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A kinetically stable plant subtilase with unique peptide mass fingerprints and dimerization properties

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A kinetically stable plant subtilase with unique peptide mass fingerprints and dimerization properties
  This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institutionand sharing with colleagues.Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third partywebsites are prohibited.In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further informationregarding Elsevier’s archiving and manuscript policies areencouraged to visit:  Author's personal copy A kinetically stable plant subtilase with unique peptide mass  󿬁 ngerprints anddimerization properties Subhash Chandra Yadav a,1 , M.V. Jagannadham b , Suman Kundu c, ⁎ , Medicherla V. Jagannadham a, ⁎ a Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, India b Proteomics Lab, Center for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India c Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India a b s t r a c ta r t i c l e i n f o  Article history: Received 23 August 2008Received in revised form 27 September 2008Accepted 27 September 2008Available online 8 October 2008 Keywords: MilinPlant subtilase homodimerizationKinetically stable protein (KSP)De novo sequencingPeptide mass  󿬁 ngerprintingDifferential subunit glycosylationMALDI TOF Milin, a potent molluscicide from the latex of   Euphorbia milii , holds promise in medicinal biochemistry.Electrophoresis, size exclusion chromatography, mass spectrometry and other biochemical characteristicsidentify milin as a homodimeric, plant subtilisin-like serine protease, the  󿬁 rst of its kind. The subunits of milin are differentially glycosylated affecting dimer association, solubility and proteolytic activity. Thedimeric dissociation is SDS-insensitive and strongly temperature dependent but does not appear to be linkedby disul 󿬁 de bridges. N-terminal sequence of acid hydrolyzed peptide fragments shows no homology toknown serine protease. Peptide mass  󿬁 ngerprinting and  de novo  sequencing of the tryptic fragments alsodid not identify putative domains in the protein. Milin seems to be a novel plant enzyme with subunitassociation partly similar to human herpes virus serine proteases and partly to penicillin binding proteins.Its behaviour on SDS-PAGE gels and other properties is like  “ kinetically stable ”  proteins. Suchsubunit association and properties might play a critical role in its physiological function and in controllingSchistosomiasis.© 2008 Elsevier B.V. All rights reserved. 1. Introduction Schistosomiasis, a communicable disease of concern especially indeveloping countries, is still a majorcause of morbidityand mortality.The risk of transmission of this disease can be controlled by the use of natural molluscicides, a relatively inexpensive treatment. However,thesearchforasuitablenaturalmolluscicidewithdesirablepropertieshas not yet yielded fruitful and con 󿬁 rmatory results. Latices of plantsbelonging to the Euphorbiaceae family are potential molluscicides,with latex of   Euphorbia milii  reported to be the most effective.Recently,wehaveshownthataputativeserineproteasefromthelatexof this plant, called milin, can be a potent molluscicide [1].For milin to be considered as an attractive natural drug for use asan anti-Schistosomiasis [1] agent, it must be thoroughlycharacterizedin several aspects. The  󿬁 nding that though there are reports of 12,000 – 35,000 species of latex producing plants [2] only those fromthe Euphorbiaceae family and especially  Euphorbia milii  are the mosteffective, points to the fact that milin could be a novel protein of medical importance. We have puri 󿬁 ed the protein to homogeneityfrom latex and the preliminary biochemical characteristics have beenreported [3]. The protein has catalytic properties of a serine protease;however, its N-terminal sequence (12 amino acids) shows nohomology with known serine proteases of either the trypsin orsubtilisin or serine-carboxypeptidase family [3]. Typical of secretedproteins, it is glycosylated. It also exhibits relatively high stabilityagainst various denaturants.Contrary to the perception that plant serine proteases are rare, thelast decade has seen several reports of such proteases in a wide rangeof tissues and organs [4]. However, most reports are only preliminaryand the enzymes are poorly characterized except cucumisin, the  󿬁 rstplant serine protease to be isolated. Their discovery in plants in largenumber means that they have important biological functions toperform.Yet,theirprimarystructure,three-dimensionalstructureandbiophysical characteristics are largely unknown. The presence of structural domains other than the proteolytic ones, their typical foldsand denaturation behavior are yet unexplored. Milin, with itstherapeutic (and yet unknown physiological) importance and uniquecharacteristics can very well serve as a model plant serine protease.The unique N-terminal amino acid sequence of milin necessitatesthat further peptide sequences of the protein be generated. Such anexercise would establish the novelty of the enzyme in its primarystructure compared to known serine proteases. Knowing furthersequenceswould alsohelp identifythe presenceof conserved domains,if any. The sequence information along with its already studiedbiochemical characteristics would probably help in placing the new Biophysical Chemistry 139 (2009) 13 – 23 ⁎  Corresponding authors. Kundu is to be contacted at Tel.: +919899007460; fax: +9111 24115270. Jagannadham, Tel.: +91 542 2367936; fax: +91 542 2367568. E-mail addresses: (S. Kundu), Jagannadham). 1 Present Address: Biotech Division, Institute of Himalayan Bioresources Technology,Palampur (H.P.) India.0301-4622/$  –  see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.bpc.2008.09.019 Contents lists available at ScienceDirect Biophysical Chemistry  journal homepage:  Author's personal copy proteaseinaproperfamilyofserineproteases.Finally,itwouldbeusefulin ongoing attempts at solving the three-dimensional structure of theprotease. Hence,  de novo  sequencingof the enzymeatvarious positionsalong its primary structure was accomplished by matrix-assisted laserdesorption/ionization time of   󿬂 ight (MALDI TOF TOF).Various serine proteases from animal and virus sources are wellstudied [5 – 7]. Many of them are multimeric and subunit associationplaysakeyroleintheirfunctionalattributesandstability.Amongothers,the trypsin-like serine protease Granzyme A, localized in the cytoplas-mic granules of activated lymphocytes and natural killer cells, wasreported in the homodimeric form [8]. Similarly, the disulphide linkeddimeric serine protease Factor IX was reported where the dimerizationis essential for its activity [7]. The dimeric forms of proteins areadvantageousin secretion machineryas well since large area of proteinsurfaces are protected in the dimer [9] and increase unusual proteaseactivity [10]. The activation of HIV 1 protease requires dimerizationwhere the dimer interfaces of protease and extra protease domainin 󿬂 uence the activation [11]. Even chymotrypsin dimerizes undercertain conditions [12]. Yet all plant serine proteases reported so farhave been found to be mainly monomeric, in spite of most of themhavinghighmolecularweight[4].Herewesuggestthefailureofnormalsodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)to detect oligomerization of highly stable plant serine protease likemilin.Inthepresentreport,theoligomerizationpropertiesofmilinhavebeen investigated in light of the fact that subunit association is provingto be vital for biological function for a wide range of protein classes,including but not limited to tumor necrosis factor receptor (TNFRs),epidermalgrowthfactorreceptors(EGFRs),serpins,GTPaseetc.[13 – 17]. 2. Materials and methods  2.1. Material Latex from  Euphorbia milii  was used to purify milin as describedpreviously[3]. Protein concentration was determined using an extinc-tion coef  󿬁 cient of   ε  2801% =29 [3]. Guanidine hydrochloride (GuHCl) wasprocured from Sigma Chemical Company, USA. All other chemicalswere of highest purity available commercially. The samples wereprepared in Millipore water and  󿬁 ltered through 0.45  μ  M  󿬁 lters.  2.2. SDS-PAGE  Electrophoresis was carried out under standard denaturing SDS-PAGE conditions where samples were heated at 95 °C for 5 min in 4% β -mercaptoethanol and 2% SDS. In addition, other harsh denaturingconditions were also used. For example, the protein (100  μ  g) wasincubatedatincreasingconcentrationofGuHCl( 󿬁 nalvolume10 μ  l)for24 h and the corresponding samples were loaded on the gel withheating (for 5 min at 95 °C) and without heating. Heating the proteininpresenceofSDSat140°Cfordifferenttimeperiods(2,5,8,10,12,15,20, 25 min) both in absence and presence of reducing agent was usedto monitor the effect of temperature on dissociation of dimer andstability of milin. Mineral oil was layered on the protein samples toprevent evaporation.  2.3. Native PAGE and gel  󿬁 ltration FornativePAGE,theproteinsampleswereloadedonthegelwithoutheating under non-denaturing and non-reducing conditions. Bovineserum albumin (BSA) and ovalbumin in the native form were usedas references. Gel  󿬁 ltration experiments were carried out on SephacrylS-200 under gravity. The column was equilibrated and protein elutedwith 50 mM potassium phosphate buffer, pH 8.0, containing 100 mMNaCl at a 󿬂 ow rate of 0.5 ml/min at 25 °C. The fractions sizewas 0.5 ml.Theelutionwasmonitoredbyabsorbancemeasurementsat280nm.Thecolumnwas pre-calibrated with standard molecular weight markers.  2.4. Mass spectrometry Mass spectrometry (MS) was used to determine the molecularmassofmilin.Thepurityandoligomerizationstateoftheproteinwerealso revealed in the process. The protein solution was extensivelydialyzed against Millipore water and 0.8  μ  l of the sample was spottedon a MALDI plate. Then 1  μ  l of sinapinic acid was used as matrix afterproteinssamplesweredried.BSAwasusedas standard.Thedatawereobtained in MALDI-TOF TOF 4800 using linear spectrum.  2.5. Deglycosylation of milin by TFMS  Deglycosylation of milin was achieved using Tri  󿬂 uoro methanesulphonic acid (TFMS), a prominent chemical deglycosylation agent[18]. Lyophilized milin (1 mg) was incubated at  − 20 °C with 150  μ  l of pre-chilled working TFMS ( − 20 °C) solution (having anisole, 1:1 v/v)for 30 min. The reaction was neutralized by adding 6% pre-chilledpyridine. The pyridine solution was diluted using 1:1 v/v methanol:water solution. The reaction solution was desalted by dialysis.Precipitated deglycosylated milin was solubilized in 1.5 M GuHCl atneutral pH and used for further studies. For SDS-PAGE, precipitateddeglycosylated milin was heated with sample buffer for 15 min at95 °C and loaded on 15% polyacrylamide gels.  2.6. Proteolytic activity of milin The ability of the enzyme to hydrolyze peptide bonds was routinelytested as described before using casein as substrate [3]. Active enzymewas indication of functional native protein while speci 󿬁 c activity asreported before was indication of the purity of the protein [3].Deglycosylated milin solubilized in 1.5 M GuHCl, as prepared above,was also assayed against casein. Native, glycosylated enzyme in 1.5 MGuHCl was used as reference for such measurements.  2.7. N-terminal sequence and homology search N-terminal sequence of the subunits of milin was determined asmentioned previously. The protein was subjected to SDS-PAGE asdescribed above. Protein bands on the gel were transferred to PVDFmembrane by Western blot method. The PVDF membrane was thenbrie 󿬂 y stained with Coomassie and the corresponding bands werecarefully and neatly excised. Homology of the N-terminal sequenceof the enzyme to known proteases was searched using NCBI blastsearch (, MEROPS database (www.merops. and manual inspection of serine protease sequences inpublished literature.  2.8. Circular dichroism Thecirculardichroism(CD)spectraofmilinundervariousconditionswererecordedonaJASCO715Aspectropolarimeter,pre-calibratedwith0.1% d-10-camphorsulfonic acid solution. Secondary structures in theprotein were monitored using far-ultraviolet CD (far-UV CD) spectra inthe wavelength range 180 – 260 nm, with a protein concentration of 0.1 mg/ml in a 1 mm path length cuvette. Whereas the changes in thetertiary structure were measured with a 10 mm path length cuvette inthe wavelength region of 260 – 340 nm with a protein concentration of 1 mg/ml.Theresultswereexpressedasmeanresidueellipticity[ θ ] MRW ,usingthe equation: θ ½  MRW   ¼  θ obs  X MRW  = 10 : c  : l  ð 1 Þ where  θ obs , c, and l represent respectively the observed ellipticity indegrees, protein concentration in mg/ml and the path length of the lightin cm. Meanweightof amino acid residues (MRW) wastaken as 110. The 14  S.C. Yadav et al. / Biophysical Chemistry 139 (2009) 13 –  23  Author's personal copy secondary structural content ( α -helix,  β -sheets) of the proteins wascalculated using software provided by JASCO.  2.9. Thermal and chemical denaturant induced unfolding of milin The protein samples were incubated in the cuvette at the desiredtemperature for 20 min prior to spectral measurements. Thetemperature was controlled using a Julabo F 25 water bath attacheddirectly to the cell holder in CD and  󿬂 uorescence. The temperature of the sample inside the cuvette was measured using a thermocoupleconnected to a digital multimeter. The  󿬂 uorescence measurementswere carried out on a Perkin-Elmer LS-50B spectro 󿬂 uorimeter. Theprotein concentration was 0.01 mg/ml for all  󿬂 uorescence measure-ments. Tryptophan was selectively excited at 292 nm. The emissionwas recorded from 300 to 400 nm with 10 and 5 nm slit widths forexcitation and emission, respectively.Calorimetricmeasurementswereperformedwitha MicrocalMC-2differential scanning calorimeter. Protein solutions (1.25 mg/ml) wereextensively dialyzed against 0.01 M buffer at the desired pH. Theprotein concentration and pH of the samples were rechecked andadjusted to desired concentration. All solutions were degassed undervacuum before being loaded in to the calorimeter cells. Thecalorimetric experiments were conducted at a scan rate of 60 °C/h.Buffer baselines were obtained under the same conditions andsubtracted from the sample curves.Urea-induced denaturation was performed by incubating theprotein sample at a desired denaturant concentration for approxi-mately 24 h at 25 °C to attain equilibrium. The  󿬁 nal concentrations of the protein and denaturant in each sample were determined byspectrophotometry and refractive index respectively. Measurementswere done using both CD and  󿬂 uorescence.  2.10. Preparation of acid hydrolysis fragments Milin(200 μ  g)washeatedinthepresenceof0.1MHClfor10minat80°C.MildacidhydrolyzedsamplewasloadedonSDS-PAGE.Differentbands obtained by acid hydrolysis were transferred to the PVDFmembrane and sequenced by N-terminal sequencing as describedabove.  2.11. Tryptic digestion of milin for MS analysis SDS-PAGE gel loaded with milin was extensively washed withwater after Coomassie Blue staining/destaining. The bands of interestwere excised and cut into ~1×1 mm squares with glass capillary. 2 – 3excised squares were taken and destained further using destainingsolution (100  μ  l of 50 mM ammonium bicarbonate in 50% acetonitrileand 0.1% TFA) for 20 min. The washing was repeated three times withfresh destaining solution each time. The supernatants werediscarded.The gel pieces were soaked in 100  μ  l of 100% acetonitrile for 5 min tillthey turned white and opaque. Acetonitrile was subsequentlyremoved and gel was dried in speed-vac for 30 min. A workingsolution of trypsinwas prepared using sequencing grade trypsin fromPromega (20  μ  g vial). Contents of this vial were dissolved in 1.0 ml of 20 mM ammonium bicarbonate and 10 aliquots (100  μ  l) wereprepared. These aliquots were frozen at  − 70 °C for future use. Thedried gel pieces were rehydrated in trypsin solution (10  μ  l per gelpiece) and incubated at 37 °C for 24 h. The digested peptides wereextracted in 50 µl of 50% acetonitrile: 0.1% TFA solution in 0.5 mlEppendorf tubes and agitated for 20 min. The supernatant wasremoved and the extraction was repeated with fresh extractionsolution. The extract was dried in the Speed-Vac and reconstituted in10 µl 0.1% TFA.1 µl of rehydrated tryptic digested sample was spottedon polished stainless steel MALDI target and left for drying. An equalvolume of a saturated solution of   α -cyano-4-hydroxycinnamic acid(HCCA) in 0.1% TFA/50% acetonitrile was spotted on the dried sample.The mixture was allowed to dry at room temperature and used forMALDI MS analysis.  2.12. de novo sequencing by MALDI TOF TOF  Peptide masses were determined using Matrix Assisted LaserDesorption Ionization -Time of Flight (MALDI-TOF) (4800 MALDI TOFTOF, Applied Biosystems) at Center for Cellular and Molecular Biology(CCMB), Hyderabad, India. Positive-ion mass spectra were recorded inboththelinearandre 󿬂 ectivemodes[19].MALDIreliesontheutilizationof a matrix compound capable of absorbing ultraviolet (UV) light. Thematriceswereofapproximately10,000-foldmolarexcessofthepeptideon  󿬁 nal deposition. The solvent was allowed to evaporate and co-crystallized analyte molecules embedded in matrix crystals wereacquired. Calibration for protein mass  󿬁 ngerprint (PMF) samples(digests) were performed both externally, using a mixture of ninepeptides ranging from  m /  z   757.40 to 3147.47 and internally by usingautolytictrypticfragments.Allsampleswereanalyzedinre 󿬂 ectormodebefore and after derivatization to obtain PMF spectra. The instrumentwasswitched to PSD mode and ion selector wasset tothe m /  z   values of precursor ions. The sample probe is then placed into the MS at highvacuum [19,20] because MALDI is a competitive process in which theionizationofananalytemaybeinhibiteddramaticallybythepresenceof others [20]. Thus, in tryptic peptide mixtures, arginine-containingpeptidesionizepreferentiallyduetothestronggasphasebasicityofthisamino acid [21,22]. 3. Results The preliminary biochemical characteristics of milin have beenreported elsewhere[3]. On classical SDS-PAGE, milin showed a singlebandat~52kDa[3].Sincetheplantserineproteasesreportedsofarareall monomeric with molecular weight in the range of 20 – 125 kDa [4],we took milin to be monomeric as well. However, discrepancies werenoticed during extensive work with the protein and we sought toinvestigate the oligomerizationproperties of the enzyme as well as itsother characteristics. Fig.1.  Electrophoreticanalysis of molecular weight and oligomerization of milin (a)Milinsubjected to SDS-PAGE. Lane 1, Protein sample prepared as per classical SDS-PAGEconditions (SDS, β -mercaptoethanol, 95 °C); Lane 2, The protein was heated to 100 °C for30 min prior to SDS-PAGE; Lane 3, Milin heated to 140 °C for 30 min prior to SDS-PAGE;Lane 4, Molecular weight marker. Milin shows temperature dependent dissociation.(b)NativePAGEofmilin.15%acrylamidegelwaspreparedwithoutSDS.Protein(15 μ  g)wasloaded on the gel without SDS,  β -mercaptoethanol and heat and electrophoresis wasperformed through cathode to anode. The molecular weight of milin is ~64 kDa. (c) Milinwas deglycosylated and the precipitated protein was heated gradually for 25 min withsamplebufferatincreasingtemperaturefrom50to95°Candloadedonthegel.Thebandat~32 kDa con 󿬁 rms the homodimeric association in milin.15 S.C. Yadav et al. / Biophysical Chemistry 139 (2009) 13 –  23  Author's personal copy  3.1. Molecular weight of milin is 64 kDa Initially, a molecular weight of ~52 kDa was observed for milin bySDS-PAGE under standard denaturing conditions (lane 1, Fig. 1a) [3]. However, native PAGE analysis revealed that the molecular weight of milinis~64kDaandnot~52kDa(Fig.1b).Further,themolecularweightbygel 󿬁 ltrationwasfoundtobe~64kDaaswell(Fig.2a),indicatingthatSDS-PAGEformilinresultsinanomalousdata.TheMSdataformilinalsoshowed two peaks indicating molecular weights of 31.593 kDaand 32.545 kDa, leading to a cumulative molecular weight of ~64 kDa(Fig. 2b). The peaks probably represent two subunits of milin.  3.2. On SDS-PAGE under harsh experimental conditions milin dissociatesinto subunits The anomaly in molecular weight and oligomerization observed formilin on SDS-PAGE and MALDI TOF were investigated further. As seenbefore[3], milinwhen heatedat95°Cshowed a singlebandat~52 kDaon SDS-PAGE (lane 1, Fig.1a). Here, theproteinwassubjected overtimeto even higher temperature (140 °C) under reducingconditionspriortoSDS-PAGE. As shown in lane 3, Fig. 3a, on being subjected to the highertemperaturefor2min,abandalsoappearedat~32kDa.Theintensityof the lower band increased and that of the upper band decreasedprogressivelywithtime(lanes4 – 10,Fig.3a),indicatingthatmilinindeedcontains subunits of molecular weight ~32 kDa thus corroborating theMS data. In absence of heat, however, the low molecular weight bandwas not observed (lane 2, Fig. 3a). The subunit association in milin thusseems to be based on a number of intricate interactions sincethe combination of lower temperature (95 °C) and  β -mercaptoethanol(lane 1, Fig. 1a), or  β -mercaptoethanol alone (lane 2, Fig. 3a) cannotdissociate the subunits. A combination of reduction ( β -mercaptoetha-nol) and high temperature is needed for complete dissociation unlikemost other oligomeric proteins. Further, the negligible contribution of disul 󿬁 de bonds to dimer association is exempli 󿬁 ed in the appearanceof similar ~32 kDa band when milin was heated to 140 °C for 30 minunder non-reducing condition (without  β -mercaptoethanol) as well(lane2,Fig.3b).Undersimilarconditions,absenceofheatdoesnotresultindissociation(lane1,Fig.3b).SuchanomalyonSDS-PAGEwasobservedfor a class of proteins known as  “ kinetically stable ”  proteins[23,24].These proteins are resistant to SDS denaturation and hence showanomalous migration on electrophoresis under reducing and non-reducing conditions.The strong association between milin subunits is further demon-strated in Fig. 4. When incubated with increasing concentrations of GuHCl (1 – 6 M) and subjected to SDS-PAGE without heating, at allconcentrations of the denaturant single bands at ~52 kDa wereobserved (Fig. 4a). Samples prepared similarly as above but heated to95°Cfor5minpriortoSDS-PAGEshowedadditionalbandsat~32kDa(Fig. 4b). In 5 M GuHCl and higher, only the low molecular weightprotein band was observed indicating complete subunit dissociationat these concentrations of the denaturant when heated (Fig. 4b). Thesubunit association thus seems to be temperature dependent as seen Fig. 2.  Molecular weight and oligomerization of milin: chromatographic and mass spectrometric analysis (a) Gel  󿬁 ltration of milin. Milin (200  μ  g) was loaded under gravity onSephacryl S-200 column of bed volume 15 ml, pre-equilibrated with 50 mM phosphate buffer, pH 8 containing 100 mM NaCl. Elutionwas performed using the same buffer at a  󿬂 owrate of 0.5 ml/min and 0.5 ml fractions were collected. The absorbance of the fractions is reported against the elution volume. Albumin, ovalbumin, chymotrypsinogen and RNasewere used as molecular weight markers under identical conditions. (b) MALDI TOFanalysis. MS analysis showed two peaks at 31,593.9 and 32,545.3 Da respectively. The cumulativemolecular weight (64 kDa) coincides with that obtained by gel  󿬁 ltration demonstrating the dimeric nature of milin. Mass resolution was obtained in the linear modes utilizingcontinuous ion extraction at threshold laser irradiance of 25 kV accelerating potential and 50 laser pulses were averaged. Fig. 3.  Effect of high temperature (140 °C) on subunit association of milin  ( a) Milin (25  μ  g) in SDS-PAGE loading buffer, layered with mineral oil on the top of the sample to preventevaporation, was heated to 140 °C over time. Lane 1. Molecular weight marker. Lanes 2 – 10. Milin heated for 0, 2, 5, 8, 10, 12, 15, 20, and 25 min, respectively. (b) SDS-PAGE wasperformed under non-reducing condition (absence of   β -mercaptoethanol). Lane 1. Sample loaded without heating; Lane 2. Sample heated for 30 min at 140 °C.16  S.C. Yadav et al. / Biophysical Chemistry 139 (2009) 13 –  23
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