Báo cáo khoa học: Characterization of membrane-bound prolyl endopeptidase from brain ppt

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Characterization of membrane-bound prolyl endopeptidasefrom brainJofre Tenorio-Laranga1, Jarkko I. Vena¨la¨inen2, Pekka T. Ma¨nnisto¨3and J. A. Garcı´a-Horsman1,31 Centro de Investigacio´n Prı´ncipe Felipe, Valencia, Spain2 Department of Pharmacology and Toxicology, University of Kuopio, Finland3 Division of Pharmacology and Toxicology, University of Helsinki, FinlandProlyl oligopeptidase (POP; EC 3.4.21.26) is a serinepeptidase with prolyl endopeptidase (PE) activity,cleaving short peptides at the C-terminal side of pro-line residues, and is highly expressed in brain. Giventhat several neuropeptides, such as substance P, argi-nine–vasopressin, thyroliberin and gonadoliberin, areputative POP substrates, the importance of this prote-ase in several brain processes has been suggested [1].However, the precise role of POP in the brain has yetto be defined. Specific inhibitors of POP increase thelevels of these neuropeptides in the brain, exert anti-amnesia effects, and reverse memory and learning defi-cits produced by certain lesions [2]. Mammalian POP,encoded by the gene Prep, has been purified and crys-tallized, and its structure has been solved; it has beenconsidered to be soluble cytoplasmic enzyme. Therehas not been any structural or sequence-derived infor-mation that would suggest that the Prep gene productKeywordsneuropeptides; neurotransmission; peptidemetabolism; prolyl endopeptidase; prolyloligopeptidaseCorrespondenceJ. A. Garcı´a-Horsman, Division ofPharmacology and Toxicology, University ofHelsinki, Viikinkaari 5E, 00014 Helsinki,FinlandFax: +358 9 191 59471Tel: +358 9 191 59459E-mail: arturo.garcia@helsinki.fi(Received 7 March 2008, revised 3 July2008, accepted 4 July 2008)doi:10.1111/j.1742-4658.2008.06587.xProlyl oligopeptidase (POP) is a serine protease that cleaves small peptidesat the carboxyl side of an internal proline residue. Substance P, arginine–vasopressin, thyroliberin and gonadoliberin are proposed physiologicalsubstrates of this protease. POP has been implicated in a variety of brainprocesses, including learning, memory, and mood regulation, as well as inpathologies such as neurodegeneration, hypertension, and psychiatric disor-ders. Although POP has been considered to be a soluble cytoplasmic pepti-dase, significant levels of activity have been detected in membranes and inextracellular fluids such as serum, cerebrospinal fluid, seminal fluid, andurine, suggesting the existence of noncytoplasmic forms. Furthermore, aclosely associated membrane prolyl endopeptidase (PE) activity has beenpreviously detected in synaptosomes and shown to be different from thecytoplasmic POP activity. Here we isolated, purified and characterized thismembrane-bound PE, herein referred to as mPOP. Although, whenattached to membranes, mPOP presents certain features that distinguish itfrom the classical POP, our results indicate that this protein has the sameamino acid sequence as POP except for the possible addition of a hydro-phobic membrane anchor. The kinetic properties of detergent-solublemPOP are fully comparable to those of POP; however, when attached tothe membranes in its natural conformation, mPOP is significantly lessactive and, moreover, it migrates anomalously in SDS ⁄ PAGE. Our resultsare the first to show that membrane-bound and cytoplasmic POP areencoded by variants of the same gene.AbbreviationsAMC, amido-4-methylcoumarin; cPOP, cytoplasmic prolyl oligopeptidase; ER, endoplasmic reticulum; HA, hydroxylapatite; mPOP,membrane-bound prolyl oligopeptidase; PE, prolyl endopeptidase; POP, prolyl oligopeptidase; PPP, pure pig recombinant prolyloligopeptidase; Z-Gly-Pro-AMC, N-carbobenzoxy-glycyl-prolyl-7-amido-4-methyl-coumarin; ZPP, N-carbobenzoxy-prolyl-prolinal.FEBS Journal 275 (2008) 4415–4427 ª 2008 The Authors Journal compilation ª 2008 FEBS 4415is present in any locations inside or outside the cellsother than the cytoplasm, and no variants have beenpredicted or reported. This has been considered para-doxical, due to the extracellular location of the puta-tive POP substrates [3]. Nevertheless, PE activity hasbeen detected in all biological fluids and in membranesfrom most of the tissues studied, especially the brain.Different extracellular proteins with PE activity havebeen described. In serum, a PE activity, insensitive tospecific POP inhibitors, has been identified as fibro-blast activation protein or seprase, but important levelsof PE activity itself, sensitive to POP-specific inhibi-tors, have also been detected [4,5], although confirma-tion of this enzyme’s identity, by direct sequencing orby antibody binding, has not been provided.Membrane-bound PE activity has been detected andmeasured, and has been considered by several authorsto be POP activity [6–8]. However, isolated prepara-tions have been analyzed and regarded as a differentpeptidase, as these preparations have shown somephysical and enzymatic features that are different fromthose of its classical cytoplasmic counterpart [9,10].Recently, we have detected binding of antibody againstPOP in internal membranes in immunohistochemistrystudies in rat brain [11]. However, no clear identifica-tion of the protein responsible for this activity has beenprovided. Here, we report the purification and identifi-cation of the membrane-bound PE (herein referred toas membrane-bound prolyl oligopeptidase, mPOP)from pig brain and characterization of the enzyme’sproperties in comparison to POP. The nature of mPOPassociation with membranes was also studied.ResultsMembrane-associated PE activity is tightly boundpreferentially to synaptosomes and endoplasmicreticulum (ER)Initial whole membrane fractionation of pig brainhomogenate resulted in the partitioning of total PEactivity between membrane-bound and soluble frac-tions with a 40 : 60 ratio. As reported previously [10],high-salt washes and a hypotonic treatment wererequired to detach loosely bound PE activity from themembranes. Accordingly, we found that a considerableamount of PE activity bound to the membranes wasreleased upon a 0.5 m NaCl wash of total membranepreparation (Table 1). A hypotonic wash and two fur-ther salt washes were necessary to ensure that all theloosely bound POP was released. Further washesreleased no detectable activity from the membranes,but detectable levels were tightly attached to them(Table 1), and those were sensitive to specific POPinhibitors (see below).Following this series of washes, the membranes werefurther fractionated by centrifugation on a sucrose gra-dient to determine which types of membrane containedPE activity. After a three-cushion gradient (0.8, 1.0and 1.2 m sucrose), we were able to separate three dif-ferent membrane fractions, low density (on top of the0.8 m layer), medium density (0.8 and 1.0 m interface)and high density (1.0 and 1.2 m interface). With theuse of specific enzyme marker assays (Fig. 1), we iden-tified the heavy membranes as ER, whereas the mem-branes of intermediate density were mainly composedof synaptosomal and mitochondrial membranes. Thelight fraction contained myelin membranes, asdescribed previously [10]. Although we detected thepresence of PE activity in all membrane fractions, thisactivity was maximal in the synaptosomal fraction(Table 2), similar to the observations reported byO’Leary & O’Connor [9]. As the activity detected inthe various membrane fractions could be attributableto other peptidases, we applied the purification proto-col (detailed in Experimental procedures) to both ERand synaptosomal membranes. All elution profilesresulting from this purification scheme were identical,regardless of the membrane fraction origin. Moreover,analysis of these preparations revealed that the kineticproperties of both fractions were also identical (datanot shown). Thus, we decided to employ the purifica-tion protocol using whole membrane preparation asstarting material, as the yield was considerably higher.A multistep protocol enriches membrane PEactivity > 2000-foldPE activity solubilization from membranes was onlyachieved by extraction with detergents, such asTriton X-100 at 0.4%. This detergent treatment wassufficient to solubilize all activity associated withTable 1. POP activity partitioning during membrane preparationfrom pig brain crude extract and membrane wash effects on recov-ered activity.SampleVolume(mL)Protein(mg)Specific activity(nmolÆmin)1Æmg)1)Total activity(nmolÆmin)1)Crude extract 1080 8704 1.0 8645Unwashedmembranes500 2354 1.4 33840.5M NaCl wash 450 305 9.0 2760Water wash 400 107 13.8 14804M NaCl wash 300 92 4.8 440Washedmembranes90 1005 0.2 161Membrane-bound prolyl oligopeptidase: mPOP J. Tenorio-Laranga et al.4416 FEBS Journal 275 (2008) 4415–4427 ª 2008 The Authors Journal compilation ª 2008 FEBSmembranes, and also produced a three-fold activationof enzymatic activity (Table 3). This increase was notdue to assay conditions, such as the presence of reduc-tants. Solubilized membrane PE activity was sub-sequently purified by several chromatography steps.The use of a DEAE column as a first step eliminatedall cationic and most weak anionic protein contami-nants, which altogether constituted more than half ofthe total protein (Table 3). Substantial further purifica-tion was achieved with phenyl–Sepharose and hydroxyl-apatite (HA) columns (see Fig. S1). Although this stepyielded more than 300-fold PE activity purification(Table 3) with respect to total membrane, SDS ⁄ PAGErevealed the presence of several protein bands (datanot shown). Thus, to further purify PE activity, wedialyzed the HA pool to decrease the salt concentra-tion and reapplied it to a DEAE column (see Fig. S1for column chromatogram). This procedure enrichedactivity 1700-fold, but SDS ⁄ PAGE analysis stillrevealed several contaminating proteins. Consequently,native gel electrophoresis was utilized to improve puri-fication. Under these conditions, a single band wasrevealed by silver staining that coincided with the PEactivity profile of the gel lane (Fig. 2). It is importantto note that once PE activity was solubilized, neitherstability nor activity was modified by detergent concen-tration. Extensive removal of Triton X-100, by seriesof dilutions and ultrafiltrations where the detergentwas undetectable (< 0.0001%), did not produce pro-tein precipitation or loss of activity.The gel filtration profile of solubilized membranePE activity varies with ionic strength, similarly tothat of cytoplasmic POP (cPOP)It has been suggested previously that the enzymeresponsible for membrane PE activity is distinct fromPOP, on the basis of the difference between theirmolecular masses (87 kDa versus 65 kDa) obtainedin gel filtration experiments [9,10]. The theoreticalmolecular mass of POP is 80 kDa, which agrees withestimates from SDS ⁄ PAGE [1]. Initially, we alsothought that membrane PE was heavier than POP,as under our conditions (20 mm potassium phos-phate) it eluted with a molecular mass of 95 kDa bygel filtration. However, when higher salt concentra-tions were used, membrane PE activity also elutedwith a molecular mass of 65 kDa (Fig. 3). Thisbehavior did not depend on the Triton X-100 con-centration, and was very similar to that of POPwhen run in the same conditions.Identification of the protein responsible formembrane PE activityPeptides produced by trypsin digestion of purifiedmembrane PE were analyzed by liquid chromatogra-phy–MS ⁄ MS on Qstar (HPLC ⁄ Q ⁄ TOF) or MALDI-TOF ⁄ TOF MS, which revealed that these fragmentscorrespond to the sequence of mammalian POP0.0750.0500.0250.000TMHMSuc. dehydrog. (units·mg–1)TM MMHMLMPSD-95CalnexinER-6090 kDa50 kDa90 kDaLMMMFig. 1. Identification of the different membrane fractions where PEactivity is bound. Various membrane fractions were obtained fol-lowing application of washed total pig brain membrane preparationto sucrose gradients. The mitochondrial marker, succinate dehydro-genase, was measured in every fraction (upper panel), as describedin Experimental procedures. PSD-95 (synaptosomal marker), calnexinand ER-60 (ER markers) were assayed by western blotting (lowerpanels). TM, total membranes; LM, low-density membranes; MM,medium-density membranes; HM, high-density membranes. Theamounts of protein loaded onto gels were as follows: for the PSD-95 blot, 70 lg of TM, 96 lg of LM, 89 lg of MM, and 93 lgofHM; for the calnexin blot, 70 lg of TM, 96 lg of LM, 89 lgof MM, and 93 lg of HM; and for the ER-60 blot, 8 lgofMMand 5 lg of HM.Table 2. Membrane-bound POP activity of different-density mem-brane fractions obtained by sucrose gradient centrifugation.SampleTotalvolume(mL)Specific activity(nmolÆmin)1Æmg)1)Total activity(nmolÆmin)1)Washed membranes 60 0.50 145Low-density membranes 2.3 0.61 9Medium-densitymembranes4.5 0.45 24High-density membranes 10 0.17 21J. Tenorio-Laranga et al. Membrane-bound prolyl oligopeptidase: mPOPFEBS Journal 275 (2008) 4415–4427 ª 2008 The Authors Journal compilation ª 2008 FEBS 4417(EC 3.4.21.26) (Fig. 4). To further confirm this, wes-tern blots were performed using an antibody specificagainst POP. All active fractions obtained during themembrane PE purification process reacted with theantibody against POP, thereby confirming the sequencesimilarity between these two variants (Fig. 5A). Analy-sis of the membrane PE tryptic peptides by MS didnot provide any insights into the membrane associa-tion mechanism, such as sugar or lipid attachments(not shown).Within the membranes, mPOP shows anomalousSDS⁄PAGE migrationPrior to Triton X-100 solubilization, different brainmembrane preparations were analyzed by western blot-ting, using POP as a control. A significant fraction ofmPOP migrated faster than POP when a whole mem-brane preparation was subjected to SDS ⁄ PAGE, andanti-POP reactive bands were detected by western blot-ting (Fig. 5B). The proportion of this ‘lighter’ form,relative to that which corresponds to the cytoplasmicpurified POP control, varied with the type of mem-brane fraction analyzed. As can be seen in Fig. 5B, themyelin fraction showed a band in the western blot atthe same size as the pure pig soluble recombinant POP(PPP), but the synaptosomal fraction showed two anti-POP reactive bands in a ratio of approximately 50%.The heavier ER membrane fraction contained almostFig. 2. Membrane PE purification by native electrophoresis fromthe concentrated HA active fractions. Three adjacent lanes of anative Triton–PAGE gel were loaded with 10 lg each of protein.After electrophoresis (see Experimental procedures), the centrallane was excised in 5 mm pieces along the lane vertical axes. Hori-zontal blade cuts were around 8 mm long such that small incisionsat the edge of adjacent lanes were produced to find the corre-sponding pieces in the western blot, made with the first lane, andthe protein stain (silver), made with the third lane.Fig. 3. Membrane PE gel filtration on Superdex-200 in 100 mMphosphate buffer ( ) and in 20 mM potassium phosphate buffer(), performed as described in Experimental procedures. Thepositions of elution of molecular mass standards are indicated:ribonuclease (Rib), 13.7 kDa; chymotrypsinogen (Chym), 25 kDa;ovoalbumin (Ovo), 43 kDa; BSA, 37 kDa; aldolase (Ald), 158 kDa;catalase (Cat), 232 kDa; ferritin (Fer), 440 kDa; and thyroglobulin(Thyr), 669 kDa.Table 3. Purification of mPOP from a total membrane preparation. POP was assayed as described in Experimental procedures. Total activityis specific activity multiplied by total protein in milligrams; after Triton X-100 extraction, there is a > 3-fold activation of activity, which isreflected by an increase in total activity. Yield percentage refers to the activity in detergent extract, which is shown in parentheses.SampleTotalvolume (mL)Totalprotein (mg)Specific activity(nmolÆmin)1Æmg)1)Total activity(nmolÆmin)1) Yield (%)FoldpurificationTotal membranes 150 855 0.5 453 100 1Triton X-100 extraction 1000 533 3.3 1759 388 (100) 6.6DEAE 680 232 7.4 1717 379 (98) 14.8Phenyl–Sepharose 160 43 39 1679 371 (95) 78HA 25 9.5 169 1595 352 (90) 338Second DEAE 6.5 0.9 857 771 170 (43) 1714Membrane-bound prolyl oligopeptidase: mPOP J. Tenorio-Laranga et al.4418 FEBS Journal 275 (2008) 4415–4427 ª 2008 The Authors Journal compilation ª 2008 FEBSexclusively the light band. This anomalous behaviorwas reproducible, and was consistently eliminated bymembrane solubilization with Triton X-100 (Fig. 5C).In these cases, the anti-POP reactive band always ranat the same molecular mass as the band that corre-sponded to cPOP, or to the pure POP control, regard-less of the membrane fraction that the sample wasprepared from.Kinetic properties of purified solublemembrane PEWe next attempted to differentiate membrane PE fromPOP on the basis of kinetic properties. This compara-tive analysis included semipure pig brain POP, PPP,and membrane PE purified by HA chromatography.Accordingly, different inhibitors and substrates weretested, and the kinetic behavior of membrane PE, incomparison to that of POP, was evaluated.It is known that POP is inhibited by some divalentcations [12,13], including the heavy metals Mn2+,Cu2+,Ni2+, and Zn2+[14]. Our results demonstratethat there were no significant differences betweenmembrane PE and POP regarding their sensitivity tothese metals (Table 4). In addition, we compared theeffects of some general serine, cysteine and metallo-protease inhibitors, specific POP inhibitors, such asN-carbobenzoxy-prolyl-prolinal (ZPP) and JTP-4819,the proteasome inhibitor N-carbobenzoxy-leucyl-leucyl-leucyl-COH, the specific dipeptidyl peptidase IVinhibitor HIV-1 tat (1–9) fragment with sequenceH-Met-Asp-Pro-Val-Asp-Pro-Asn-Ile-Glu-OH, and thePOP inhibitor a2-gliadin 33-mer peptide [15] (seeABCFig. 5. Western blots of different membrane PE preparationsobtained using an antibody against POP. (A) Cross-reactivity againstmembrane PE and recombinant POP of antibody against POP.Membrane PE, 8 lg of protein; PPP, 120 ng of protein. (B, C) Bloton total membranes (TM), light-density membranes (LM), medium-density membranes (MM) and high-density membranes (HM) frompig brain before (B) or after (C) Triton X-100 solubilization and com-pared with PPP. Protein amounts were as follows: (B) PPP0.15 lg, TM 30 lg, LM and HM 60 lg, and HM 120 lg. (C) PPP0.15 lg, TM 12 lg, LM 23 lg, MM 23 lg, and HM 30 lg.Fig. 4. POP amino acid sequence from Sus scrofa (accession num-ber NP_001004050 Ver NP_001004050.1 GI:51592147). Peptidesfrom tryptic digestion of purified membrane PE are highlighted (seeDoc. S1 in Supporting information also).Table 4. Effects of divalent cations, ionic strength and chelators onPOP activity of membrane PE (A) and on cPOP (B).0 lM 1 lM 10 lM 100 lM 1mM 10 mM(A) Membrane PEMg2+100 100 101 104 101 98Ca2+100 97 99 103 101 97Mn2+100 98 96 86 80 28Cu2+100 102 98 90 11 8Ni2+100 96 94 78 8 8Zn2+100 93 94 13 8 80mM 100 mM 200 mM 400 mM 600 mM 800 mMNaCl 100 124 127 153 138 1330mM 2.5 mM 5mM 10 mM 25 mM 125 mMEDTA 100 89 78 62 42 26(B) cPOPMg2+100 101 99 96 99 98Ca2+100 96 98 104 93 95Zn2+100 96 86 4 3 30mM 2.5 mM 5mM 10 mM 25 mM 125 mMEDTA100 86 85 64 46 24J. Tenorio-Laranga et al. Membrane-bound prolyl oligopeptidase: mPOPFEBS Journal 275 (2008) 4415–4427 ª 2008 The Authors Journal compilation ª 2008 FEBS 4419Table S1). As expected, both membrane PE and POPwere very sensitive to the specific POP inhibitors. Fur-thermore, the kinetic constants produced by carefultitration of ZPP, a very specific and potent POP inhib-itor, were equivalent for both preparations (Fig. 6).Both membrane PE and POP were partially resistantto phenylmethanesulfonyl fluoride, a generic serineprotease inhibitor that has a low efficiency in inhibit-ing POP [1]. We also found that membrane PE wasinhibited by SH-reactive compounds such as malei-mides, similarly to POP, and that both were similarlyresistant to dipeptidyl peptidase IV and proteasomeinhibitors. In addition, membrane PE showed the samesensitivity to the 33-mer peptide (see Table S1) as hasbeen reported for POP [15].Previous reports suggest that membrane PE andPOP display similar specificity for several proline-containing neuropeptides, including bradykinin, angio-tensin II, neurotensin, substance P, and gonadoliberin[10]. In addition to these substrates, we assayed severalother peptides in an effort to define functional differ-ences between membrane PE and POP. However, asshown in Table 5, no differences were observed forany of the substrates tested.Membrane PE probably associates with themembrane by nonprotein hydrophobic anchoringThe nature of the association of membrane PE withmembranes is not known. We tried to address thisquestion in several ways. Analysis of the hydrophobic-ity profile of the POP protein sequence revealed thatthe presence of membrane-spanning segments is highlyimprobable (see Doc. S2 in Supporting information).Our experiments with membrane PE extracted withTriton X-114 demonstrated that this enzyme activity isquantitatively partitioned in the hydrophilic phase,arguing against any important hydrophobic proteindomain that would link the protein to the intermem-brane milieu.To further confirm this, we ran a temperaturedependence assay with membrane PE-containing mem-branes (Fig. 7). Intrinsic membrane proteins display abreak in the Arrhenius plot due to membrane phasetransition, and delipidation of these particulateenzymes eliminates the discontinuity in the plots [16].We did not observe any break in the membrane PEArrhenius plot; however, there was a significant changein the slope of the temperature dependency curve whencompared with that obtained for POP (Fig. 7). Fur-thermore, the membrane PE Arrhenius plot resembledthat of POP when the experiment was performed afterdetergent solubilization of membrane PE.POP has been implicated in axonal transport [17].Soluble protein elements in these processes are recruitedto membranes, through interaction with other proteinsand SH-bonding and ⁄ or divalent cation (Ca2+)-depen-dent mechanisms. To test whether a similar processwould mediate the membrane PE association withmembranes, we evaluated the expression levels of PEin membranes after homogenization or washes withN-ethylmaleimide or EDTA. The presence of N-ethyl-Fig. 6. Inhibitory effect of ZPP (Z-Pro-Prolinal) on POP (s) or mem-brane PE () activity. The assay was performed as described inExperimental procedures in the presence of the correspondinginhibitor concentrations during preincubation. The estimated IC50values for membrane PE (continuous line) and POP (broken line)were 0.48 nM (± 0.005 nM) and 0.52 nM (± 0.005 nM) respectively.Table 5. Substrate specificity studies on membrane PE as compared with POP. +, ZPP-sensitive cleavage of the peptide occurred;), cleavage of the peptide did not occur.Peptide Sequence Membrane PE POP12-mer H2N-QLQPFPQPQLPY-OH ++33-mer LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF ))HIV H-MDPVDPNIE-OH ++PEP-3 YGRKKRRQRRRG-NH2))PEP-26 RGTICKKTMLDGLNNYCTGVGR-NH2))PEP-48_2 Ac-LINEEEFFDAVEAALDRQ-NH2))PEP-50 Ac-PYSRSSSMSSIDLVSASDDVHRFSSQ-NH2))PEP-52 Ac-CDPGYIGSR-NH2++Membrane-bound prolyl oligopeptidase: mPOP J. Tenorio-Laranga et al.4420 FEBS Journal 275 (2008) 4415–4427 ª 2008 The Authors Journal compilation ª 2008 FEBSmaleimide or EDTA, or both, during homogenizationor membrane washes did not alter the amount of PEdetected in the membrane fraction (data not shown).An alternative explanation is that membrane PE isattached to membranes through a hydrophobic anchorthat has been added to the protein post-translationally.A web-based (expasy) analysis of the POP sequencefor post-translational modifications related to glycosyl-ation, myristoylation, prenylation and glycosylphos-phatidyl inositol anchoring returned very low scores(see Doc. S2 in Supporting information). Furthermore,POP lacks a signal sequence required for some of thesemodifications. The only possibility that was found ispalmitoylation of the Cys563 within the sequenceGGLLVATCANQRPDL(556–570), which, accordingto the css-palm server (http://bioinformatics.lcd-ust-c.org/css_palm/), has a relatively good score for modifi-cation (see Doc. S2 in Supporting information andFig. 8).Using [3H]palmitate, or [3H]palmitoyl-CoA, we havetried to measure in vitro or metabolic palmitoylation,but our attempts have been unsuccessful, in partbecause of the relatively low expression levels ofendogenous membrane PE. These results, however, donot rule out this possibility.DiscussionThis article reports the identification of the proteinresponsible for PE activity in membranes isolatedfrom pig brain, which we now call mPOP. For morethan 20 years, it has been known that some PEactivity it associated with membranes from almostall tissues and especially from brain [18–20]. Further-more, mPOP activity has been found to change withage [6,21]. Purification of mPOP from bovine brainhas been attempted previously [9,10], and on thebasis of those studies, it was concluded that thispeptidase is expressed mainly in the synaptosomalfraction and has a heavier mass (87 kDa) than POP(65 kDa). Furthermore, on the basis of sensitivity tothiol-reactive inhibitors, mPOP was thought to be athiol-dependent metallopeptidase [10], but the basicproblem was that this enzyme has never been readilyidentified before.In an attempt to clarify the identity of mPOP, weundertook the task of purifying and characterizingparticulate POP from pig brain. We have confirmedthat a significant amount of PE activity can be mea-sured in the particulate fraction from crude pig brainhomogenates. In our preparations, this activityaccounted for around 40% of the total homogenateactivity, similar to the 50% reported previously forthe corresponding fraction from bovine brain [9].However, after osmotic shock and high-salt treat-ment, our total membrane preparation contained lessthan 5% of total activity, as compared with 20%recovery reported earlier for washed synaptosomalmembranes from bovine brain [9,10]. This discrep-ancy may be species-related, as the preparation con-ditions were essentially the same. The PE activitypresent in our washed membrane preparation wasvery tightly bound, and was solubilized only afterdetergent treatment. Upon sucrose gradient fraction-ation, some PE activity was present in the heaviermembrane fraction containing ER, but the majoritywas detected within the synaptosomal membranefraction, consistent with a role for mPOP in synapsefunction. This is consistent with the finding of bind-ing of antibody against POP to internal neuronalmembranes in rat brain slices [11].Kinetic experiments demonstrated that the substratepreference and the inhibitor sensitivity of purifiedmPOP are identical to those of POP (see Table 4 andTable S1). Furthermore, POP-specific antibodiescross-reacted with mPOP on western blots. Analysisof peptide fragments generated by trypsin digestionidentified mPOP protein as POP. Thus, we foundonly two features that distinguish the two forms, andthose are attributed to the membrane milieu wheremPOP resides. One was the tight membrane associa-tion, which could only be disrupted by detergent solu-bilization. The second feature was the differentFig. 7. Arrhenius plots of membrane PE ( ), POP ( ) and solubi-lized membrane PE (d). Activity assays were performed in dupli-cate as described in Experimental procedures, but reaction tubeswere preincubated at every temperature (every 2 °C between 10and 40 °C) and the reaction was incubated at the correspondingtemperature for 40 min. Solid lines represent the linear regres-sions.J. Tenorio-Laranga et al. Membrane-bound prolyl oligopeptidase: mPOPFEBS Journal 275 (2008) 4415–4427 ª 2008 The Authors Journal compilation ª 2008 FEBS 4421temperature dependence between mPOP activity,when is bound to membranes, and POP activity. Fortransmembrane enzymes, Arrhenius plots generallyshow a break that corresponds to the membranemelting point. At temperatures above this point, themore fluid environment produces a decrease of activa-tion energy, producing a convex Arrhenius plot [22].However, we found that the Arrhenius plot of mPOPdid not show any break but did reflect an increase ofactivation energy in relation to POP or Triton-solubi-lized mPOP. Interestingly, these two latter forms werevery similar to each other (Fig. 6). We interpret thisas an inhibitory effect of the membranous milieu, orof the putative anchor, or both, on mPOP activity, asthe Arrhenius profile switched to a POP profile oflower activation energy upon detergent solubilization.It is remarkable that Triton X-100 activated mPOPactivity at least by a factor of 3 (Table 3). It was alsoobserved that the SDS ⁄ PAGE migration of mPOP,when still attached to the native membranes, wasatypical. Consistently, different membrane fractionsshowed a reactive band for antibody against POPthat migrated at a slightly, but perceptibly, lowermolecular mass, present at different proportions forthe different fractions (Fig. 5B). In total membranes,this lighter band was a major component, becomingalmost exclusive in the heavy membrane fraction.Medium-density membranes presented two bands: thelight band and the one that matched the soluble POPcontrol. On the other hand, in the light membranefraction, the latter band was the only one appearing.It was remarkable that this lower molecular massband completely disappeared from all membrane frac-tions when the very same samples were solubilizedwith Triton X-100. After this treatment, only oneband around 80 kDa, as with the cPOP control, wasdetected in all cases. It can be argued that the mem-brane-associated state induces a more compact andtighter conformation in which the anchor site is lessaccessible to reduction, thereby resulting in fastermigration. After solubilization and membrane disrup-tion, the protein anchor might be more accessible toreducing agents, and thus could be readily dissociatedto yield the soluble form with normal migration inSDS ⁄ PAGE (Fig. 5B,C). The fact that both normallymigrating and abnormally migrating bands, from thedifferent membrane fractions, appear in differentproportions could be explained by bringing into playFig. 8. Localization of Cys563 on the 3Dmodel of pig POP. Peptidase_S9 domainresidues are shown in magenta; propellerdomain chains are represented in navy,brown, green, gray and orange; Cys563 isshown in yellow. Modeled from file mmd-bId:21074, for POP from porcine brain, fromthe NCBI’s Entrez Structure database, andhandled byCN3D v. 4.1 (NCBI) software.Membrane-bound prolyl oligopeptidase: mPOP J. Tenorio-Laranga et al.4422 FEBS Journal 275 (2008) 4415–4427 ª 2008 The Authors Journal compilation ª 2008 FEBSthe different physical and chemical properties of thecorresponding membranes, which would obviouslyaffect the interactions with mPOP.Several studies indicate that POP interacts with cyto-skeleton proteins and that it is probably involvedin axonal transport [17]. Components of neuronalmembrane trafficking are tightly associated withmembrane-bound organelles in a chaperone-mediatedor chaperone-sensitive way, and are therefore resistantto in vitro treatments, including high salt, which releasemost peripheral membrane proteins [23]. Homogeni-zation with buffers containing millimolar levels ofSH-modifying agents, such as N-ethylmaleimide, orchelators of divalent cations, such as EDTA, is knownto significantly release these proteins from membranes.Proteins such as annexins associate with membranes inaCa2+-dependent way, and EDTA disrupts this asso-ciation and solubilizes the proteins [24]. We did notnote any effects of N-ethylmaleimide or EDTA duringhomogenization or membrane washing on the mem-brane PE levels (not shown). These data indicate thatthe mechanism of mPOP membrane association ismost probably not mediated by SH interaction or bydivalent cations.On the whole, our results may suggest that POPundergoes a post-translational modification in which amembrane anchor is added to the protein, attaching itto membranes. The nature of this putative anchorremains to be determined, but it is most likely a hydro-phobic chain, as POP does not contain any substantialhydrophobic domain that could be used to embed it incellular membranes (see Doc. S2 in Supporting infor-mation). Our analysis of the POP amino acid sequencesuggests palmitoylation as a possible post-translationalmodification; the well-conserved sequence of pig POP,GGLLVATCANQRPDL(556–570), yields a very goodscore for this kind of modification according to css-palm software [25]. Examination of the 3D structureof pig POP revealed that a critical cysteine is situatedvery near the surface of the enzyme, on the bottomside of the catalytic domain, making it a good candi-date for the putative palmitoylation (Fig. 8). In fact,this cysteine residue is totally conserved among alleukaryotic POP genes sequenced [26]. AlthoughS-palmitoylation is theoretically sensitive to SH group-reducing agents, this site could be buried when mPOPis anchored to the membrane. Although our attemptsto measure in vitro or metabolic palmitoylation havenot succeeded, the possibility of this post-translationalmodification is still open.It is also important to note that we did not use anyinhibitors for lytic enzymes during mPOP preparation,and it is possible that the putative membrane anchorwas removed during the process of isolation and purifi-cation, or during trypsinization and peptide extraction,preventing its identification by MS. Additionally,membrane-bound proteins, which interact directly withthe hydrophobic inner membrane phase, require thepresence of an amphiphilic compound, such as a deter-gent or a phospholipid, for stability and activity. Wefound that once mPOP is solubilized from membranes,the concentration of Triton X-100 can be considerablydecreased without any protein precipitation or loss ofactivity. This strongly points to the intrinsically solublenature of mPOP, as strongly hydrophobic proteinstend to aggregate and lose activity during lipid ordetergent removal.The cytoplasmic location of POP is paradoxicalwhen considering its major role in the metabolism ofextracellular neuropeptides [3]. Recently, other roleshave been suggested for POP, such as axonal transportand ⁄ or modulation of intracrine peptide regulation [1].The existence of an alternative particulate form withPE activity, a different enzyme but with the same func-tional properties as POP, that is responsible for thedegradation of neuropeptides in the synaptosomalcleft, would solve the localization problem. However,attempts to identify this enzyme have been unsuccess-ful [9,10]. Contrary to expectations, we found severalpieces of evidence suggesting that the particulate formis, in fact, a variant of POP (EC 3.4.21.26); mPOP hasthe same gel filtration, immunological, activity andinhibitory properties as soluble POP, and even MSdata provided high confidence in the identification.Our results additionally suggest that a post-transla-tional modification is necessary for POP to be associ-ated with membranes. Furthermore, the data reportedhere also suggest that this modification is sensitive tothe reducing state of the environment, and this mayindicate the existence of specific cell machinery thatcontrols the association–dissociation event. One funda-mental question is the orientation of mPOP in thesynaptosomes. Fast and effective neuropeptide degra-dation in the synaptosomal cleft would only be possi-ble if mPOP was facing that side. Another interestingaspect invoked is the possibility that the membraneassociation of POP is connected to its transport out ofthe cell, the membrane-bound form being only a tran-sitory stage.Experimental proceduresTotal membrane preparationEighty grams of freshly isolated pig brain were homoge-nized in 320 mL of ice-cold 0.32 m sucrose in 100 mmJ. Tenorio-Laranga et al. Membrane-bound prolyl oligopeptidase: mPOPFEBS Journal 275 (2008) 4415–4427 ª 2008 The Authors Journal compilation ª 2008 FEBS 4423potassium phosphate buffer (pH 7.4), sonicated, and centri-fuged at 1000 g for 10 min. The resulting supernatant wassonicated again, and centrifuged at 155 100 g for 30 min.The pellet was washed first with 0.5 m NaCl, once with dis-tilled water, twice with 4 m NaCl, and finally with distilledwater. The resulting membranes were resuspended in100 mm potassium phosphate buffer (pH 7.4).Membrane fractionationAn aliquot of the total membrane preparation was broughtto 0.32 m sucrose and 100 mm potassium phosphate buffer(pH 7.4), and layered on a discontinuous sucrose gradient(1.2, 1 and 0.8 m sucrose). The tubes were centrifuged at80 000 g for 90 min, in a swing rotor. After centrifugation,the membrane layers were carefully collected by pipetting.Each fraction was diluted to 0.32 m sucrose, centrifugedat 155 100 g, and resuspended in 100 mm potassiumphosphate buffer (pH 7.4).Enzymatic assaysPE activity was assayed by measuring the fluorescencereleased from the substrate N-carbobenzoxy-glycyl-prolyl-7-amido-4-methyl-coumarin (Z-Gly-Pro-AMC) (200 lm), aspreviously reported [27], by incubating protein samples in100 mm sodium phosphate buffer (pH 7.0). The assay wasstopped by the addition of 1 m sodium acetate buffer(pH 4.2). A succinate dehydrogenase assay was performed in0.3 mL containing 0.01 m sodium succinate, 0.05 m phos-phate (pH 7.5), and 0.4 mg (in 50 lL) of each membranefraction. The mixtures were incubated at 37 °C for 15 min,0.1 mL of 2.5 mgÆmL)1p-iodotetrazolium violet was added,and incubation was continued for a further 10 min. Thereaction was stopped with 1 mL of ethyl acetate ⁄ ethanol ⁄trichloroacetic acid (5 : 5: 1), and centrifuged at 16 000 g for1 min; absorbance was measured at 490 nm.Protein determinationProtein was determined by the Bradford method (Bio-Rad,Hercules, CA, USA) using BSA (Sigma-Aldrich, St Louis,MO, USA) as standard, and in the presence of 0.1%Triton X-100 when required.SDS ⁄ PAGE and western blottingSamples were diluted 1 : 1 with loading buffer (100 mmTris ⁄ HCl, pH 6.8, 70% glycerol, 2% SDS, 0.005% bromo-phenol blue, 10 mm b-mercaptoethanol) and separated on8% or 10% polyacrylamide ⁄ bis-acrylamide Tris ⁄ HCl dis-continuous gels. Gels were stained for protein or trans-ferred to nitrocellulose for blotting. For protein staining,gels were fixed with methanol ⁄ acetic acid ⁄ water (4 : 5 : 4)for 30 min, washed with water for 30 min (two changes),sensitized with 0.02% sodium thiosulfate for 1–2 min, andincubated with 0.1% silver nitrate for 20 min at room tem-perature. Gels were then rinsed twice with distilled water,and bands were visualized by incubating for 1–2 min with2% sodium carbonate and 0.04% formaldehyde. Westernblotting was performed under standard conditions usingprimary antibody [28] diluted 1 : 5000 (with 0.5 m NaCl,20 mm Tris-HCL pH 7.5 and 0.05% Tween 20), and theanti-(chicken horseradish peroxidase) complex diluted1 : 50 000 (Pierce, Rockford, IL, USA). Protein visualiza-tion was performed using an ECL kit (Amersham-Biosci-ence, Little Chalfont, UK), following the manufacturer’sinstructions.Purification of mPOPThe total membrane preparation (see above) wasextracted with 0.4% Triton X-100 (at 1 mg of TritonX-100 per mg of protein) in 20 mm buffer and 100 mmNaCl on ice for 1 h. After the extraction, the sampleswere centrifuged at 155 100 g for 30 min and the pelletwas discarded. Buffer was exchanged by dilution andultrafiltration (CentriPrep 50; Amicon, Millipore Corp.Billerica, MA, USA), with DEAE equilibration buffer[50 mm Tris, pH 7.4, 1 mm EDTA, 5 mm dithiothreitol,0.05% Triton X-100], bound to an equilibrated DEAE–Sepharose Fast Flow column (1.6 · 10 cm; Amersham,Uppsala, Sweden), washed with DEAE equilibration buf-fer, and step-eluted with 500 mm NaCl in the same buf-fer. The eluted pool was concentrated and the buffer wasexchanged (as stated above) for phenyl–Sepharose equili-bration buffer [900 mm (NH4)2SO4,50mm Tris ⁄ HCl,pH 7.4, 5 mm dithiothreitol, 1 mm EDTA], and loadedonto a phenyl–Sepharose High Performance column(0.7 · 2.5 cm; Amersham). Activity eluted within theflow-through. Most of the contaminating protein wasretained in the column and eluted by a wash without(NH4)2SO4. Peak fractions were pooled, the buffer wasexchanged for HA equilibration buffer (10 mm potassiumphosphate, pH 7.4, 5 mm dithiothreitol, 1 mm EDTA,0.05% Triton X-100), and the sample was loaded onto a0.59 · 3.6 cm HA column (Bio-Rad). Activity was elutedat around 250 mm potassium phosphate over a 10–500 mm linear gradient (see Fig. S1). The buffer of theHA pool was exchanged with DEAE buffer 2 (EDTA1mm,5mm dithiothreitol, 50 mm Tris ⁄ HCl, pH 6.6),loaded onto an equilibrated DEAE HiTrap Fast Flowcolumn (0.7 · 2.5 cm; Amersham), and eluted with a0–500 mm NaCl gradient. Activity eluted at around200 mm salt. All chromatographic steps were accom-plished using an A¨KTA prime system and monitoredwith primeview plus software (both from Amersham).All chromatography profiles are shown in Fig. S1.Membrane-bound prolyl oligopeptidase: mPOP J. Tenorio-Laranga et al.4424 FEBS Journal 275 (2008) 4415–4427 ª 2008 The Authors Journal compilation ª 2008 FEBS[...]... Ontogeny of soluble and particulate prolyl endopeptidase activity in several areas of the rat brain and in the pituitary gland Dev Neurosci 25, 316–323 7 Irazusta J, Larrinaga G, Gonzalez-Maeso J, Gil J, Meana JJ & Casis L (2002) Distribution of prolyl endopeptidase activities in rat and human brain Neurochem Int 40, 337–345 8 Agirregoitia N, Casis L, Gil J, Ruiz F & Irazusta J (2007) Ontogeny of prolyl endopeptidase. .. 9 O’Leary RM & O’Connor B (1995) Identification and localisation of a synaptosomal membrane prolyl endopeptidase from bovine brain Eur J Biochem 227, 277– 283 10 O’Leary RM, Gallagher SP & O’Connor B (1996) Purification and characterization of a novel membrane- 4426 15 16 17 18 19 20 21 22 23 bound form of prolyl endopeptidase from bovine brain Int J Biochem Cell Biol 28, 441–449 Myohanen TT, Venalainen... distribution of rat brain prolyl oligopeptidase and its association with specific neuronal neurotransmitters J Comp Neurol 507, 1694–1708 Bausback HH & Ward PE (1986) Vascular, post proline cleaving enzyme: metabolism of vasoactive peptides Adv Exp Med Biol A 198, 397–404 Kato T, Nakano T, Kojima K, Nagatsu T & Sakakibara S (1980) Changes in prolyl endopeptidase during maturation of rat brain and hydrolysis of. .. Miettinen R & Mannisto PT (2007) ¨ ¨ Distribution of immunoreactive prolyl oligopeptidase in human and rat brain Neurochem Res 32, 1365– 1374 Venalainen JI, Juvonen RO, Forsberg MM, Garcı´ a¨ ¨ Horsman A, Poso A, Wallen EA, Gynther J & Man¨ nisto PT (2002) Substrate-dependent, non-hyperbolic ¨ kinetics of pig brain prolyl oligopeptidase and its tight Membrane-bound prolyl oligopeptidase: mPOP binding inhibition... P (1984) Inactivation of neurotensin by rat brain synaptic membranes Cleavage at the Pro10-Tyr11 bond by endopeptidase 24.11 (enkephalinase) and a peptidase different from proline -endopeptidase J Neurochem 43, 1295–1301 Dalmaz C, Netto CA, Volkmer N, Dias RD & Izquierdo I (1986) Distribution of proline endopeptidase activity in sub-synaptosomal fractions of rat hypothalamus Braz J Med Biol Res 19,... O’Connor B (2003) Characterisation of the active site of a newly-discovered and potentially significant post-proline cleaving endopeptidase called ZIP using LC-UV-MS Analyst 128, 670– 675 5 Cunningham DF & O’Connor B (1997) Identification and initial characterisation of a N-benzyloxycarbonylprolyl-prolinal (Z-Pro-prolinal)-insensitive 7-(N-benzyloxycarbonyl-glycyl -prolyl- amido)-4-methylcoumarin (Z-Gly-Pro-NH-Mec)-hydrolysing... cPOP Purification of PPP PPP was expressed in Escherichia coli and purified as described previously [29] Peptide digestion assay The assay mixture (140 lL) was composed of 50 mm Tris ⁄ HCl (pH 7.0) and cPOP, or mPOP, to 4 nmolÆmin)1 of activity Each peptide was added (prewarmed) at a final concentration of 140 lm The reaction was carried out at 30 °C for 60 min and stopped by the addition of trifluoroacetic... Juvonen RO & Mannisto PT (2004) ¨ ¨ ¨ ¨ Evolutionary relationships of the prolyl oligopeptidase family enzymes Eur J Biochem 271, 2705–2715 Venalainen JI, Garcı´ a-Horsman JA, Forsberg MM, ¨ ¨ Jalkanen A, Wallen EA, Jarho EM, Christiaans JA, Gynther J & Mannisto PT (2006) Binding kinetics and ¨ ¨ duration of in vivo action of novel prolyl oligopeptidase inhibitors Biochem Pharmacol 71, 683–692 Myohanen... Venalainen JI ¨ ¨ ¨ ¨ (2007) On the role of prolyl oligopeptidase in health and disease Neuropeptides 41, 1–24 2 Mannisto PT, Venalainen J, Jalkanen A & Garcı´ a¨ ¨ ¨ ¨ Horsman JA (2007) Prolyl oligopeptidase: a potential target for the treatment of cognitive disorders Drug News Perspect 20, 293–305 3 Brandt I, Scharpe S & Lambeir AM (2007) Suggested functions for prolyl oligopeptidase: a puzzling paradox... J Neurochem 35, 527–535 Kato T, Okada M & Nagatsu T (1980) Distribution of post-proline cleaving enzyme in human brain and the peripheral tissues Mol Cell Biochem 32, 117–121 Garcı´ a-Horsman JA, Venalainen JI, Lohi O, Auriola ¨ ¨ IS, Korponay-Szabo IR, Kaukinen K, Maki M & ¨ Mannisto PT (2007) Deficient activity of mammalian ¨ ¨ prolyl oligopeptidase on the immunoactive peptide digestion in coeliac . Characterization of membrane-bound prolyl endopeptidase from brain Jofre Tenorio-Laranga1, Jarkko I. Vena¨la¨inen2,. identifi-cation of the membrane-bound PE (herein referred toas membrane-bound prolyl oligopeptidase, mPOP) from pig brain and characterization of the enzyme’sproperties
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