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.     

Purification and characterization of new mating pheromones of the ciliate Euplotes raikovi

Cell union in mating pairs in the ciliate Euplotes raikovi is controlled by a system of multiple mating types which are inherited with alleles codominant at the genetic locus mat and expressed via diffusible mating pheromones. The mating pheromones Er-2, Er-3, and Er-11 were purified from cells homozygous for the mat-2, mat-3, and mat-11 alleles, respectively. These pheromones are proteins of similar Mr (11,000-12,000) and acidity (pI 3.7-4.0) and are active at a concentration that varies from 2.9 X 10(-12) to 1.2 X 10(-11) M. Data on amino acid composition revealed that an unusually high amount of cysteine (12-15.7%) and poor contents of basic amino acids are common to every pheromone. On the basis of this uniformity in the main biochemical traits, which also holds for the previously purified pheromone Er-1, it was concluded that E. raikovi mating pheromones are members of a family of proteins structurally diversified from each other to varying extents.     

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.     

Molecular Structures and Coding Genes of the Water‐Borne Protein Pheromones of Euplotes petzi,an Early Diverging Polar Species of Euplotes

Euplotes is diversified into dozens of widely distributed species that produce structurally homologous families of water‐borne protein pheromones governing self‐/nonself‐recognition phenomena. Structures of pheromones and pheromone coding genes have so far been studied from species lying in different positions of the Euplotes phylogenetic tree. We have now cloned the coding genes and determined the NMR molecular structure of four pheromones isolated from Euplotes petzi, a polar species which is phylogenetically distant from previously studied species and forms the deepest branching clade in the tree. The E. petzi pheromone genes have significantly shorter sequences than in other congeners, lack introns, and encode products of only 32 amino acids. Likewise, the three‐dimensional structure of the E. petzi pheromones is markedly simpler than the three‐helix up‐down‐up architecture previously determined in another polar species, Euplotes nobilii, and in a temperate‐water species, Euplotes raikovi. Although sharing the same up‐down‐up architecture, it includes only two short α‐helices that find their topological counterparts with the second and third helices of the E. raikovi and E. nobilii pheromones. The overall picture that emerges is that the evolution of Euplotes pheromones involves progressive increases in the gene sequence length and in the complexity of the three‐dimensional molecular structure.     

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.     

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.     

Evidence for Gene Duplication and Allelic Codominance (not Hierarchical Dominance) at the Mating‐Type Locus of the Ciliate,Euplotes crassus

The high‐multiple mating system of Euplotes crassus is known to be controlled by multiple alleles segregating at a single locus and manifesting relationships of hierarchical dominance, so that heterozygous cells would produce a single mating‐type substance (pheromone). In strain L‐2D, now known to be homozygous at the mating‐type locus, we previously identified two pheromones (Ec‐α and Ec‐1) characterized by significant variations in their amino acid sequences and structure of their macronuclear coding genes. In this study, pheromones and macronuclear coding genes have been analyzed in strain POR‐73 characterized by a heterozygous genotype and strong mating compatibility with L‐2D strain. It was found that POR‐73 cells contain three distinct pheromone coding genes and, accordingly, secrete three distinct pheromones. One pheromone revealed structural identity in amino acid sequence and macronuclear coding gene to the Ec‐α pheromone of L‐2D cells. The other two pheromones were shown to be new and were designated Ec‐2 and Ec‐3 to denote their structural homology with the Ec‐1 pheromone of L‐2D cells. We interpreted these results as evidence of a phenomenon of gene duplication at the E. crassus mating‐type locus, and lack of hierarchical dominance in the expression of the macronuclear pheromone genes in cells with heterozygous genotypes.     

Organization of the Euplotes crassus micronuclear genome

Euplotes crassus, like other hypotrichous ciliated protozoa, eliminates most of its micronuclear chromosomal DNA in the process of forming the small linear DNA molecules that comprise the macronuclear genome. By characterizing randomly selected lambda phage clones of E. crassus micronuclear DNA, we have determined the distribution of repetitive and unique sequences and the arrangement of macronuclear genes relative to eliminated DNA. This allows us to compare the E. crassus micronuclear genome organization to that of another distantly related hypotrichous ciliate, Oxytricha nova. The clones from E. crassus segregate into three prevalent classes: those containing primarily eliminated repetitive DNA (Class I); those containing macronuclear genes in addition to repetitive sequences (Class II); and those containing only eliminated unique sequence DNA (Class III). All of the repetitive sequences in these clones belong to the same highly abundant repetitive element family. Our results demonstrate that the sequence organization of the E. crassus and O. nova micronuclear genomes is related in that the macronuclear genes are clustered together in the micronuclear genome and the eliminated unique sequences occur in long stretches that are uninterrupted by repetitive sequences. In both organisms a single repetitive element family comprises the majority of the eliminated interspersed middle repetitive DNA and appears to be preferentially associated with the macronuclear sequence clusters. The similarities in the sequence organization in these two organisms suggest that clustering of macronuclear genes plays a role in the chromosome fragmentation process.     

Structural requirements for activity of the pheromones of Ustilago hordei

Ustilago hordei, the cause of barley-covered smut, initiates mating with pheromones. Gene sequence analysis suggested that these pheromones, Uhmfa1 and Uhmfa2, would be farnesylated peptides. Although isolation of mating-type-specific activity was rarely possible, chromatographic separations of culture supernatants yielded fractions that stimulated or inhibited mating. Based on predicted amino acid sequences and mass spectra of stimulating fractions, a series of pheromone analogs were synthesized and their activities were determined. Underivatized Uhmfa1 (PGKSGSGLGYSTC) or Uhmfa2 (EGKGEPAPYC) peptides were inactive, while peptides that were farnesylated and/or methyl esterified specifically induced conjugation tubes by cells of the opposite mating type. Uhmfa1 truncated from the amino terminus beyond the lysine lost activity, while truncated Uhmfa2 remained partially active. In mating bioassays, a pheromone concentration-dependent default mating response was observed. In competition studies, shorter Uhmfa1 peptides lacking pheromone activity inhibited activity of full-length peptides most effectively when both had the same functional groups.     

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.     

Peptide and protein pheromones in amphibians

Purification, characterization and biological activity of urodele and anuran sex-pheromones were reviewed. Female-attracting pheromones obtained from the abdominal gland of Cynops pyrrhogaster and C. ensicauda males are peptides consisting of 10 amino acid residues being designated sodefrin and silefrin, respectively. Each pheromone attracted only conspecific females. Molecular cloning of cDNAs encoding sodefrin and silefrin revealed that both are generated from precursor proteins. Synthesis of these pheromones is regulated by prolactin (PRL) and androgen. Responsiveness of the female vomeronasal epithelium to sodefrin is enhanced by PRL and estrogen. The submandibular gland of the male terrestrial salamander, Plethodon jardani secretes a 22-kD proteinaceous pheromone that enhances female receptivity. It was revealed that every salamander synthesizes multiple isoforms of this pheromone, Plethodontid receptivity factor. The magnificent tree frog, Litoria splendida breed in an aquatic environment. The skin glands of the male secrete a female-attracting peptide pheromone, splendipherin, comprising 25 amino acid residues. The significance of the structure of the amphibian sex-pheromone as peptide and protein is discussed in terms of their species specificity.     

Peptide pheromones in newts

This article reviews the current state of understanding of reproductive pheromones in amphibians, focusing mainly on the purification and characterization of peptide pheromones in newts of the genus Cynops, molecular cloning of cDNAs encoding the pheromone molecules, and hormonal control of secretion of these pheromones. Pheromones that attract sexually developed female Cynops pyrrhogaster and C. ensicauda newts were isolated from the male abdominal glands. The C. pyrrhogaster and C. ensicauda pheromones are peptides, designated sodefrin and silefrin, with the amino acid sequences SIPSKDALLK and SILSKDAQLK, respectively. Each pheromone attracts only conspecific females. Molecular cloning of cDNAs encoding sodefrin and silefrin revealed the presence of precursor proteins that are considered to generate these pheromone peptides. Pheromone precursor mRNA levels and radioimmunoassayable pheromone concentrations in the abdominal glands were elevated by prolactin and androgen. Sexual dimorphism and hormone dependency of the responsiveness of vomeronasal epithelium to sodefrin were noted. Significance of pheromones in the form of peptide for those performing reproductive behavior in an aquatic environment was also discussed.     

Characterization of the pheromone gene family of an Antarctic and Arctic protozoan ciliate, Euplotes nobilii

Allelic genes encoding water-borne signal proteins (pheromones) were amplified and sequenced from the somatic (macronuclear) sub-chromosomic genome of Antarctic and Arctic strains of the marine ciliate, Euplotes nobilii. Their open reading frames appeared to be specific for polypeptide sequences of 83 to 94 amino acids identifiable with cytoplasmic pheromone precursors (pre-pro-pheromones), requiring two proteolytic steps to remove the pre- and pro-segments and secrete the mature pheromones. Differently from most of the macronuclear genes that have so far been characterized from Euplotes and other hypotrich ciliates, the 5′ and 3′ non-coding regions of all the seven E. nobilii pheromone genes are much longer than the coding regions (621 to 700 versus 214 to 285 nucleotides), and the 5′ regions in particular show nearly identical sequences across the whole set of pheromone genes. These structural peculiarities of the non-coding regions are likely due to the presence of intron sequences and provide presumptive evidence that they are site of basic, conserved activities in the mechanism that regulates the expression of the E. nobilii pheromone genes.     

Causes and consequences of variability in peptide mating pheromones of ascomycete fungi

The reproductive genes of fungi, like those of many other organisms, are thought to diversify rapidly. This phenomenon could be associated with the formation of reproductive barriers and speciation. Ascomycetes produce two classes of mating type-specific peptide pheromones. These are required for recognition between the mating types of heterothallic species. Little is known regarding the diversity or the extent of species specificity in pheromone peptides among these fungi. We compared the putative protein-coding DNA sequences of the 2 pheromone classes from 70 species of Ascomycetes. The data set included previously described pheromones and putative pheromones identified from genomic sequences. In addition, pheromone genes from 12 Fusarium species in the Gibberella fujikuroi complex were amplified and sequenced. Pheromones were largely conserved among species in this complex and, therefore, cannot alone account for the reproductive barriers observed between these species. In contrast, pheromone peptides were highly diverse among many other Ascomycetes, with evidence for both positive diversifying selection and relaxed selective constraint. Repeats of the α-factor-like pheromone, which occur in tandem arrays of variable copy number, were found to be conserved through purifying selection and not concerted evolution. This implies that sequence specificity may be important for pheromone reception and that interspecific differences may indeed be associated with functional divergence. Our findings also suggest that frequent duplication and loss causes the tandem repeats to experience "birth-and-death" evolution, which could in fact facilitate interspecific divergence of pheromone peptide sequences.     

Structural characterization of En-1, a cold-adapted protein pheromone isolated from the Antarctic ciliate Euplotes nobilii

The second of two diffusible cell signal proteins (pheromones) purified from a wild-type strain of the Antarctic ciliate, Euplotes nobilii, has been determined by automated Edman degradation of the whole molecule and peptides generated by its chymotryptic digestion. The proposed sequence of 52 amino acids of this new pheromone, designated En-1, is: NPEDWFTPDT(10)CAYGDSNTAW(20)TTCTTPGQTC(30)YTCCSSCFDV(40)VGEQACQMSA(50)QC. In common with the previously determined 60-amino-acid sequence of the other pheromone, En-2, it bears eight cysteines in conserved positions (presumably linked into four conserved intrachain disulfide bonds), and physicochemical features of potential significance for cold adaptation, such as a reduced hydrophobicity, an increased solvent accessibility, and an improved local backbone flexibility. However, En-1 diverges from En-2 for having evolved a threonine cluster in the place of a glycine cluster to apparently make more flexible a region that is likely functionally important.     

NMR solution structure of attractin,a water-borne protein pheromone from the mollusk Aplysia californica

Water-borne protein pheromones are essential for coordination of reproductive activities in many marine organisms. In this paper, we describe the first structure of a pheromone protein from a marine organism, that of attractin (58 residues) from Aplysia californica. The NMR solution structure was determined from TOCSY, NOESY, and DQF-COSY measurements of recombinant attractin expressed in insect cells. The sequential resonance assignments were done with standard manual procedures. Approximately 90% of the 949 unambiguous NOESY cross-peaks were assigned automatically with simultaneous three-dimensional structure calculation using our NOAH/DIAMOD/FANTOM program suite. The final bundle of energy-refined structures is well-defined, with an average rmsd value to the mean structure of 0.72 +/- 0.12 A for backbone and 1.32 +/- 0.11 A for heavy atoms for amino acids 3-47. Attractin contains two antiparallel helices, made up of residues Ile9-Gln16 and I30-S36. The NMR distance constraints are consistent with the three disulfide bonds determined by mass spectroscopy (C4-C41, C13-C33, and C20-C26), where the first two could be directly determined from NOESY cross-peaks between CH beta protons of the corresponding cysteines. The second helix contains the (L/I)(29)IEECKTS(36) sequence conserved in attractins from five species of Aplysia that could interact with the receptor. The sequence and structure of this region are similar to those of the recognition helix of the Er-11 pheromone of the unicellular ciliate Euplotes raikovi, suggesting a possible common pathway for intercellular communication of these two distinct pheromone families.     

Quantitative Analyses of Interbreeding in Populations of vannus-Morphotype Euplotes, with Special Attention to the Nominal Species E. vannus and E. crassus

ABSTRACT. Classical genetic techniques were applied to clonal cultures of the Euplotes vannus-crassus-minuta sibling species complex in an effort to provide some resolution to the species problem among these hypotrichs. Complex mating interactions were observed among clonal stock cultures derived from samples collected from sympatric and allopatric populations in a wide geographic survey. These results suggested that the classical model for the mating type inheritance and determination in these Euplotes is necessary but not sufficient to describe the mating mating interactions among populations of these ciliates. Successful conjugation between the nominal species E. crassus and E. vannus was observed routinely, and crosses between these two nominal species did not differ significantly from those among the other clonal stock cultures with respect to mating intensity and exconjugant survival. Data from backcrosses suggests that E. vannus and E. crassus can and do exchange genes. Based upon these data, we conclude that E. vannus and E. crassus comprise a single, highly polymorphic species with countless small populations, among which incomplete genetic exchange takes place.     

Variation in mate-recognition pheromones of the fungal genus Microbotryum

Mate recognition is an essential life-cycle stage that exhibits strong conservation in function, whereas diversification of mating signals can contribute directly to the integrity of species boundaries through assortative mating. Fungi are simple models, where compatibility is based on the recognition of pheromone peptides by corresponding receptor proteins, but clear patterns of diversification have not emerged from the species examined, which are few compared with mate signaling studies in plant and animal systems. In this study, candidate loci from Microbotryum species were used to characterize putative pheromones that were synthesized and found to be functional across multiple species in triggering a mating response in vitro. There is no significant correlation between the strength of a species'' response and its genetic distance from the pheromone sequence source genome. Instead, evidence suggests that species may be strong or weak responders, influenced by environmental conditions or developmental differences. Gene sequence comparisons reveals very strong purifying selection on the a₁ pheromone peptide and corresponding receptor, but significantly less purifying selection on the a₂ pheromone peptide that corresponds with more variation across species in the receptor. This represents an exceptional case of a reciprocally interacting mate-recognition system in which the two mating types are under different levels of purifying selection.