International Journal for Parasitology 46 (2016) 253–262

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International Journal for Parasitology

journal homepage: www.elsevier .com/locate / i jpara

The distinct C-terminal acidic domains of HMGB proteinsare functionally relevant in Schistosoma mansoni

http://dx.doi.org/10.1016/j.ijpara.2015.12.0070020-7519/� 2016 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

⇑ Corresponding author at: Instituto de Bioquímica Médica Leopoldo de Meis,Universidade Federal do Rio de Janeiro, CCS, Ilha do Fundão, Rio de Janeiro 21941-902, Brazil. Tel.: +55 21 3938 6608; fax: +55 21 2270 8647.

E-mail address: fantappie@bioqmed.ufrj.br (M.R. Fantappié).

Isabel Caetano de Abreu da Silva a, Vitor Coutinho Carneiro a, Amanda Roberta Revoredo Vicentino a,Estefania Anahi Aguilera b, Ronaldo Mohana-Borges b, Silvana Thiengo c, Monica Ammon Fernandez c,Marcelo Rosado Fantappié a,⇑a Instituto de Bioquímica Médica Leopoldo de Meis, Programa de Biologia Molecular e Biotecnologia, Universidade Federal do Rio de Janeiro, Brazilb Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brazilc Laboratório de Malacologia, Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Brazil

a r t i c l e i n f o

Article history:Received 19 September 2015Received in revised form 8 December 2015Accepted 10 December 2015Available online 26 January 2016

Keywords:Schistosoma mansoniHMGB proteinsDNA binding proteinsDNA transactions

a b s t r a c t

The Schistosoma mansoni High Mobility Group Box (HMGB) proteins SmHMGB1, SmHMGB2 andSmHMGB3 share highly conserved HMG box DNA binding domains but have significantly differentC-terminal acidic tails. Here, we used three full-length and tailless forms of the S. mansoni HMGB proteinsto examine the functional roles of their acidic tails. DNA binding assays revealed that the different lengthsof the acidic tails among the three SmHMGB proteins significantly and distinctively influenced their DNAtransactions. Spectroscopic analyses indicated that the longest acidic tail of SmHMGB3 contributes to thestructural stabilisation of this protein. Using immunohistochemical analysis, we showed distinct patternsof SmHMGB1, SmHMGB2 and SmHMGB3 expression in different tissues of adult worms. RNA interferenceapproaches indicated a role for SmHMGB2 and SmHMGB3 in the reproductive system of female worms,whereas for SmHMGB1 no clear phenotype was observed. Schistosome HMGB proteins can be phospho-rylated, acetylated and methylated. Importantly, the acetylation and methylation of schistosome HMGBswere greatly enhanced upon removal of the acidic tail. These data support the notion that the C-terminalacidic tails dictate the differences in the structure, expression and function of schistosome HMGBproteins.

� 2016 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction

Schistosomes are parasitic blood flukes, infecting approxi-mately 240 million people globally (Committee, 2002). Severalattempts to fight this disease have included infrastructure andeducation programs, as well as the development of vaccines andnew drugs (DeMarco and Verjovski-Almeida, 2009; Hotez et al.,2010; Pierce et al., 2012; Carneiro et al., 2014). In addition to theseefforts, a better understanding of the fundamental molecular biol-ogy of this parasite is needed.

Schistosoma mansoni presents different developmental forms,separate sexes and a complex life cycle with two hosts: the snailas the intermediate host and human as the definitive host(DeMarco and Verjovski-Almeida, 2009). During its life cycle,

S. mansoni is exposed to different environmental and biologicalconditions such as fresh water, haemolymph in mollusks, andblood in the mammalian host. As a consequence, this parasiteundergoes many morphological and physiological transformations.Thus, this species represents an interesting but challenging biolog-ical system in which to investigate gene regulatory processes (El-Ansary and Al-Daihan, 2005; Fantappie et al., 2008).

The eukaryotic genome must be highly condensed and organ-ised into chromatin (a conserved structural polymer of DNA), his-tones and non-histone proteins (Kornberg and Thomas, 1974;Kornberg and Lorch, 1999; Happel and Doenecke, 2009). Dynamicchanges in the local or global organisation of chromatin arerequired to perform most nuclear activities (Groth et al., 2007; Liet al., 2007), and these changes profoundly impact the access ofregulatory factors to the corresponding target DNA sites, which isrequired to regulate the fidelity of gene expression and establisha gene phenotype. Chromatin structure can be remodelled throughthe disruption of the nucleosome core particle, the bending andunwinding of DNA, the incorporation of specific histone variants

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and the chemical modification, such as phosphorylation, acetyla-tion and methylation, of histones (Happel and Doenecke, 2009).

Major structural changes in chromatin can be mediated throughnon-histone proteins, known as High Mobility Group Box proteinsor HMGBs (Thomas and Stott, 2012; Watson et al., 2014). Thecanonical HMGB proteins comprise two DNA-binding motifs, i.e.,HMG boxes A/B, joined by a basic linker to an acidic C-terminus,i.e., the acidic tail (Stott et al., 2010). Vertebrates contain fourHMGB proteins, namely HMGB1, 2, 3 and 4 (Malarkey andChurchill, 2012; Park and Lippard, 2012), which share highly con-served HMG boxes but significantly differ in the C-terminal tails(Lee and Thomas, 2000). The well-studied HMGB1 is an abundantprotein proposed to primarily act as an architectural factor forthe assembly of nucleoprotein complexes (Watson et al., 2007).

It is generally assumed that the acidic tail of HMGB1 down-regulates the binding of the HMG boxes to linear and distortedDNA (Sheflin et al., 1993; Stros et al., 1994; Stros, 1998), but thisregion has no effect on HMGB1 binding to high affinity substrates,such as DNA minicircles (Bianchi et al., 1989; Pil and Lippard,1992; Stros et al., 1994; Pasheva et al., 1998). The acidic tail hasalso been shown to interact with HMG boxes (Ramstein et al.,1999; Watson et al., 2007; Stott et al., 2010), suggesting a potentialmechanism by which the tail negatively regulates HMGB1–DNAinteractions (Stott et al., 2010).

We have previously identified three HMGB cDNAs in S. mansoni(de Oliveira et al., 2006). The three HMGB proteins from S. mansoni(SmHMGB1, SmHMGB2 and SmHMGB3) are highly homologouswithin the HMG boxes but have important differences in thelengths and sequences of their acidic tails (de Oliveira et al.,2006). SmHMGB1 is currently the only characterised HMGB pro-tein (de Oliveira et al., 2006; Carneiro et al., 2009; de Abreu daSilva et al., 2011), which revealed an important functional rolefor the short acidic tail of this protein (de Abreu da Silva et al.,2011). In the present study, we conducted structural and func-tional comparisons among SmHMGB1, SmHMGB2 and SmHMGB3to determine the functional roles possibly attributed to their differ-ent C-terminal tails.

2. Materials and methods

2.1. Ethics statement

Animals used for polyclonal antibody production were handledfollowing the guidelines of the institutional care and use commit-tee (Committee for Evaluation of Animal Use for Research from theFederal University of Rio de Janeiro, CEUA-UFRJ, Brasil). The proto-cols were approved by CEUA-UFRJ under registry IBqM # 030. Theprocedures were conducted in adherence to the institutionalguidelines for animal husbandry.

2.2. Plasmids, protein expression and purification

Complementary DNAs encoding recombinant SmHMGB1 andSmHMGB1DC have been previously described (de Oliveira et al.,2006). The available sequences for SmHMGB2 (GenBank accessionnumber BN000821) and SmHMGB3 (GenBank accession numberKF383283) were used to design oligonucleotides (SupplementaryTable S1) to amplify the full-length cDNA or fragments with acidictail deletions (DC). The restriction sites for BamHI and HindIII wereadded to the 50 end of the sense and antisense primers, respec-tively, for pQE80L sub-cloning (Qiagen, USA). We performedreverse transcription PCR (RT-PCR) on male and female S. mansoniadult worm cDNA and pGEM T-Easy (Promega, USA) was used asan entry vector. All three SmHMGB proteins were expressed asN-terminal hexa-histidine (6His)-tagged proteins. It is worth

mentioning that the DNA activities assayed with tagged recombinantHMGB proteins have been previously reported (Stros et al., 2004,2009). Escherichia coli BL-21 (kDE3) cells (Novagen, USA) containedthe pQE-80L constructs (Qiagen) encoding SmHMGB1, SmHMGB2,SmHMGB3, SmHMGB1DC, SmHMGB2DC or SmHMGB3DC. Proteinexpression and purification was carried out as previously reported(de Oliveira et al., 2006).

2.3. Ligase-mediated circularisation assay and fluorescence resonanceenergy transfer (FRET)

The circularisation assay was performed as previouslydescribed (de Oliveira et al., 2006). Briefly, the 32P-labelled123 bp DNA fragment (1 nM) with cohesive ends was pre-incubated with appropriate amounts of schistosome recombinantHMGB proteins (1–2 pmol) and subsequently ligated with T4DNA ligase (Promega). One of the ligation mixtures was digestedafter ligation termination with Exonuclease III (Promega). Beforeelectrophoresis, all DNA samples were deproteinized. The DNAwas extracted with chloroform/isoamyl alcohol (24:1) and precip-itated in the presence of 0.02% linear polyacrylamide (LPA; Sigma,USA), and ethanol. The DNA samples were fractionated on a 6%non-denaturing polyacrylamide gel. DNA bending efficiencies andangles were determined by fluorescence resonance energy transfer(FRET) analysis using a 20 bp double stranded (ds)DNA as previ-ously described (Belgrano et al., 2013).

2.4. DNA supercoiling

DNA supercoiling assays were performed as previouslydescribed (de Oliveira et al., 2006). In brief, the supercoiled plas-mid pTZ19R (Thermo Scientific, USA) was relaxed at a DNA concen-tration of �170 lg/mL with wheat germ topoisomerase I.Topoisomerase I was added to the relaxed DNA followed by theaddition of recombinant SmHMGB proteins and reactions wereincubated at 37 �C for 1 h. The distribution of DNA topoisomerswas analysed through electrophoresis on agarose gels. The gelswere stained with ethidium bromide and destained in water. Thesupercoiling efficiency by SmHMGB1, SmHMGB2, SmHMGB3 ortheir acidic tail deletion forms were quantitated by densitometryanalysis using the Image J (National Institutes of Health (NIH) soft-ware, USA).

2.5. Gel retardation assay

The gel retardation experiments were performed using anequimolar mixture of supercoiled plasmid DNA pTZ19R (ThermoScientific) and the HindIII-linearised plasmid, as previouslydescribed (de Oliveira et al., 2006). Briefly, 0.5 lg of each plasmidDNA was mixed with increasing amounts of SmHMGB proteins.The DNA–protein complexes were resolved through electrophore-sis on 1% agarose gels. The gels were stained with ethidium bro-mide and destained in water.

2.6. In vitro post-translational modification assay

For the phosphorylation assays, recombinant SmHMGB proteins(1 lg) were phosphorylated using commercial rat protein kinaseCK2, protein kinase A (PKA) and protein kinase C (PKC) (Promega)and 0.5 lCi [c 32P]ATP (PerkinElmer, USA). For the acetylationassays, the histone acetyltransferase enzyme (6His-SmGCN5/HAT)was expressed and purified (de Moraes Maciel et al., 2004). Recom-binant SmHMGB proteins (1 lg) were acetylated using 1 lg ofSmGCN5 in the presence of 0.25 lCi [3H] acetyl-CoA (PerkinElmer).Negative controls were performed using heat inactivated HATenzyme. For the methylation assays, the GST-SmPRMT1 (protein

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arginine methyltransferase) enzyme was expressed and purified aspreviously described (Mansure et al., 2005). For the methylationassays, 1 lg of GST-SmPRMT1 was incubated in the presence of7 lM S-adenosyl-l-[methyl-3H]methionine ([3H]SAM) (PerkinEl-mer). Negative controls were performed using heat-inactivatedenzymes. The reactions were terminated with SDS–PAGE samplebuffer. Samples were analysed with SDS–PAGE, stained with Coo-massie Blue, destained (digitalized images were used as the inputprotein concentrations) and vacuum dried. The gels were exposedto Hyperfilm (GE HealthCare, USA) and stored at �80 �C until visu-alisation. For the acetylation and methylation reactions, the gelswere subjected to fluorography using Amplify FluorographicReagent (GE HealthCare) for 1 h prior to vacuum drying.

2.7. Circular dichroism (CD) and fluorescence spectroscopy

The CD experiments were conducted using a Chirascan CircularDichroism Spectropolarimeter (Applied Photophysics, UK) and thefluorescence spectroscopy measurements were performed using aVarian Cary Eclipse spectrofluorometer (Australia). The CD and flu-orimetry were carried out as previously described in detail (Ribeiroet al., 2012; Belgrano et al., 2013). The final protein concentrationof each sample used in the measurements was quantitated usingthe Bradford Assay kit (Sigma).

2.8. Polyclonal antibody production and Western blotting

The schistosome HMGB antigens used to immunise the animalswere SmHMGB1, amino acid (aa) 84–169; SmHMGB2, aa 169–226;and SmHMGB3, aa 1–293 (full-length). Mice were inoculated with50 lg of protein mixed with FCA (Sigma) and boosted four timeswith 50 lg of protein mixed with incomplete Freund’s adjuvant(Sigma). Pre-immune serum was collected before the first immuni-sation. Western blots were carried out with 10–100 lg of total pro-tein extract from adult worms or 250 ng of the full-lengthrecombinant SmHMGB proteins. The reacted bands were analysedon an ImageQuantTM LAS 4000 (GE HealthCare).

2.9. dsRNA interference (dsRNAi) and microscopy analysis

Adult worms were obtained by whole body perfusion of ham-sters following a 6-week infection and cultivated as previouslyreported (Smithers and Terry, 1965). In preparation for the dsRNAsynthesis, 500 bp fragments of the firefly luciferase (dsLUC, nega-tive control), SmHMGB1, SmHMGB2 or SmHMGB3 cDNAs wereamplified by PCR using the gene-specific primers listed in Supple-mentary Table S1. The dsRNAs were synthesized using the MEGA-script RNA transcription kit (Ambion, USA). Eight worm coupleswere used in each experimental procedure (quantitative RT-PCR(qRT-PCR), Confocal Laser Scanning Microscopic (CLSM) or Wes-tern blot) and were electroporated using a single square wavepulse of 125 V of 20 ms duration, using 4 mm electroporation cuv-ettes (Bio-Rad GenePulser XCell, USA). The worms were then culti-vated for 72 h, and the media was refreshed every 24 h. The eggswere counted and worm motility and morphologies were moni-tored daily. The mRNA levels of SmHMGB1, SmHMGB2 orSmHMGB3 knock-downs were determined by qRT-PCR, nor-malised by S. mansoni glyceraldehyde 3-phosphate dehydrogenase(SmGAPDH) transcription levels. The results are depicted in rela-tion to the non-specific dsRNAi (dsLUC). Protein levels of thesilenced worms were evaluated by Western blot analysis usingpolyclonal antibodies against SmHMGB1, SmHMGB2 orSmHMGB3. The anti-tubulin antibody was used as a loading con-trol. For the CLSM analysis, the adult worms were fixed and stainedas previously described (Carneiro et al., 2014). The RNAi experi-ments were repeated three times.

2.10. Immunohistochemistry (IHC)

IHC for histological sections of male and female worms was per-formed as previously described (Pinlaor et al., 2009). The primarypolyclonal antibodies dilutions were SmHMGB1 (1:1000),SmHMGB2 (1:100) and SmHMGB3 (1:500). Bright-field images ofsections were recorded using a digital camera (Leica, Germany)fitted on an Olympus Model compound microscope. For negativecontrols, worm sections were incubated with mouse and rabbitpre-immune serum (Supplementary Fig. S1A, B) and/or anti-mouseor anti-rabbit secondary antibodies (Supplementary Fig. S1C, D).

3. Results

3.1. Sequence comparison of schistosome HMGB proteins

The comparison of the full aa sequences of SmHMGB1,SmHMGB2 and SmHMGB3 (Fig. 1A) revealed a high degree of con-servation among their DNA binding domains, the HMG boxes A andB (Fig. 1A, residues 1–86 and residues 98–166, respectively). Alter-natively, these proteins showed important differences in their C-terminal acidic tails (Fig. 1A, residues 209–253), with SmHMGB1having the shortest tail, which is a stretch of only five acidic resi-dues (aspartic or glutamic acid), followed by SmHMGB2, whichhas a tail of 16 acidic residues, and SmHMGB3, with the longestacidic tail of 33 acidic residues. Interestingly, SmHMGB3 has twoextra sequences flanking the acidic region (Fig. 1A, residues 186–208 and 254–293). This structure can be better interpreted in theschematic diagram (Fig. 1B) showing SmHMGB1, SmHMGB2 andSmHMGB2 proteins and their respective acidic tail mutants. Wealso performed SDS–PAGE of the recombinant proteins used in thisstudy (Fig. 1C) to illustrate their purity.

3.2. The role of the acidic tails in schistosome HMGB secondary andtertiary structures

We obtained the Far-UV CD spectra of schistosome HMGB pro-teins and the respective tailless forms of these proteins, showingthat all six proteins had a high a-helical content (Fig. 2A, B). Whenwe compared the tailless forms of SmHMGB1, SmHMGB2 andSmHMGB3, only SmHMGB3, which has the longest acidic tail,had a slight increase in the negative mean residue ellipticity at208 and 220 nm (Fig. 2B), indicating an increase in the proportionof a-helices in the proteins with deleted acidic tails. To furthercharacterise this difference, we compared only the full-lengthand tailless forms of SmHMGB3 and confirmed that removal ofthe acidic tail resulted in a significant increase in a-helix content(Fig. 2C). We used the intrinsic fluorescence of the tryptophan resi-dues of schistosome HMGB proteins (SmHMGB1 box A: W48, boxB: W121, W134; SmHMGB2 box A: W50, W104; box B: W125,W138; SmHMGB3 box A: W49, W103, box B: W124, W137) todetermine the contributions of the acidic tails to the tertiary struc-tures. The data obtained from the maximum fluorescence intensi-ties (Fig. 2D) showed significant differences among SmHMGB1(193 arbitrary units (a.u.)), SmHMGB2 (460 a.u) and SmHMGB3(135 a.u.). Notably, SmHMGB1 contains three tryptophan residueswithin the boxes, whereas SmHMGB2 and SmHMGB3 each containfour tryptophan residues (Fig. 1A) that are conserved in their posi-tions. Interestingly, when we analysed the maximum fluorescenceintensities of the proteins lacking acidic tails, the signal forSmHMGB2 was significantly reduced (Fig. 2E). When the full-length and tailless forms of the three schistosome HMGB proteinswere subjected to denaturing conditions prior to analysis, the max-imum absorption values were observed (Fig. 2F, G), indicating thecomplete exposure of the tryptophan residues and reflecting anunfolded state.

Fig. 1. Comparison of sequence and structure alignments of Schistosoma mansoni High Mobility Group Box (HMGB) proteins. (A) Amino acid sequence alignment ofSmHMGB1, SmHMGB2 and SmHMGB3 proteins (GenBank accession numbers AAY44045, CAJ29301, AGR51647, respectively). (B) Schematic diagram of HMGB proteins fromS. mansoni. SmHMGB1: full-length; SmHMGB1DC: lacking the acidic tail; SmHMGB2: full-length; SmHMGB2DC: lacking the acidic tail; SmHMGB3: full-length; andSmHMGB3DC: lacking the acidic tail. Note that the sizes of the acidic tails are proportionally represented. (C) SDS–PAGE of recombinant hexa-histidine (6His)-taggedSmHMGB proteins used in this study.

Fig. 2. Analysis of the secondary and tertiary structures of Schistosoma mansoni (Sm)HMGB proteins and equivalent proteins (DC) which lack the acidic tail. Circulardichroism spectra of (A) full-length SmHMGB1, SmHMGB2 and SmHMGB3; (B) SmHMGB1DC, SmHMGB2DC and SmHMGB3DC or (C) SmHMGB3 and SmHMGB3DC. Thespectra were averaged from three scans at a 30 nm/min speed recorded from 190 to 260 nm, and the buffer baselines were subtracted from the respective sample spectra. (D–G) SmHMGB1, SmHMGB2, SmHMGB3, SmHMGB1DC, SmHMGB2DC and SmHMGB3DC were analysed using fluorescence spectroscopy, either in the absence (native state) (D,E) or presence of 8 M Urea (denatured state) (F, G), to evaluate tertiary structure content. The excitation wavelength was fixed at 280 nm, and the emission spectrum wasrecorded from 300 to 420 nm. SmHMGB1 (black line), SmHMGB2 (blue line), SmHMGB3 (red line), SmHMGB1DC (black dashed line), SmHMGB2DC (blue dashed line) andSmHMGB3DC (red dashed line). All experiments were performed at 25 �C. (For interpretation of the references to colour in this figure legend, the reader is referred to the webversion of this article.)

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3.3. The role of the acidic tails in schistosome HMGB–DNA binding

Due to the significant differences observed in the acidicC-terminal regions of schistosome HMGB proteins, we comparedthe ability of the full-length and acidic-tail truncation forms ofSmHMGB1, SmHMGB2 and SmHMGB3 to bind and change DNAtopology (Fig. 3A–I). The results of the gel retardation assaysclearly showed that all three schistosome HMGBs preferentiallybound to supercoiled DNA (‘S’ on Fig. 3A) compared with lineardsDNA (‘L’ on Fig. 3A). Importantly, SmHMGB1, SmHMGB2 andSmHMGB3 had different levels of binding activities to supercoiledDNA, revealed by the different profiles of the shifted bands, withSmHMGB1 showing the lowest affinity (Fig. 3A, lanes 2–4) andSmHMGB3 showing the highest affinity (Fig. 3A, lanes 8–10). Next,we determined whether these differences in activities reflected thedistinct acidic tails of these proteins. The gel retardation assaysshowed that the binding activities of the tailless proteins to super-coiled DNA were significantly lower (Fig. 3B) than the full-lengthform, even though the concentrations of the tailless forms weretwice that of the concentrations of the full-length forms; whenthe same equimolar amounts were used, the bands were over-shifted and distorted Fig. 3B, lanes 7, 10). To further characterisethe influence of the acidic tails of schistosome HMGBs on bindingto supercoiled DNA, we compared the activities of each full-length

Fig. 3. DNA transactions using recombinant Schistosoma mansoni (Sm)HMGB proteins. (proteins to supercoiled DNA. (A) Equimolar mixtures of supercoiled and linearised plas1.5 lM) of SmHMGB1, SmHMGB2 or SmHMGB3, or (B) increasing amounts (1, 2 and 3respective tailless forms of each protein were compared individually as follows: (C) SmHM4 lM); and (E) SmHMGB3 and SmHMGB3DC (0.4, 0.8 and 1.6 lM). C, controls withoutsupercoiling by full-length or tailless SmHMGB proteins. Circular relaxed plasmid pTZ1SmHMGB proteins (2.75 and 5.5 lM). Deproteinized DNA topoisomers were resolved osupercoiled DNA. (H, I) DNA bending by full-length and tailless SmHMGB proteins. Thamounts of the recombinant proteins (50–100 nM), followed by ligation with T4 DNdeproteinized DNA ligation products were subjected to electrophoresis on 6% non-dexonuclease III; L, linear DNA; C, circular DNA. Agarose gels were photographed through asupercoiling efficiency (F and G) and the formation of minicircles (H and I; average of thtail deletion forms, were quantitated by densitometry analysis (Fb–Ib), using Image J (N

protein with the acidic tail deletion mutant form (Fig. 3C–E). ForSmHMGB1, SmHMGB2 and SmHMGB3, the removal of the acidictails slightly reduced their DNA-binding capacity (Fig. 3C–E; com-pare lanes 2–4 with lanes 5–7). Because SmHMGB3 has a higheraffinity for supercoiled DNA than SmHMGB1 or SmHMGB2 (shownin Fig. 3A, B), the results presented in Fig. 3E were obtained usingless than half of the concentration of SmHMGB3 (see figure legendfor details). We also compared the ability of the three schistosomeHMGB proteins to bind circular DNA and promote DNA supercoil-ing. All three proteins promoted DNA supercoiling (Fig. 3F), withSmHMGB1 being the most active (Fig. 3F, lanes 3, 4). SmHMGB2showed intermediate supercoiling activity (Fig. 3F, lanes 5, 6),and SmHMGB3 was the least active protein (Fig. 3F, lanes 7, 8). Sur-prisingly, when we compared the full-length and tailless forms ofthe three proteins (Fig. 3, compare panels F, G), the supercoilingactivities of SmHMGB2 and SmHMGB3 were remarkably enhanced(Fig. 3G, lanes 5–8), whereas the activity of truncated SmHMGB1remained unaltered (Fig. 3G, lanes 3, 4). The quantification of thesupercoiling data is provided in Fig. 3Fb, Gb (densitometry ofbands). Next, we compared the ability of the full-length or taillessforms of the schistosome HMGB proteins to bend DNA (Fig. 3H, I).All proteins were able to bend DNA, and no significant differencesin the bending activities were observed among SmHMGB1,SmHMGB2 and SmHMGB3 (Fig. 3H), or among the tailless forms

A–E) Preferential binding of the full-length or tailless forms (DC) of the SmHMGBmid pTZ19R (�10 nM) were pre-incubated with increasing amounts (0.5, 1.0 andlM) of SmHMGB1DC, SmHMGB2DC or SmHMGB3DC. (C–E) The full-length andGB1 and SmHMGB1DC (2, 3 and 4 lM); (D) SmHMGB2 and SmHMGB2DC (2, 3 and

proteins; ⁄, relaxed circular DNA; L, linear DNA; S, supercoiled DNA. (F and G) DNA9R DNA (�10 nM) was incubated in the presence of topoisomerase I (Topo I) andn 1% agarose gels, followed by staining with ethidium bromide. I, Linear DNA; II,e 32P-labelled-123 bp DNA fragment (�1 nM) was pre-incubated with increasingA ligase. Exonuclease III was used to verify the identity of DNA minicircles. Theenaturing polyacrylamide gels and visualised through autoradiography. Exo III,red filter using a UV-transilluminator (Mini-Bis Pro, Bio Imaging Systems, USA). Theree independent experiments) by SmHMGB1, SmHMGB2, SmHMGB3 or their acidicIH software, USA).

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of these proteins (Fig. 3I). Bending efficiencies and angles were alsodetermined by FRET and confirmed that all three schistosome full-length HMGB proteins bend DNA in a similar manner (Supplemen-tary Table S2).

3.4. The role of the acidic tails in schistosome HMGB post-translationalmodifications

CK2, PKA and PKC phosphorylate SmHMGB1 (de Abreu da Silvaet al., 2011), and our results showed that in addition to SmHMGB1(Fig. 4A, lane 1), SmHMGB3 can also be phosphorylated byCK2 (Fig. 4A, lane 5). However, SmHMGB2 was not a target ofCK2 (Fig. 4A, lane 3). Alternatively, we clearly showed thatSmHMGB1, SmHMGB2 and SmHMGB3 were all substrates of eitherPKA or PKC (Fig. 4B, C, lanes 1, 3 and 5). When we analysed the roleof the acidic tails in the phosphorylation status of the threeschistosome HMGB proteins, we observed that only in the case ofCK2 was phosphorylation abolished by SmHMGB1 (de Abreu daSilva et al., 2011) or SmHMGB3 acidic tails (Fig. 4A, lanes 2 and6, respectively) but not by the SmHMGB2 acidic tail (Fig. 4A, lane4). In addition to phosphorylation, we showed that SmHMGB1,SmHMGB2 and SmHMGB3 were modified through methylation(Fig. 4D) or acetylation (Fig. 4E) by the schistosome proteinarginine methyltransferase 1, SmPRMT1 (Mansure et al., 2005) orthe histone acetyltransferase, SmGCN5 (de Moraes Maciel et al.,2004), respectively. Remarkably, the tailless forms of SmHMGB2or SmHMGB3 revealed a significant increase in methylation(Fig. 4D, compare lanes 3 and 4 for SmHMGB2 or lanes 5 and 6for SmHMGB3) or acetylation levels (Fig. 4E, compare lanes 1 and2 for SmHMGB2 or 3 and 4 for SmHMGB3). For SmHMGB1, the tail-less form had no effect on the methylation level of the protein(Fig. 4D, compare lane 1 and 2). However, we have previously

Fig. 4. Post-translational modification analysis of Schistosoma mansoni (Sm)HMGB prrecombinant full-length or tailless (DC) SmHMGB proteins and commercial protein kinas1 lg of full-length or tailless SmHMGB proteins, and GST-protein arginine methyltransSAM). (E) In vitro acetylation assays were performed with 1 lg of full-length or tailless(6�His-tagged-SmGCN5) in the presence of [3H] acetyl-CoA. (Aa–Ea) Coomassie blue stainor tailless SmHMGB2 forms, respectively. The asterisk indicates the auto acetylation of

reported that the tailless form of SmHMGB1 showed significantlyenhanced acetylation (Carneiro et al., 2009). For phosphorylationand acetylation, control reactions were performed using heat-inactivated enzymes (Supplementary Fig. S1A–C, E). For methyla-tion, the control reaction was performed against the GST moietyonly (Supplementary Fig. S1D).

3.5. Expression and function of native schistosome HMGB proteins inadult worms

Immunohistochemical localisation of the three schistosomeHMGB proteins (Fig. 5) revealed distinct patterns of expression.Notably, SmHMGB1 and SmHMGB2 were mainly and highlyexpressed in the testis, ovary and vitellaria (Fig. 5A–D), whereasfor SmHMGB3, high expression was observed in testis and vitel-laria but not in the ovary (Fig. 5E, F). However, no reaction wasobserved when using the secondary antibody or pre-immuneserum, alone (Supplementary Fig. S2). Expression of SmHMGB2and SmHMGB3, but not SmHMGB1, was observed in the tegumenttubercles (Fig. 5A–F). Importantly, we showed using Western blotsthat the antibodies against SmHMGB1, SmHMGB2 or SmHMGB3had high specificity and no cross-reaction among the three pro-teins (Supplementary Fig. S3A, B).

We performed dsRNAi (Fig. 6) with the aim of understandingthe role of schistosome HMGB proteins on worm physiology and/or ultrastructure. We were able to significantly (Fig. 6A, C, F andG) and specifically (Supplementary Fig. S4A) knock-downSmHMGB1, SmHMGB2 or SmHMGB3 in adult worms, both at themRNA levels (Fig. 6A, C, F) and protein levels (Fig. 6G). Thebehaviour of the dsRNAi-treated worms was monitored on a dailybasis, and a prominent reduction in egg laying was observed forSmHMGB2 or SmHMGB3 in the worms that received the dsRNAi

oteins. (A–C) In vitro phosphorylation assays were performed using 1 lg of thees CK2 (A), PKA (B) or PKC (C). (D) In vitro methylation assays were performed usingferase 1 (SmPRMT1) in the presence of S-adenosyl-l-[methyl-3H]methionine ([3H]SmHMGB proteins and hexa-histidine tagged S. mansoni histone acetyltransferaseed-gels. (Ab–Eb) Autoradiographies. The arrows or arrowheads depict the full-lengthSmGCN5.

Fig. 5. Immunolocalisation of schistosome HMGB proteins. Representative thin sections of paraffin embedded male and female adult worms were probed with polyclonalantibodies against SmHMGB1 (A, B), SmHMGB2 (C, D) or SmHMGB3 (E, F). Abbreviations were used for seminal vesicle (sv), testicular lobes (tl), tegument (t), ovaries (ov) andvitellaria (v). Overall, the SmHMGB proteins showed strong immunoperoxidase brown staining to the reproductive organs of male and female schistosomes. Originalmagnification �40.

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(Fig. 6D, F). For the worms that received SmHMGB1-dsRNAi, egglaying was as efficient as in worms that received the controlLUC-dsRNAi (Fig. 6B). Other parameters, such as worm pairing,motility and adhesion to the plate by their suckers, were also mon-itored daily. Again, for the worms treated with SmHMGB1-dsRNAi,all of these parameters were considered normal during the 4 daysof culture (Supplementary Table S3). Alternatively, 65% of theworms that were treated with dsRNAi for SmHMGB2 or SmHMGB3were uncoupled, with only 15% of these worms adhering to theplate, revealing a significant reduction in motility (SupplementaryTable S3). In addition, our functional dsRNAi assays revealedimportant phenotypic alterations in females. By CLSM microscopy,we observed a significant disorganisation of the ovaries and areduction of their size when SmHMGB2 or SmHMGB3 wereknocked down (Fig. 6O, R). Worms treated with the double-stranded luciferase control gene (dsLUC) showed normal ultra-structures, as revealed by their integral tegumental structures(Fig. 6R), the size of the ovaries with expected number and appear-ance of the immature and mature oocytes (Fig. 6I), and the typicalmorphology and number of testicular lobes, and sperm production(Fig. 6J). Notably, these ovaries contained significantly fewermature oocytes (Fig. 6, compare the oocytes in I, with those in Oand R). For the SmHMGB1-dsRNAi, no apparent phenotypicchanges were observed (Fig. 6K–M).

4. Discussion

Schistosomes contain three members of the HMGB superfamily,and they basically differ in their C-terminal regions, in terms ofboth length and the number of acidic residues. Importantly, thesame pattern has been observed in their mammalian counterparts,which express four HMGB proteins, with important differences inthe C-terminal acidic regions (Stros et al., 2007).

HMGB proteins play a role in essentially all DNA transactionssuch as transcription, replication, recombination, DNA repair andchromatin remodelling (Thomas and Travers, 2001; Agresti and

Bianchi, 2003; Travers, 2003; Jaouen et al., 2005; Stros, 2010).Although HMG boxes act as major DNA-binding platforms, theacidic tails are responsible for regulating these interactions andthe subsequent changes in the DNA topology (Lee and Thomas,2000; Ueda et al., 2004; Wang et al., 2007; Watson et al., 2007;Stott et al., 2010; Belgrano et al., 2013). Due to the importance ofthe acidic tail in the function of HMGB proteins, in the presentstudy we characterised the influence of the diverse acidic taillengths of schistosome HMGB proteins on the (i) changes in theirprotein structure; (ii) binding to different DNA ligands; (iii) promo-tion of post-translational modifications and (iv) tissue-specificfunctions.

We compared the full-length forms of schistosome HMGB pro-teins that naturally and significantly differ in the acidic tails withthe respective tailless constructs, and the CD data showed thatthe distinct lengths of the acidic tails played a role in the overallstructure of these proteins. In the case of SmHMGB3, which con-tains the longest acidic tail, the removal of the tail increased theproportion of a-helices, consistent with a predominant randomcoil conformation of the tail. Similarly, the fluorescence datashowed that the spectra were distinct among SmHMGB1,SmHMGB2 and SmHMGB3, suggesting a role for the differentacidic tails in the final folding of the proteins. Because the tail ofmammalian HMGB1 interacts with both boxes (Watson et al.,2007), the distinct fluorescence patterns obtained from SmHMGB1,SmHMGB3 or SmHMGB3 might reflect different modes of interac-tions between the acidic tails and the box(es).

The acidic tail of mammalian HMGB1 modulates the binding ofthe HMG boxes to different topological DNA substrates. In general,the acidic tail down-regulates the binding and/or decreases theselectivity to DNA (Bianchi et al., 1989; Pil and Lippard, 1992;Stros et al., 1994; Pasheva et al., 1998). We used three DNA sub-strates avidly recognised and bound by HMGB proteins to gaininsight into the role of the acidic tails of schistosome HMGB pro-teins in DNA transactions. Remarkably, depending on the natureof DNA ligand used, the full-length and tailless forms of SmHMGB1,SmHMGB2 or SmHMGB3 acted distinctly. For example, a

Fig. 6. RNA interference (RNAi)-induced knock-down and phenotype in schistosome adult worms. A double stranded (ds)RNAi experiment was carried out on adult wormpairs cultivated for 3 days. Worms were electroporated, followed by 24 h soaking with 25 lg of dsRNAi from firefly luciferase (LUC) and Schistosoma mansoni (Sm) HighMotility Group Box proteins (SmHMGB1, SmHMGB2 or SmHMGB3). On the third day of culture, the mRNA levels of SmHMGB1 (A), SmHMGB2 (C) or SmHMGB3 (E) weredetermined by quantitative reverse transcription-PCR, normalised by the glyceraldehyde 3-phosphate dehydrogenase (SmGAPDH) transcription levels. The results aredepicted in relation to the non-specific dsRNAi (dsLUC). After 24 h of each dsRNAi treatment, the eggs were counted on a daily basis (B, D, F). (G) Protein levels of the silencedworms were evaluated by Western blot analysis, using polyclonal antibodies against SmHMGB1 (silencing of 46%), SmHMGB2 (silencing of 100%) or SmHMGB3 (silencing of100%) The anti-tubulin antibody was used as a loading control. The teguments, ovaries, vitellaria and testis were analysed by Confocal Laser Scanning Microscopic. Wormstreated with the double-stranded luciferase control gene (dsLUC, H–J), dsSmHMGB1 (K–M), dsSMHMGB2 (N–P) and dsSmHMGB3 (Q–S). Tg, tegument; io, immature oocytes;mo, mature oocytes; ov, ovary; v, vitellaria; tl, testicular lobes; sv, sperm vesicle. Scale bar = 10 lm.

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comparison of the structure-selectivity of the three full-length pro-teins revealed that SmHMGB1, SmHMGB2 or SmHMGB3, despitetheir naturally distinct acidic tails, showed a preference for circularsupercoiled DNA over linear DNA. Interestingly, using supercoiledDNA as a substrate, we observed that this affinity increasedwith increasing acidic tail length, with SmHMGB1 (shortest tail)showing only modest binding activity at the highest concentration,

and SmHMGB3 (longest tail) showing significant binding activity,even at lower concentrations. Remarkably, the removal of theacidic tails of these proteins significantly decreased binding tosupercoiled DNA. Interestingly, a similar approach, using minicir-cle DNA as a high-affinity substrate for mammalian HMGB1,revealed that the removal of the HMGB1 acidic tail did not alterthe binding pattern of this protein (Lee and Thomas, 2000).

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Importantly, a different scenario was observed when we testedthe ability of the schistosome HMGB proteins to promote DNAsupercoiling or bending. Overall, the data indicated that the acidictails of each schistosome HMGB protein played an inhibitory rolein supercoiling DNA in the presence of topoisomerase I, particu-larly in the case of SmHMGB2 and SmHMGB3. However, otherparts of the proteins, outside the acidic tails, might also be con-tributing to the supercoiling activity, as suggested from the dataobtained with the tailless forms. In the case of DNA bending bySmHMGB1, SmHMGB2 and SMHMGB, the removal of the acidictails did not significantly alter the bending activity of these pro-teins. These data are consistent with reports attributing the bend-ing capacity to the HMG boxes of HMGB proteins (Grosschedl et al.,1994; Stros et al., 1994; Ribeiro et al., 2012).

HMGB1 proteins undergo post-translational modificationsthrough phosphorylation, acetylation, methylation, glycosylationand poly (ADP)-ribosylation (Stros, 2010), which mediate DNA/chromatin binding, subcellular localisation and/or secretion ofHMGB1. We have previously reported that SmHMGB1 can beacetylated and phosphorylated, and both modifications influencethe nuclear-cytoplasmic translocation and secretion of this protein(Carneiro et al., 2009; de Abreu da Silva et al., 2011). In this con-text, CK2 phosphorylation of SmHMGB1 mediates its secretionfrom the nucleus to the extracellular milieu in the liver of infectedmice (de Abreu da Silva et al., 2011), likely acting as an alarmineand playing a role in the pathogenesis of the hepatic granuloma.We have also previously demonstrated that, similarly to the mam-malian HMGB1, acetylation might contribute to the extracellularrelease of SmHMGB1 (Carneiro et al., 2009). Here we showed thatSmHMGB2 and SmHMGB3 were also modified through phosphory-lation and acetylation. In addition, this study presents, to ourknowledge, the first evidence of the arginine methylation of HMGBproteins. In this regard, we showed here that the acidic tails ofSmHMGB2 and SmHMGB3, but not of SmHMGB1, negatively regu-late the methylation or acetylation of these proteins, because theremoval of their tails significantly enhanced these modifications.One feasible explanation for SmHMGB2 and SmHMGB3 enhancedacetylation and methylation upon tail removal is the fact thatlonger acidic tails could adopt a flexible structure for interactionswith the boxes, shielding potential methylation and/or acetylationsites present in SmHMGB2 and SmHMGB3. This hypothesis is sub-stantiated by previous data showing the enhanced acetylation ofhuman HMGB1 lacking the acidic tail (Pasheva et al., 2004), andseveral models proposing that the C-tail forms intra-molecularinteractions with the N-terminal DNA binding domains coveringpost-translational putative sites (Wang et al., 2007; Watsonet al., 2007; Stott et al., 2010).

The mammalian HMGB1 function has been extensively charac-terised, showing ubiquitous expression and having a pleiotropicrole in a variety of cells and tissues (Lee and Thomas, 2000; Uedaet al., 2004; Wang et al., 2007; Watson et al., 2007; Stott et al.,2010; Belgrano et al., 2013). Alternatively, HMGB2, HMGB3 andHMGB4 seem to have a more restricted pattern of expression, sug-gesting that those are required in specific cells (Ronfani et al.,2001; Agresti and Bianchi, 2003). In this regard, HMGB2 andHMGB3 have been shown to play important roles in spermatogen-esis (Ronfani et al., 2001; Catena et al., 2009). In addition, HMGB2and HMGB3 have been implicated in the regulation of haematopoi-esis (Nemeth et al., 2006; Pinlaor et al., 2009). In the case ofS. mansoni, our immunolocalisation and RNAi data suggested thatSmHMGB1, SmHMGB2 and SmHMGB3 might participate in molec-ular events involved in adult worm sexual physiology. This hypoth-esis is based on the high specific expression levels of SmHMGB1,SmHMGB2 and/or SmHMGB3 in testis, vitellaria and/or ovaries.The fact that schistosome HMGB proteins might be functionallyrelevant in sexual physiology can be concluded by the phenotypic

alterations observed in the structures of the ovaries and oocytes,and in egg laying, when SMHMGB2 or SmHMGB3 were partiallydeleted. The simplest interpretation of the schistosome HMGBexpression and knock-down data is that all three proteins areindeed functionally equivalent, and a minimum total amount ofSmHMGB1, SmHMGB2 and SmHMGB3 is required for sexual devel-opment. Perhaps the lack of phenotypic changes by the SmHMGB1knock-downmight simply indicate that its pattern of expression inspecific cell types or under specific developmental conditions isvariable rather than constant. In addition, we believe that due tothe high expression levels of SmHMGB1 in these organs, its partialreduction by the RNAi assay could not have been sufficient to gen-erate a phenotype. In addition, Western blot analysis of theSmHMGB1-silenced worms suggested that SmHMGB1 might bemore stable and, therefore, a long-lived protein.

In summary, these data indicated that the different lengths and/or the aminoacid composition of the acidic tails of nativeschistosome HMGB proteins could introduce variability instructure-specific DNA binding, mediate differential protein-protein interactions andpromote tissue-specificexpressionpatterns,which would ultimately lead to distinct biological activity patternsduring the life cycle and/or infection duration of the parasite.

Acknowledgements

We thank Mr. Paulo Cesar dos Santos (Fiocruz, Rio de Janeiro,Brasil) for providing S. mansoni cercariae. This work was financiallysupported through grants from the Conselho Nacional de Desen-volvimento Científico e Tecnológico (CNPq – grant number470099/20143), Brasil and Fundação Carlos Chagas Filho deAmparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ – grantnumber: E-26/010.001554/2014), Brasil to MRF.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.ijpara.2015.12.007.

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