toxins

Article Serrulin: A Glycine-Rich Bioactive Peptide from the Hemolymph of the Yellow Tityus serrulatus Scorpion

Thiago de Jesus Oliveira 1,2,*, Ursula Castro de Oliveira 1 and Pedro Ismael da Silva Junior 1,2,*

1 Special Laboratory for Applied Toxinology (LETA), Butantan Institute, São Paulo CEP 05503-900, SP, Brazil 2 Institute of Biomedical Sciences, University of São Paulo, São Paulo CEP 05508-900, SP, Brazil * Correspondence: [email protected] or [email protected] (T.d.J.O.); [email protected] (P.I.d.S.J.); Tel.: +55-11-96830-5482 (T.d.J.O.); +55-11-2627-9731 (P.I.d.S.J.)

 Received: 26 June 2019; Accepted: 5 August 2019; Published: 6 September 2019 

Abstract: Antimicrobial peptides (AMPs) are small molecules, which have a potential use as antibiotic or pharmacological tools. In chelicerate organisms, such as scorpions, these molecules constitute an alternative defense system against microorganisms. The aim of this work was to identify AMPs in the hemolymph of the Tityus serrulatus scorpion. Fractions of plasma and hemocytes were subjected to high-performance liquid chromatography (HPLC) and then analyzed to determine their activity in inhibiting microbial growth. One of the fractions from the hemocytes presents antimicrobial activity against microorganisms, such as Gram-negative and Gram-positive bacteria, fungi, and yeast. These fractions were analyzed by mass spectrometry, and a fragment of 3564 Da. was identified. The peptide was called serrulin, because it is derived from the species T. serrulatus. A comparison of the amino acid sequence of serrulin with databases shows that it has a similarity to the glycine-rich peptides described in Cupienius salai and gomesiana (). Furthermore, serrulin has no hemolytic activity against human erythrocytes. While the presence of AMPs in T. serrulatus venom has been described in other works, this is the first work to characterize the presence of these molecules in the hemolymph (hemocytes) of this species and show its potential use as an alternative to conventional antibiotics against different species of microorganisms.

Keywords: antimicrobial peptide; glycine-rich peptide; innate immune system; scorpions; Tityus serrulatus

Key Contribution: Antimicrobial peptide from the hemolymph of the scorpion, Tityus serrulatus, shows activity against different microorganisms and a similarity to glycine-rich AMPs from spiders.

1. Introduction In Brazil, there have been many studies on the species of yellow scorpion, Tityus serrulatus, which belongs to the Buthidae family. Scorpion stings are the most commonly reported accident of the reported accidents relating to venomous in Brazil, and the species T. serrulatus is considered one of the most dangerous species in Brazil [1]. T. serrulatus has a high proliferation by parthenogenesis [2] and is very common, mainly in Southeastern Brazil. T. serrulatus venom is composed mainly of neurotoxins that interact in molecular complexes that are essential cell membrane components (ion channels) [3]. The antimicrobial action of two T. serrulatus toxins (TsAP-1 and TsAP-2) and their analogs (TsAP-S1 and TsAP-S2) against microorganisms (bacteria and yeast), and their ability to inhibit the proliferation of human cancer cells [4,5] have been evaluated. While there have been studies that elucidate the composition and toxicity of the venom, there are no researches that describe the functioning of the immune system or characterize the antimicrobial peptides (AMPs) from the hemolymph (plasma and hemocytes) of T. serrulatus.

Toxins 2019, 11, 517; doi:10.3390/toxins11090517 www.mdpi.com/journal/toxins Toxins 2019, 11, 517 2 of 13

The innate immune system in invertebrates is basically composed of events involving cellular responses, including phagocytosis, nodulation, and encapsulation, and humoral responses, including the production of molecules from clotting and phenoloxidase cascade and the synthesis of AMPs [6,7]. AMPs can be classified according to their amino acid sequence, three-dimensional (3D) structure, and action spectrum. Defensins are a group of AMPs that are amphipathic with cysteine residues, and they have patterns of conserved bonds that ensure their stability [8]. The overall similarity between the defensins of ticks and those of scorpions is up to 70%, while the similarity between defensins of scorpions and those of insects is much lower [9]. Defensins have been described in the hemolymph from three different scorpions of the species Leiurius quinquestriatus, which was characterized as a defensin of 4321 Da and shown to have activity against Gram-positive bacteria [10]. In the scorpion Androctonus australis, an androctonin molecule shows activity against Gram-negative and Gram-positive bacteria and fungi. Androctonin has two disulfide bridges and a molecular mass of 3076 Da. In the same study using Androctonus australis, buthinin, an antimicrobial peptide with a molecular mass of 4605 Da, and which has activity against Gram-positive and Gram-negative bacteria and similarity to a defensin described in insects (Aeschna cyanea), was also described [11]. The most current work describing AMPs in the hemolymph of scorpions concerns a defensin found in the Centruroides limpidus limpidus. This defensin is denominated Cll-dlp (C. limpidus limpidus defensin-like peptide), presenting activity against Gram-positive and Gram-negative bacteria, with a molecular mass of 3821 Da [12]. These studies were performed with the total hemolymph of scorpions, without separating the peptides present in the plasma and those present in the hemocytes. Therefore, it was not possible to indicate the location of these peptides. The second AMP group contains the small open-end cyclic peptide, for example, the gomesin, isolated from the hemocytes of the Acanthoscurria gomesiana. Gomesin forms two internal disulfide bridges and adopts a β-hairpin-like fold. This peptide consists of 18 amino acids and has been tested against a variety of microorganisms [13]. The third group contains glycine-rich peptides. These AMPs are cationic and characterized by a high glycine content, for example, acanthoscurrins. These peptides are two isoforms isolated from the hemocytes of the spider Acanthoscurria gomesiana, and they have a positive charge and act against Gram-negative bacteria, Escherichia coli, and the yeast Candida albicans [14]. Ctenidins are glycine-rich AMPs from the hemocytes of the spider Cupiennius salei, and they are distinguished from acanthoscurrins by a sequence of 10 amino acids: VIDGKDDVGL. They also act against E. coli and Candida albicans [15]. The discovery of new AMPs from natural sources is of great importance for public health, since these molecules are pharmacological candidates due to their effective antimicrobial activity and low resistance rates [16]. AMPs have already been characterized in the hemolymph of different animals, such as acanthoscurrins and gomesin from the Acanthoscurria gomesiana spider [13,14], Rondonin from the Acanthoscurria rondoniae spider [17], Lacrain from the “centipede” Scolopendra viridicornis [18], and a fragment of a human fibrinogen peptide found in the hemolymph of Triatoma infestans [19]. All of these molecules are involved in the protection of these species and show how the discovery and characterization of bioactive peptides is very important and has a wide applicability, for example, selectivity towards cancer cells, with effects on cell proliferation [20]. In the present study, we isolated an AMP, serrulin, from the hemocytes of the scorpion T. serrulatus. We evaluated the antimicrobial and hemolytic activity, and mass spectrometry revealed a high percentage of glycine residues (G) and a similarity to other glycine-rich peptides from spiders.

2. Results and Discussion

2.1. Fractionation of Peptides from the Hemocytes After extraction of the hemolymph from the Tityus serrulatus scorpion, hemocytes and plasma were separated by centrifugation. The acid extract from hemocytes was applied to Sep-Pak® C18 cartridges for pre-fractionation. The molecules were eluted by three successive concentrations of Toxins 2019, 11, x FOR PEER REVIEW 3 of 13

Toxinscartridges2019, 11 for, 517 pre-fractionation. The molecules were eluted by three successive concentrations3 of of 13 acetonitrile (ACN) (5%, 40%, and 80%) in acidified water (0.05% TFA). All the material eluted at 40% acetonitrileACN was subjected (ACN) (5%, to RP-HPLC, 40%, and 80%)and all in fractions acidified we waterre tested (0.05% against TFA). different All the material microorganisms eluted at 40%in a ACNliquid was growth subjected inhibitory to RP-HPLC, assay. The and search all fractions for the hemocytes were tested ofagainst the scorpion different T. serrulatus microorganisms showed inthe a liquidpresence growth of five inhibitory antimicrobial assay. fractions The search (6, 2, for 30, the 34, hemocytes and 40) (Table of the 1), scorpion and the T.numbers serrulatus correspondshowed the to presencethe fraction of fivenumber antimicrobial from a set fractions of fractions. (6, 2, 30, 34, and 40) (Table1), and the numbers correspond to the fraction number from a set of fractions. Table 1. Antimicrobial activity fractions from hemocytes. Table 1. Antimicrobial activity fractions from hemocytes. Microorganism Escherichia coli MicroorganismMicrococcus luteus Candida albicans Aspergillus Hemocytes Fractions SBS363Escherichia MicrococcusA270 CandidaMDM8 albicans Aspergillusniger Hemocytes Fractions 5% 6 –coli SBS363 luteus–A270 MDM8– niger + 5%2 6+ – –+ – + + + 30 2 + + ++ ++ + + 40% 34 30 + + ++ ++ + + 40%40 34 + + ++ ++ + + 40 + + + +

Antimicrobial fractions from the hemocytes of the scorpion Tityus serrulatus present activity againstAntimicrobial microorganisms. fractions The from fractions the hemocytes were pre-fracti of the scorpiononated Tityusin three serrulatus concentrationspresent activityof acetonitrile against microorganisms.(ACN) (5%, 40%, The and fractions 80%) wereusing pre-fractionated a Sep-Pak® Column, in three concentrationsthen the samples of acetonitrile were subjected (ACN) (5%, to ® 40%,high-performance and 80%) using liquid a Sep-Pak chromatographyColumn, then using the a samples reverse-phase were subjected column to(RP-HPLC). high-performance The fractions liquid chromatographywere analyzed in usinga liquid a reverse-phasegrowth inhibition column assay, (RP-HPLC). and we found The five fractions antimicrobial were analyzed fractions in (6, a liquid 2, 30, growth34, and 40). inhibition assay, and we found five antimicrobial fractions (6, 2, 30, 34, and 40). Samples eluted in 5% (Figure 11)) andand 40%40% (Figure(Figure2 2A)A) ACN, ACN, with with 0.05% 0.05% TFA, TFA, presented presented fractions fractions with antimicrobial activity (marked with squaressquares andand/or/or circles).circles). No fractions with antimicrobial activity were identifiedidentified in the samples elutedeluted inin 80%80% ACN.ACN.

Figure 1.1.Reverse-phase Reverse-phase high-performance high-performance liquid liquid chromatography chromatography (RP-HPLC) (RP-HPLC) of the hemocyteof the hemocyte samples elutedsamples at eluted 5% acetonitrile at 5% acetonitrile (ACN). After (ACN). the fractionation After the fractionation step using astep Sep-Pak using® aC18 Sep-Pak column,® C18 the column, samples elutedthe samples in 5% eluted ACN were in 5% subjected ACN were to a Jupitersubjected® C18 to a semi-preparative Jupiter® C18 semi-preparative column, with acolumn, linear gradient with a fromlinear0% gradient to 20% from ACN 0% (dotted to 20% line) ACN in (dotted acidified line) water in acidified for 60 min water (1.5 mLfor /60min). min The(1.5 fractionsmL/min). were The collected manually and submitted to a test of the inhibition of microbial growth in a liquid medium. Peaks with squares indicate the antimicrobial activities against Aspergillus niger ().

Toxins 2019, 11, x FOR PEER REVIEW 4 of 13

fractions were collected manually and submitted to a test of the inhibition of microbial growth in a liquid medium. Peaks with squares indicate the antimicrobial activities against Aspergillus niger (□).

The presence of the antimicrobial peptides characterized in other and eluted at 40% ACN may be justified by the amphipathic characteristic of many antimicrobial peptides [11,12]. One of the fractions from hemocytes eluted at 40% ACN in 0.05% TFA was selected. This fraction, with antimicrobial activity and a higher absorbance, was chosen for a forward analysis. This fraction was the major peak, compared with the other fractions (Figure 2A), and it was named serrulin, referring to the species T. serrulatus. The homogeneity of the fraction was verified by RP-HPLC (Figure 2B). ToxinsThe peaks2019, 11 were, 517 hand-collected, and the antimicrobial activity of the major peak was reconfirmed4 of by 13 the inhibition of microbial growth in a liquid medium.

Figure 2.2.Reverse-phase Reverse-phase high-performance high-performance liquid liquid chromatography chromatography (RP-HPLC) (RP-HPLC) of the hemocyteof the hemocyte samples sampleseluted at eluted 40% acetonitrile at 40% acetonitrile (ACN). After (ACN). the fractionation After the fractionation step using astep Sep-Pak using® aC18 Sep-Pak column,® C18 the column, samples theeluted samples in 40% eluted ACN werein 40% subjected ACN were to a subjected Jupiter® C18 to a semi-preparative Jupiter® C18 semi-preparative column, witha column, linear gradient with a linearfrom 2% gradient to 60% from ACN 2% (dotted to 60% line) ACN in acidified (dotted waterline) in for acidified 60 min. Thewater fractions for 60 min. were The collected fractions manually were collectedand submitted manually to a testand ofsubmitted the inhibition to a test of microbialof the inhibition growth of in microbial a liquid medium. growth in Peaks a liquid with medium. squares Peaksand circles with indicatesquares antimicrobialand circles indicate activities antimicrobial against the testedactivities microorganisms: against the testedEscherichia microorganisms: coli SBS 363 (Escherichia), Microccocus coli SBS luteus 363 A270(●), Microccocus ( ), Candida luteus albicans A270MDM (○), Candida 8 (), and albicansAspergillus MDMniger 8 (■),( and)(A Aspergillus). Serrulin, nigerindicated (□) (A with). Serrulin, an arrow, indicated was# submitted with an to arrow, RP-HPLC was usingsubmitted a Jupiter to RP-HPLC® C18 analytic-column, using a Jupiter with® C18 a analytic-column,linear gradient from with 13% a linear to 43% gradient ACN from (dotted 13% line) to 43% in 0.05%ACN (dotted TFA, for line) 60 in min. 0.05% The TFA, fractions for 60 weremin. Thecollected fractions manually were collected and submitted manually toa and test submitted of the inhibition to a test of of microbial the inhibition growth of microbial in a liquid growth medium in aand liquid mass medium analyses and (B ).mass analyses (B).

The other presence fractions of the of antimicrobial the hemocytes peptides that showed characterized antimicrobial in other activity arthropods (fraction and 6 eluted eluted at in 40% 5% ACNand fractions may be justified2, 30, and by 40 the eluted amphipathic in 40%) characteristic were submitted of many to the antimicrobial same processes peptides but [were11,12 ].not One as homogeneousof the fractions as from fraction hemocytes 34 (serrulin). eluted The at 40% analyses ACN inby 0.05%mass spectrometry TFA was selected. were Thisnot as fraction, satisfactory with asantimicrobial the results activityobtained and for a higherfraction absorbance, 34. We believ wase chosenthat these for afractions forward could analysis. be Thisother fraction AMPs, wasfor example,the major the peak, small compared open-end with cyclic the other peptide fractions or defensins, (Figure2 A),which and have it was already named serrulin,been described referring in relationto the species to otherT. scorpions. serrulatus. The homogeneity of the fraction was verified by RP-HPLC (Figure2B). The peaks were hand-collected, and the antimicrobial activity of the major peak was reconfirmed by the inhibition of microbial growth in a liquid medium.

The other fractions of the hemocytes that showed antimicrobial activity (fraction 6 eluted in 5% and fractions 2, 30, and 40 eluted in 40%) were submitted to the same processes but were not as homogeneous as fraction 34 (serrulin). The analyses by mass spectrometry were not as satisfactory as the results obtained for fraction 34. We believe that these fractions could be other AMPs, for example, the small open-end cyclic peptide or defensins, which have already been described in relation to other scorpions. Toxins 2019, 11, 517 5 of 13

2.2. Bioassay

2.2.1. Antimicrobial Activity, Minimal Inhibitory Concentration (MIC) The antimicrobial activity was measured by liquid growth inhibition in a liquid medium [21]. The native peptide of the T. serrulatus scorpion hemolymph, called serrulin, was screened against a species of Gram-positive and Gram-negative bacteria, filamentous fungus, and yeast. The minimal inhibitory concentration (MIC) of serrulin was: 1.87–3.75 µg/mL (0.5–1µM) for Micrococcus luteus A270, 30–60 µg/mL (9–16 µM) for Echerichia coli SBS 363, 0.05–0.1 µg/mL (0.01–0.1 µM) for Pseudomonas aeruginosas, 12–24 µg/mL (3–6 µM) for Aspergillus niger, and 6–12 µg/mL (1.5–3 µM) for Candida albicans MDM8 (Table2). Compared with the other peptides isolated in the hemolymph of scorpions, serrulin shows a higher antimicrobial activity, mainly in the low concentrations that inhibited the growth of the bacteria Pseudomonas aeruginosas (MIC = 0.01–0.3). The molecule acted against Gram-positive and Gram-negative bacteria, filamentous fungus, and yeast, but it was not possible to confirm if the target of the action was common for all of the microorganisms. Further experiments would be needed to elucidate the mechanisms of action of this molecule for the different tested microorganisms.

Table 2. Antimicrobial activity spectrum of serrulin.

Strain MIC (µg/mL) µM Gram-positive bacteria Micrococcus luteus A270 1.87–3.75 (0.5–1) Gram-negative bacteria Escherichia coli SBS 363 30–60 (9–16) Pseudomonas aeruginosa ATCC 27853 0.05–0.1 (0.01–0.3) Fungi Aspergillus niger 12–24 (3–6) Yeast Candida albicans MDM8 6–12 (1.5–3) MIC, minimum inhibitory concentration (µg/mL): µM. The MIC refers to the concentration necessary to achieve a growth inhibition of 100%.

2.2.2. Hemolytic Activity No hemolytic activity of serrulin was identified at high concentrations (up to 60 µg/mL) against human erythrocytes. These results demonstrate that serrulin is not able to lyse human red blood cells (Figure S1).

2.3. Sodium Dodecyl Sulfate-Polyacrylamide Gel (SDS-PAGE) Serrulin’s action against microorganisms was detected by the inhibition of microbial growth in a liquid medium, and this fraction was submitted to another step of purification. The chromatographic profile (Figure2B) revealed a single absorbed protein peak, which was eluted at 33% ACN. The sample (20 µg) was applied to SDS-PAGE 20%, and the masses were compared to a molecular weight marker. Serrulin corresponds to a single peptide band between 3 and 6 kDa. (Figure3), thereby confirming its homogeneity and approximate molecular mass. It is interesting to highlight that AMPs with masses of 4 kDa have been described in the hemolymph of other scorpions, such as L. quinquestriatus [10], A. australis [11], and C. limpidus limpidus [12]. Toxins 2019, 11, 517 6 of 13 ToxinsToxins 20192019,, 1111,, xx FORFOR PEERPEER REVIEWREVIEW 66 ofof 1313

FigureFigure 3.3.3. Sodium SodiumSodium Dodecyl DodecylDodecyl Sulfate SulfateSulfate Polyacrylamide PolyacrylamidePolyacrylamide Gel ElectrophoresisGelGel ElectrophoresisElectrophoresis 20% (SDS-PAGE-20%), 20%20% (SDS-PAGE-20%),(SDS-PAGE-20%), stained stainedwithstained Coomasie-R withwith Coomasie-RCoomasie-R Blue. Line Blue.Blue. 1, molecular LineLine 1,1, momo weightlecularlecular marker weightweight (invitrogen markermarker (invitrogen(invitrogen SeeBlue® SeeBlue),SeeBlue expressed®®),), expressedexpressed in kDa; line inin kDa;2,kDa; serrulin lineline 2,2, in serrulinserrulin a non-reducing inin aa non-reducingnon-reducing condition conditioncondition (20 µg). (20(20 µg).µg).

2.4.2.4. MassMass SpectrometrySpectrometry anan anddd BioinformaticsBioinformatics AnalysesAnalyses AliquotsAliquots ofof samples samples digested digested (by(by trypsin) trypsin) andand not not digested digested were were analyzed analyzed separately separately by by mass mass spectrometryspectrometry in in an an LC-MS/MS,LC-MS/MS, LC-MS/MS, coupled coupled withwith anan LTQ-LTQ- LTQ-OrbitrapOrbitrapOrbitrap Velos. Velos. The The analysis analysis of of the the undigested undigested samplesample revealedrevealed aa singlesingle moleculemolecule ofof m/zm/z m/z 3564.0 3564.0 Da Da (Figure (Figure 4 4),4),), thusthusthus corroboratingcorroboratingcorroborating thethethe resultresultresult thatthatthat thethethe observedobserved mass mass in in SDS-PAGE SDS-PAGE 20% 20% waswas aboveabove 33 kDakDa (Figure(Figure3 3).3).).

FigureFigure 4. 4. SerrulinSerrulin spectrum. spectrum. MassMass spectrometryspectrometry analysesanalyses (LC-MS/MS,(LC-MS/MS, (LC-MS/MS, coupledcoupled withwith aa LTQ-OrbitrapLTQ-Orbitrap ® Velos)Velos) ofofof the thethe peptide peptidepeptide serrulin serrulinserrulin revealed revealedrevealed an m anan/z ofm/m/ 3564.0zz ofof 3564.03564.0 Da. Ions Da.Da. were IonsIons submitted werewere submittedsubmitted to MagTran toto MagTranMagTransoftware.®® software.software.

Toxins 2019, 11, 517 7 of 13 Toxins 2019, 11, x FOR PEER REVIEW 7 of 13

TheThe analysis analysis of serrulin of serrulin spectra spectra by PEAKS by Studio PEAKS software Studio revealed software a 3047 revealed kDa fragment, a 3047 with kDa afragment, 97% withcoverage, a 97%showing coverage, a showingsequence a sequencecomposed composed of 37 of 37amino amino acids acids (GFGGGRGGFGGGRGGFGGGGIGGGGFGGGYGGGKIKG)(GFGGGRGGFGGGRGGFGGGGIGGGGFGGGYGGGKIKG) (Figure (Figure 55),), deposited in the database of transcriptstranscripts of of the the T.T. serrulatus serrulatus scorpionscorpion (midgut) (midgut) [22] [22] and and its its venom venom gland gland (telson) (telson) [23]. [23]. A A mass mass near near ® 30473047 kDa kDa was was identified identified in in this this sample sample by by the the MagTran MagTran® softwaresoftware (Figure (Figure 4),4), indicating that some aminoamino acids acids were were not not identified identified in in PEAKS PEAKS Studio Studio software. software. In In addition, addition, the the PEAKS PEAKS Studio Studio software software identifiedidentified a a post-translational post-translational modification modification (PTM) (PTM) in in serrulin serrulin and and an an amidated amidated Lysine–Lys Lysine–Lys (K) (K) in in the the C-terminalC-terminal region region (Figure (Figure 55).).

Figure 5. Collision-induced dissociation (CID) spectrum of the novo sequence from serrulin. The ions Figurebelonging 5. Collision-induced to the -y (red) and dissociation -b (blue) series, (CID) indicated spectrum in of the the spectrum, novo sequence correspond from serrulin. to the amino The ions acid belongingsequence ofto thethe peptide:-y (red) and GFGGGRGGFGGGRGGFGGGGIGGGGFGGGYGGGKIKG. -b (blue) series, indicated in the spectrum, correspond to The the fragmentsamino acid of sequencethe sequenced of the peptidepeptide: are GFGGGRGGFGGGRGGFGGGGIGGGGFGGGYGGGKIKG. represented by standard amino acid code letters. The fragments of the sequenced peptide are represented by standard amino acid code letters. A BLAST (basic local alignment search tool) was used to identify similar sequences, using the 10 UniProtA BLAST as a database (basic local (), alignment with search an E-valuetool) was threshold used to ofidentify 10− . similar It is relevant sequences, to highlight using twothe UniProtgroups ofas moleculesa database that (Arthropod), showed similarity with an toE-value serrulin. threshold The first of result 10−10. isIt ais fragment relevant fromto highlight the secreted two groupsglycine-rich of molecules protein found that inshowed ticks of similarity the species toIxodes serrulin. scapularis The ,first with result an identity is a fragment of 68.1% (Figurefrom the S2). secretedMany peptides glycine-rich were protein identified, found especially in ticks of in the the species saliva ofIxodes the genusscapularisIxodes, with[24 an,25 identity]. While of none 68.1% of (Figurethese sequences S2). Many corresponded peptides were to identified AMPs or, especially had known in activities,the saliva theyof the were genus proteins Ixodes [24,25]. obtained While by a nonedirect of submission these sequences from the correspondedIxodes scapularis toGenome AMPs Projector had and known did not activities, have confirmed they were antimicrobial proteins obtainedactivity [26by]. a direct submission from the Ixodes scapularis Genome Project and did not have confirmed antimicrobial activity [26]. The second result, obtained by a database search, corresponds to two AMPs from the hemolymph (hemocytes) of the spider Acanthoscurria gomesiana (Theraphosidae), acanthoscurrin 1

Toxins 2019, 11, 517 8 of 13

ToxinsThe 2019 second, 11, x FOR result, PEER obtainedREVIEW by a database search, corresponds to two AMPs from the hemolymph8 of 13 (hemocytes) of the spider Acanthoscurria gomesiana (Theraphosidae), acanthoscurrin 1 (Acantho 1) and acanthoscurrin(Acantho 1) and 2 (Acantho acanthoscurrin 2). Both 2 are(Acantho molecules 2). classifiedBoth are as molecules glycine-rich classified antimicrobial as glycine-rich peptides. Serrulinantimicrobial has a peptides similarity. Serrulin of 63% has to Acanthoa similarity 2. of The 63% alignment to Acantho of 2. serrulin The alignment with the of last serrulin 37 amino with acidsthe last of 37 Acantho amino 1acids and of Acantho Acantho 2 1 shows and Acantho a conserved 2 shows sequence a conserved of amino sequence acids inof theamino C-terminal acids in regionthe C-terminal (Figure6 ).region It is very (Figure interesting 6). It tois notevery that, interesting in both, serrulinto note andthat, acanthoscurins in both, serrulin have and an amidationacanthoscurins of the have lysine an residue amidation in the of C-terminal.the lysine residue Acanthoscurrins in the C-terminal. has a marked Acanthoscurrins activity against hasC. a albicansmarked(MIC activity= 1.15–2.3 against µC.M) albicans and E. (MIC coli (MIC = 1.15–2.3= 2.3–5.6 µM)µ M)and [14 E.]. coli Despite (MIC the= 2.3–5.6 similarity µM) in [14]. the Despite glycine contentthe similarity and activity in the ofglycine serrulin content and acanthoscurrins,and activity of serrulin serrulin and presents acanthoscurrins, remarkable serrulin differences presents from acanthoscurrins,remarkable differences for example, from acanthoscurrins, the mass of serrulin for isexam 3564.0ple, Da, the while mass thatof serru of Acantholin is 3564.0 1 and Da, Acantho while 2that is 10169of Acantho Da and 1 10225and Acantho Da, respectively. 2 is 10169 InDa addition, and 10225 serrulin’s Da, respectively. activity against In addition,M. luteus serrulin’sand A. nigeractivitycould against be detected. M. luteus It isand interesting A. niger tocould note be that detected. there is anIt is amidation interesting of theto note lysine that residue therein is thean C-terminalamidation of in boththe lysine serrulin residue and acanthoscurins in the C-terminal (Figure in both5), suggesting serrulin and that acanthoscurins this modification (Figure may be5), importantsuggesting for that the this activity modification of these may molecules. be important for the activity of these molecules.

FigureFigure 6.6. Alignment of the amino acid sequencesequence ofof serrulinserrulin withwith otherother antimicrobialantimicrobial glycine-richglycine-rich peptides.peptides. UsingUsing the the UNIProt UNIProt alignment, alignment, the serrulin the sequenceserrulin wassequence aligned was with AMPs:aligned Acanthoscurrin with AMPs: 1,Acanthoscurrin Acanthoscurrin 1, 2,Acanthoscurrin and Ctenidin. Amino2, and Ctenidin. acids that Amino were identical acids that in allwere sequences identical are in shown all sequences in blue (orare *),shown and minorin blue modifications (or *), and minor are shown modifications in yellow are or shown (:). in yellow or (:).

CtenidinsCtenidins areare antimicrobial antimicrobial glycine-rich glycine-rich peptides peptides from from the hemocytesthe hemocytes of the of mature the mature female female spider Cupienniusspider Cupiennius salei. Three salei molecules. Three molecules were isolated were and isolated denominated and denominated ctenidins 1–3. ctenidins The monoisotopic 1–3. The molecularmonoisotopic masses molecular were 8810, masses 9507, were and 95648810, Da. 9507, Ctenidins and 9564 presented Da. Ctenidins antimicrobial presented activity antimicrobial against E. coliactivity(MIC against= 2.5–5 E.µ coliM), (MIC and, with= 2.5–5 ctenidins, µM), and, the with growth ctenidins, of Staphylococcus the growth aureusof Staphylococcuswas reduced aureus but was not inhibitedreduced but at concentrationsnot inhibited at of concentrations up to 10 µM. Theof up amidation to 10 µM. of The the amidation C-terminal of of the the C-terminal mature peptide of the ctenidinmature peptide is an ordinary ctenidin modification is an ordinary of somemodificati AMPson from of some other AMPs organisms, from ot suchher asorganisms, chelicerates such and as crustaceanschelicerates [and27–29 crustaceans], making these[27–29], peptides making more these stable. peptides In addition, more stable. the amidation In addition, is present the amidation in both acanthoscurrinsis present in both and acanthoscurrins serrulin. and serrulin. InIn T.T. serrulatusserrulatus transcriptstranscripts databases,databases, thethe fragmentfragment correspondingcorresponding toto serrulinserrulin waswas foundfound toto bebe aa “protein“protein notnot characterized”.characterized”. This is is because because the the identi identificationfication of of the the molecules molecules is is directly directly related related to to a astructure structure research research (midgut (midgut and and venom venom gland), gland), and and serrulin serrulin is is not not a a toxin toxin or a protein involvedinvolved inin digestion.digestion. However, it may be present in these structures,structures, because it is presentpresent inin thethe hemolymphhemolymph (whole(whole body)body) ofof thisthis species.species. AnalysesAnalyses usingusing the the ExPASy ExPASy ProtParam ProtParam [30 ][30] tool tool indicate indicate that serrulinthat serrulin is, like is, other like AMPs, other aAMPs, cationic a molecule,cationic molecule, containing containing a positive a charge positive (+4) charge (Figure (+4) S3). (Figure Generally, S3). cationicGenerally, antimicrobial cationic antimicrobial peptides act onpeptides the membrane act on the of membrane microorganisms of microorganisms by an electrostatic by an di electrostaticfference [31], difference but there are[31], AMPs but there that actare internallyAMPs that and act can internally interact and on ribosome, can interact internal on ribosome, proteins, orinternal nucleic proteins, acids (DNA or nucleic or RNA) acids [32– (DNA34]. Like or ctenidinsRNA) [32–34]. and acanthoscurrins, Like ctenidins theand mechanism acanthoscurrins, of action the of mechanism serrulin was of not action identified of serrulin in this work,was not so itidentified is not possible in this to work, compare so itsit is characteristics not possible with to compare those of similarits characteristics molecules. with those of similar molecules.The purification processes (Sep-Pak and HPLC), biological activities (antimicrobial activity, MIC, and hemolyticThe purification assay), characterizationprocesses (Sep-P (SDS-Page),ak and HPLC), and computational biological activities data acquisition (antimicrobial (mass analysis)activity, yieldedMIC, and quantities hemolytic of theassay), native characterization molecule that allowed(SDS-Page), us to and initially computational identify and data characterize acquisition serrulin. (mass Toanalysis) elucidate yielded its mechanisms quantities of of action, the itnative would molecu be necessaryle that to allowed extract more us to hemolymph initially identify and perform and thecharacterize purification serrulin. processes To elucidate again. its mechanisms of action, it would be necessary to extract more hemolymph and perform the purification processes again. It would be interesting to identify the site and mode of action of serrulin in the different microorganisms, but due to the difficult process of obtaining this molecule, we intend to carry out such studies with synthetic sequences of its conserved regions in the future.

3. Conclusions

Toxins 2019, 11, 517 9 of 13

It would be interesting to identify the site and mode of action of serrulin in the different microorganisms, but due to the difficult process of obtaining this molecule, we intend to carry out such studies with synthetic sequences of its conserved regions in the future.

3. Conclusions We isolated and characterized an antimicrobial peptide from the hemolymph of the Tityus serrulayus scorpion, and we named this peptide serrulin, referring to the name of the species. Serrulin exhibits a similar primary structure to glycine-rich antimicrobial peptides, which have already been described in relation to other , such as ticks and spiders, and it has a molecular mass of 3564 Da. Serrulin shows antimicrobial activity against Gram-positive and Gram-negative bacteria, filamentous fungi, and yeast. However, serrulin does not present cytotoxicity against human erythrocytes at the tested concentrations. In this work, the first antimicrobial peptide from the hemolymph of the yellow scorpion, Tityus serrulatus, was described. This acts on the innate immune system of the scorpion to aid in its self-protection. Moreover, the study of this molecule can be of great interest for the discovery of a new antibiotic molecule that acts against resistant bacteria or a pharmacological tool.

4. Material and Methods

4.1. Animals and Hemolymph Extraction Scorpions from the species Tityus serrulatus (, Buthidae) were collected in São Paulo and the Minas Gerais states and kept alive in a vivarium in the Special Laboratory for Applied Toxinology, under the licenses: Permanente Zoological Material Licenses 11024-3 IBAMA and Special Authorization for Access to Genetic Patrimony: 010345/2014-0, Instituto Butantan, São Paulo, Brazil. This project was accepted by the Commission of Ethics regarding the use of Animals of the Butantan Institute (Comissão de Ética no Uso de Animais do Instituto Butantan), number: 1839260716, on 5 November 2018. The total hemolymph (about 1 mL) was extracted from 50 individuals by cardiac puncture using a hypodermic needle. The hemolymph was extracted with a sodium citrate buffer (0.45 M NaCI; 0.1 M glucose; 30 mM trisodium citrate; 26 mM citric acid; and 10 mM EDTA), pH 4.6, to prevent hemocyte degranulation and clotting. The separation of the plasma and hemocytes was achieved through centrifugation in 800 g for 15 min at 4 C. The hemocytes were acidified in 5 mL of × ◦ acetic acid 2 M (Synth, Diadema, Brazil), homogenized using a Dounce® homogenizer, and maintained under agitation on ice for 30 min. Then, the sample was centrifuged at 16,000 g for 10 min at 4 C. × ◦ The plasma was acidified in 20 mL of acidified ultrapure water [trifluoroacetic acid (TFA) 0.05%]. The solution was maintained under agitation on ice for 30 min, and the sample was centrifuged at 16,000 g for 10 min at 4 C[35]. × ◦ 4.2. Fractionation of Antimicrobial Peptides The supernatant of the plasma and hemocytes (separated) was loaded in