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 .
 small ubiquitin - related modifier ( sumo ) is involved in numerous cellular processes including protein localization , transcription , and cell cycle control . 
 sumoylation is a dynamic process , catalyzed by three sumo - specific enzymes and reversed by sentrin / sumo - specific proteases ( senps ) . 
 here we report the characterization of these proteases in schistosoma mansoni . using in silico analysis , we identified two senps sequences , orthologs of mammalian senp1 and senp7 , confirming their identities and conservation through phylogenetic analysis . 
 in addition , the transcript levels of smsenp1/7 in cercariae , adult worms , and in vitro cultivated schistosomula were measured by qrt - pcr . 
 our data revealed upregulation of the smsenp1/7 transcripts in cercariae and early schistosomula , followed by a marked differential gene expression in the other analyzed stages . however , 
 no significant difference in expression profile between the paralogs was observed for the analyzed stages . 
 furthermore , in order to detect desumoylating capabilities in crude parasite extracts , smsenp1 enzymatic activity was evaluated using sumo-1-amc substrate . 
 the endopeptidase activity related to sumo-1 precursor processing did not differ significantly between cercariae and adult worms . 
 taken together , these results support the developmentally regulated expression of sumo - specific proteases in s. mansoni . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion