Ciliate
pheromones: primordial self/non-self recognition signals
Running title: Ciliate pheromones
Adriana Vallesi,
Claudio Alimenti, Pierangelo Luporini
Laboratory of Eukaryotic Microbiology and Animal
Biology, School of Biosciences and Veterinary Medicine, University of Camerino,
62032 Camerino (MC) Italy
E-mail:
Summary
As is common
among multi-cellular life forms, also single-cell organisms use pheromones to
communicate among members of the same species. In protist ciliates, pheromones
have been identified in functional association with mating systems. Consistently
with this association, they have for long time been regarded only as non-self
signals committed to elicit a mating response of cells to which they bind in
paracrine-like (heterologous) fashion. However, their spectrum of activity has
revealed wider borders. It also includes a self activity which promotes the
vegetative growth of the same cells from which pheromones are secreted, and to
which they continuously bind in autocrine fashion. This double self and
non-self activity is made possible by the pheromone ability to compete with one
another in cell binding reactions. In Euplotes species, which synthesize
pheromone families under the control of multiple series of alleles at a single
locus, this ability is ensured by the relationships of the structural homology
that link these molecules into species-specific globular, disulfide-rich
protein families having a common three-helix fold.
Key Words
Diffusible
signaling proteins; globular helical proteins; cell-cell communication;
protein-protein interactions; ciliate mating and growth
Introduction
Independently of their prokaryotic or eukaryotic nature, cells are
evolutionarily driven to socialize to acquire the temporary or stable multi-cellular
organization necessary to increase their size and engage in functional
specialization and partition of labor (1). This ancient impulse to socialize implicates
a very early evolution of the cell’s ability to discriminate between self and
non-self, which is a sine qua non condition for pursuing effective cell-cell communication,
cooperation and structure-function integration. In its most basic aspects, this
ability is primarily directed to the recognition of self signaling molecules, as
exemplified by the quorum sensing phenomena in which bacteria perceive their
environmental population densities and activities and, consistently, vary their
behavior and metabolism (2, 3). It then undergoes vast functional
diversification among the unicellular eukaryotes in which it governs impressive
phenomena of cell aggregation based on kin discrimination in social amoebas (4,
5). Its maximal complexity is eventually shown by animals with the evolution of
innate and adaptive immunity systems that are primarily directed to recognizing
and processing non-self molecules for the defense of body integrity (6, 7).
This chapter focuses attention on the biology and
structure of diffusible protein signals, nowadays known as pheromones and
earlier as mating-type factors/substances or gamones, that control
self/non-self recognition in protist ciliates. Together with Dinoflagellates
and Apicomplexa, ciliates (Ciliophora) form the group of Alveolata which
clusters with those of Stramenopiles (diatoms) and Rhizaria (foraminifers and
radiolarians) into the so called SAR supergroup (8).
Pheromone identification
Ciliate pheromones are the chemical markers of genetically distinct
vegetative cell classes—only two like sexes in some species, or multiple with
indefinite numbers in others (9, 10)—that have been described as mating types
because their mixing determines a ciliate-specific mating phenomenon of
conjugation. Ciliate conjugation is a sexual phenomenon as it involves a
temporary union in mating pairs between cells which mutually exchange
gamete-nuclei, undergo fertilization, and develop new micro- and macronuclei
from the synkarya. However, it has nothing to do with general phenomena of
fertilization between gametes of opposite sexes. One major reason for opposing
this parallelism is that many species of ciliates may form mating pairs between
genetically different cells (heterotypic pairs), as well as between genetically
alike (clonal) cells (homotypic mating pairs). Although destined to generate
self-fertilization, these homotypic pairs are fully fertile (the Blepharisma case excepted) just like the
heterotypic ones.
It is precisely by virtue of this unique capability that
ciliates have to form homotypic (intraclonal, or selfing) mating pairs that ciliate
pheromones were identified more than half a century ago (11). By investigating mating
interactions in Euplotes patella—the first Euplotes species used to study the Mendelian genetics of ciliate multiple
mating systems controlled by series of alleles co-dominantly expressed at a
single genetic locus (annotated as mat locus)--Kimball (11) observed that an experimental condition sufficient to
induce the formation of homotypic mating pairs was the simple suspension of
cell cultures with cell-free filtrates from other cultures of different mating
types. From this observation it became evident that in E. patella, and ciliates in general, the mating-type factors (pheromones) can be freely released into
the extracellular environment and that the presence of these molecules in
solution can be promptly detected by assaying the mating induction activity of
cell-free filtrates.
By applying this
mating-induction assay to cell-free filtrates, pheromones have been identified
in species of Blepharisma, Dileptus, Oxytricha and Tokophrya besides
that in other Euplotes species (12,
13, as reviews). However, studies of structure-function characterization have
been carried out to varying degrees of complexity only on pheromones isolated
from B. japonicum, E. raikovi, E. octocarinatus, E. nobilii and E. crassus. These studies are
reviewed in this chapter with particular attention to the cross-reactions that
some Euplotes pheromones show with the
signaling system components of more modern organisms.
Pheromone
structures
The first two pheromones to be purified and
structurally characterized belong to the pink-colored freshwater ciliate, B. japonicum. Although they have been
reported as being distinctive of two mating types (I and II) defined as
“complementary”, of which nothing is known about their genetic determination, their
molecular structures are chemically unrelated. The pheromone isolated from the
type-I cells (originally designated as Gamone 1, or Blepharmone) is an unstable
glycoprotein of 272 amino acids and six N-linked
sugars (14). It is particularly rich in polar and aromatic amino acids (Asn
10.3%, Ser 10.3%, Tyr 8.1%, Trp 5.1%), synthesized in the form of a cytoplasmic
precursor, and active in inducing mating between type II cells at picogram
concentrations. In contrast, the second pheromone distinctive of type-II cells (also
designated as Gamone 2, or Blepharismone) is a very stable tryptophan
derivative, namely a calcium-3-(2’-formyl-amino-5’-hydroxybenzoil) lactate (15).
This has also been obtained by chemical synthesis and is active at nano-molar
concentrations in attracting type-I cells besides that for inducing their mate
(16).
Quite different is the picture that emerges for the pheromone
structures determined in four species of Euplotes,
namely E. raikovi, E. nobilii, E. octocarinatus and E.
crassus. In all these species, pheromones have been shown to form species-specific
families of structurally homologous proteins. Among these pheromone families, a
more in-depth structural knowledge has been obtained for E. raikovi and E. nobilii,
which secrete comparatively larger amounts of pheromones. Up to 200 mg of pure protein/liter can be recovered from E. raikovi cell-free filtrates (17).
This has first greatly facilitated chemical and genetic approaches to the
determination of significant numbers of pheromone primary amino acid sequences.
It subsequently opened the way to NMR and crystallographic analyses of the
pheromone three-dimensional structures based on the use of proteins at natural
isotope composition purified from cell-free filtrate preparations.
In E. raikovi,
nine distinct amino acid pheromone sequences in their form of cytoplasmic
precursors (pre-pro-pheromones) and six three-dimensional structures of the
secreted molecules are known (18_24) (Fig. 1). In contrast to the
tight structural conservation of the strongly hydrophobic pre segment (or signal
peptide) of 19 amino acids and (to a slightly lesser extent) of the pro segment
of 16_18 amino acids, the secreted pheromones of 37_40
amino acids (51 only in pheromone Er-23)
show minimal sequence conservation. Only the positions of six cysteines are fully
conserved and the sequence dissimilarity between any two pheromones may be as high
as 74%. In any case, despite this wide variability in amino acid sequence all
the pheromones (pheromone Er-23 in
part excepted) mimic one another in their three-dimensional conformation. This
is based on a fold of three nearly parallel, right-handed a helices (increased to five with the addition of two single-turn
helices in Er-23), that remain tightly
associated with each other by the close proximity and strategic disposition of
their disulfide bonds. As is usually required for water-borne signaling
molecules that must maintain long-term activity in the environment, the stability
of the globular conformation of E.
raikovi pheromones is such that even heating these proteins up to 95 °C
does not cause unfolding and disruption of their secondary structures (25). However,
in spite of showing closely comparable molecular architectures and equivalent stabilities,
each pheromone has its own unmistakable cell-type structural earmarks that are
likely deputed to confer specificity to its signaling activity. These earmarks are
imposed by the evolution of variations that are most apparent for the shape,
extension and spatial arrangement of the carboxy-terminal domain, as well as for the geometry of the inter-helix
loops of the molecules.
The overall structural
organization of E. nobilii pheromones
strictly matches the E. raikovi pheromone organization, consistently with the close phylogenetic kinship that
links these two species (26_27). Nevertheless, E. nobilii pheromones, for which seven amino acid sequences of the
precursor forms (28) and four NMR structures have been determined (29_31)
(Fig. 2), have longer sequences (from 52 to 63 amino acids), include eight
instead of six cysteines and, more important, show structural specificities that
appear closely correlated with the different ecology that distinguishes E. nobilii from E. raikovi. While E. raikovi thrives in temperate waters, E. nobilii has colonized the Antarctic and Arctic seas (32_33). Hence, E. nobilii pheromones are cold-adapted, psychrophilic
proteins. With respect to E. raikovi pheromones, their overall contents of polar and hydrophobic amino acids is markedly
different, most likely in functional correlation with their improved
interactions with the solvent (34). They are much richer in Thr (11.7% vs 5.7%),
Asn (7.7% vs 4.2%), and Ser (8.6% vs 5.7%) residues, and poorer in Leu (1.1% vs
7.3%), Pro (4.7% vs 8.9%) and Ile (1.6% vs 5.7%). Structurally more apparent
are however the reduced extensions that E.
nobilii pheromones show for the three helices of the molecular architecture
with respect to E. raikovi pheromones. These reductions determine a spatial preponderance of regions
devoid of a regular secondary organization, implying that the activity of E. nobilii pheromones in the
thermodynamically adverse environment of the polar waters is facilitated by an
improved flexibility of their molecular backbones. Indeed, E. nobilii pheromones have been measured to be much less thermo-stable
than E. rakovi pheromones, unfolding
upon heating to only 55_70 °C (25_35).
Much less information is available on the structures
of E. octocarinatus and E. crassus pheromones for which only the
amino acid sequences are known on the basis of their respective DNA coding
sequences. The determination of three-dimensional conformations of these
pheromones has been made impractical by the very low concentrations in which they
are secreted; 0.5 mg of pure protein/liter of cell-free filtrate are usually obtained in E. ocrocarinatus and 15_20 mg in E. crassus (36_37).
Nine distinct pheromone
amino acid sequences have been determined in E. octocarinatus (Fig. 3). They are twice as long as the E. raikovi and E. nobilii pheromone sequences, extending from 85 to 109 amino
acids with ten cysteines (eight in the pheromone Phr4) (38_39). The
positions of only four of the ten cysteines have counterparts in E. raikovi and E. nobilii pheromones, implying that the E. octocarinatus pheromones have only two disulfide bonds and few
structural motifs in common with those of E.
raikovi and E. nobilii. They also
appear to be unique among Euplotes pheromones
in functioning as chemo-attractants between cells of different mating types (40).
In E. crassus,
pheromones have been structurally characterized only recently (37, 41). They were
a long time thought to be represented by insoluble membrane-bound proteins, and
as such difficult to extract and characterize from cell-membrane preparations. Evidence
that E. crassus pheromones are,
instead, constitutively secreted into the extracellular environment as in other Euplotes species was obtained from
inter-specific mating induction assays (37), suggested by the notion that cross-mating
reactions are relatively frequent between Euplotes species in correlation with their high-multiple (virtually open) mating systems
(9, 42). The genetic control of these E.
crassus pheromones appears not to conform with the general Euplotes pattern provided by multiple series
of alleles at a single mat locus. Results
from molecular approaches have been found to be more consistently explained by assuming
that this control involves a phenomenon of mat gene duplication responsible for the production of two distinct E. crassus pheromone sub-families. One includes
the cell type-specific pheromones, and the other includes a new pheromone species
that appears to be synthesized in common among cells of compatible mating types
(37, 41). The functions of this ‘shared’ pheromone, of which only a single
56-amino acid sequence with eight cysteines (designated Ec-a)
has been determined, are still obscure. Preliminary evidence suggests that it
might behave like an adaptor, or scaffold protein that selectively interacts
with the cell type-specific pheromones to mediate and/or reinforce their
receptor binding reactions (41). This possibility is supported by an unusually
strong propensity of the Ec-a pheromone to oligomerize
when it is purified by chromatographic separation, and is reinforced by the comparison
of its sequence with the three 45-amino acid sequences with ten cysteines (designated
Ec-1, Ec-2 and Ec-3) that have been
determined for the sub-family of the cell type-specific pheromones (Fig. 4). This
comparison makes it evident that a
major distinctive structural trait of the Ec-a pheromone is the evolution
of an unusually extended hydrophilic domain lying centrally in the molecule.
This domain lacks Cys residues, abounds in Gly residues and likely takes a random-coil
conformation which is presumptive for a specific capability of the Ec-a pheromone to interact with the other proteins.
Pheromone activity
As outlined above, it is
essentially due to the application of mating induction assays that ciliate
pheromones have been identified. This application has thus decisively
contributed to support the generalization that ciliate pheromones function
exclusively as “sexual factors” committed to stimulate cell mating by binding
in paracrine (or heterologous) fashion only to cells other than those from
which they are synthesized and secreted (12). However, observations on
the physiology of pheromone secretion in E. raikovi turned out
to counter this view, because cells were shown to secrete their pheromones
throughout their life cycle independently of their incompetence to mate during
the immaturity stage, to vary their rates of pheromone secretion in relation to
the environmental concentrations of their pheromone, as well as to refrain from
forming homotypic mating pairs in the presence of environmental excess of their
pheromone (43). A more coherent view with these observations thus
appeared to be one predicting that ciliate pheromones, in addition to acting as
non-self mating signals, also (and most likely, primarily) act as self signals
committed to promoting the vegetative proliferation (mitotic growth) of their
same source cells to which they bind in autocrine (or autologous)
fashion (43, 44).
A decisive
support for an autocrine, cell growth-promoting activity of ciliate pheromones
was provided by the finding that E. raikovi cells bind their
pheromones to membrane-bound isoforms that arise from a splicing mechanism of
intron-like sequences of the same genes which encode the pheromones in the
transcriptionally active cell macronucleus (45, 46). These isoforms
(functionally regarded as pheromone-receptors) incorporate the full sequence of
the cytoplasmic pheromone precursor and, using the signal (pre) peptide
(normally expected to be proteolytically removed before the pheromone
secretion) as trans-membrane anchoring domain, take a spatial orientation
typical of membrane proteins of type-II which have the C-terminal end directed
to the cell outside and the N-terminal end directed to the cell inside. Given
this structural equivalence between pheromones and extracellular binding
domains of the twin receptor molecules, the way pheromones interact with their
receptors on the cell surface has been thought to be fully mimicked
by the protein-protein interactions that determine the molecular packing of the E.
raikovi pheromone Er-1 into crystals (22). In the
bi-dimensional plane of the crystal, the Er-1 molecules (that may be figured
as pyramids with a triangular base) associate with one another by means of
all three of their faces (a, b and c,
delimited by helices 1 and 2, 2 and 3, and 1 and 3, respectively), through the
cooperative utilization of initially weak interactions that arise from the
formation of two distinct types of dimer. One of these involves face a of
each monomer, and is symmetrical with the two monomers related by a twofold
axis; the other involves faces b and c, and is
asymmetrical with the two monomers related by a twofold screw axis (Fig.
5). This association is such that half of the molecules direct
their carboxy-terminus to one side of the plane, and may thus reflect the
spatial orientation of the pheromone-receptors on the cell surface. The second
half of the molecules, instead, direct their amino-terminus to the opposite
side of the plane and may thus be regarded as free pheromone molecules that
bind to their receptors. In line with a cooperative model of
protein-protein interactions, the homotypic pheromone/pheromone-receptor
complexes formed by cells which grow suspended with their own (self) pheromone
have been observed to undergo clustering and internalization via endocytotic
vesicles, whereas the heterotypic complexes formed by cells that have been
suspended with a non-self pheromone for being induced to mate are not
internalized and believed to be in some way involved in bridging cells
into mating pairs (47).
Pheromone
structure/function relationships with other signaling proteins
Evolutionary relationships of proteins are commonly inferred using traditional
automatic sequence/structure comparison methods such as PSI-BLAST and Dali. The
reliability of these methods is however generally regarded as sound only for
globular proteins that are larger than 100 residues, which is not the case of Euplotes pheromones characterized by
quite short polypeptide chains. To assess small globular proteins for their
evolutionary relationships, an alternative method has been thought to be provided
by a comparative analysis of the variety of structural folds that functionally
diverse proteins (encompassing growth factors, toxins, enzyme inhibitors and
others) have adopted for their small (an average of 57 ± 29 residues) disulfide-rich
(an average of 3 ± 1) domains which are either individual or included within
larger polypeptide chains, and characterized by global folds that are
stabilized primarily by disulfide bonds and, in second place, by secondary
structure elements and hydrophobic interactions (48). In this analysis and with
the exception of pheromone Er-23 that
presents an its unique five-helix globular array, Euplotes pheromones have been classified together with
disulfide-rich domains of other four protein families represented by
anaphylotoxins (C5a), oncogene-encoded proteins (p8MTCPI), sea
anemone toxins K, and cysteine-rich secretory proteins (CRISP) within a fold
group identified by a disulfide-bonded three-helix bundle with right-handed
connections between the a-helices. While classified within the same fold group, these protein
families however show quite disparate functions and mechanisms of action with
respect to Euplotes pheromones, and lacking
a functional analogy their structural relationships with Euplotes pheromones thus appear to be better interpreted as a
result of phenomena of convergent evolution rather than of homology.
More significant
structure/function relationships have been observed between the E. raikovi pheromone family and another
family of long-distance water-borne protein
pheromones that various species of the common marine mollusk Aplysia freely release from their yolk gland into the environment, and use in phenomena
of intra- and inter-specific attraction of sexually mature individuals towards their
egg cordons (49). In their active form, these 56_58 amino acid proteins,
designated as attractins in relation to their activity, have a compact
folded structure (annotated as “a-Hairpin” in the above classification of protein fold groups) that is stabilized by three tightly conserved disulfide bonds and dominated by two anti-parallel helices, the second of which is essentially formed by the
heptapeptide Ile-Glu-Glu-Cys-Lys-Thr-Ser (50). This building block is common to
all attractins of the different species of Aplysia (which well accounts for attractin inter-specific attractiveness), and finds a
close counterpart in the third helix of E. raikovi pheromones in terms of both the
conformation of its helical backbone and orientation of its side chains. In
addition, it is likely directly involved in receptor-binding interactions, as is
shown to be the case for the third helix of E.
raikovi pheromones, since the attractin activity is abolished by mutating the
charged residues that are exposed on its surface (51).
A
last intriguing point on the relationships of Euplotes pheromones with other signaling proteins has been quite
fortuitously revealed by experiments in which some prototypic animal growth
factors and cytokines were used to competitively inhibit the autocrine cell
binding interactions of the E. raikovi pheromone Er-1 (52). Human
interleukin-2 (hIL-2) and, to a lesser extent, epidermal growth factor were surprisingly
observed to exert strong inhibitory activity. At a 50-fold molar excess, hIL-2 appeared
able to inhibit almost completely the pheromone binding to its target cell
surface receptors. Considering the disparity in molecular dimensions that separates
hIL-2 and Er-1 and precludes reliable
comparisons between the two protein families, a possible explanation for this
hIL-2 competitive activity has tentatively been identified with some degree of the
sequence similarity that hIL-2 and Er-1
bear at the level of two their sequence segments, one extended for five amino
acids (Leu56-Gln-Cys-Leu-Glu60 in hIL-2 and Iso8-Gln-Cys-Val-Glu12 in Er-1) and the other for seven (Thr102-Phe-Met-Cys-Glu-Tyr-Ala108 in hIL-2 and Arg25-Thr-Gly-Cys-Tyr-Met-Tyr31 in Er-1). The Cys residue included in each of
the two segments is involved in the formation of a disulfide bond known to be
essential for the hIL-2 biological activity (53), and the shorter segment represents
a substantially conserved motif within both the hIL-2 and Er-1 protein families in addition to being well exposed on the
molecular surface.
Cross-reactions
between the IL-2 and E. raikovi pheromone systems have supported a possible functional significance of these
common sequence motifs. It has initially been shown (54) that Er-1 binds specifically to the IL-2
receptor on the surface of CTLL-2 cells, that are a mouse T lymphocyte line totally
dependent on IL-2 for survival and proliferation. The binding involves the a and b subunits of the trimeric IL-2 receptor in particular,
and occurs with an appreciable higher affinity to the a subunit that has a leading role in the ligand binding
activity of the IL-2 receptor being the first subunit that interacts with
extracellular IL-2. More recently it has been observed that the pheromone Er-1 also exerts strong
effects on some cellular functions that IL-2 regulates in the human lymphoid T cells of the Jurkat line (55). These cells, suspended in a low-serum containing medium enriched with the addition of
Er-1 nano-molar concentrations, were initially reported to significantly increase
their proliferation and secretion
rates, beside that of IL-2, of a wide spectrum of cytokines including IFN-γ,
TNF-α, IL-1β and IL-13 (Fig. 6). They were later observed to undergo a significant decrease in the expression of the a-subunit of the IL-2 receptor due to the reversible and time-dependent endocytosis of this receptor, and activate the
transduction pathway that is regulated by the protein kinases Erk1 and Erk2.
Conclusions
Significant numbers of molecular structures of ciliate
pheromones have been determined from distinct species of Euplotes. This determination has made manifest that these water-borne
signaling molecules form species-specific families of small, disulfide-rich
globular proteins in full accord with their genetic determination at a single multi-allelic
locus. Within each family, the global pheromone structure has been shown to acquire
virtually unlimited polymorphisms from variations in shape and geometry that interest
individual structural elements. The varying degrees of intra- and
inter-specific relationships of structural homology that link Euplotes pheromones to one another
closely recall the relationships of homology which exist within and between families
of signal proteins of higher life forms. In these families, each component can
compete and cross-react with its family members to elicit multiple and
context-dependent cell responses. Similarly, Euplotes pheromones have been shown to be fully able to bind cells
in mutual competition (56), and to carry out a spectrum of activities that is
not exclusively limited to eliciting cell mating as hypothesized for the
eccentric case of the structurally unrelated glycoprotein and
tryptophan-derivative pheromones of B.
japonicum (12). In addition to including a paracrine-like (non-self)
activity, this spectrum also includes an autocrine (self) activity which is
probably primary and directed to promoting the vegetative proliferation of the
same cells from which pheromones are constitutively synthesized and secreted
throughout the life cycle (44). The similarities of structure that some Euplotes pheromones have revealed with Aplysia attractins and the mammalian
cytokine IL-2 most likely represent fortuitous instances of convergent
molecular evolution. Nevertheless, they provide evidence that functionally
important sequence motifs may be evolutionarily conserved from ancient to more
modern cell signaling proteins, and stimulate investigations on their potential
into biotechnological and finalized perspectives.
References
1. Grosberg RK, Strathmann RR. The evolution of multicellularity:
a minor major transition? Ann Rev Ecol
Evol S 2007; 38:621–54.
2. Waters CM,
Bassler BL. Quorum sensing: cell-to-cell communication in bacteria. Ann Rev Cell Dev Biol 2005; 21:319–46.
3. Gibbs KA, Urbanowski ML, Greenberg EP. Genetic determinants of self
identity and social recognition in bacteria. Science 2008; 321:256_9.
4. Benabentos R,
Hirose S, Sucgang R, Curk T, Katoh M, Ostrowski EA, et al. Polymorphic members
of the lag gene family mediate kin
discrimination in Dyctiostelium. Curr Biol 2009; 19:567–72.
5. Hirose S,
Benabentos R, Ho H-I., Kuspa A, Schaulsky G. Self recognition in social amoebae
is mediated by allelic pairs of Tiger genes. Science 2011; 333:467–70.
6. Cooper MD, Alder MN. The evolution of adaptive immune
systems. Cell 2006; 124:815–22.
7. Rosengarten RD, Nicotra ML. Model systems of
invertebrate allorecognition. Curr Biol 2011; 21:R82–R92.
8. Adl SM, Simpson AGB, Lane CE, Lukes J, Bass D, Bowser
SS, et al. The revised classification
of eukaryotes. J Eukaryot Microbiol 2012;
59:429–93.
9. Valbonesi A, Ortenzi C, Luporini P. The species
problem in a ciliate with a high-multiple mating type system, Euplotes crassus. J Protozool 1992; 39:45–54.
10. Phadke SS,
Zufall RA. Rapid diversification of mating systems in ciliates. Biol J Linnean Soc 2009; 98:187–97.
11. Kimball RF. The nature and inheritance of mating types
in Euplotes patella. Genetics 1942; 27:269–85.
12. Miyake A 1996. Fertilization and sexuality in
ciliates. In: Hausmann K, Bradbury PC, editors. Ciliates, cells as organisms. Stuttgart: Gustav Fisher; 1996, p.
243–90.
13. Luporini P,
Alimenti C, Vallesi A. Ciliate pheromone structures and activity: a review. It J Zool 2015; 82:3–15.
14. Sugiura M, Harumoto T. Identification,
characterization, and complete amino acid sequence of the conjugation-inducing
glycoprotein (blepharmone) in the ciliate Blepharisma
japonicum. Proc Natl Acad Sci USA 2001; 98:14446–51.
15. Kubota T, Tokoroyama T, Tsukuda J, Koyama H, Miyake A.
Isolation and structure determination of blepharismin, a conjugation initiating
gamone in the ciliate Blepharisma. Science 1973; 179:400–2.
16. Sugiura M, Shiotani H, Suzaki T, Harumoto T.
Behavioural changes induced by the conjugation-inducing pheromones, gamones 1
and 2, in the ciliate Blepharisma
japonicum. Eur J Protistol 2010;
46:143–9.
17. Raffioni S, Miceli C, Vallesi A, Chowdhury SK, Chait BT,
Luporini P, Bradshaw RA. Primary structures of Euplotes raikovi pheromones from cells with variable mating
interactions. Proc Natl Acad Sci USA 1992; 89:2071–5.
18. Brown
LR, Mronga S, Bradshaw RA, Ortenzi C, Luporini P, Wüthrich K. Nuclear magnetic
resonance solution structure of the pheromone Er-10 from the ciliated protozoan Euplotes raikovi. J Mol Biol 1993; 231:800–16.
19. Mronga S, Luginbühl P, Brown LR, Ortenzi C, Luporini
P, Bradshaw RA, Wüthrich K. The NMR solution structure of the pheromone Er-1 from the ciliated protozoan Euplotes raikovi. Prot Sci 1994; 3:1527–36.
20. Ottiger M, Szyperski T, Luginbühl P, Ortenzi C,
Luporini P, Bradshaw RA, Wüthrich K. The NMR solution structure of the
pheromone Er-2 from the ciliated
protozoan Euplotes raikovi. Prot Sci 1994; 3:1515–26.
21. Luginbühl P, Ottiger M, Mronga S, Wüthrich K.
Structure comparison of the NMR structures of the pheromones Er-1, Er-10, and Er-2 from Euplotes raikovi. Prot Sci 1994; 3:1537–46.
22. Weiss MS, Anderson DH, Raffioni S, Bradshaw RA,
Ortenzi C, Luporini P, Eisenberg D. A cooperative model for ligand recognition
and cell adhesion: evidence from the molecular packing in the 1.6 Å crystal
structure of the pheromone Er-1 from
the ciliate protozoan Euplotes raikovi. Proc
Natl Acad Sci USA 1995; 92:10172–6.
23. Liu A, Luginbühl P, Zerbe O, Ortenzi C, Luporini P,
Wüthrich K. NMR structure of
the pheromone Er-22 from Euplotes raikovi. J Biomol NMR 2001; 19:75–8.
24. Zahn R, Damberger F, Ortenzi C, Luporini P, Wüthrich
K. NMR structure of the Euplotes raikovi pheromone Er-23 and identification of its five
disulfide bonds. J Mol Biol 2001; 313:923–31.
25. Geralt M, Alimenti C, Vallesi A, Luporini P, Wüthrich K. Thermodynamic stability of
psychrophilic and mesophilic pheromones of the protozoan ciliate Euplotes. Biology 2013; 2:142–50.
26. Jiang J, Zhang Q, Warren A,
Al-Rasheid KAS, Song S. Morphology and SSU rRNA gene based phylogeny of two
marine Euplotes species, E. orientalis spec. nov. and E. raikovi Agamaliev, 1966 (Ciliophora,
Euplotida). Eur J Protistol 2010; 46:121–32.
27. Di Giuseppe G, Erra F, Frontini FP, Dini F, Vallesi A, Luporini P. Improved description of the bipolar ciliate, Euplotes petzi, and definition of its
basal position in the Euplotes phylogenetic
tree. Eur J Protistol 2014;
50:402–11
28. Vallesi A, Alimenti C, Pedrini B, Di Giuseppe G, Dini F,
Wüthrich K, Luporini P. Coding genes and molecular structures of the diffusible
signalling proteins (pheromones) of the polar ciliate, Euplotes nobilii. Mar Genomics 2012; 8:9–13.
29. Pedrini B,
Placzek WJ, Koculi E, Alimenti C, La Terza A, Luporini P, Wuthrich K.
Cold-adaptation in sea-water-borne signal proteins: sequence and NMR structure
of the pheromone En-6 from the
Antarctic ciliate Euplotes nobilii. J Mol Biol 2007; 372:277–86.
30. Placzek WJ,
Etezady-Esfarjani T, Herrmann T, Pedrini B, Peti W, Alimenti C, Luporini P,
Wuthrich K. Cold-adapted signal proteins: NMR structures from the Antarctic
ciliate Euplotes nobilii. IUBMB Life 2007; 59:578–85.
31. Di Giuseppe G, Erra F, Dini F, Alimenti C,
Vallesi A, Pedrini B, Wüthrich K, Luporini P. Antarctic and Arctic populations of the ciliate Euplotes nobilii show common pheromone-mediated cell-cell signaling
and cross-mating. Proc Natl Acad Sci USA 2011; 108:3181–6.
32. Valbonesi
A, Luporini P. Description of two new species of Euplotes and Euplotes
rariseta from Antarctica. Polar Biol 1990; 11:47–53.
33. Di Giuseppe G, Barbieri
M, Vallesi A, Luporini P, Dini F. Phylogeographical pattern of Euplotes nobilii, a protist ciliate with
a bipolar biogeographical distribution. Mol
Ecol 2013; 22:4029–37.
34. Alimenti C, Vallesi A, Pedrini B, Wüthrich
K, Luporini P. Molecular cold-adaptation: comparative analysis of two homologous families of psychrophilic and
mesophilic signal proteins of the protozoan ciliate, Euplotes. IUBMB Life 2009; 61:838–45.
35. Cazzolli G, Skrbic T, Guella G, Faccioli P. Unfolding
thermodynamics of cysteine-rich proteins and molecular thermal-adaptation of
marine ciliates. Biomolecules 2013;
3:967_85.
36. Schulze-Dieckhoff H, Freiburg M, Heckmann K. The
isolation of gamone 3 and 4 of Euplotes
octocarinatus. Eur J Biochem 1987; 168:89–94.
37. Alimenti C, Vallesi A, Federici S, Di Giuseppe G, Dini F,
Carratore V, Luporini P. Isolation and structural characterization of two
water-borne pheromones from Euplotes
crassus, a ciliate commonly known to carry membrane-bound pheromones. J Eukaryot Microbiol 2011; 58:234–41.
38. Brünen-Nieveler C, Weiligmann JC, Hansen B, Kuhlmann
HW, Möllenbeck M, Heckmann K. The pheromones and pheromone genes of new stocks
of the Euplotes octocarinatus species complex. Eur
J Protistol 1998; 34:124–32.
39. Möllenbeck M, Heckmann K. Characterization of two
genes encoding a fifth so far unknown pheromone of Euplotes octocarinatus. Eur J
Protistol 1999; 35:225–30.
40. Kuhlmann HW, Brünen-Nieveler C, Heckmann K. Pheromones of the ciliate Euplotes octocarinatus not only induce
conjugation but also function as chemoattractants. J Exp Zool 1997; 277:38–48.
41. Vallesi A, Alimenti C, Federici S, Di Giuseppe G, Dini
F, Guella G, Luporini P. Evidence
for gene duplication and allelic
codominance (not hierarchical dominance) at
the mating type locus of the ciliate, Euplotes crassus. J Eukaryot Microbiol 2014; 61:620–9.
42. Nobili R,
Luporini P, Dini F. Breeding system, species relationships and evolutionary
trends in some marine species of Euplotidae (Ciliata Hypotrichida). In:
Battaglia B, Beardmore J, editors. Marine
Organisms: Genetics, Ecology and Evolution. New York: Plenum Press; 1978,
p. 591–616.
43. Luporini P, Miceli C. Mating pheromones. In: Gall JG, editor. The Molecular Biology of Ciliated Protozoa. New York: Academic
Press; 1986, p.
263–99.
44. Vallesi A, Giuli G, Bradshaw RA, Luporini P. Autocrine
mitogenic activity of pheromones produced by the protozoan ciliate Euplotes raikovi. Nature 1995; 376:522–4.
45. Miceli C, La Terza A, Bradshaw RA, Luporini P. Identification and structural characterization of a
cDNA clone encoding a membrane-bound form of the polypeptide pheromone Er-1 in the ciliate protozoan Euplotes raikovi. Proc Natl Acad Sci USA 1992; 89:1988–92.
46. Ortenzi C, Alimenti C, Vallesi A, Di Pretoro B, La Terza
A, Luporini P. The autocrine mitogenic loop of the ciliate Euplotes raikovi: the pheromone membrane-bound forms are the cell
binding sites and potential signaling receptors of soluble pheromones. Mol Biol Cell 2000; 11:1445–55.
47. Luporini P, Alimenti C, Ortenzi C, Vallesi A. Ciliate mating types and their specific protein
pheromones. Acta Protozool 2005; 44:89–101.
48. Cheek S, Krisna SS, Grishin NV.
Structural classification of small, disulfide-rich protein domains. J Mol Biol 2006; 359:215–37.
49. Painter SD, Chong MG, Wong MA, Gray
A, Cormier JG, Nagle GT. Relative contributions of the egg layer and egg cordon
to pheromonal attraction and the induction of mating and egg-laying behavior in Aplysia. Biol Bull 1991; 181:81–94.
50. Schein CH, Nagle GT, Page JS,
Sweedler JV, Xu Y, Painter SD, et al. Aplysia attractin : biophysical
characterization and modeling of a water-borne pheromone. Biophys J 2001 ; 81 :463–72.
51. Painter SD, Cummuns SF, Nichols AE,
Akalal DBG, Schein CH, Braun W, et al. Structural and functional analysis of Aplysia attractins, a family of
water-borne protein pheromones with interspecific attractiveness. Proc Natl Acad Sci USA 2004; 101:6929–33.
52. Ortenzi C, Miceli C, Bradshaw RA, Luporini P. Identification
and initial characterization of an autocrine pheromone receptor in the
protozoan ciliate Euplotes raikovi. J
Cell Biol 1990; 111:607–14.
53. Wang X, Rickert M, Garcia KC. Structure of the
quaternary complex of interleukin-2 with its alpha, beta,
and gamma receptors. Science 2005; 310:1159–63.
54. Vallesi A,
Giuli G, Ghiara P, Scapigliati G, Luporini P. Structure-function relationships
of pheromones of the ciliate Euplotes
raikovi with mammalian growth factors: cross-reactivity between Er-1 and IL-2 systems. Exp Cell Res 1998; 241: 253–9.
55. Cervia D, Catalani E,
Belardinelli MC, Perrotta C, Picchietti S, Alimenti C, et al. The protein pheromone Er-1 of
the ciliate Euplotes raikovi stimulates human T-cell activity: involvement of interleukin-2 system. Exp Cell Res 2013;
319:56–67.
56. Ortenzi C, Luporini P. Competition among homologous
polypeptide pheromones of the ciliated Euplotes
raikovi for binding to each other’s cell receptor. J Eukaryot Microbiol 1995; 42:242–8.
Figure
Legends
Figure
1. Euplotes
raikovi pheromone family. (A)
Multiple alignment of nine distinct sequences of pheromone precursors, as
deduced from their DNA coding sequences. Sequence alignment is based on the
Clustal W algorithm and optimized manually by gap insertions. Residues
conserved among all sequences, or all but one sequence are shadowed. The
positions of the cysteine residues are highlighted in bold. The numbers of
residues of the pre, pro and secreted regions are reported on the right of the
sequences. (B) Ribbon presentations
(as visualized by the program MOLMOL) of the six pheromone structures (side
view) that have been determined by NMR analysis of native protein preparations.
The Protein Data Bank (PDB) codes are the following ones: 1erc (Er-1), 1erd (Er-2), 1erp (Er-10), 1ery
(Er-11), 1hd6 (Er-22), and 1ha8 (Er-23).
The amino and carboxyl termini of each molecule are labeled N and C,
respectively, and the sulfur atoms involved in the disulfide bonds are
indicated as spheres. The three more regular a-helices common to all six molecules are labeled h1 to
h3 starting from the N-terminus and shown in dark shading.
Figure
2. Euplotes
nobilii pheromone family. (A)
Multiple alignment of seven distinct sequences of pheromone precursors, as
deduced from their DNA coding sequences. (B)
Ribbon presentations (as visualized by the program MOLMOL) of the four
pheromone structures that have been determined by NMR analysis of native
protein preparations. The Protein Data Bank (PDB) codes are the following ones:
2nsv (En-1), 2nsw (En-2), 2jms (En-6), 2kk2 (En-A1).
Indications and symbols as in Fig. 1.
Figure
3. Euplotes octocarinatus pheromone family. Multiple
alignment of nine distinct sequences of pheromone precursors, as deduced from
the determination of their DNA coding sequences. Indications as in Fig. 1.
Figure
4. Euplotes
crassus pheromones. Multiple alignment of four distinct sequences of
pheromone precursors, as deduced from their DNA coding sequences. Three
sequences (Ec-1, Ec-2 and Ec-4) are cell-type specific and one (Ec-a)
is shared identical among different cell types. Indications as in Fig. 1.
Figure
5. Intermolecular helix-helix interactions in the
crystal structure of the
E. raikovi pheromone E
r-1. (
A) Structure of the symmetrical dimer 1 in which two molecules are related
by a twofold axis (
![]()
). Each monomer involves helices 1 and 2, delimiting face a, in the
formation of a four-helix bundle. (
B)
Structure of the asymmetrical dimer 2 in which two molecules are related by a
twofold screw axis (
![]()
). Each monomer stacks helix 3, and its adjacent faces
b and c, in an anti-↑parallel fashion to form a linear structure without
symmetrical contacts. The E
r-1
molecule packing into the crystal lattice is shown in the boxed diagrammatic
presentation. Shadowed molecules mimic ligand binding (pheromone-receptor)
molecules oriented with their C-terminus towards the cell outside, while light
molecules mimic soluble pheromone molecules oriented with their C-terminus
towards the cell inside. After Weiss et al. (22).
Figure
6. Effects of E.
raikovi pheromones on human lymphocyte Jurkat cells. (A) Viability of Jurkat cells cultured for 48h in the absence
(control), or in the presence of increasing concentrations of pheromone Er-1. Histograms represent the mean ± SEM
of data from at least four independent experiments. Data are expressed by
setting the control samples as 100%. (B)
Measurements by quantitative PCR of the mRNA levels of different cytokines in
Jurkat cells grown in the presence of 10ng/ml Er-1 for 48h in restrictive culture conditions. Each histogram
represents the mean ± SEM of data from at least three independent experiments.
Fold changes were determined putting equal to 1 the mRNA levels in cells grown
in the absence of Er-1 (control).
After Cervia et al. (55)