Analysis of the Effects of Polymorphism on Pollen Profilin Structural Functionality and the Generation of Conformational,T- and B-Cell Epitopes

An extensive polymorphism analysis of pollen profilin, a fundamental regulator of the actin cytoskeleton dynamics, has been performed with a major focus in 3D-folding maintenance, changes in the 2-D structural elements, surface residues involved in ligands-profilin interactions and functionality, and the generation of conformational and lineal B- and T-cell epitopes variability.Our results revealed that while the general fold is conserved among profilins, substantial structural differences were found, particularly affecting the special distribution and length of different 2-D structural elements (i.e. cysteine residues), characteristic loops and coils, and numerous micro-heterogeneities present in fundamental residues directly involved in the interacting motifs, and to some extension these residues nearby to the ligand-interacting areas. Differential changes as result of polymorphism might contribute to generate functional variability among the plethora of profilin isoforms present in the olive pollen from different genetic background (olive cultivars), and between plant species, since biochemical interacting properties and binding affinities to natural ligands may be affected, particularly the interactions with different actin isoforms and phosphoinositides lipids species.Furthermore, conspicuous variability in lineal and conformational epitopes was found between profilins belonging to the same olive cultivar, and among different cultivars as direct implication of sequences polymorphism. The variability of the residues taking part of IgE-binding epitopes might be the final responsible of the differences in cross-reactivity among olive pollen cultivars, among pollen and plant-derived food allergens, as well as between distantly related pollen species, leading to a variable range of allergy reactions among atopic patients. Identification and analysis of commonly shared and specific epitopes in profilin isoforms is essential to gain knowledge about the interacting surface of these epitopes, and for a better understanding of immune responses, helping design and development of rational and effective immunotherapy strategies for the treatment of allergy diseases.     

Profilin is essential for tip growth in the moss Physcomitrella patens

The actin cytoskeleton is critical for tip growth in plants. Profilin is the main monomer actin binding protein in plant cells. The moss Physcomitrella patens has three profilin genes, which are monophyletic, suggesting a single ancestor for plant profilins. Here, we used RNA interference (RNAi) to determine the loss-of-function phenotype of profilin. Reduction of profilin leads to a complete loss of tip growth and a partial inhibition of cell division, resulting in plants with small rounded cells and fewer cells. We silenced all profilins by targeting their 3' untranslated region sequences, enabling complementation analyses by expression of profilin coding sequences. We show that any moss or a lily (Lilium longiflorum) profilin support tip growth. Profilin with a mutation in its actin binding site is unable to rescue profilin RNAi, while a mutation in the poly-l-proline binding site weakly rescues. We show that moss tip growing cells contain a prominent subapical cortical F-actin structure composed of parallel actin cables. Cells lacking profilin lose this structure; instead, their F-actin is disorganized and forms polarized cortical patches. Plants expressing the actin and poly-l-proline binding mutants exhibited similar F-actin disorganization. These results demonstrate that profilin and its binding to actin are essential for tip growth. Additionally, profilin is not needed for formation of F-actin, but profilin and its interactions with actin and poly-l-proline ligands are required to properly organize F-actin.     

MAP kinase phosphorylation of plant profilin

Profilin is a small actin-binding protein and is expressed at high levels in mature pollen where it is thought to regulate actin filament dynamics upon pollen germination and tube growth. The majority of identified plant profilins contain a MAP kinase phosphorylation motif, P-X-T-P, and a MAP kinase interaction motif (KIM). In in vitro kinase assays, the tobacco MAP kinases p45(Ntf4) and SIPK, when activated by the tobacco MAP kinase kinase NtMEK2, can phosphorylate the tobacco profilin NtProf2. Mutagenesis of the threonine residue in this motif identified it as the site of MAP kinase phosphorylation. Fractionation of tobacco pollen extracts showed that p45(Ntf4) is found exclusively in the high-speed pellet fraction while SIPK and profilin are predominantly cytosolic. These data identify one of the first substrates to be directly phosphorylated by MAP kinases in plants.     

Plant profilin isovariants are distinctly regulated in vegetative and reproductive tissues

Profilin is a low-molecular weight, actin monomer-binding protein that regulates the organization of actin cytoskeleton in eukaryotes, including higher plants. Unlike the simple human or yeast systems, the model plant Arabidopsis has an ancient and highly divergent multi-gene family encoding five distinct profilin isovariants. Here we compare and characterize the regulation of these profilins in different organs and during microspore development using isovariant-specific monoclonal antibodies. We show that PRF1, PRF2, and PRF3 are constitutive, being strongly expressed in all vegetative tissues at various stages of development. These profilin isovariants are also predominant in ovules and microspores at the early stages of microsporogenesis. In contrast, PRF4 and PRF5 are late pollen-specific and are not detectable in other cell types of the plant body including microspores and root hairs. Immunocytochemical studies at the subcellular level reveal that both the constitutive and pollen-specific profilins are abundant in the cytoplasm. In vegetative cell types, such as root apical cells, profilins showed localization to nuclei in addition to the cytoplasmic staining. The functional diversity of profilin isovariants is discussed in light of their spatio-temporal regulation during vegetative development, pollen maturation, and pollen tube growth.     

The profilin multigene family of maize: differential expression of three isoforms

Profilin is a small (12–15 kDa) actin- and phospholipid-binding protein previously known only from studies on animals and lower eukaryotes but recently identified as a birch pollen allergen. Here we have identified and characterized three members of the profilin multigene family from the plant Zea mays . Two cDNAs isolated from a maize pollen library ( ZmPRO 1 and ZmPRO 3) each have a single, large open reading frame encoding a putative polypeptide 131 amino acids long with a predicted molecular weight of approximately 14 kDa. A third maize pollen cDNA ( ZmPRO 2) has two in-frame translation initiation codons. Use of the first ATG would result in a polypeptide 137 amino acids long with a molecular weight of 14.8 kDa. The three maize profilins are highly homologous to each other (>90% nucleotide and amino acid sequence identity) as well as other plant profilins but show far less similarity (30–40% amino acid sequence identity) to animal and lower eukaryote profilins. Multiple sequence alignments indicate that only nine residues are shared by all eukaryotic profilins examined. However, limited comparisons reveal domains in the NH₂ and COOH termini that have a high degree of similarity suggesting functional conservation. The maize gene family size is estimated to contain three to six members based on Southern blot experiments with gene-specific and coding region probes. Northern blot analysis demonstrates that the three maize profilin cDNAs characterized here are utilized in a tissue-specific manner and are anther or pollen specific.     

Maize profilin isoforms are functionally distinct

Profilin is an actin monomer binding protein that, depending on the conditions, causes either polymerization or depolymerization of actin filaments. In plants, profilins are encoded by multigene families. In this study, an analysis of native and recombinant proteins from maize demonstrates the existence of two classes of functionally distinct profilin isoforms. Class II profilins, including native endosperm profilin and a new recombinant protein, ZmPRO5, have biochemical properties that differ from those of class I profilins. Class II profilins had higher affinity for poly-l-proline and sequestered more monomeric actin than did class I profilins. Conversely, a class I profilin inhibited hydrolysis of membrane phosphatidylinositol-4,5-bisphosphate by phospholipase C more strongly than did a class II profilin. These biochemical properties correlated with the ability of class II profilins to disrupt actin cytoplasmic architecture in live cells more rapidly than did class I profilins. The actin-sequestering activity of both maize profilin classes was found to be dependent on the concentration of free calcium. We propose a model in which profilin alters cellular concentrations of actin polymers in response to fluctuations in cytosolic calcium concentration. These results provide strong evidence that the maize profilin gene family consists of at least two classes, with distinct biochemical and live-cell properties, implying that the maize profilin isoforms perform distinct functions in the plant.     

Molecular cloning and characterization of profilin from tobacco (Nicotiana tabacum): increased profilin expression during pollen maturation

Profilin has recently been identified as an actin-binding protein in higher plants. A cDNA coding for tobacco profilin, which shared an average sequence identity of 75% with other plant profilins, was isolated from a tobacco pollen cDNA library by antibody screening. Tobacco profilin was expressed in Escherichia coli and purified by affinity to poly-(L-proline) Sepharose. A rabbit antiserum was raised against recombinant tobacco profilin and used to estimate the amount of profilin expressed in different tobacco tissues. Profilin can be detected in different somatic tissues, but the expression is 50–100 fold higher in mature pollen. Immunofluorescence and confocal laser scanning microscopy showed a homogeneous distribution of profilin in the cytoplasm of in vitro cultured pollen grains and pollen tubes of tobacco whereas some growing pollen tubes were stained more intensively a their tip. A possible role of pollen profilin as a developmentally upregulated microfilament precursor in mature pollen is discussed.     

Pollen profilin function depends on interaction with proline-rich motifs.

The actin binding protein profilin has dramatic effects on actin polymerization in vitro and in living cells. Plants have large multigene families encoding profilins, and many cells or tissues can express multiple profilin isoforms. Recently, we characterized several profilin isoforms from maize pollen for their ability to alter cytoarchitecture when microinjected into living plant cells and for their association with poly-L-proline and monomeric actin from maize pollen. In this study, we characterize a new profilin isoform from maize, which has been designated ZmPRO4, that is expressed predominantly in endosperm but is also found at low levels in all tissues examined, including mature and germinated pollen. The affinity of ZmPRO4 for monomeric actin, which was measured by two independent methods, is similar to that of the three profilin isoforms previously identified in pollen. In contrast, the affinity of ZmPRO4 for poly-L-proline is nearly twofold higher than that of native pollen profilin and the other recombinant profilin isoforms. When ZmPRO4 was microinjected into plant cells, the effect on actin-dependent nuclear position was significantly more rapid than that of another pollen profilin isoform, ZmPRO1. A gain-of-function mutant (ZmPRO1-Y6F) was created and found to enhance poly-L-proline binding activity and to disrupt cytoarchitecture as effectively as ZmPRO4. In this study, we demonstrate that profilin isoforms expressed in a single cell can have different effects on actin in living cells and that the poly-L-proline binding function of profilin may have important consequences for the regulation of actin cytoskeletal dynamics in plant cells.     

Molecular characterization of profilin isoforms from tobacco (Nicotiana tabacum) pollen

Profilins are actin-binding proteins in eukaryotes which participate in the phosphoinositide pathway via binding to PIP₂. Using polyclonal rabbit sera raised against plant profilins, the occurrence of several profilin isoforms is demonstrated in two-dimensionally analyzed tobacco pollen extracts. The cDNAs coding for two novel tobacco profilin isoforms (ntPro2, ntPro3) were isolated from a pollen cDNA library by antibody screening. When the cDNA and deduced amino acid sequences of the two isoforms were compared with a previously isolated tobacco pollen profilin cl)NA (ntPro1), significant differences were noted in the non-coding regions, whereas the coding sequences, in particular the functional domains, showed little variation. The cDNAs coding for the three tobacco profilin isoforms were expressed inEscherichia coli and shown to bind comparably to different anti-profilin antisera. The high degree of similarity among the different tobacco pollen profilin isoforms points to functional equivalence. Assuming that the presence of profilin is indispensable to the control of the large amounts of actin present in pollen, the occurrence of different profilin isoforms in pollen is interpreted to represent a protective mechanism against loss of profilin functions.     

The affinities of human platelet and Acanthamoeba profilin isoforms for polyphosphoinositides account for their relative abilities to inhibit phospholipase C.

In light of recent work implicating profilin from human platelets as a possible regulator of both cytoskeletal dynamics and inositol phospholipid-mediated signaling, we have further characterized the interaction of platelet profilin and the two isoforms of Acanthamoeba profilin with inositol phospholipids. Profilin from human platelets binds to phosphatidylinositol-4-monophosphate (PIP) and phosphatidylinositol-4,5-bisphosphate (PIP2) with relatively high affinity (Kd approximately 1 microM for PIP2 by equilibrium gel filtration), but interacts only weakly (if at all) with phosphatidylinositol (PI) or inositol trisphosphate IP3) in small-zone gel-filtration assays. The two isoforms of Acanthamoeba profilin both have a lower affinity for PIP2 than does human platelet profilin, but the more basic profilin isoform from Acanthamoeba (profilin-II) has a much higher (approximately 10-microM Kd) affinity than the acidic isoform (profilin-I, 100 to 500-microM Kd). None of the profilins bind to phosphatidylserine (PS) or phosphatidylcholine (PC) in small-zone gel-filtration experiments. The differences in affinity for PIP2 parallel the ability of these three profilins to inhibit PIP2 hydrolysis by soluble phospholipase C (PLC). The results show that the interaction of profilins with PIP2 is specific with respect to both the lipid and the proteins. In Acanthamoeba, the two isoforms of profilin may have specialized functions on the basis of their identical (approximately 10 microM) affinities for actin monomers and different affinities for PIP2.     

On the origin and evolution of vertebrate and viral profilins

The three dimensional structures of profilins from invertebrates and vertebrates are remarkably similar despite low sequence similarity. Their evolutionary relationship remains thus enigmatic. A phylogenetic analysis of profilins from Deuterostoma indicates that profilin III and IV isoforms each form distinct groups. Profilin IV is most related to invertebrate profilins and originated prior to vertebrate evolution whereas separation of profilin I, II and III isoforms occurred early in vertebrate evolution. Viral profilins are most similar to profilin III. In silico analysis of representative profilin gene structures corroborates the phylogenetic result and we discuss this in terms of biochemical differences.     

Localization of profilin- and actin-like immunoreactivity in in vitro-germinated tobacco pollen tubes by electron microscopy after special water-free fixation techniques

Double immunogold labeling of profilin and actin was performed on ultrathin sections of in vitro germinated tobacco pollen using different anti-profilin and anti-actin antibodies. Since profilin, besides its role as an actin-binding protein, is known as an allergen, water-free fixation in p-formaldehyde vapor was used. Profilin labeling occurs throughout the cytoplasm of the pollen tube. There is no profilin in the pollen tube wall. Actin reactivity is found in the cytoplasm and extracellularly in the pollen tube wall where three out of four different anti-actin antibodies give a positive signal. This labeling of the pollen tube wall may result from a wall-bound actin, an isoform of actin not yet described or from the presence of a molecule immunologically indistinguishable from actin.     

Structure and functions of profilins

Profilins are small actin-binding proteins found in eukaryotes and certain viruses that are involved in cell development, cytokinesis, membrane trafficking, and cell motility. Originally identified as an actin sequestering/binding protein, profilin has been involved in actin polymerization dynamics. It catalyzes the exchange of ADP/ATP in actin and increases the rate of polymerization. Profilins also interact with polyphosphoinositides (PPI) and proline-rich domains containing proteins. Through its interaction with PPIs, profilin has been linked to signaling pathways between the cell membrane and the cytoskeleton, while its role in membrane trafficking has been associated with its interaction with proline-rich domain-containing proteins. Depending on the organism, profilin is present in a various number of isoforms. Four isoforms of profilin have been reported in higher organisms, while only one or two isoforms are expressed in single-cell organisms. The affinity of these isoforms for their ligands varies between isoforms and should therefore modulate their functions. However, the significance and the functions of the different isoforms are not yet fully understood. The structures of many profilin isoforms have been solved both in the presence and the absence of actin and poly-L-proline. These structural studies will greatly improve our understanding of the differences and similarities between the different profilins. Structural stability studies of different profilins are also shedding some light on our understanding of the profilin/ligand interactions. Profilin is a multifaceted protein for which a dramatic increase in potential functions has been found in recent years; as such, it has been implicated in a variety of physiological and pathological processes.     

Primary structure of profilins from two species of Echinoidea and Physarum polycephalum

Profilin is a small G-actin-binding protein, the amino acid sequence of which was previously reported for calf, human, Acanthamoeba and yeast. Here the amino acid sequences of three profilins obtained from eggs of two species of Echinoidea, Clypeaster japonicus (order, Clypeasteroida) and Anthocidaris crassispina (order, Echinoida), and plasmodium of Physarum polycephalum were determined. Two echinoid profilins were composed of 139 amino acid residues, N-termini were acylated and the molecular mass was calculated to be 14.6 kDa, slightly larger than that of 13 kDa estimated by SDS/PAGE [Mabuchi, I. & Hosoya, H. (1982) Biomed. Res. 3, 465-476]. On the other hand, Physarum profilin was composed of 124 amino acid residues, the N-terminus was acylated, and the calculated molecular mass was 13132 Da. The sequences of C. japonicus and A. crassispina profilins were homologous (84% identical). However, the similarity of these profilins with those form other organisms was low. The sequence of Physarum profilin was homologous with Acanthamoeba profilin isoforms (51% identical) and with yeast profilin (42% identical), but not with other profilins. The relatively conservative sequence of profilins from yeast, Physarum, Acanthamoeba, echinoid eggs and mammalian cells was found in the N-terminal region, which was suggested to be a common actin-binding region. The C-terminal region was also conserved, although to a lesser extent than the N-terminal region.     

Biochemical characterization of actin assembly mechanisms with ALS-associated profilin variants

Eight separate mutations in the actin-binding protein profilin-1 have been identified as a rare cause of amyotrophic lateral sclerosis (ALS). Profilin is essential for many neuronal cell processes through its regulation of lipids, nuclear signals, and cytoskeletal dynamics, including actin filament assembly. Direct interactions between profilin and actin monomers inhibit actin filament polymerization. In contrast, profilin can also stimulate polymerization by simultaneously binding actin monomers and proline-rich tracts found in other proteins. Whether the ALS-associated mutations in profilin compromise these actin assembly functions is unclear. We performed a quantitative biochemical comparison of the direct and formin mediated impact for the eight ALS-associated profilin variants on actin assembly using classic protein-binding and single-filament microscopy assays. We determined that the binding constant of each profilin for actin monomers generally correlates with the actin nucleation strength associated with each ALS-related profilin. In the presence of formin, the A20T, R136W, Q139L, and C71G variants failed to activate the elongation phase of actin assembly. This diverse range of formin-activities is not fully explained through profilin-poly-L-proline (PLP) interactions, as all ALS-associated variants bind a formin-derived PLP peptide with similar affinities. However, chemical denaturation experiments suggest that the folding stability of these profilins impact some of these effects on actin assembly. Thus, changes in profilin protein stability and alterations in actin filament polymerization may both contribute to the profilin-mediated actin disruptions in ALS.     

Distinct roles of the first introns on the expression of Arabidopsis profilin gene family members

Profilin is a small actin-binding protein that regulates cellular dynamics of the actin cytoskeleton. In Arabidopsis (Arabidopsis thaliana), five profilins were identified. The vegetative class profilins, PRF1, PRF2, and PRF3, are expressed in vegetative organs. The reproductive class profilins, PRF4 and PRF5, are mainly expressed in pollen. In this study, we examined the role of the first intron in the expression of the Arabidopsis profilin gene family using transgenic plants and a transient expression system. In transgenic plants, we examined PRF2 and PRF5, which represent vegetative and reproductive profilins. The expression of the PRF2 promoter fused with the beta-glucuronidase (GUS) gene was observed in the vascular bundles, but transgenic plants carrying the PRF2 promoter-GUS with its first intron showed constitutive expression throughout the vegetative tissues. However, the first intron of PRF5 had little effect on the reporter gene expression pattern. Transgenic plants containing PRF5 promoter-GUS fusion with or without its first intron showed reproductive tissue-specific expression. To further investigate the different roles of the first two introns on gene expression, the first introns were exchanged between PRF2 and PRF5. The first intron of PRF5 had no apparent effect on the expression pattern of the PRF2 promoter. But, unlike the intron of PRF5, the first intron of PRF2 greatly affected the reproductive tissue-specific expression of the PRF5 promoter, confirming a different role for these introns. The results of a transient expression assay indicated that the first intron of PRF1 and PRF2 enhances gene expression, whereas PRF4 and PRF5 do not. These results suggest that the first introns of profilin genes are functionally distinctive and the first introns are required for the strong and constitutive gene expression of PRF1 and PRF2 in vegetative tissues.     

Oligomerization of profilins from birch, man and yeast. Profilin, a ligand for itself?

 Profilins are structurally well conserved low molecular weight (12–15 kDa) eukaryotic proteins which interact with a variety of physiological ligands: (1) cytoskeletal components, e.g., actin; (2) polyphosphoinositides, e.g., phosphatidylinositol-4,5-bisphosphate; (3) proline-rich proteins, e.g., formin homology proteins and vasodilatator-stimulated phosphoprotein. Profilins may thus link the microfilament system with signal transduction pathways. Plant profilins have recently been shown to be highly crossreactive allergens which bind to IgE antibodies of allergic patients and thus cause symptoms of type I allergy. We expressed and purified from Escherichia coli profilins from birch pollen (Betula verrucosa), humans (Homo sapiens) and yeast (Schizosaccharomyces pombe) and demonstrated that each of these profilins is able to form stable homo- and heteropolymers via disulphide bonds in vitro. Circular dichroism analysis of oxidized (polymeric) and reduced (monomeric) birch pollen profilin indicates that the two states have similar secondary structures. Using ¹²⁵I-labeled birch pollen, yeast and human profilin in overlay experiments, we showed that disulphide bond formation between profilins can be disrupted under reducing conditions, while reduced as well as oxidized profilin states bind to actin and profilin-specific antibodies. Exposure of profilin to oxidizing conditions, such as when pollen profilins are liberated on the surface of the mucosa of atopic patients, may lead to profilin polymerization and thus contribute to the sensitization capacity of profilin as an allergen. Received: 25 February 1998 / Revision accepted: 12 May 1998