reversible posttranslational modification by ubiquitin and ubiquitin - like proteins ( ubls ) plays a crucial role in dynamic regulation of protein function , fate , binding partners , activity , and localization . small ubiquitin - related modifier ( sumo ) is a member of ubls family that is covalently attached to lysine residues of their protein targets in cells , altering function and subcellular localization . sumoylation has been implicated in the regulation of numerous biological processes including transcription   and cell cycle control . the sequential actions of e1 , e2 , and e3 enzymes catalyze the attachment of sumo to target proteins , while deconjugation is promoted by sumo - specific proteases . in addition , like other ubls , sumo polypeptide is synthesized as an inactive precursor , which requires a c - terminal cleavage to expose the glycine residue for substrate conjugation . the catalytic activity is maintained within a highly conserved 200 amino acid region at the c - terminus of the proteases [ 46 ] . excessive sumo conjugation using knockouts for either senp1 or senp2 induced an embryonic lethal phenotype [ 7 , 8 ] and deregulation of either sumo conjugation or deconjugation can contribute to cancer progression as reviewed by . in humans there are six senp enzymes ( senp1 , -2 , -3 , -5 , -6 , and -7 ) that deconjugate mono - sumoylated proteins or disassemble polymeric sumo side chains , while in saccharomyces cerevisiae there are only two desumoylating enzymes , ulp1 and ulp2 . based on these activities and affinities for sumo isoforms , senps can be categorized into three independent subfamilies : senp1 and senp2 ; senp3 and senp5 ; senp6 and senp7 . the initial molecular characterization of sumoylation pathway in s. mansoni showed the presence of two sumo paralogs called smt3c and smt3b , suggesting a variety of targeted substrates for these modifications . recently , our group reported the conservation of sumo conjugating enzyme smubc9 by phylogeny , primary and modeled tertiary structures , as well as mrna transcription and protein levels throughout the s. mansoni life cycle . our data showed that smubc9 transcription levels are upregulated in early schistosomula , suggesting the utilization of this posttranslational modification mechanism s. mansoni development , particularly at the initial phase of invasion of the vertebrate host . to extend the investigation of the sumoylation pathway in s. mansoni , we firstly retrieved through homology - based searches putative smsenps using the publically available s. mansoni databases . furthermore , the levels of smsenp1/7 transcripts were evaluated by qrt - pcr using mrna from cercariae , adult worms and mechanically - transformed schistosomula ( mts ) in vitro - cultivated during 3.5  h , 1 , 2 , 3 , 5 , and 7 days . we also measured the endopeptidase activities of smsenps in cercariae and adult worm crude extracts . the present study reveals differences in the gene expression profile of smsenp1/7 during schistosomula development . in addition , similar smsenp1 activity was observed in cercariae and adult worms , suggesting the importance of this proteolytic activity during the parasite 's life cycle . all experiments involving animals were authorized by the ethical committee for animal care of federal university of ouro preto , and were in accordance with the national and international regulation accepted for laboratory animal use and care . le strain was maintained by routine passage through biomphalaria glabrata snails and balb / c mice . the infected snails were induced to shed cercariae under light exposure for 2  h and the cercariae were recovered by sedimentation on ice . adult worm parasites were obtained by liver perfusion of mice after 50 days of infection . briefly , cercariae were recovered , and washed in rpmi 1640 medium ( invitrogen ) , before vortexing at maximum speed for 90  s and immediately cultured during 3.5  h at 37c , 5% co2 . then the recovered schistosomula were washed with rpmi 1640 until no tails were detected . for subsequent incubations , the parasites were maintained in m169 medium supplemented with 10% fbs , penicillin , and streptomycin at 100  g / ml and 5% schneider 's medium   at 37c on 5% co2 during 3.5  h , 1 , 2 , 3 , 5 , and 7 days . smsenp1/7 sequences were retrieved from the s. mansoni genome database version 5.0 available at http://www.genedb.org/genedb/smansoni/ through blast searches . amino acid sequences from drosophila melanogaster , caenorhabditis elegans and homo sapiens senp orthologs were used as queries . the blastp algorithm , underpinned by the pfam ( v26.0 ) , allowed detection of conserved protein domains or motifs from s. mansoni sequences . multiple alignments of smsenp1/7 were performed by clustalx 2.0 and phylogenetic analysis was conducted in mega 5 . the bootstrap consensus tree inferred from 1000 replicates was used to represent the evolutionary history of the taxa analyzed . the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test ( 1000 replicates ) is shown next to the branches . the tree was drawn to scale , with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree . total rna from adult worms , cercariae , and schistosomula was obtained using a combination of the trizol reagent ( gibco - brl ) and chloroform for extraction , and then on column purified using the  sv total rna isolation system  ( promega , belo horizonte , brazil ) . the preparation was treated with rnase - free dnase i in 3 different rounds by decreasing enzyme concentration ( rq1 dnase ; promega ) . rna was quantified using a spectrophotometer and an aliquot containing 1  g of total rna reverse transcribed using an oligodt primer from the thermoscript rt - pcr system ( invitrogen so paulo , brazil ) as described by the manufacturer . the efficiency of dnase i treatment was evaluated by pcr amplification of the cdna reaction mix without the addition of the thermoscript enzyme . s. mansoni specific senp1/7 primers were designed using the program generunner for smsenp1 ( genedb access number smp_033260.2 ) ( forward 5-aggaaacggaggcgggattc-3 , reverse 5-acactggagacacgggatgagc-3 ) and for smsenp7 ( genedb access number smp_159120 ) ( forward 5-tcagttacacggccctttatc-3 , reverse 5-cctgagaagtggatgcgatc-3 ) . reverse - transcribed cdna samples were used as templates for pcr amplification using sybr green master mix udg - rox ( invitrogen ) and 7300 real time pcr system ( applied biosystems , rio de janeiro , brazil ) . specific primers for s. mansoni-tubulin were used as an endogenous control ( genbank access number m80214 ) ( forward 5-cgtattcgcaagttggctgacca-3 , reverse 5-ccatcgaagcgcagtgatgca-3 ) . the efficiency of each pair of primers was evaluated according to the protocol developed by the applied biosystems application ( cdna dilutions were 1  :  10 , 1  :  100 and 1  :  1000 ) . for all investigated transcripts three biological replicates were performed and their gene expression normalized against the -tubulin transcript according to the 2 method   using the applied biosystems 7300 software . to determine the enzymatic activity of senp proteases present in adult worms and cercariae crude extracts we used the fluorogenic substrate sumo-1-amc that is specific for senp1 ( enzo life sciences , so paulo , brazil ) . in these assays 10  g of total protein were used and 0,45  m of the fluorogenic substrate in 50  mm tris - hcl ph 8 , 10  mm mgcl2  0,45  m sumo-1-aldehyde ( enzo life sciences ) to control for specific enzyme inhibition . each enzymatic assay was conducted in a final volume of 100  l for both extracts , followed by 60 minutes incubation at 37c . the fluorometric readings were taken at wavelengths of 380  nm ( excitation ) and 440  nm ( emission ) in a spectrofluorimeter ( turner quantechtm fluorometer ) , and the results expressed in fluorescence units per g of total protein . statistical analysis was performed using graphpad prism version 5.0 software package ( irvine , ca , usa ) . normality of the data was established using one way analysis of variance ( anova ) . tukey post tests were used to investigate significant differential expression of sumo proteases throughout the investigated stages . in all cases , in silico analysis of smsenps revealed the conserved putative domains of sumo proteases in s. mansoni . the parasite database has two sequences encoding putative senps : smp_033260.2 ( putative senp1 ) and smp_159120 ( putative senp7 ) . our results showed that each senp was grouped in 2 distinct clades , reinforcing their structural conservation among the thirteen analyzed orthologs ( figure 1 ) . to confirm that these predicted proteases are cysteine - family members , analyses using the pfam protein domain database were conducted . for both smsenps entries our in silico analysis demonstrated smsenp1 more closely related to hssenp1 ( genbank access number np_055369.1 ) , whereas smsenp7 is related to hssenp7 ( genbank access number np_065705.3 ) . alignment of their catalytic domains with the human senps showed the presence of the three essential catalytic residues ( cys - his - asp ) , highlighted in figure 2 . we designed specific primers to amplify smsenp1 and smsenp7 transcripts in the cercariae - schistosomula transition and adult worm ( figure 3 ) . we observed that smsenp1 and smsenp7 transcripts are expressed in basal levels from mts-2d to 7 days and adult worms . the levels of the senps transcripts were significantly higher in mts-3.5  h being approximately 10-fold higher when compared to levels in adult worms and during schistosomula development ( mts-2 , 3 , 5 , and 7 days ) and 3-fold higher when compared to cercariae and mts-1d . we also observed that smsenp1 and smsenp7 transcription levels were not significantly different ( p < 0.05 ) within a given stage , except for mts-3.5  h where smsenp7 levels are 3 fold - higher than smsenp1 . 
 in vitro endopeptidase assays the smsenp1 enzymatic activity was slightly higher in cercariae compared to adult worms , although the levels were not significantly different ( p < 0.05 ) . enzyme activities were also recorded in the presence of a commercially available hssenp1 inhibitor but significant differences were not observed in the analyzed stages . previous reports from our group showed the presence of two sumo paralogs ( smt3c and smt3b ) in s. mansoni , exhibiting differential expression between larvae and adult worms . furthermore , our group demonstrated a differential expression profile for ubc9 throughout the parasite life cycle . the structural sumo conservation in s. mansoni reinforces sumo as a candidate for a transcription regulator and a degradation signal by facilitating ubiquitylation of certain target proteins during parasite development . in order to understand the role of sumoylation in s. mansoni , we evaluated the conservation of sumo proteases and their gene expression profile in cercariae , schistosomula , and adult worm . firstly , we retrieved , through homology - based searches , two sequences containing conserved domains of sumo proteases . a phylogenetic approach then revealed two putative proteases closely associated to human senp1 and senp7 . alignment of parasite sequences with their human counterparts also demonstrated conservation of the catalytic triad for both proteases . the active site residues within the core domain of ulp1 and ulp2 have been proven to be necessary for catalytic activity and in vivo function in yeast [ 19 , 20 ] . furthermore , ulp1 lacks obvious sequence similarity to any known deubiquitinating enzyme so it is unlikely that it is able to process ubiquitin - linked substrates . considering that the sumoylation pathway dictates the function of a variety of substrates , and the existence of two sumo paralogs in s. mansoni , it is plausible that the worm utilizes distinct sumo proteases for sumo maturation and desumoylation of target substrates . discrimination between sumo-1 and sumo-2/3 paralogs may also account for distinct functions of senp proteases in s. mansoni . in mammalian cells , it is well established that senp1 processes sumo-1 in preference to sumo-2 , and has a very low catalytic constant for sumo-3 . in contrast , the recent characterization of the catalytic domain of senp7 in humans demonstrated a greater deconjugating activity for sumo-2/3 chains . as discussed by shen   senp7 acts as a sumo-2/3 specific protease that is likely to regulate the metabolism of polysumo-2/3 rather than sumo-1 conjugation in vivo . in the present investigation , the transcriptional profile of sumo proteases was also evaluated by qpcr , using total rna from distinct stages of the s. mansoni life cycle . the gene expression data revealed similar expression profile for smsenp1 and smsenp7 at any given stage , but with a remarkable differential expression for both proteases comparing the larvae and adult stages . the differential gene expression profile observed for smsenp1/7 throughout the s. mansoni life cycle attests for the distinct patterns of sumo conjugates observed during parasite development . polysumoylation has been reported to increase during heat - shock treatment and stress conditions , with senp7 being responsible for dismantling sumo polymers [ 25 , 27 ] . given that the levels of smsenp1/7 transcripts were significantly higher in cercariae and mts-3.5  h , we suggest a stress - induced expression of the sumoylation machinery during the parasite 's body adaptation to the new metabolic and morphological alterations it undergoes . our findings are also in agreement with pereira   which demonstrated a similar pattern of gene expression profile for smubc9 in the aforementioned parasite stages . we have not observed a positive correlation between differential transcriptional levels of smsenp1 and enzymatic activity evaluated using synthetic sumo-1-amc substrate . the levels of enzymatic activity for smsenp1 were slightly higher in extracts of cercariae compared to adult worms . in the presence of a specific inhibitor , no significant change in enzymatic activity was detected . this may account for the limited amino acid sequence identity ( 28% ) shared between smsenp1 and its human ortholog , from which the synthesis of sumo-1 aldehyde is based . additional studies aimed at elucidating substrate selection of smsenp1/7 in s. mansoni and a detailed kinetic analysis of their enzymatic activities , particularly during the cercarie to schistosomula transition may provide further insights into the biological activities of these enzymes and the role of sumo conjugation in s. mansoni .