Recombinant Profilin Research Articles

Expression, purification and crystallization of wheat profilin (Tri a 12)

Wheat profilin, designated Tri a 12, has recently been found to be recognized by specific IgE antibodies in patients suffering from bakers' asthma, wheat induced food allergy and also in patients with grass pollen allergy. Since profilin sequences are highly conserved among plants, individuals who are sensitized to profilin commonly show allergic symptoms to a large number of unrelated plants. Knowledge about the three dimensional structure of these allergenic proteins is necessary to gain information on the surface structure required to predict cross-reactivity. However, in contrast to other plant profilins, the three dimensional structure of Tri a 12 has not been resolved yet. In the present study Tri a 12 was expressed in E. coli and purified from the soluble fraction by affinity chromatography. The molecular weight and the structural integrity of the recombinant protein were verified by mass spectrometry and circular dichroism, respectively. Recombinant Tri a 12 showed intact secondary structures of mixed alpha helices and beta sheet elements characteristic for members of the profilin protein family. This well characterized batch of purified recombinant wheat profilin was then used for crystallization. Crystallization conditions were screened with the sitting-drop vapour-diffusion method. The best crystals of wheat profilin with maximal dimensions between 0.1 and 0.3 mm were observed after one week at 290 K for reservoir solutions containing 3.2-3.7 mol L-1 sodium formate and 50 mmol L-1 HEPES–NaOH buffer, pH = 7.5. A data set diffracting to a resolution of 3.3 A was collected in-house from a single crystal. The crystals belonged to space group P3221, with unit-cell parameters a = b = 58.9 A, c = 82.5 A, α = β = 90° and γ = 120°. Model building and refinement of the crystal structure, as well as further optimization of crystal diffraction quality is under way.

Montanide™ IMS 1313 N VG PR nanoparticle adjuvant enhances antigen-specific immune responses to profilin following mucosal vaccination against Eimeria acervulina

This study investigated protection against Eimeria acervulina (E. acervulina) following vaccination of chickens with an Eimeria recombinant profilin in conjunction with different adjuvants, or by changing the route of administration of the adjuvants. Day-old broilers were immunized twice with profilin emulsified in Montanide™ IMS 1313 N VG PR adjuvant (oral, nasal, or ocular routes), Montanide™ ISA 71 VG adjuvant (subcutaneous route), or Freund's adjuvant (subcutaneous route) and orally challenged with virulent E. acervulina parasites. Birds orally immunized with profilin plus IMS 1313 N VG PR, or subcutaneously immunized with profilin plus ISA 71 VG, had increased body weight gains compared with animals nasally or ocularly immunized with profilin plus IMS 1313 N VG PR, or subcutaneously immunized with profilin plus Freund's adjuvant. All adjuvant formulations, except for IMS 1313 N VG PR given by the nasal or ocular routes, decreased fecal parasite excretion and/or reduced intestinal lesions, compared with non-vaccinated and infected controls. Compared with animals vaccinated with profilin plus Freund's adjuvant, chickens immunized with profilin plus IMS 1313 N VG PR or ISA 71 VG showed higher post-infection intestinal levels of profilin-reactive IgY and secretary IgA antibodies. Finally, immunization with profilin in combination with ISA 71 VG was consistently better than profilin plus IMS 1313 N VG PR or Freund's adjuvant for increasing the percentages of CD4+, CD8+, BU1+, TCR1+, and TCR2+ intestinal lymphocytes. These results indicate that experimental immunization of chickens with the recombinant profilin subunit vaccine in conjunction with IMS 1313 or ISA 71 VG adjuvants increases protective mucosal immunity against E. acervulina infection.

Mucosal immunity against Eimeria acervulina infection in broiler chickens following oral immunization with profilin in Montanide™ adjuvants

The present study was conducted to compare aqueous nanoparticle-based Montanide™ IMS 1313 N VG PR (IMS 1313) and oil-based ISA 71 VG (ISA71) adjuvants in combination with an Eimeria subunit protein vaccine on protection against avian coccidiosis. Male broiler chicks were vaccinated twice with an Eimeria recombinant profilin protein alone or in conjunction with IMS 1313 or ISA 71 prior to infection with live, sporulated Eimeria acervulina oocysts. For comparison, chickens were immunized with a commercial live coccidiosis vaccine (Coccivac-B). The following parameters were assessed as measures of protective immunity: body weight gain, fecal oocyst output, profilin-specific intestinal secretary IgA (sIgA) or IgY antibody levels, and percentages of CD4+, CD8+, TCR1+, or TCR2+ intestinal intraepithelial lymphocytes (IELs). Birds vaccinated with profilin plus ISA 71 had increased body weight gains, equivalent to Coccivac-B vaccination, compared with the profilin-only group. Immunization with profilin plus IMS 1313, or with profilin plus ISA 71, reduced fecal oocysts shedding compared with the profilin-only group. Intestinal sIgA levels were greater in the profilin plus IMS 1313 or ISA 71 groups, and IgY levels were greater in the profilin plus ISA 71 group, compared with profilin alone. Birds vaccinated with profilin plus IMS 1313 or ISA 71 had higher percentages of CD4+, CD8+, and TCR1+, but not TCR2+, intestinal IELs compared with the profilin-only vaccinated group. Taken together, these results indicate that immunization of chickens with the recombinant profilin subunit vaccine in conjunction with IMS 1313 or ISA 71 adjuvants increases protective immunity against experimental E. acervulina infection.

An approach for detection and quantification of fruits' natural profilin: natural melon profilin as a model

Anaphylactic reactions are of great importance in clinical allergology. Measurement of allergen concentration in foods is helpful to estimate their allergenicity. In this study, a sandwich ELISA for the evaluation of profilin concentration in fruits was designed. A mixture of monoclonal antibodies against saffron profilin was used as a capture antibody. The recombinant melon profilin was expressed in Escherichia coli BL-21 and applied as a standard antigen along with different fruit extracts to measure their profilin concentration. Furthermore, a polyclonal antibody against the recombinant melon profilin was produced and applied as the secondary antibody. In this way, the profilin concentration of various fruits including banana, tomato, kiwi, mulberry, cantaloupe, peach, watermelon and zucchini was measured. The results showed that the designed ELISA had a sensitivity threshold of about 1 ng/ml for the melon profilin and it is therefore suggested for determination of profilin concentration in other fruits.

Chenopodium album pollen profilin (Che a 2): homology modeling and evaluation of cross-reactivity with allergenic profilins based on predicted potential IgE epitopes and IgE reactivity analysis

The inhalation of Chenopodium album (C. album) pollen has been reported as an important cause of allergic respiratory symptoms. The aim of this study was to produce the recombinant profilin of C. album (rChe a 2) pollen and to investigate its cross-reactivity with other plant-derived profilins based on potential conformational epitopes and IgE reactivity analysis. Che a 2-coding sequence was cloned, expressed, and purified using one step metal affinity chromatography to recover high-purity target protein. We assessed cross-reactivity and predicted IgE potential epitopes among rChe a 2 and other plant-derived profilins. Immunodetection and inhibition assays using sixteen individual sera from C. album allergic patients demonstrated that purified rChe a 2 could be the same as that in the crude extract. The results of inhibition assays among rChe a 2 and other plant-derived profilins were in accordance with those of the homology of predicted conserved conformational regions. In this study, amino acid sequence homology analysis showed that a high degree of IgE cross-reactivity among plant-derived profilins may depend on predicted potential IgE epitopes.

Embryo vaccination of chickens using a novel adjuvant formulation stimulates protective immunity against Eimeria maxima infection

Our previous study demonstrated that chickens immunized subcutaneously with an Eimeria recombinant profilin protein vaccine emulsified in a Quil A/cholesterol/DDA/Carbopol (QCDC) adjuvant developed partial protection against experimental avian coccidiosis compared with animals immunized with profilin alone. Because in ovo vaccination is presently used in commercial applications worldwide throughout the poultry industry, the current study was undertaken to investigate chicken embryo vaccination with profilin plus QCDC adjuvant. Eighteen day-old embryos were immunized with isotonic saline (control), profilin alone, QCDC alone, or profilin plus QCDC, and orally challenged with live Eimeria maxima at 7 days post-hatch. Body weight gain, fecal oocyst output, and intestinal cytokine transcript levels were assessed as measures of protective immunity. While immunization with profilin alone or QCDC alone did not alter body weight gain of infected chickens compared with the saline control group, vaccination with profilin plus QCDC increased body weight gain such that it was equal to the uninfected controls. Immunization with profilin plus QCDC also reduced fecal oocyst shedding compared with unimmunized controls, although in this case QCDC failed to provide an adjuvant effect since no difference was observed between the profilin-only and profilin/QCDC groups. Finally, increased levels of transcripts encoding IL-1β, IL-15, and IFN-γ were seen in the intestinal tissues of animals given profilin plus QCDC compared with the profilin-only or QCDC-only groups. In summary, this study demonstrates an adjuvant effect of QCDC on body weight gain and intestinal cytokine responses following in ovo vaccination of chickens with an Eimeria profilin vaccine.

Montanide™ ISA 71 VG adjuvant enhances antibody and cell-mediated immune responses to profilin subunit antigen vaccination and promotes protection against Eimeria acervulina and Eimeria tenella

The present study was conducted to investigate the immunoenhancing effects of Montanide™ ISA 71 VG adjuvant on profilin subunit antigen vaccination. Broiler chickens were immunized subcutaneously with a purified Eimeria acervulina recombinant profilin protein, either alone or mixed with ISA 71 VG, and host immune responses were evaluated. After secondary immunization, antigen-specific antibody and T-cell responses were higher in the group which received profilin plus ISA 71 VG compared with the other groups. Furthermore, body weight gains and fecal oocyst shedding were evaluated following oral challenge infection with live E. acervulina or Eimeria tenella oocysts. Vaccination with profilin plus ISA 71 VG reduced oocyst shedding compared with animals immunized with profilin alone. These results demonstrate that the recombinant profilin subunit vaccine, when given in combination with Montanide™ ISA 71 VG, augments protective immunity against E. acervulina and E. tenella.

The effects of a novel adjuvant complex/ Eimeria profilin vaccine on the intestinal host immune response against live E. acervulina challenge infection

The effects of a novel adjuvant composed of Quil A, cholesterol, dimethyl dioctadecyl ammonium bromide, and Carbopol (QCDC) on protective immunity against avian coccidiosis following immunization with an Eimeria recombinant protein were determined. Broiler chickens were subcutaneously immunized with isotonic saline (control), Eimeria recombinant profilin alone, or profilin emulsified with QCDC at 1 and 7 days post-hatch, and orally challenged with live Eimeria acervulina at 7 days following the last immunization. Body weight gains, gut lesion scores, fecal oocyst outputs, profilin serum antibody titers, lymphocyte proliferation, and intestinal cytokine transcript levels were assessed as measures of protective immunity. Chickens immunized with profilin plus QCDC showed increased body weight gains and decreased intestinal lesion scores compared with the profilin only or control groups. However, no differences were found in fecal oocyst shedding among the three groups. Profilin serum antibody titers and antigen-induced peripheral blood lymphocyte proliferation in the profilin/QCDC group were higher compared with the profilin only and control groups. Finally, while immunization with profilin alone or profilin plus QCDC uniformly increased the levels of intestinal transcripts encoding all cytokines examined (IL-1β, IL-10, IL-12, IL-15, IL-17F, and IFN-γ) compared with the control group, transcripts for IL-10 and IL-17F were further increased in the profilin/QCDC group compared with the profilin only group. In summary, this study provides the first evidence of the immunoenhancing activities of QCDC adjuvant in poultry.

Watermelon Profilin: Characterization of a Major Allergen as a Model for Plant-Derived Food Profilins

Background: Plant profilins have been reported as minor allergens. They are a well-known pan-allergen family responsible for cross-reactivity between plant-derived foods and pollens. Watermelon profilin has been reported to be a major allergen in watermelon (Citrullus lanatus).The aim of this study was to characterize recombinant watermelon profilin, confirming its reactivity for diagnostic purposes and the development of immunotherapy. Methods: Native profilin was purified from watermelon extract by affinity chromatography using poly-L-proline. Recombinant His-tagged profilin was produced in Pichia pastoris yeast using pPICZαA vector and purified by metal chelate affinity chromatography. ELISA and immunoblot were carried out with sera from 17 watermelon-allergic patients. Biological activity was tested by the basophil activation test. Results: Native profilin and recombinant profilin were purified and identified by mass spectrometry. Both show similar IgE reactivity in vitro and are biologically active. Conclusions: Similarities were found in the IgE-binding patterns and biological activity of recombinant profilin and native profilin. Recombinant profilin may be a powerful tool for specific diagnosis.

Immunoenhancing effects of Montanide™ ISA oil-based adjuvants on recombinant coccidia antigen vaccination against Eimeria acervulina infection

The current study was conducted to investigate the immunoenhancing effects of Montanide™ adjuvants on protein subunit vaccination against avian coccidiosis. Broiler chickens were immunized subcutaneously with a purified Eimeria acervulina recombinant profilin protein, either alone or mixed with one of four adjuvants (ISA 70 VG, ISA 71 VG, ISA 201 VG or ISA 206 VG), and body weight gains, fecal oocyst shedding, and humoral and innate immune responses were evaluated following oral challenge infection with live E. acervulina oocysts. Immunization with profilin plus ISA 70 VG or ISA 71 VG increased body weight gains compared with vaccination with profilin alone. Profilin plus ISA 71 VG also reduced fecal oocyst shedding compared with vaccination in the absence of adjuvant. All adjuvants enhanced profilin serum antibody titers. Increased levels of gene transcripts encoding IL-2, IL-10, IL-17A, and IFN-γ, but decreased levels of IL-15 mRNAs, were seen in intestinal intraepithelial lymphocytes of chickens immunized with profilin plus adjuvants compared with immunization with profilin alone. Finally, increased infiltration of lymphocytes, especially CD8 + lymphocytes at the site of immunization was observed in birds given profilin plus ISA 71 VG compared with profilin alone. These results demonstrate that vaccination with the E. acervulina profilin subunit vaccine in combination with Montanide™ adjuvants enhances protective immunity against avian coccidiosis.

Component‐resolved diagnosis from latex allergy by microarray

A positive specific IgE (sIgE) result for latex does not always mirror the clinical situation and is frequently found in individuals without overt latex allergy. We sought to investigate the potential of component-resolved diagnosis (CRD) of latex allergy by microarray and to assess whether the technique allows discriminating genuine allergy from asymptomatic sensitization. Twenty-six healthy controls without a history of latex allergy with a negative latex sIgE and skin test, 22 latex-allergic patients with a compelling history of latex allergy with a positive latex sIgE and prick test and 20 latex-sensitized individuals with a frequent asymptomatic exposure to natural rubber latex-containing devices with a negative latex skin test but a positive sIgE were also included. CRD was performed with the ImmunoCAP ISAC microarray and traditional singleplexed ImmunoCAP. In all patients, the diagnosis of latex allergy could be established by the combination of recombinant latex components present on the microarray (Hev b 1, Hev b 3, Hev b 5 and Hev b 6.02). Over three-quarters of our patients were sensitized for Hev b 5 and/or Hev b 6.02. Some patients also displayed reactivity for Hev b 1 and/or Hev b 3. In contrast, none of the individuals sensitized to natural rubber latex or control individuals demonstrated IgE reactivity for rHev b 1, rHev b 3, rHev b 5 or rHev b 6.02. Three-quarters of the patients sensitized to latex displayed a positive microarray result for recombinant latex profilin (rHev b 8). In contrast to the results obtained by traditional ImmunoCAP for bromelain, almost no sensitization for cross-reactive carbohydrates was demonstrated by bromelain spotted on the microarray. CRD by traditional singleplexed ImmunoCAP showed highly comparable results. CRD by microarray is a reliable tool for diagnosing latex allergy. In addition, the technique allows discrimination between genuine allergy and sensitization. CRD by microarray can improve the diagnosis of IgE-mediated latex allergy by discriminating between genuine allergy and sensitization. CRD by microarray is a reliable tool to diagnose latex allergy. In addition, the technique allows discrimination between a genuine allergy and simple sensitization.

The Interaction of Proline-Rich Ligands with Profilin Probed with an Enzyme-Linked Immunosorbent Assay

To detect interactions of different proline-rich ligands with profilins, the authors developed a simple analytical antibody-based screening method. Profilin I or profilin IIa was coated in microplates, and ligand binding was monitored via antibody detection. Using purified components, the authors show that the assay is very sensitive as nanomolar concentrations of recombinant profilin ligands can be used. They further apply this technique to detect interaction of profilin with various proline-rich partners, either endogenously present or ectopically expressed as tagged fusions, using lysates. With this assay, the authors identify Shootin1 as a novel profilin IIa partner. In addition, they demonstrate that this assay can be used for studying competition or ternary complex formation. In conclusion, they developed a sensitive, easy-to-use, and versatile method for the study of the interaction between profilin and different ligands.

Cloning and Expression of the Allergen Cro s 2 Profilin from Saffron (Crocus sativus)

Profilin is a panallergen that is recognized by IgE in allergic patients. Allergy to saffron (Crocus sativus) pollen has been described in people exposed to its pollen. Saffron contains a profilin that may cause allergic reactions in atopic subjects. The aim of this study was to describe the cloning, expression and purification of saffron profilin from pollen. Cloning of saffron profilin was performed by polymerase chain reaction using specific primers from saffron pollen RNA. Expression was carried out in Escherichia coli BL21 (DE3) using a vector pET-102- TOPO. A recombinant fusion protein was expressed and the recombinant profilin was purified by metal precipitation. Immunological characterization was performed by immunoblotting experiments. The 34kDa- recombinant saffron profilin, Cro s 2, as a fusion protein was purified. Immunoblotting tested with the sera of allergic patients showed a specific reaction with the recombinant Cro s 2 band. The sequence of Cro s 2 showed a high degree of identity and similarity to other plant profilins and the recombinant saffron profilin, Cro s 2, may be used for target-specific diagnosis and structural analyses and investigation of cross reactivity of Cro s 2 with other plant profilins.

Expression Analysis and Characteristics of Profilin Gene from Silkworm, Bombyx mori

A recombinant Bombyx mori profilin protein (rBmPFN) was overexpressed in Escherichia coli BL21. Purified rBmPFN was used to generate anti-BmPFN polyclonal antibody, which were used to determine the subcellular localization of BmPFN. Immunostaining indicated that profilin can be found in both the nucleus and cytoplasm but is primarily located in the cytoplasm. Real-time RT-PCR and Western blot analyses indicated that, during the larvae stage, profilin expression levels are highest in the silk gland, followed by the gonad, and are lowest in the fatty body. Additionally, BmPFN expression begins during the egg stage, increases during the larvae stage, reaches a peak during the pupa stage, and decreases significantly in the moth. Therefore, we propose that BmPFN may play an important role during larva stage development, especially in the silk gland.

One‐step purification of histidine‐tagged profilin with high purity and yield by using metal precipitation

A simple one-step method for the purification of recombinant His-tagged profilin from the bacterial cell lysate is reported. Noting the greater ease with which hexahistidine-tagged proteins can be metalprecipitated compared with unwanted protein impurities, we investigated the effect of lysis-buffer additives and optimization of other conditions to recover selectively desired proteins in a one-step metal precipitation without using biopolymers. Purification of the His-tagged melon (Cucumis melo) profilin was used to demonstrate the utility of this method and up to 80% recovery with a purity of 98% was achieved. This method obtained a yield of the protein nearly comparable with that obtained using metal-affinity chromatography. This purification procedure can reduce the time and cost of the purification process, especially on a large scale.

Cloning of the panallergen profilin from lychee fruit and its cross-reactivity with birch pollen profilin Bet v 2

Lychee fruits can cause severe anaphylactic reactions and has cross-reactivity with other allergens. Panallergen profilin is partly responsible for the cross-reactivity between allergens. In the present study, the cDNA of lychee profilin was performed by RT-PCR and 3′RACE from total RNA. The lychee profilin cDNA includes an open reading frame coding for 131 amino acids. The deduced amino acid sequence of the corresponding protein show high identity with other allergenic profilins. Expression of the recombinant lychee profilin was carried out in Escherichia coli BL21 (DE3) using vector PET-28a, and the purification of the recombinant protein was performed via affinity chromatography with Ni+ coupled to sepharose. IgE reactivity of recombinant lychee profilin was investigated by immunoblot, and five of 15 lychee-allergic patients tested presented specific IgE antibodies to recombinant lychee profilin. IgE inhibition and ELISA inhibition experiments were performed to analyze lychee profilin cross-reactivity with profilins from birch pollen, and a high cross-reactivity between lychee profilin and birch profilin has been found in sera from lychee allergic patients.

Mango profilin: cloning, expression and cross-reactivity with birch pollen profilin Bet v 2

Mango can cause severe anaphylactic reactions. Profilin has been assumed partly responsible for the cross-reactivity between mango fruit and other allergens but has not been finally clarified. In this study, two isoforms of mango fruits profilin were amplified by RT-PCR and 3'RACE from total RNA. Each mango profilin cDNA includes an open reading frame coding for 131 amino acids. The deduced amino acid sequence of the corresponding protein show high identity with other allergenic profilins. Expression of the recombinant mango profilin was carried out in Escherichia coli BL21(DE3) using vector PET28a and the purification of the recombinant protein was performed via affinity chromatography with Ni+ coupled to sepharose. IgE reactivity of recombinant mango profilin was investigated by immunoblot and 8 of 18 mango-allergic patients tested presented specific IgE-antibodies to recombinant mango profilin. IgE-inhibition and ELISA inhibition experiments were performed to analyze mango profilin cross-reactivity with profilins from birch pollen and high cross-reactivities have been found.

Characterization of recombinant Mal d 4 and its application for component‐resolved diagnosis of apple allergy

Profilins are ubiquitous panallergens that have been extensively characterized; yet, their clinical relevance is still unclear. The aim of the present study was to produce recombinant apple profilin (rMal d 4) and to evaluate its allergenic activity and its potency for component-resolved allergy diagnosis. Complementary DNA-derived Mal d 4 was cloned, expressed in Escherichia coli and subsequently purified via poly (l-proline) sepharose. A total of 28 sera from apple-allergic patients were used for IgE-ELISA, immunoblot, RAST and basophil histamine release (BHR) test. In addition, skin prick tests (SPTs) were performed in five patients. Four different complementary DNA coding for apple profilin, Mal d 4, each with an open reading frame of 393 nucleotides, were identified. One isoform Mal d 4.0101 was expressed in Escherichia coli and subsequently purified. Mass spectroscopy revealed the expected mass of 13.826 for rMal d 4.0101, and circular dichroism analysis data were typical for a folded protein and small-angle X-ray scattering measurement identified the protein as a monomer. All the serum samples displayed IgE binding to rMal d 4.0101 in IgE ELISA, immunoblot and RAST. In immunoblotting, IgE binding to natural Mal d 4 was partially/completely inhibited by preincubation with rMal d 4.0101, and RAST values to apple extract were significantly reduced upon serum pretreatment with rMal d 4.0101. SPTs and BHR assays using purified rMal d 4.0101 were positive. Purified rMal d 4.0101 was destroyed within seconds when subjected to pepsin digestion. Apple profilin complementary DNAs were identified. The physicochemical and allergenic properties of purified recombinant Mal d 4.0101 were evaluated showing that the recombinant protein was equal to the natural protein as shown by inhibition assays. Thus, Mal d 4 represents another example suitable for component-resolved diagnosis of food allergy.

Cloning and characterization of profilin (Pru du 4), a cross-reactive almond ( Prunus dulcis) allergen

The identity of allergenic almond proteins is incomplete. Our objective was to characterize patient IgE reactivity to a recombinant and corresponding native almond allergen. An almond cDNA library was screened with sera from patients with allergy for IgE binding proteins. Two reactive clones were sequenced, and 1 was expressed. The expressed recombinant allergen and its native counterpart (purified from unprocessed almond flour) were assayed by 1-dimensional and 2-dimensional gel electrophoresis, dot blot, and ELISA, and screened for cross-reactivity with grass profilin. The 2 selected clones encoded profilin (designated Pru du 4) sequences that differed by 2 silent mutations. By dot-blot analyses, 6 of 18 patient sera (33%) reacted with the recombinant Pru du 4 protein, and 8 of 18 (44%) reacted with the native form. ELISA results were similar. Almond and ryegrass profilins were mutually inhibitable. Two-dimensional immunoblotting revealed the presence of more than 1 native almond profilin isoform. The strength of reactivity of some patients' serum IgE differed markedly between assays and between native and recombinant profilins. Almond nut profilin is an IgE-binding food protein that is cross-reactive with grass pollen profilin and is susceptible to denaturation, resulting in variable reactivity between assay types and between patients. Serum IgE of nearly half of the tested patients with almond allergy reacts with almond nut profilin. Because most patients also had pollinosis, the well-known cross-reactivity between pollen and food profilins could account for this pattern of reactivity.

Mouse Profilin 2 Regulates Endocytosis and Competes with SH3 Ligand Binding to Dynamin 1

Mammalian profilins are abundantly expressed actin monomer-binding proteins, highly conserved with respect to their affinities for G-actin, poly-L-proline, and phosphoinositides. Profilins associate with a large number of proline-rich proteins; the physiological significance and regulation of which is poorly understood. Here we show that profilin 2 associates with dynamin 1 via the C-terminal proline-rich domain of dynamin and thereby competes with the binding of SH3 ligands such as endophilin, amphiphysin, and Grb2, thus interfering with the assembly of the endocytic machinery. We also present a novel role for the brain-specific mouse profilin 2 as a regulator of membrane trafficking. Overexpression of profilin 2 inhibits endocytosis, whereas lack of profilin 2 in neurons results in an increase in endocytosis and membrane recycling. Phosphatidylinositol 4,5-bisphosphate releases profilin 2 from the profilin 2-dynamin 1 complex as well as from the profilin 2-actin complex, suggesting that profilin 2 is diverging the phosphoinositide signaling pathway to actin polymerization as well as endocytosis.