taken to suggest that a pheromone-receptor production is stimulated during immaturity by autocrine pheromone binding to cells and that this production might be essential for the development of a pheromone-receptor density high enough to transform the cell from "immature" to "adult," that is competent to respond as well to pheromones of conspecific, genetically different cells.     

Self from nonself discrimination in single-celled eukaryotes

A mechanism for self recognition has been proposed to control the mating type interactions in the marine ciliate Euplotes raikovi. The relevant molecules involved in this mechanism are the mating pheromones inherited via high-multiple alleles codominant at the Mendelian locus mat. Four of these mating pheromones have been isolated, purified, and characterized. They are relatively small and acidic proteins (Mr, 11,000-12,000; pI, 3.7-4.0), which are still active at the concentration of approximately 10(-12) molar. Data, yet preliminary, of amino acid sequencing of the purified mating pheromones showed extensive structural homologies and the common presence of the aspartic acid at the amino terminal.     

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.     

A structurally deviant member of the Euplotes raikovi pheromone family: Er-23

Pheromones of Euplotes raikovi form a homologous family of proteins with 37- to 40-amino acid residues, including six cysteines that form three strictly conserved disulfide bridges. The determination of the primary structure of the pheromone Er-23, which was isolated from cells derived from natural populations of E. raikovi that secrete the other known pheromones, has now revealed a novel structure type. The polypeptide chain of this pheromone contains 51 residues, 10 of which are cysteines presumably involved in the formation of five disulfide bridges, and lacks a carboxyl-terminal tail following the last cysteine of the sequence. The elongation of the Er-23 molecule is presumed to result from multiple events of gene duplication starting from an ancestral motif Xxx(2-4)-Cys-Xxx(5-7)-Cys.     

Autocrine, mitogenic pheromone receptor loop of the ciliate Euplotes raikovi: pheromone-induced receptor internalization

The ciliate Euplotes raikovi produces a family of diffusible signal proteins (pheromones) that function as prototypic growth factors. They may either promote cell growth, by binding to pheromone receptors synthesized by the same cells from which they are secreted (autocrine activity), or induce a temporary cell shift from the growth stage to a mating (sexual) one by binding to pheromone receptors of other, conspecific cells (paracrine activity). In cells constitutively secreting the pheromone Er-1, it was first observed that the expression of the Er-1 receptor "p15," a type II membrane protein of 130 amino acids, is quantitatively correlated with the extracellular concentration of secreted pheromone. p15 expression on the cell surface rapidly and markedly increased after the removal of secreted Er-1 and gradually decreased in parallel with new Er-1 secretion. It was then shown that p15 is internalized through endocytic vesicles following Er-1 binding and that the internalization of p15/Er-1 complexes is specifically blocked by the paracrine p15 binding of Er-2, a pheromone structurally homologous to, and thus capable of fully antagonizing, Er-1. Based on previous findings that the p15 pheromone-binding site is structurally equivalent to Er-1 and that Er-1 molecules polymerize in crystals following a pattern of cooperative interaction, it was proposed that p15/Er-1 complexes are internalized as a consequence of their unique property (not shared by p15/Er-2 complexes) of undergoing clustering.     

Structural characterization of mating pheromone precursors of the ciliate protozoan Euplotes raikovi. High conservation of pre and pro regions versus high variability of secreted regions.

The precursors of Euplotes raikovi pheromones Er-2 and Er-10 have been structurally characterized from the sequences of their coding regions that were amplified and cloned using the polymerase chain reaction and oligonucleotide primers corresponding to conserved sequences of the gene for pheromone Er-1. The predicted amino acid sequences contain 75 residues distributed through three domains: signal peptide, pro segment and mature pheromone. Despite the conservation of the overall length, there is variation in the size of the pro segments and of the mature pheromones. The comparison of the sequences shows a gradient of identity from the amino to the carboxyl terminus; the signal sequences are identical (with greater than or equal to 95% identity in the nucleotide sequences), the pro segments more variable and the mature pheromones quite diverse. The processing site of the pro pheromones, to produce the mature forms, is apparently characterized by the unusual Xaa-Asp sequence.     

游仆虫信息素Er-1和Er-2对四膜虫的种间作用

信息素是生物体向外释放的化学物质,在细胞及生物体中具有种内信息传递的生理学功能。信息素这一类分子广泛分布于系统发生史中,它们的特异活性在单细胞生物、昆虫以及脊椎动物中均有报道。脊椎动物中信息素的信号传输已被证实是一嗅觉依赖过程,7TM-受体被认为是信号传输过程中的信号转换器。在低等单细胞生物(例如:来可夫游仆虫)的细胞膜上存在有信息素异构体,作为信息素分子的有效结合位点而行使其功能。本研究主要探讨单细胞的信息素(Er-1和Er-2)的基础细胞生理学作用是仅限于产生该信息素的物种,还是对其它的原生动物(例如:四膜虫)或对系统发育中分类地位较高的细胞(例如:MRC5成纤维细胞或J774巨噬细胞)均具有调节活性。研究结果表明,游仆虫的两种信息素对梨形四膜虫GL的生长调节有显著不同的作用:当信息素浓度为10-11M时,Er-1具有正调控作用,而Er-2具有抑制剂的作用。这两种配体的趋化作用也有很不同:Er-1具有一种广范的化学排斥特性,而Er-2具有一个双峰的化学吸引剂的性质。计算机检测发现,与Er-2的作用不同,Er-1可略微降低被测细胞的游动速率。趋化现象的选择特性表明Er-2信息素的受体有一种“短期”的特性;而Er-1是不能选择任何亚种群的,这也支持了我们先前的研究数据,即这两种信息素在四膜虫GL内产生两种不同的信号。四膜虫对信息素特异性的反应表明四膜虫能辨别非常近似但带有微小差异的配体(如Er-1和Er-2的电荷差异)。     

The NMR solution structure of the pheromone Er-11 from the ciliated protozoan Euplotes raikovi.

The NMR solution structure of the pheromone Er-11, a 39-residue protein from the ciliated protozoan Euplotes raikovi, was calculated with the distance geometry program DIANA from 449 NOE upper distance constraints and 97 dihedral angle constraints, and the program OPAL was employed for structure refinement by molecular mechanics energy minimization in a water bath. For a group of 20 conformers used to characterize the solution structure, the average of the pairwise RMS deviations from the mean structure calculated for the backbone heavy atoms N, C alpha, and C' of residues 2-38 was 0.30 A. The molecular architecture is dominated by an up-down-up bundle of three short helices with residues 2-9, 12-19, and 22-32, which is closely similar to the previously determined structures of the homologous pheromones Er-1, Er-2, and Er-10. This finding provides structural evidence for the capability shown by these pheromones to compete with each other in binding reactions to their cell-surface receptors.     

The disulfide bond pairing of the pheromones Er-1 and Er-2 of the ciliated protozoan Euplotes raikovi.

The disulfide pairings of the two Euplotes raikovi pheromones Er-1 and Er-2 have been determined by chemical and mass spectrometric analyses. Cystine-linked peptides from thermolytic digestions of the native molecules were purified by reverse-phase high performance liquid chromatography and identified in the known sequences to make the assignments. The same pairing, Cys(I)-Cys(IV), Cys(II)-Cys(VI), and Cys(III)-Cys(V), was found in both pheromones, suggesting that this pattern occurs commonly throughout this family of molecules. This arrangement of disulfides indicates that the three-dimensional structure is defined by three loops, which can vary in size and charge distribution from one pheromone to another.     

Structure comparison of the pheromones Er-1, Er-10, and Er-2 from Euplotes raikovi.

The NMR structures of the homologous pheromones Er-1, Er-10, and Er-2 from the ciliated protozoan Euplotes raikovi are compared. For all 3 proteins the molecular architecture is made up of an antiparallel 3-helix bundle. The preservation of the core part of the structure is directly manifested by similar patterns of slowed backbone amide proton exchange rates, hydrogen bond formation, and relative solvent accessibility. To align the 6 half-cystine residues in the individual sequences within the preserved 3-dimensional core structure, several deletions and insertions had to be introduced that differ from those previously proposed on the basis of the primary structures. Of special interest is a deletion in the second helix of Er-2, which is accommodated by a transition from an alpha-helix in Er-1 and Er-10 to a 3(10)-helix in Er-2. The most significant structural differences are located in the C-terminal part of the proteins, which may have an important role in specific receptor recognition.     

The NMR solution structure of the pheromone Er-2 from the ciliated protozoan Euplotes raikovi.

The NMR structure of the pheromone Er-2 from the ciliated protozoan Euplotes raikovi has been determined in aqueous solution. The structure of this 40-residue protein was calculated with the distance geometry program DIANA from 621 distance constraints and 89 dihedral angle constraints; the program OPAL was employed for the energy minimization. For a group of 20 conformers used to characterize the solution structure, the average pairwise RMS deviation from the mean structure calculated for the backbone heavy atoms N, C alpha, and C' of residues 3-37 was 0.31 A. The molecular architecture is dominated by an up-down-up bundle of 3 short helices of residues 5-11, 14-20, and 23-33, which is similar to the structures of the homologous pheromones Er-1 and Er-10. Novel structural features include a well-defined N-cap on the first helix, a 1-residue deletion in the second helix resulting in the formation of a 3(10)-helix rather than an alpha-helix as found in Er-1 and Er-10, and the simultaneous presence of 2 different conformations for the C-terminal tetrapeptide segment, i.e., a major conformation with the Leu 39-Pro 40 peptide bond in the trans form and a minor conformation with this peptide bond in the cis form.     

The NMR solution structure of the pheromone Er-1 from the ciliated protozoan Euplotes raikovi.

The 3-dimensional structure of the pheromone Er-1 isolated from the ciliated protozoan Euplotes raikovi has been determined in aqueous solution by 1H NMR spectroscopy. The structure of this 40-residue protein was calculated with the distance geometry program DIANA on the basis of 503 upper distance constraints derived from nuclear Overhauser effects and 77 dihedral angle constraints derived from spin-spin coupling constants, and refined by restrained energy minimization with the program OPAL. The Er-1 solution structure is represented by a group of 20 conformers with an average RMS deviation relative to the mean structure of 0.55 A for the backbone atoms N, C alpha, and C', and 0.93 A for all heavy atoms of the complete polypeptide chain, residues 1-40. The molecular architecture is dominated by an up-down-up bundle of 3 alpha-helices formed by residues 2-9, 12-19, and 24-33. Although this core part coincides closely with the previously determined structure of the homologous pheromone Er-10, the C-terminal peptide segment adopts a novel conformation. This is of interest in view of previous suggestions, based on sequence comparisons, that this molecular region may be important for the different specificity of receptor recognition by different pheromones.     

Cold-adaptation in sea-water-borne signal proteins: sequence and NMR structure of the pheromone En-6 from the Antarctic ciliate Euplotes nobilii

Ciliates of Euplotes species constitutively secrete pleiotropic protein pheromones, which are capable to function as prototypic autocrine growth factors as well as paracrine inducers of mating processes. This paper reports the amino acid sequence and the NMR structure of the pheromone En-6 isolated from the antarctic species Euplotes nobilii. The 63-residue En-6 polypeptide chain forms three alpha-helices in positions 18-25, 36-40 and 46-56, which are arranged in an up-down-up three-helix bundle forming the edges of a distorted trigonal pyramid. The base of the pyramid is covered by the N-terminal heptadecapeptide segment, which includes a 3(10)-turn of residues 3-6. This topology is covalently anchored by four long-range disulfide bonds. Comparison with the smaller pheromones of E. raikovi, a closely related species living in temperate waters, shows that the two-pheromone families have the same three-helix bundle architecture. It then appears that cold-adaptation of the En proteins is primarily related to increased lengths of the chain-terminal peptide segments and the surface-exposed loops connecting the regular secondary structures, and to the presence of solvent-exposed clusters of negatively charged side-chains.     

Cold-adapted signal proteins: NMR structures of pheromones from the Antarctic ciliate Euplotes nobilii

Cell type-specific signal proteins, known as pheromones, are synthesized by ciliated protozoa in association with their self/nonself mating-type systems, and are utilized to control the vegetative growth and mating stages of their life cycle. In species of the most ubiquitous ciliate, Euplotes, these pheromones form families of structurally homologous molecules, which are constitutively secreted into the extracellular environment, from where they can be isolated in sufficient amounts for chemical characterization. This paper describes the NMR structures of En-1 and En-2, which are members of the cold-adapted pheromone family produced by Euplotes nobilii, a species inhabiting the freezing coastal waters of Antarctica. The structures were determined with the proteins from the natural source, using homonuclear (1)H NMR techniques in combination with automated NOESY peak picking and NOE assignment. En-1 and En-2 have highly homologous global folds, which consist of a central three-alpha-helix bundle with an up-down-up topology and a 3(10)-helical turn near the N-terminus. This fold is stabilized by four disulfide bonds and the helices are connected by bulging loops. Apparent structural specificity resides in the variable C-terminal regions of the pheromones. The NMR structures of En-1 and En-2 provide novel insights into the cold-adaptive modifications that distinguish the E. nobilii pheromone family from the closely related E. raikovi pheromone family isolated from temperate waters.     

Pheromones trigger filamentous growth in Ustilago maydis.

Cell recognition and mating in the smut fungus Ustilago maydis have been proposed to involve specific pheromones and pheromone receptors. The respective structural genes are located in the a mating type locus that exists in the alleles a1 and a2. We demonstrate that binding of pheromone to the receptor can induce a morphological switch from yeast-like to filamentous growth in certain strains. Using this as biological assay we were able to purify both the a1 and a2 pheromone. The structure of the secreted pheromones was determined to be 13 amino acids for a1 and nine amino acids for a2. Both pheromones are post-translationally modified by farnesylation and carboxyl methyl esterification of the C-terminal cysteine. An unmodified a1 peptide exhibits dramatically reduced activity. The pheromone alone is able to induce characteristic conjugation tubes in cells of opposite mating type and confers mating competence; even cells of the same mating type undergo fusion. We discuss the role of pheromones in initiating filamentous growth and pathogenic development.