Experimental Parasitology 164 (2016) 1e4
Contents lists available at ScienceDirect
Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr
Research brief
Echinococcus granulosus fatty acid binding proteins subcellular localization Gabriela Alvite*, Adriana Esteves 4225, C.P. 11400 Montevideo, Uruguay n Bioquímica, Facultad de Ciencias, Universidad de la República, Igua Seccio
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Echinococcus granulosus FABPs subcellular localization was analyzed by 2D-PAGE, WB and MS. Cytosolic, nuclear, mitochondrial and microsomal larvae fractions were purified. Three EgFABP1 isoforms were identified in each fraction. Probably EgFABP2 protein expression is low or absent in protoscoleces. EgFABP1 isoforms could be involved in several cellular processes.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 July 2015 Received in revised form 4 February 2016 Accepted 5 February 2016 Available online 10 February 2016
Two fatty acid binding proteins, EgFABP1 and EgFABP2, were isolated from the parasitic platyhelminth Echinococcus granulosus. These proteins bind fatty acids and have particular relevance in flatworms since de novo fatty acids synthesis is absent. Therefore platyhelminthes depend on the capture and intracellular distribution of host's lipids and fatty acid binding proteins could participate in lipid distribution. To elucidate EgFABP's roles, we investigated their intracellular distribution in the larval stage by a proteomic approach. Our results demonstrated the presence of EgFABP1 isoforms in cytosolic, nuclear, mitochondrial and microsomal fractions, suggesting that these molecules could be involved in several cellular processes. © 2016 Elsevier Inc. All rights reserved.
Keywords: Fatty acid binding proteins Echinococcus granulosus
1. Introduction Fatty acid binding proteins (FABPs) are mainly intracellular proteins that belong to a multigene family of low molecular weight proteins (14e15 kDa) with a wide phylogenetic distribution (Esteves and Ehrlich, 2006; Storch and Thumser, 2010). These molecules bind non-covalently long chain fatty acids (FA) and other hydrophobic ligands. FABPs are named according to the tissue in which they were first identified or are predominantly expressed
* Corresponding author. E-mail address: [email protected] (G. Alvite). http://dx.doi.org/10.1016/j.exppara.2016.02.002 0014-4894/© 2016 Elsevier Inc. All rights reserved.
(Storch and Thumser, 2010), e.g. H-FABP for heart fatty acid binding protein. FABP family members exhibit low similarity in terms of primary structure but have a highly conserved tridimensional structure (Storch and Thumser, 2010). They are implicated in FA capture and intracellular transport; however the specific function of each FABP is still under investigation. Recently, different experimental approaches indicate that they could be involved in gene expression regulation (Hostetler et al., 2009) and lipid metabolism regulation (Smith et al., 2007). Several FABPs have been isolated and characterized in parasitic platyhelminthes (Zheng et al., 2013). These parasites are unable to synthesize de novo their own fatty acids and sterols (Smyth and McManus, 1989). In this scenario, parasitic platyhelminthes
2
G. Alvite, A. Esteves / Experimental Parasitology 164 (2016) 1e4
depend on the capture and intracellular distribution of host's lipids (Smyth and McManus, 1989) and an efficient lipid binding system is required. In this respect, FABPs could be key molecules involved in intracellular distribution of host's fatty acids (Alvite and Esteves, 2012). Our research group has isolated and characterized two highly similar fatty acid binding proteins from the cestode Echinococcus granulosus, named EgFABP1 and EgFABP2 (Esteves et al., 1993, 2003). These proteins share 76% of identical residues and exhibit significant sequence similarity to the vertebrate H-FABP type (Esteves and Ehrlich, 2006). In addition, EgFABP1 binds unsaturated long-chain FA with high affinity (Alvite et al., 2001). In order to decipher EgFABP's roles we investigated their intracellular distribution in the larval stage. Subcellular fractions were submitted to two-dimensional electrophoresis and analyzed by Western blot and mass spectrometry. The study of EgFABPs intracellular localization could help to elucidate the role of these proteins in lipid metabolism, gene expression regulation, and hosteparasite relationships.
2. Materials and methods Fertile hydatid cysts were collected from lungs and livers of naturally infected cattle in Uruguay. The protoscoleces (larvae stage) were aseptically aspirated, settled by gravity, and extensively washed in phosphate-buffered saline to remove dead protoscolex debris. They were observed under a light microscope for viability and immediately used. Protoscoleces nuclear, mitochondrial, microsomal and cytosolic subcellular fractions were obtained by sequential centrifugation according to Alvite and collaborators (Alvite et al., 2014). The enrichment of the subcellular fractions was monitored as described previously (Alvite et al., 2014). Subcellular fractions were purified with PlusOne 2-D Clean-Up Kit (Amersham Biosciences, USA) and concentrated for further studies. Protein extracts of each enriched fraction (20 mg) were analyzed by two two-dimensional electrophoresis (2D) replicas using Western Blot and mass spectrometry MALDI/TOFeTOF (MS). Protein quantifications were performed using BCA kit (Sigma Aldrich Co., USA). 2D were performed according Alvite and collaborators (Alvite et al., 2014). After the electrophoresis, one gel was electrotransferred to a PVDF Hybond-P membrane (Amersham Biosciences) and the membrane was blocked and incubated with the
rabbit polyclonal anti-EgFABP1 serum. The signal was developed by the alkaline phosphatase reaction. The second gel was stained with silver nitrate as described previously (Alvite et al., 2014). In some cases, the spots identified by Western Blots with the expected mass and isoelectric point (IP) were excised from de second gel for MS identification. Peptide mass fingerprints were submitted to MASCOT (Matrix Science http://www.matrixscience.com/search_ form_select.html) software. Criteria to protein identification include: MASCOT scores, sequence coverage, molecular mass and IP of EgFABPs. 2D and MS were performed in the UByPA service of Institut Pasteur de Montevideo.
3. Results and discussion Since parasitic platyhelminthes depend on lipids uptake from the host, an efficient mechanism of transport between cells and intracellular compartments should exist. To investigate the putative role of EgFABPs in FA subcellular distribution, we performed EgFABPs Western Blot identification of nuclear, mitochondrial, microsomal and cytosolic enriched fractions. Fractions enrichment is shown in Table 1. Previously, we demonstrated that anti-EgFABP1 serum recognizes EgFABP1 and EgFABP2 recombinant proteins (Alvite, 2014; supplementary material). Theoretical molecular mass and isoelectric point of EgFABP1 and EgFABP2 are 15 065.4 Da and 7.7, and 15 408.7 Da and 6.37, respectively. The anti-EgFABP1 serum identified three spots with similar molecular mass and with isoelectric points of 8 (15 kDa), 7.7 (15 kDa) and 6.5 (15.2 kDa) in the cytosolic, nuclear, mitochondrial and microsomal enriched fractions (Fig. 1). The three spots of the cytosolic enriched fraction were statistically identified as EgFABP1 by MS with a coverage of 40%. In addition, the nuclear 7.7 isoform was identified by MS. In all fractions the 7.7 spots corresponded to the most abundant EgFABP1 isoform (Fig. 1). We suggest that isoforms with IP of 8 and 6.5 should have posttranslational modifications. The isoform with IP 6.5 could have a phosphorylation, as was reported for rat H-FABP (Nielsen and Spener, 1993). A phosphorylation consensus motif was detected in EgFABP1, where Thr 29 could be the phosphorylated residue (Paulino et al., 1998). Six cytosolic (Fig. 1E) and two nuclear proteins (data not shown) were analyzed by MS, taking into account the molecular mass and the isoelectric point of EgFABP2. However, neither of them matched with EgFABP2. One of the cytosolic spots (IP 6.3, 16.3 kDa) was
Table 1 Purity of enriched fractions.
Lactate Dehydrogenase Homogenate Nuclear fraction Mitochondrial fraction Microsomal fraction Cytosolic fraction Succinate Dehydrogenase Homogenate Nuclear fraction Mitochondrial fraction Microsomal fraction Cytosolic fraction Peroxidase Homogenate Nuclear fraction Mitochondrial fraction Microsomal fraction Cytosolic fraction
Enz. act. (U/ml)
[Prot] (mg/ml)
Sp. act. (U/mg)
Purific. f.
0.0080 ± 0.0016 0 0 0 0.0169 ± 0.0007
1.68 0.688 0.943 1.143 0.871
0.0048 ± 0.0010 0 0 0 0.0194 ± 0.0008
1.00 ± 0.21 0 0 0 4.04 ± 0.17
± ± ± ±
1.68 0.688 0.943 1.143 0.871
0.0053 0.0007 0.0201 0.0012 0
0.0089 0.0005 0.0190 0.0014 0
0.0005 0.0007 0.0010 0.0003
9.7 ± 0.27 0 0 12.45 ± 0.34 0
1.68 0.688 0.943 1.143 0.871
± ± ± ±
0.0003 0.0010 0.0011 0.0003
5.77 ± 0.16 0 0 10.89 ± 0.30 0
1.00 0.13 3.79 0.22 0
± ± ± ±
0.06 0.19 0.21 0.06
1.00 ± 0.03 0 0 1.89 ± 0.05 0
The enzymatic activity (Enz. act.), specific activity (Sp. act.) and the purification factor (Purif. f.) are shown with the standard deviations. [Prot]: protein concentration.
G. Alvite, A. Esteves / Experimental Parasitology 164 (2016) 1e4
3
Fig. 1. 2D electrophoresis and EgFABPs Western Blots. A) Cytosolic, B) nuclear, C) mitochondrial, and D) microsomal protoscoleces enriched fractions. Black and dashed arrows: spots identified by MS and Western Blot, respectively. E) Cytosolic enriched fraction 2D electrophoresis. Black and dotted arrows: spots identified as EgFABP1 by MS and proteins unidentified, respectively. Thick black arrow: protein similar to the hypothetical protein from E. multilocularis. Load: 100 mg of protein extract.
identified as a protein similar to a putative E. multilocularis protein (Em_CW_03A07_T7) (Fig. 1E). This result suggests that EgFABP2 protein expression is low or absent in protoscoleces. It is noteworthy that EgFABP2 cDNA was cloned from protoscoleces (Esteves et al., 2003), for this reason a post-transcriptional regulation mechanism could be possible. These findings differ from those obtained previously for Mesocestoides vogae, where two MvFABPs
were identified in the analyzed compartments and no isoforms were found (Alvite et al., 2014). This fact could be the result of a subtle metabolic regulation since M. vogae larvae were cultured prior protein extraction while E. granulosus larvae were not. Several vertebrates’ FABP members have been localized in the nuclei, mitochondria and Golgi of cultured cells (Hostetler et al., 2009; Thumser and Storch, 2007). Mitochondrial localization of
4
G. Alvite, A. Esteves / Experimental Parasitology 164 (2016) 1e4
EgFABP1 isoforms could be indicative of a relationship with the oxidation processes. This result is rather unexpected because the beta-oxidation pathway is considered to be inactive in parasitic €rting, platyhelminthes (Smyth and McManus, 1989; Barrett and Ko 1976). However, it has been reported that some platyhelminthes € rting, 1976). express beta-oxidation enzymes (Barrett and Ko Vinaud and co-workers showed that Taenia crassiceps is capable of producing energy from lipids as an alternative energy source in glucose shortage (Vinaud et al., 2007). We cannot discard the hypothesis that FABPs could be a source of FAs for mitochondrial energetic metabolism in E. granulosus. EgFABP1 was also detected in the microsomal enriched fraction. This fraction comprises small heterogeneous vesicles composed by endoplasmic reticulum, Golgi, plasma membrane, peroxisomes and lysosomes. This complexity makes difficult to attribute a putative role to these proteins. The identification of EgFABP1 isoforms in the nuclear fraction suggests that they could be involved in nuclear lipids synthesis, lipid droplets formation, or gene expression regulation. Nuclear phospholipid biosynthetic pathways and nuclear lipid droplets have been identified (Layerenza et al., 2013). Various experimental approaches have shown that different FABPs are targeted to nuclei € rchers et al., 1989; Hostetler et al., 2009), and many FABPs (Bo activate PPARs by direct interaction with these transcription factors (Hostetler et al., 2009; Tan et al., 2002). Members of the nuclear receptor family have been identified in cestodes (www.genedb. org), so we cannot discard a role of EgFABP1 in gene expression regulation. 4. Conclusions Our results show the presence of three EgFABP1 isoforms in the cytosolic, nuclear, mitochondrial and microsomal fraction of E. granulosus protoscoleces. These findings extend the canonical role of taenia FABPs as simple cytoplasmic FA carriers. The ancestral FABP could play roles related to FA oxidation and gene expression regulation. The reduced diversity in invertebrates, in combination with our localization results, suggests that these molecules have a larger repertoire of interactions in the cell than vertebrate FABPs. Therefore, it is likely that the ancestral FABP could satisfy all the functions of the actual vertebrate's proteins of the family (Esteves and Ehrlich, 2006). Future work will be performed to describe with more accuracy the localization in each cellular compartment as well as to identify putative molecular partners. Acknowledgements This work was supported by Programa de Desarrollo de las sicas (www.pedeciba.edu.uy), Comisio n Sectorial de Ciencias Ba n Científica (www.csic.edu.uy, NO 4003, 2008e2010; Investigacio n e CAP fellowship) and Agencia Nacional de Investigacio n (www.anii.org.uy, FCE2007_57). Innovacio
Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.exppara.2016.02.002.
References Alvite, G., 2014. FABPs de cestodos: interacciones y destinos. PhD thesis. PEDECIBA, Uruguay. Alvite, G., Esteves, A., 2012. Lipid binding proteins from parasitic platyhelminthes. Front. Physio. 3, 363. , J.A., Ehrlich, R., Esteves, A., 2001. Binding Alvite, G., Di Pietro, S.M., Santome properties of Echinococcus granulosus fatty acid binding protein. Biochim. Biophys. Acta 1533, 293e302. Alvite, G., Garrido, N., Kun, A., Paulino, M., Esteves, A., 2014. Towards an understanding of Mesocestoides vogae fatty acid binding proteins' roles. PLOS ONE 9, e111204. € rting, W., 1976. Studies on beta-oxidation in the adult liver fluke Barrett, J., Ko Fasciola hepatica. Int. J. Parasitol. 6, 155e157. €rchers, T., Unterberg, C., Rtidel, H., Robenek, H., Spener, F., 1989. Subcellular Bo distribution of cardiac fatty acid-binding protein in bovine heart muscle and quantitation with an enzyme-linked immunosorbent assay. Biochim. Biophys. Acta 1002, 54e61. Esteves, A., Dallagiovanna, B., Ehrlich, R., 1993. A developmentally regulated gene of Echinococcus granulosus codes for a 15.5 kilodalton polypeptide related to fatty acid binding proteins. Mol. Biochem. Parasitol. 58, 215e222. Esteves, A., Ehrlich, R., 2006. Invertebrate intracellular fatty acid binding proteins. Comp. Biochem. Physiol. C 142, 262e274. Esteves, A., Portillo, V., Ehrlich, R., 2003. Genomic structure and expression of a gene coding for a new fatty acid binding protein from Echinococcus granulosus. Biochim. Biophys. Acta 1631, 26e34. Hostetler, H.A., McIntosh, A.L., Atshaves, B.P., Atshaves, B.P., Storey, S.M., Payne, H.R., Kier, A.B., Schroeder, F., 2009. L-FABP directly interacts with PPAR in cultured primary hepatocytes. J. Lipid Res. 50, 1663e1675. lez, P., García, M.M., Polo, M.P., Sisti, M.S., Ves-Losada, A., 2013. Layerenza, J.P., Gonza Nuclear lipid droplets: a novel nuclear domain. Biochim. Biophys. Acta 1831, 327e340. Nielsen, S.U., Spener, F., 1993. Fatty acid-binding protein from rat heart is phosphorylated on Tyr19 in response to insulin stimulation. J. Lipid Res. 34, 1355e1366. Paulino, M., Esteves, A., Vega, M., Tabares, G., Ehrlich, R., Tapia, O., 1998. Modelling a 3D structure for EgDf1 from Echinococcus granulosus: putative epitopes, phosphorylation motifs and ligand. J. Comput. Aided Mol. Des. 12, 351e360. Smith, B.R., Thompson, B.R., Sanders, M.A., Bernlohr, D.A., 2007. Interaction of the adipocyte fatty acid-binding protein with the hormone-sensitive lipase: regulation by fatty acids and phosphorylation. J. Biol. Chem. 44, 32424e32432. Smyth, J.D., McManus, D.P., 1989. The adult: general metabolism and chemical composition. In: The Physiology and Biochemistry of Cestodes. Cambridge University Press, Cambridge, pp. 53e76. Storch, J., Thumser, A.E., 2010. Tissue-specific functions in the fatty acid-binding protein family. J. Biol. Chem. 285, 32679e32683. Tan, N.S., Shaw, N.S., Vinckenbosch, N., Liu, P., Yasmin, R., Desvergne, B., Wahli, W., Noy, N., 2002. Selective cooperation between fatty acid binding proteins and peroxisome proliferator-activated receptors in regulating transcription. Mol. Cell. Biol. 22, 5114e5127. Thumser, A.E., Storch, J., 2007. Characterization of a BODIPY-labeled fluorescent fatty acid analogue. Binding to fatty acid-binding proteins, intracellular localization, and metabolism. Mol. Cell. Biochem. 299, 67e73. Vinaud, M.C., de Souza, R., Barreto, J.C., 2007. Taenia crassiceps organic acids detected in cysticerci. Exp. Parasitol. 116, 335e339. Zheng, Y., Blair, D., Bradley, J., 2013. Phyletic distribution of fatty acid-binding protein genes. PLOS ONE 8, e77636.
Contents lists available at ScienceDirect
Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr
Research brief
Echinococcus granulosus fatty acid binding proteins subcellular localization Gabriela Alvite*, Adriana Esteves 4225, C.P. 11400 Montevideo, Uruguay n Bioquímica, Facultad de Ciencias, Universidad de la República, Igua Seccio
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Echinococcus granulosus FABPs subcellular localization was analyzed by 2D-PAGE, WB and MS. Cytosolic, nuclear, mitochondrial and microsomal larvae fractions were purified. Three EgFABP1 isoforms were identified in each fraction. Probably EgFABP2 protein expression is low or absent in protoscoleces. EgFABP1 isoforms could be involved in several cellular processes.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 July 2015 Received in revised form 4 February 2016 Accepted 5 February 2016 Available online 10 February 2016
Two fatty acid binding proteins, EgFABP1 and EgFABP2, were isolated from the parasitic platyhelminth Echinococcus granulosus. These proteins bind fatty acids and have particular relevance in flatworms since de novo fatty acids synthesis is absent. Therefore platyhelminthes depend on the capture and intracellular distribution of host's lipids and fatty acid binding proteins could participate in lipid distribution. To elucidate EgFABP's roles, we investigated their intracellular distribution in the larval stage by a proteomic approach. Our results demonstrated the presence of EgFABP1 isoforms in cytosolic, nuclear, mitochondrial and microsomal fractions, suggesting that these molecules could be involved in several cellular processes. © 2016 Elsevier Inc. All rights reserved.
Keywords: Fatty acid binding proteins Echinococcus granulosus
1. Introduction Fatty acid binding proteins (FABPs) are mainly intracellular proteins that belong to a multigene family of low molecular weight proteins (14e15 kDa) with a wide phylogenetic distribution (Esteves and Ehrlich, 2006; Storch and Thumser, 2010). These molecules bind non-covalently long chain fatty acids (FA) and other hydrophobic ligands. FABPs are named according to the tissue in which they were first identified or are predominantly expressed
* Corresponding author. E-mail address: [email protected] (G. Alvite). http://dx.doi.org/10.1016/j.exppara.2016.02.002 0014-4894/© 2016 Elsevier Inc. All rights reserved.
(Storch and Thumser, 2010), e.g. H-FABP for heart fatty acid binding protein. FABP family members exhibit low similarity in terms of primary structure but have a highly conserved tridimensional structure (Storch and Thumser, 2010). They are implicated in FA capture and intracellular transport; however the specific function of each FABP is still under investigation. Recently, different experimental approaches indicate that they could be involved in gene expression regulation (Hostetler et al., 2009) and lipid metabolism regulation (Smith et al., 2007). Several FABPs have been isolated and characterized in parasitic platyhelminthes (Zheng et al., 2013). These parasites are unable to synthesize de novo their own fatty acids and sterols (Smyth and McManus, 1989). In this scenario, parasitic platyhelminthes
2
G. Alvite, A. Esteves / Experimental Parasitology 164 (2016) 1e4
depend on the capture and intracellular distribution of host's lipids (Smyth and McManus, 1989) and an efficient lipid binding system is required. In this respect, FABPs could be key molecules involved in intracellular distribution of host's fatty acids (Alvite and Esteves, 2012). Our research group has isolated and characterized two highly similar fatty acid binding proteins from the cestode Echinococcus granulosus, named EgFABP1 and EgFABP2 (Esteves et al., 1993, 2003). These proteins share 76% of identical residues and exhibit significant sequence similarity to the vertebrate H-FABP type (Esteves and Ehrlich, 2006). In addition, EgFABP1 binds unsaturated long-chain FA with high affinity (Alvite et al., 2001). In order to decipher EgFABP's roles we investigated their intracellular distribution in the larval stage. Subcellular fractions were submitted to two-dimensional electrophoresis and analyzed by Western blot and mass spectrometry. The study of EgFABPs intracellular localization could help to elucidate the role of these proteins in lipid metabolism, gene expression regulation, and hosteparasite relationships.
2. Materials and methods Fertile hydatid cysts were collected from lungs and livers of naturally infected cattle in Uruguay. The protoscoleces (larvae stage) were aseptically aspirated, settled by gravity, and extensively washed in phosphate-buffered saline to remove dead protoscolex debris. They were observed under a light microscope for viability and immediately used. Protoscoleces nuclear, mitochondrial, microsomal and cytosolic subcellular fractions were obtained by sequential centrifugation according to Alvite and collaborators (Alvite et al., 2014). The enrichment of the subcellular fractions was monitored as described previously (Alvite et al., 2014). Subcellular fractions were purified with PlusOne 2-D Clean-Up Kit (Amersham Biosciences, USA) and concentrated for further studies. Protein extracts of each enriched fraction (20 mg) were analyzed by two two-dimensional electrophoresis (2D) replicas using Western Blot and mass spectrometry MALDI/TOFeTOF (MS). Protein quantifications were performed using BCA kit (Sigma Aldrich Co., USA). 2D were performed according Alvite and collaborators (Alvite et al., 2014). After the electrophoresis, one gel was electrotransferred to a PVDF Hybond-P membrane (Amersham Biosciences) and the membrane was blocked and incubated with the
rabbit polyclonal anti-EgFABP1 serum. The signal was developed by the alkaline phosphatase reaction. The second gel was stained with silver nitrate as described previously (Alvite et al., 2014). In some cases, the spots identified by Western Blots with the expected mass and isoelectric point (IP) were excised from de second gel for MS identification. Peptide mass fingerprints were submitted to MASCOT (Matrix Science http://www.matrixscience.com/search_ form_select.html) software. Criteria to protein identification include: MASCOT scores, sequence coverage, molecular mass and IP of EgFABPs. 2D and MS were performed in the UByPA service of Institut Pasteur de Montevideo.
3. Results and discussion Since parasitic platyhelminthes depend on lipids uptake from the host, an efficient mechanism of transport between cells and intracellular compartments should exist. To investigate the putative role of EgFABPs in FA subcellular distribution, we performed EgFABPs Western Blot identification of nuclear, mitochondrial, microsomal and cytosolic enriched fractions. Fractions enrichment is shown in Table 1. Previously, we demonstrated that anti-EgFABP1 serum recognizes EgFABP1 and EgFABP2 recombinant proteins (Alvite, 2014; supplementary material). Theoretical molecular mass and isoelectric point of EgFABP1 and EgFABP2 are 15 065.4 Da and 7.7, and 15 408.7 Da and 6.37, respectively. The anti-EgFABP1 serum identified three spots with similar molecular mass and with isoelectric points of 8 (15 kDa), 7.7 (15 kDa) and 6.5 (15.2 kDa) in the cytosolic, nuclear, mitochondrial and microsomal enriched fractions (Fig. 1). The three spots of the cytosolic enriched fraction were statistically identified as EgFABP1 by MS with a coverage of 40%. In addition, the nuclear 7.7 isoform was identified by MS. In all fractions the 7.7 spots corresponded to the most abundant EgFABP1 isoform (Fig. 1). We suggest that isoforms with IP of 8 and 6.5 should have posttranslational modifications. The isoform with IP 6.5 could have a phosphorylation, as was reported for rat H-FABP (Nielsen and Spener, 1993). A phosphorylation consensus motif was detected in EgFABP1, where Thr 29 could be the phosphorylated residue (Paulino et al., 1998). Six cytosolic (Fig. 1E) and two nuclear proteins (data not shown) were analyzed by MS, taking into account the molecular mass and the isoelectric point of EgFABP2. However, neither of them matched with EgFABP2. One of the cytosolic spots (IP 6.3, 16.3 kDa) was
Table 1 Purity of enriched fractions.
Lactate Dehydrogenase Homogenate Nuclear fraction Mitochondrial fraction Microsomal fraction Cytosolic fraction Succinate Dehydrogenase Homogenate Nuclear fraction Mitochondrial fraction Microsomal fraction Cytosolic fraction Peroxidase Homogenate Nuclear fraction Mitochondrial fraction Microsomal fraction Cytosolic fraction
Enz. act. (U/ml)
[Prot] (mg/ml)
Sp. act. (U/mg)
Purific. f.
0.0080 ± 0.0016 0 0 0 0.0169 ± 0.0007
1.68 0.688 0.943 1.143 0.871
0.0048 ± 0.0010 0 0 0 0.0194 ± 0.0008
1.00 ± 0.21 0 0 0 4.04 ± 0.17
± ± ± ±
1.68 0.688 0.943 1.143 0.871
0.0053 0.0007 0.0201 0.0012 0
0.0089 0.0005 0.0190 0.0014 0
0.0005 0.0007 0.0010 0.0003
9.7 ± 0.27 0 0 12.45 ± 0.34 0
1.68 0.688 0.943 1.143 0.871
± ± ± ±
0.0003 0.0010 0.0011 0.0003
5.77 ± 0.16 0 0 10.89 ± 0.30 0
1.00 0.13 3.79 0.22 0
± ± ± ±
0.06 0.19 0.21 0.06
1.00 ± 0.03 0 0 1.89 ± 0.05 0
The enzymatic activity (Enz. act.), specific activity (Sp. act.) and the purification factor (Purif. f.) are shown with the standard deviations. [Prot]: protein concentration.
G. Alvite, A. Esteves / Experimental Parasitology 164 (2016) 1e4
3
Fig. 1. 2D electrophoresis and EgFABPs Western Blots. A) Cytosolic, B) nuclear, C) mitochondrial, and D) microsomal protoscoleces enriched fractions. Black and dashed arrows: spots identified by MS and Western Blot, respectively. E) Cytosolic enriched fraction 2D electrophoresis. Black and dotted arrows: spots identified as EgFABP1 by MS and proteins unidentified, respectively. Thick black arrow: protein similar to the hypothetical protein from E. multilocularis. Load: 100 mg of protein extract.
identified as a protein similar to a putative E. multilocularis protein (Em_CW_03A07_T7) (Fig. 1E). This result suggests that EgFABP2 protein expression is low or absent in protoscoleces. It is noteworthy that EgFABP2 cDNA was cloned from protoscoleces (Esteves et al., 2003), for this reason a post-transcriptional regulation mechanism could be possible. These findings differ from those obtained previously for Mesocestoides vogae, where two MvFABPs
were identified in the analyzed compartments and no isoforms were found (Alvite et al., 2014). This fact could be the result of a subtle metabolic regulation since M. vogae larvae were cultured prior protein extraction while E. granulosus larvae were not. Several vertebrates’ FABP members have been localized in the nuclei, mitochondria and Golgi of cultured cells (Hostetler et al., 2009; Thumser and Storch, 2007). Mitochondrial localization of
4
G. Alvite, A. Esteves / Experimental Parasitology 164 (2016) 1e4
EgFABP1 isoforms could be indicative of a relationship with the oxidation processes. This result is rather unexpected because the beta-oxidation pathway is considered to be inactive in parasitic €rting, platyhelminthes (Smyth and McManus, 1989; Barrett and Ko 1976). However, it has been reported that some platyhelminthes € rting, 1976). express beta-oxidation enzymes (Barrett and Ko Vinaud and co-workers showed that Taenia crassiceps is capable of producing energy from lipids as an alternative energy source in glucose shortage (Vinaud et al., 2007). We cannot discard the hypothesis that FABPs could be a source of FAs for mitochondrial energetic metabolism in E. granulosus. EgFABP1 was also detected in the microsomal enriched fraction. This fraction comprises small heterogeneous vesicles composed by endoplasmic reticulum, Golgi, plasma membrane, peroxisomes and lysosomes. This complexity makes difficult to attribute a putative role to these proteins. The identification of EgFABP1 isoforms in the nuclear fraction suggests that they could be involved in nuclear lipids synthesis, lipid droplets formation, or gene expression regulation. Nuclear phospholipid biosynthetic pathways and nuclear lipid droplets have been identified (Layerenza et al., 2013). Various experimental approaches have shown that different FABPs are targeted to nuclei € rchers et al., 1989; Hostetler et al., 2009), and many FABPs (Bo activate PPARs by direct interaction with these transcription factors (Hostetler et al., 2009; Tan et al., 2002). Members of the nuclear receptor family have been identified in cestodes (www.genedb. org), so we cannot discard a role of EgFABP1 in gene expression regulation. 4. Conclusions Our results show the presence of three EgFABP1 isoforms in the cytosolic, nuclear, mitochondrial and microsomal fraction of E. granulosus protoscoleces. These findings extend the canonical role of taenia FABPs as simple cytoplasmic FA carriers. The ancestral FABP could play roles related to FA oxidation and gene expression regulation. The reduced diversity in invertebrates, in combination with our localization results, suggests that these molecules have a larger repertoire of interactions in the cell than vertebrate FABPs. Therefore, it is likely that the ancestral FABP could satisfy all the functions of the actual vertebrate's proteins of the family (Esteves and Ehrlich, 2006). Future work will be performed to describe with more accuracy the localization in each cellular compartment as well as to identify putative molecular partners. Acknowledgements This work was supported by Programa de Desarrollo de las sicas (www.pedeciba.edu.uy), Comisio n Sectorial de Ciencias Ba n Científica (www.csic.edu.uy, NO 4003, 2008e2010; Investigacio n e CAP fellowship) and Agencia Nacional de Investigacio n (www.anii.org.uy, FCE2007_57). Innovacio
Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.exppara.2016.02.002.
References Alvite, G., 2014. FABPs de cestodos: interacciones y destinos. PhD thesis. PEDECIBA, Uruguay. Alvite, G., Esteves, A., 2012. Lipid binding proteins from parasitic platyhelminthes. Front. Physio. 3, 363. , J.A., Ehrlich, R., Esteves, A., 2001. Binding Alvite, G., Di Pietro, S.M., Santome properties of Echinococcus granulosus fatty acid binding protein. Biochim. Biophys. Acta 1533, 293e302. Alvite, G., Garrido, N., Kun, A., Paulino, M., Esteves, A., 2014. Towards an understanding of Mesocestoides vogae fatty acid binding proteins' roles. PLOS ONE 9, e111204. € rting, W., 1976. Studies on beta-oxidation in the adult liver fluke Barrett, J., Ko Fasciola hepatica. Int. J. Parasitol. 6, 155e157. €rchers, T., Unterberg, C., Rtidel, H., Robenek, H., Spener, F., 1989. Subcellular Bo distribution of cardiac fatty acid-binding protein in bovine heart muscle and quantitation with an enzyme-linked immunosorbent assay. Biochim. Biophys. Acta 1002, 54e61. Esteves, A., Dallagiovanna, B., Ehrlich, R., 1993. A developmentally regulated gene of Echinococcus granulosus codes for a 15.5 kilodalton polypeptide related to fatty acid binding proteins. Mol. Biochem. Parasitol. 58, 215e222. Esteves, A., Ehrlich, R., 2006. Invertebrate intracellular fatty acid binding proteins. Comp. Biochem. Physiol. C 142, 262e274. Esteves, A., Portillo, V., Ehrlich, R., 2003. Genomic structure and expression of a gene coding for a new fatty acid binding protein from Echinococcus granulosus. Biochim. Biophys. Acta 1631, 26e34. Hostetler, H.A., McIntosh, A.L., Atshaves, B.P., Atshaves, B.P., Storey, S.M., Payne, H.R., Kier, A.B., Schroeder, F., 2009. L-FABP directly interacts with PPAR in cultured primary hepatocytes. J. Lipid Res. 50, 1663e1675. lez, P., García, M.M., Polo, M.P., Sisti, M.S., Ves-Losada, A., 2013. Layerenza, J.P., Gonza Nuclear lipid droplets: a novel nuclear domain. Biochim. Biophys. Acta 1831, 327e340. Nielsen, S.U., Spener, F., 1993. Fatty acid-binding protein from rat heart is phosphorylated on Tyr19 in response to insulin stimulation. J. Lipid Res. 34, 1355e1366. Paulino, M., Esteves, A., Vega, M., Tabares, G., Ehrlich, R., Tapia, O., 1998. Modelling a 3D structure for EgDf1 from Echinococcus granulosus: putative epitopes, phosphorylation motifs and ligand. J. Comput. Aided Mol. Des. 12, 351e360. Smith, B.R., Thompson, B.R., Sanders, M.A., Bernlohr, D.A., 2007. Interaction of the adipocyte fatty acid-binding protein with the hormone-sensitive lipase: regulation by fatty acids and phosphorylation. J. Biol. Chem. 44, 32424e32432. Smyth, J.D., McManus, D.P., 1989. The adult: general metabolism and chemical composition. In: The Physiology and Biochemistry of Cestodes. Cambridge University Press, Cambridge, pp. 53e76. Storch, J., Thumser, A.E., 2010. Tissue-specific functions in the fatty acid-binding protein family. J. Biol. Chem. 285, 32679e32683. Tan, N.S., Shaw, N.S., Vinckenbosch, N., Liu, P., Yasmin, R., Desvergne, B., Wahli, W., Noy, N., 2002. Selective cooperation between fatty acid binding proteins and peroxisome proliferator-activated receptors in regulating transcription. Mol. Cell. Biol. 22, 5114e5127. Thumser, A.E., Storch, J., 2007. Characterization of a BODIPY-labeled fluorescent fatty acid analogue. Binding to fatty acid-binding proteins, intracellular localization, and metabolism. Mol. Cell. Biochem. 299, 67e73. Vinaud, M.C., de Souza, R., Barreto, J.C., 2007. Taenia crassiceps organic acids detected in cysticerci. Exp. Parasitol. 116, 335e339. Zheng, Y., Blair, D., Bradley, J., 2013. Phyletic distribution of fatty acid-binding protein genes. PLOS ONE 8, e77636.
