Int J Pept Res Ther DOI 10.

1007/s10989-013-9351-2

Proteolytic Activity of the MMGP1 Antifungal Peptide Derived from Marine Metagenome
Muthuirulan Pushpanathan Jeyaprakash Rajendhran Paramasamy Gunasekaran

Accepted: 20 May 2013 Springer Science+Business Media New York 2013

Abstract An antifungal peptide, MMGP1 with direct cell penetrating property was recently reported from marine metagenome. The peptide showed efcient in vitro proteolytic activity, which could be associated with its antifungal activity. The proteolytic activity of MMGP1 was conrmed by tricine SDS-PAGE and gel ltration chromatography. Liquid chromatography-mass spectrometry analysis of MMGP1 treated bovine serum albumin (BSA), RNaseA and casein substrates revealed that the peptide does not have common cleavage position and it cleaves the substrates non-specically at all peptide bonds. The proteolytic activity of MMGP1 was enhanced in the presence of Mn2?. Molecular docking studies revealed that the predicted active site residues of MMGP1 could interact with BSA, RNaseA and casein. Keywords Marine metagenome Antifungal peptide Proteolytic activity Substrate specicity

Introduction Candidiasis is a potential life threatening infection to humans caused by an opportunistic fungal pathogen, Candida albicans. Due to the increasing frequency of candidiasis in immunocompromised patients together with strain resistant to currently available classes of antifungal drugs, there is an increased need
M. Pushpanathan J. Rajendhran (&) P. Gunasekaran Department of Genetics, Centre for Excellence in Genomic Sciences, School of Biological Sciences, Madurai Kamaraj University, Madurai 625021, India e-mail: jrajendhran@gmail.com Present Address: P. Gunasekaran Thiruvalluvar University, Vellore 632106, India

for identication and characterization of newer classes of anticandidal drugs as treatment alternatives (Sangamwar et al. 2008). Antimicrobial peptides are promising drug candidate for treatment of fungal infections as they possess mechanisms of action distinct from available antifungal agent and have the ability to regulate the host immune defense response as well (Danesi et al. 2002). Increasingly through evidences suggest that the antimicrobial peptides (AMPs) have internal targets by which they exert their antimicrobial action. The internal targets for AMPs include inhibition of macromolecular processes such as protein, nucleic acid and cell wall synthesis and enzymatic activities (Brogden 2005). Several short AMPs, like LL-37 (Sandgren et al. 2004), PAF26 (Munoz et al. 2006), lactoferrin and histatin5 (Huo et al. 2011) have been shown to have the ability to interact with nucleic acids inhibits the macromolecular synthesis of the target cells and causes cell death. Certain other AMPs, like BAC2A, indolicidin, sub3 and sub5 interacts with ATP and ATP dependent enzymes (Hilpert et al. 2010). Recently, we have identied a novel peptide [MLWSASMRIFASAFSTRGLGTRMLMYCSLPSRCWRK (MMGP1)], with direct cell penetrating property from marine metagenome (Pushpanathan et al. 2012a, b). The peptide showed efcient antifungal activity against C. albicans. In the present study, we investigated the proteolytic activity of MMGP1 that could be associated with its antifungal activity.

Materials and Methods Peptide Synthesis MMGP1 peptide was synthesized with [98 % purity employing solid phase methods using N-(9-uorenyl)methoxycarbonyl (Fmoc) Chemistry (Genscript Corporation, Piscataway,

123

Int J Pept Res Ther

NJ, USA). The peptide (5 mg) was dissolved in 1 ml of 50 mM Tris buffer (pH 7.4) and appropriately diluted sample was used for subsequent analysis. In Vitro Proteolytic Activity and Gel Filtration Chromatography The bovine serum albumin (100 lg) and RNaseA (100 lg) (Sigma-Aldrich, CA, USA) was incubated with MMGP1 (5 lM) in 50 mM Tris buffer (pH 7.4) for 1 h at 37 C. The samples treated with same volume of 50 mM Tris buffer without peptide was used as a negative control. The treated samples were then resolved on 16 % tricine-SDSPAGE. To further validate the proteolytic activity, RNaseA treated with MMGP1 was resolved by gel ltration chromatography using biologic duoow fast performance liquid chromatography system (BioRad, CA, USA). The treated sample was loaded on Biogel-P6 column (BioRad) equilibrated with 50 mM potassium phosphate buffer (pH 7.4) with 0.5 M NaCl and eluted with the same buffer at ow rate of 0.5 ml/min for 30 min. The protein and short peptide fragments were monitored at wave lengths 280 and 214 nm, respectively. LCMS Analysis Liquid chromatographymass spectrometry (LCMS) was used to study the substrate specicity for the protease activity. The substrates such as bovine serum albumin (BSA), RNaseA and casein was incubated with MMGP1 (5 lM) for 1 h at 37 C and the peptide fragments were separated by reverse phase-ultra high performance liquid chromatography (uHPLC) followed by mass spectrometry using Thermo Scientic LCQ Fleet Ion Trap LC/MSn (Thermo Fisher Instruments Limited, CA, USA). The mass LCMS data was collected in an elevated energy mode of acquisition with the spectral acquisition time of 0.019 s scan and 0.014 s interscan delay. All analyses were performed in positive mode of ESI. The TOF analyzer of the mass spectrometer was externally calibrated with a sodium formate mixture from m/z 100 to 2,000 Da. The mass data were post acquisition lock mass corrected using the Leucine-encephalin. The mass data were analyzed and the substrate specicity of MMGP1 was studied using Protein Lynx Global server (PLGS) version 2.2.5. Proteolytic Assay The proteolytic assay was performed using the azocasein assay method described by Reichard et al. (1990) with minor modications. Azocasein is the casein conjugated with azodyes, which liberates free dyes on action with substrates by proteolytic enzymes that could be assayed

quantitatively. For quantitative measurement of MMGP1 proteolytic activity, an appropriate dilution of peptide in 120 ll of 50 mM Tris buffer (pH 7.4) was mixed with 480 ll of 1 % (w/v) azocasein and the reaction mixture was incubated at 37 C for 30 min. The reaction was terminated by adding 600 ll of 10 % trichloroacetic acid and incubated on ice for 15 min followed by centrifugation at 15,0009g for 10 min at 4 C. The 800 ll of supernatant was neutralized with 200 ll of 1.8 N NaOH, and the absorbance was measured at 420 nm using spectrophotometer (Hitachi U-2000, Japan). One unit of protease activity was dened as the amount of protein required to yield an absorbance of 0.01 at A420 in 30 min at 37 C. The 50 mM Tris buffer (pH 7.4) without peptide was used as negative control. Effect of Metal Ions and Inhibitors The proteolytic activity of the peptide was assayed in the presence of different metal ions (Na2?, Mn2?, Mg2?, Fe2?, Cu2?, Co2? and Zn2?), inhibitors (PMSF and EDTA) and reducing agents (dithiothreitol and b-mercaptoethanol) at a nal concentration of 5 mM for 15 min at 37 C. The residual activity was measured under standard assay conditions. Computational Methods The active site of the peptide was predicted using computer atlas of surface topography of proteins (CASTp) (Dundas et al. 2006). Molecular docking of MMGP1 with BSA, RNaseA and casein was carried out in Hex server (http:// hexserver.loria.fr/) (Macindoe et al. 2010). The interaction of active site residues with the substrates were viewed using molecular visualization tool PyMOL.

Results Proteolytic Activity of MMGP1 The proteolytic activity of MMGP1 was studied using BSA and RNaseA as the substrate. The RNaseA and BSA treated with MMGP1 were resolved in tricine-SDS-PAGE. The partial degradation was observed for BSA, whereas presence of multiple protein bands was observed for RNaseA, which conrms the proteolytic activity of the peptide (Fig. 1a, b). The partial degradation of BSA could be due to the ratio between the MMGP1 and substrate used for analysis. The gel ltration of untreated and MMGP1 treated RNaseA was performed on Bio-Gel P-6 column. The untreated RNaseA was observed as a single major

123

Int J Pept Res Ther

different substrates produced by proteolytic activity of MMGP1 revealed that the peptide act substrates non-specically at all peptide bonds. Effect of Metal Ions and Additives The proteolytic activity of MMGP1 was estimated in the presence of metal ions and different additives (Fig. 4). The metal ions such as Fe2?, Mg2? and NaCl did not affect the proteolytic activity of the peptide even at a concentration of 5 mM, whereas Cu2?, Zn2? and Co2? showed below 50 % reduction in the proteolytic activity of MMGP1. However, Mn2? showed twofold increases in the proteolytic activity of MMGP1. The proteolytic activity was not signicantly affected in the presence of serine protease inhibitor, PMSF, whereas b-mercaptoethanol, DTT and EDTA showed slight reduction in the proteolytic activity. Active Site Prediction and Molecular Docking The active site of the MMGP1 was predicted using CASTp server. The aminoacid residues predicted in the active sites of MMGP1 are SER6, MET7, PHE10, MET25, TRP34 and ARG35, respectively (Fig. 5a). Molecular docking of MMGP1 with different substrates such as BSA, RNaseA and casein revealed that the predicted active site residues of MMGP1 favorably interact with these substrates as shown in Fig. 5b, which could attribute for its non-specic proteolytic activity.

Fig. 1 Proteolytic activity of MMGP1. The protein substrates BSA and RNaseA were treated with MMGP1 and the treated samples were resolved on 16 % tricine-SDS-PAGE a BSA treated with MMGP1, C-untreated BSA; 1-BSA treated with MMGP1 for 1 h b RNaseA treated with MMGP1, C-untreated RNaseA; 1-RNaseA treated with MMGP1 for 1 h

peak at 280 nm, whereas, the MMGP1 treated RNaseA showed a major peak at 214 nm proceeded by minor peaks represent the short peptide fragment and proteins of RNaseA produced by the proteolytic activity of MMGP1 (Fig. 2). Substrate Specicity of MMGP1 Substrate specicity of MMGP1 was studied using BSA, RNaseA and casein as substrates by LCMS. The mass spectra for the MMGP1 treated different substrates are shown in Fig. 3. The treatment of BSA, RNaseA and casein with MMGP1 produced proteins and short peptides fragments of masses range from 100 to 1500 Da. The analysis of proteins and peptide fragments of three

Discussion Previously, we have reported that MMGP1 induces cell death in C. albicans through energy independent direct cell penetration mechanism. The peptide was found to be

Fig. 2 Gel ltration analysis RNaseA treated with MMGP1. RNaseA was treated with MMGP1 for 1 h and the treated protein sample was resolved on Biogel P-6 column. The eluted proteins and

short peptide fragments were monitored at 280 nm (green) and 214 nm (blue), respectively. Control-untreated RNaseA; TreatedRNaseA treated with MMGP1 for 1 h period (Color gure online)

123

Int J Pept Res Ther

Fig. 3 LCMS analysis of different substrates treated with MMGP1. Mass spectra for different substrates such as BSA (a), RNaseA (b), casein (c) treated with MMGP1

123

Int J Pept Res Ther

Fig. 4 Effect of inhibitors and metal ions on the proteolytic activity of MMGP1. The peptide was pre incubated with inhibitors and metal ions for 15 min at 37 C. The proteolytic activity measured without additive was set as 100 %

initially localized on the cell membrane after 2 h and gets accumulated within the cytosol of C. albicans cells after 6 h without disrupting the cell membrane (Pushpanathan et al. 2012a, b). After entering into the target cells, the

peptide may affect the normal cell functions, which eventually lead to cell death. In present study, we investigated the proteolytic activity of MMGP1 that could be associated with its antifungal activity against C. albicans. The MMGP1 showed efcient in vitro proteolytic activity on three protein substrates. We found that the peptide does not have any common cleavage position and it cleaves the substrate non-specically at all peptide bonds. Since, there are no previous reports available on the proteolytic activity of an antifungal peptide. But certain proteases such as Solanum tuberosum aspartic proteases (StAP), Artocarpus heterophyllus AMP-48, Sarcophaga peregrine 26-kDa serine protease were reported to exhibit antifungal activity. Earlier, the hydrolytic specicity of StAPs was studied using oxidized insulin b-chain. Three cleavage sites (Leu15-Tyr16; Leu17-Val18 and Phe25-Tyr26) were reported for stAP1 and two cleavage sites ((Leu15-Tyr16 and Phe24Phe25) were reported for stAP3 (Guevara et al. 2004). AMP-48 is a serine protease, which efciently hydrolyzed a subunit followed by partially hydrolysis of b and c subunits of human brinogen (Siritapetawee et al. 2012). 26-kDa serine protease is a trypsin like serine proteinase serves to disintegrate the gut of immature stages of esh y and reshape it to form adult insects gut (Tsuji et al. 2001). The antimicrobial actions of all such proteases are mainly

Fig. 5 Active site prediction and molecular docking. a Active site of MMGP1 was predicted using CASTp server and the active site residues were marked as pink colour. b Molecular docking of MMGP1(green) with three different substrates such as BSA (grey), RNaseA (pink) and casein (brown) and the active site of MMGP1 were marked as red colour (Color gure online)

123

Int J Pept Res Ther

due to their proteolytic activity. Similarly, MMGP1 has non-specic proteolytic activity, which could attribute for its efcient antifungal activity. The active site of MMGP1 was predicted using CASTp server. It uses a semi-global-pairwise-sequence alignment method to obtain sequence mapping entries in Swiss-Prot, Online Mendelian Inheritance in Man (OMIM) and Protein Data Bank (PDB) and it locates and measures the pockets and voids in 3D-structure (Dundas et al. 2006). Molecular docking of MMGP1 with different substrates was performed using Hex server. It is a rst Fourier transform (FFT)-based protein docking server powered by graphics processors, which performs a typical 6D docking runs for*15 s and provides the docking prediction (Macindoe et al. 2010). The docking prediction of MMGP1 with different protein substrates such as BSA, RNaseA and casein revealed that the predicted active residues of MMGP1 could interact favorably with the substrate in exerting its proteolytic activity. It is interesting to note that the proteolytic activity of MMGP1 was found to be increased more than twofold in the presence of Mn2? suggesting that this metal ion could be used as a cofactor to improve its proteolytic activity. PMSF is a serine protease inhibitor, which acts by binding with serine residues at the active sites of the proteases. In our study, predicted active site of MMGP1 contains serine residue (at position 6) and the PMSF did not much affects the proteolytic activity of MMGP1, which showed that the serine residue in the active site of MMGP1 is not crucial for its proteolytic activity. Earlier, it was reported that the transition metal, Titanium (Ti) was known to inhibit the protease activity by direct binding to enzyme active sites and thereby prevents the subsequent binding of substrates to active sites (Duffy et al. 1998). Similarly, Zn2? Co2? and Cu2? showed decrease in the proteolytic activity of MMGP1, which could be due to direct binding of these metal ions to MMGP1 active sites. MMGP1 contains two cysteine residues at position 27 and 33. DTT and b-mercaptoethanol showed slight decrease in the proteolytic activity, which could be associated with the disruption of disulphide bond in MMGP1. In summary, this study shows the proteolytic activity of a direct cell penetrating antifungal peptide, MMGP1, which could be associated with its mechanisms of antifungal action.
Acknowledgments MP gratefully acknowledges the LADY TATA memorial trust, Mumbai for providing nancial support. JR acknowledges the Department of Science and Technology, New Delhi for providing nancial support under SERC Fast Track Scheme for Young Scientists (No. SR/FT/LS-004/2008). Authors gratefully acknowledge the Department of Biotechnology, New Delhi for providing nancial support (No. BT/PR-10486/BCE/08/657/2008). Authors also acknowledge the central facilities, CAS, CEGS, NRCBS, DBT-IPLS, DST-CEFC, DST-PURSE at MKU.

Conict of interests

Authors declare no conict of interest.

References
Brogden KA (2005) Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat Rev Microbiol 3:238250 Danesi R, Senesi S, Wout JW, Dissel JT, Lupetti A, Nibbering PH (2002) Antimicrobial peptides: therapeutic potential for the treatment of Candida infection. Expert Opin Investig Drugs 11:309318 Duffy B, Schwietert C, France A, Mann N, Culberston K et al (1998) Transition metals as protease inhibitors. Biol Trace Element Res 64:197213 Dundas J, Ouyang Z, Tseng J, Binowski A, Turzap Y et al (2006) CASTp: computed atas of surface topography of proteins with structural and topographical mapping of functionally annotated residues. Nucl Acids Res 34:116118 ssimo P, Pires E, Faro C, Daleo GR (2004) Potato Guevara MG, Ver aspartic proteases: induction, antimicrobial activity and substrate specicity. J Plant Pathol 86:233238 Hilpert K, McLeod B, Yu J, Elliott MR, Rautenbach M et al (2010) Short cationic antimicrobial peptides interact with ATP. Antimicrob Agents Chemother. doi:10.1128/AAC.01664-09 Huo L, Zhang K, Ling J, Peng Z, Huang X et al (2011) Antimicrobial and DNA-binding activities of the peptide fragments of human lactoferrin and histatin 5 against Streptococcus mutans. Arch Oral Biol. doi:10.1016/j.archoralbio.2011.02.004 Macindoe G, Mavridis L, Venkatraman V, Devignes MD, Ritchie DW (2010) HexServer: an FFT-based protein docking server powered by graphics processors. Nucleic Acids Res 38:W445 W449 Munoz A, Lopez-Garcia B, Marcos JF (2006) Studies on the mode of action of the antifungal hexapeptide PAF26. Antimicrob Agents Chemother 50:38473855 Pushpanathan M, Rajendhran J, Jayashree S, Sundarakrishnan B, Jayachandaran et al (2012a) Identication of a novel antifungal peptide with chitin-binding property from marine metagenome. Prot Pept Lett 19:12891296 Pushpanathan M, Rajendhran J, Jayashree S, Sundarakrishnan B, Jayachandaran S (2012b) Direct cell penetration of the antifungal peptide, MMGP1, in Candida albicans. J Pept Sci 18:657660 Reichard U, Buttner S, Eiffert H, Staib F, Ruchel R (1990) Purication and characterisation of an extracellular serine proteinase from Aspergillus fumigatus and its detection in tissue. J Med Microbiol 33:243251 Sandgren S, Wittrup A, Cheng F, Jonsson M, Eklund E (2004) The human antimicrobial peptide LL-37 transfers extracellular DNA plasmid to the nuclear compartment of mammalian cells via lipid rafts and proteoglycan-dependent endocytosis. J Biol Chem 279:1795117956 Sangamwar AT, Despande UD, Pekamwar SS (2008) Antifungals: need to search for a new molecular target. Indian J Pharma Sci 70:423430 Siritapetawee J, Thammasirirak S, Samosornsuk W (2012) Antimicrobial activity of a 48-kDa protease (AMP48) from Artocarpus heterophyllus latex. Eur Rev Med Pharmacol Sci 16:132137 Tsuji Y, Aoyama T, Takeuchi K, Homma K, Takahashi H et al (2001) Identication and characterization of an antibacterial peptide of the 26-kDa protease of Sarcophaga peregrina with antibacterial activity. J Biol Chem 130:313318

123