Báo cáo khoa học: Molecular characterization and gene disruption of mouse lysosomal putative serine carboxypeptidase 1 ppt

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Molecular characterization and gene disruption of mouselysosomal putative serine carboxypeptidase 1Katrin Kollmann1, Markus Damme1, Florian Deuschl1,Jo¨rg Kahle2, Rudi D’Hooge3,Renate Lu¨llmann-Rauch4and Torben Lu¨bke11 Abteilung Biochemie II, Georg-August Universita¨tGo¨ttingen, Germany2 Abteilung Molekularbiologie, Georg-August Universita¨tGo¨ttingen, Germany3 Laboratory of Biological Psychology, KU Leuven, Belgium4 Anatomisches Institut, Universita¨t Kiel, GermanyThe lysosomal compartment plays a pivotal role in thedegradation of macromolecules within the cell. Todate, over 60 soluble lysosomal hydrolases and acces-sory proteins and 25 lysosomal membrane proteinshave been identified [1–3]. Defects in the lysosomalproteins mostly result in one of about 50 lysosomalstorage diseases (LSDs) which are characterized by theaccumulation of undigested materials in the lysosomes.As a result of the clinical relevance of soluble lyso-somal proteins in LSDs and a notable number ofLSD-like diseases of unknown etiology, there is a com-mon interest in the identification of the proteome ofthe lysosomal compartment and of the soluble luminallysosomal mannose 6-phosphate (M6P)-containingKeywordsgene disruption; lysosomes; processing;Scpep1; serine carboxypeptidaseCorrespondenceT. Lu¨bke, Zentrum Biochemie undMolekulare Zellbiologie, AbteilungBiochemie II, Georg-August Universita¨tGo¨ttingen, Heinrich-Du¨ker-Weg 12,D-37073 Go¨ttingen, GermanyFax: +49 551 395979Tel: +49 551 395932E-mail: tluebke@gwdg.de(Received 4 July 2008, revised 18December 2008, accepted 23 December2008)doi:10.1111/j.1742-4658.2009.06877.xThe retinoid-inducible serine carboxypeptidase 1 (Scpep1; formerly RISC)is a lysosomal matrix protein that was initially identified in a screen forgenes induced by retinoic acid. Recently, it has been spotlighted by severalproteome analyses of the lysosomal compartment, but its cellular functionand properties remain unknown to date. In this study, Scpep1 from micewas analysed with regard to its intracellular processing into a mature dimerconsisting of a 35 kDa N-terminal fragment and a so far unknown 18 kDaC-terminal fragment and the glycosylation status of the mature Scpep1fragment. Although Scpep1 shares notable homology and a number ofstructural hallmarks with the well-described lysosomal carboxypeptidaseprotective protein ⁄ cathepsin A, the purified recombinant 55 kDa precursorand the homogenates of Scpep1-overexpressing cells do not show proteo-lytic activity or increased serine carboxypeptidase activity towards artificialserine carboxypeptidase substrates. Hence, we disrupted the Scpep1 gene inmice by a gene trap cassette, resulting in a Scpep1 ⁄ b-galactosidase ⁄ neo-mycin phosphotransferase fusion protein. The fusion protein is devoid ofthe C-terminal half of Scpep1, including two amino acids of the assumedcatalytic triad which is indispensable for its predicted serine carboxypepti-dase activity. However, Scpep1-deficient mice were viable and fertile, anddid not exhibit either lysosomal storage or reduced lysosomal SC activityunder any tested condition.AbbreviationsAEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride; CBZ, benzyloxycarbonyl; Cpvl, carboxypeptidase vitellogenic-like; CPY, carboxypeptidase Y;Ctsa, protective protein ⁄ cathepsin A; FA, furylacryloyl; geo, b-galactosidase ⁄ neomycin phosphotransferase; Lamp1, lysosomal associatedmembrane protein 1; LSD, lysosomal storage disease; M6P, mannose 6-phosphate; MEFs, mouse embryonic fibroblasts; MPR, mannose6-phosphate receptor; PNGase F, peptide N-glycosidase F; RISC, retinoid-inducible serine carboxypeptidase; SC, serine carboxypeptidase;Scpep1, serine carboxypeptidase 1; Scpep1-gt, Scpep1 gene trap.1356 FEBS Journal 276 (2009) 1356–1369 ª 2009 The Authors Journal compilation ª 2009 FEBSproteins in particular. Soluble lysosomal proteinsreceive M6P residues on their N-linked oligosaccha-rides [4], which are recognized in the trans-Golgi net-work by M6P receptors (MPRs) required forlysosomal transport [5]. By exploiting the M6P recog-nition marker of the soluble lysosomal proteins forMPR-dependent affinity chromatography, followed bytheir identification by mass spectrometry, we andothers have identified a novel putative serine carboxy-peptidase 1 (Scpep1) [6–9]. Originally, Scpep1 wasidentified in rat aortic smooth muscle cells by a screen-ing for retinoid-inducible genes, as reflected by its ini-tial name, ‘retinoid-inducible serine carboxypeptidase’(RISC) [10]. Northern blot analyses demonstrated hightranscript levels in kidney and aorta in rat and lowerlevels in heart, spleen and lung, whereas the humantranscript was detected strongly in the kidney andheart but at a low level in a number of other tissues[10]. In mice, Scpep1 is expressed in embryonic heartand vasculature, as well as in a broad range of adulttissues [10]. The mouse Scpep1 gene (GeneID: 74617;cDNA Accession No. NM_029023) encodes a productof 452 amino acids (Protein Accession No.NP_0832299) that localizes to the lysosomes [11]. Fur-thermore, it has been demonstrated that, in mice, a55 kDa Scpep1 precursor is processed into a 35 kDaform [11]. Although no peptidase activity has beendemonstrated so far, Scpep1 has been assigned to theserine carboxypeptidase (SC) family S10 because ofreasonable sequence homology to members of thisfamily, such as the lysosomal protective pro-tein ⁄ cathepsin A (official gene name Ctsa; 35% simi-larity) and four conserved domains that are predictedto constitute the substrate-binding site and three cata-lytic sites. Each of these catalytic sites accounts forone amino acid of the catalytic triad Ser-Asp-His[10,12]. To obtain an insight into the physiological andcellular function of the putative lysosomal SC Scpep1,we analysed the molecular properties of Scpep1 andgenerated an Scpep1 gene trap (Scpep1-gt) mousemodel.ResultsMolecular forms of Scpep1In order to generate Scpep1-specific antisera, wepurified a C-terminally His-tagged version of full-length mouse Scpep1 from secretions of stablyexpressing HT1080 cells (HT1080-Scpep1). Coomassieand silver staining after SDS-PAGE revealed anapparent molecular size of 55 kDa for the secretedand purified His-tagged Scpep1 starting with Ile29,as identified by N-terminal sequencing. We derivedScpep1-specific antisera from rat and rabbit. Bothantisera were suitable for confirming the lysosomallocalization of endogenous Scpep1 by immunofluores-cence (see Fig. S1).Western blot analysis using an antibody directedagainst the C-terminal His-tag detected the 55 kDaScpep1 in cell extracts and in the medium of HT1080-Scpep1 cells (Fig. 1A, lanes 3 and 4), but not inuntransfected HT1080 cells (lanes 1 and 2).Unexpectedly, an additional 18 kDa form of Scpep1was detectable by the anti-His IgG1 in homogenates ofHT1080-Scpep1 cells (lane 3), thus representing theC-terminal fragment of processed Scpep1.Both of our Scpep1-specific antisera detected the55 kDa precursor in homogenates and secretions ofHT1080-Scpep1 cells (lanes 7, 8 and 11, 12), as well asa strong signal at 35 kDa in the homogenates (lanes 7and 11), representing the N-terminal moiety ofprocessed Scpep1. Rabbit antiserum showed lowBαα1pepcS-tibbarStandard(kDa)Retention time(min)20 2530403518mn082DO160 67 43 13.7α-His α-Scpep1 α-Scpep1rabbit rat15075503725201510C M C M C M C M C M C M553518kDakDa0801TH-0801THsiH-1pepcS0801TH-0801THsiH-1pepcS0801TH-0801THsiH-1pepcSALane 1 2 3 4 5 6 7 8 9 10 11 12Fig. 1. Molecular forms of Scpep1. (A) Analysis of molecular formsof Scpep1: 100 lg of cell lysates (C) and 50 lL of medium (M) ofHT1080 and HT1080-Scpep1 were separated by SDS-PAGE, blottedand probed with the a-His antibody and the a-Scpep1 antisera fromrabbit and rat, respectively. (B) Gel filtration analysis of a lysosome-enriched fraction (F2): 50 lg of F2 were buffered in 20 mM Mes(pH 4.5) containing 150 mM NaCl, loaded onto a Superdex 75 col-umn on an analytic SMARTÔ system (Pharmacia) and eluted in20 lL fractions at a flow rate of 40 lLÆmin)1, which were analysedby western blot using the rabbit a-Scpep1 antibody. A mixture ofmolecular mass standard proteins, including IgG (160 kDa), albumin(67 kDa), ovalbumin (43 kDa) and ribonuclease A (13.7 kDa), wasapplied to gel filtration under the same conditions.K. Kollmann et al. Functional characterization of lysosomal Scpep1FEBS Journal 276 (2009) 1356–1369 ª 2009 The Authors Journal compilation ª 2009 FEBS 1357cross-reactivity with polypeptides in homogenates ofuntransfected human HT1080 (lane 5), whereas nospecific signal could be detected in the medium (lane 6)or with rat antiserum in HT1080 cells (lanes 9 and 10).Omitting the reducing agents did not alter the mobilityproperties in SDS-PAGE (data not shown). However,gel filtration chromatography with a lysosome-enrichedfraction from mouse liver showed that the 35 and18 kDa subunits of Scpep1 co-eluted in the samefractions with an apparent size between 43 and 67 kDa(Fig. 1B), suggesting that both fragments are linked toeach other noncovalently.Processing of Scpep1To investigate the processing of the endogenousScpep1 precursor in mice, mouse embryonic fibroblasts(MEFs) were pulse labelled with [35S]methionine for1 h and chased for up to 72 h. After immunoprecipita-tion, Scpep1 was separated by SDS-PAGE and analy-sed by autoradiography (Fig. 2A). In MEFs which hadbeen labelled for 1 h (0 h chase), only the  55 kDaprecursor was detected. After 2 h of chase, half of the55 kDa precursor had been processed into 37 and20 kDa intermediates. Between 4 and 6 h of chase, the55 kDa precursor disappeared, whereas the 37 kDaintermediate and the final 18 kDa fragment which wasderived from the 20 kDa fragment showed constantsignals. At 24 h of chase, the 35 kDa fragment wasfinally processed, and both fragments remained detect-able even after 72 h of chase (Fig. 2A). The mediumdid not show any specific Scpep1 signal (data notshown).Some lysosomal peptidases, such as cathepsin B andcathepsin D, undergo autoproteolytic activation[13,14]. To investigate the autoproteolytic activation ofScpep1, we incubated 0.16–3.2 lm of recombinant55 kDa Scpep1 precursor at varying pH (pH 4.5 andpH 7.5), temperature (4 °C, room temperature, 37 °C)and incubation time (1–16 h), but could not detect anyprocessing of the precursor into mature forms bywestern blot analysis (data not shown).In order to further define the Scpep1-processing pro-tease, MEFs were pulse labelled in the presence orabsence of various protease inhibitors. The conversionof the 55 kDa Scpep1 precursor into the matureform was sensitive to the serine protease inhibitor4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF)(Fig. 2B). However, two other serine protease inhibi-tors, aprotinin and antipain, had no effect, althoughthe latter interferes with cathepsin A activity [15]. Pep-statin A, an aspartic protease inhibitor, and the cyste-ine protease inhibitor E-64, as well as the metal0 2 4 6 24 48 72ABCChase (h)5535150100755037kDa18∗∗∗250252015MEFkDa∗MEFChase (h)––ATDE46-EFSBEAxiM-IP150100755037kDa250252015kDa0 4∗∗553518Chase (h) 0 6 0 65535NH4Cl –+150100755037kDaHT1080-Scpep1C M C M C M C M% of t0100 – 15 45 100 – 20 55AntipainAprotininPepstatin AFig. 2. Processing of Scpep1. (A) Immunoprecipitation of Scpep1from MEFs using the rat-derived Scpep antiserum after pulse label-ling with [35S]methionine for 1 h. Cells were chased for up to 72 h.Nonspecific signals are marked with asterisks (*) at chase time 0.(B) Effects of protease inhibitors on the processing of Scpep1.MEFs were pulse labelled and chased for 0 or 4 h in the absence()) or presence of the following inhibitors: EDTA (2 mM final con-centration), E-64 (10 lM), pepstatin A (100 lM), AEBSF (1 mM),aprotinin (0.3 lM), antipain (75 lM) and a mixture containing allinhibitors in the assigned concentrations. Scpep1 was immunopre-cipitated and visualized by autoradiography. (C) Processing ofScpep1 in NH4Cl-treated HT1080-Scpep1 cells. Intracellular (C) orsecreted (M) Scpep1 was immunoprecipitated after pulse labellingand 0 or 6 h of chase in the absence ()) and presence (+) of thelysosomotropic agent NH4Cl.Functional characterization of lysosomal Scpep1 K. Kollmann et al.1358 FEBS Journal 276 (2009) 1356–1369 ª 2009 The Authors Journal compilation ª 2009 FEBSchelator EDTA, had no effect on Scpep1 maturation.These results suggest that, under our test conditions, aserine protease different from cathepsin A mediatesScpep1 processing.In order to investigate whether Scpep1 maturationoccurs in the endosomal–lysosomal compartment,HT1080-Scpep1 cells were pulse labelled in the absenceor presence of NH4Cl (Fig. 2C). In untreated HT1080-Scpep1 cells, the 55 kDa precursor was processed intothe 35 kDa fragment, but the 18 kDa fragment wasnot detected. In addition, HT1080-Scpep1 cellssecreted large amounts of the 55 kDa precursor after6 h of chase (45% of the total Scpep1 signal at chase0 h). NH4Cl interfered with the intracellular processingof the precursor to the 35 kDa form, but only moder-ately enhanced the secretion of the precursor (55% ofthe Scpep1 signal at chase 0 h). These results indicatethat Scpep1 matures in late endosomes or lysosomesand could be targeted in an M6P-dependent manner.Glycosylation of Scpep1 in MEFsThe amino acid sequence of Scpep1 contains five puta-tive N-glycosylation sites, four of which are locatedwithin the 35 kDa N-terminal fragment (Asn64,Asn102, Asn126, Asn192) and one within the 18 kDaC-terminal fragment (Asn362). MEFs were pulselabelled for 1 h and chased for 4 h, and immunopre-cipitated Scpep1 was subjected to peptide N-glycosi-dase F (PNGase F) treatment for 1 h and separated bySDS-PAGE (Fig. 3). The major form of the Scpep1precursor from MEFs migrated at an apparent molec-ular mass of 55 kDa (lane 1). In addition, a minorsignal was detected with a slightly reduced molecularmass of  52 kDa that was partially covered by the55 kDa form and most probably represents a less gly-cosylated Scpep1. PNGase F treatment of the precur-sor resulted in a shift towards  40 kDa in size(lane 2). After 4 h of chase, the  55 kDa precursorwas completely converted into two major maturesubunits of 35 and 18 kDa in size and a minor signalof  30 kDa that might arise from the 52 kDa precur-sor form (lane 3). The limited deglycosylation of the35 kDa subunit led to a deglycosylated  25 kDa frag-ment via the  30 kDa fragment and an intermediateof 28 kDa (lane 4). PNGase F treatment of the18 kDa subunit resulted in a partial shift towards alower molecular mass of  16 kDa (lane 4). Consider-ing an apparent molecular mass of about 2–2.5 kDaper N-linked oligosaccharide [16,17], the results suggestthat, in MEFs, all N-glycosylation sites are utilized.SC activity of Scpep1Like carboxypeptidase Y (CPY) and Ctsa, Scpep1 isclassified as a member of the SC type C family(S10.013, MEROPS database) and should preferen-tially exhibit proteolytic activity at acidic pH towardshydrophobic amino acids in the P1¢ position [18]. Puri-fied Scpep1 protein, mainly consisting of the 55 kDaprecursor, recombinant CPY as a positive control andBSA were tested for SC activity towards differentN-terminal blocked peptides, such as CBZ-Phe-Leuand FA-Phe-Phe (CBZ, benzyloxycarbonyl; FA, furyl-acryloyl), representing SC type C substrates, FA-Ala-Lys, as an SC type D substrate, and the non-SCsubstrate CBZ-Gly-Leu [19]. Although CPY cleavedSC substrates such as FA-Phe-Phe in a pH-dependentmanner, neither purified Scpep1 precursor nor BSAshowed proteolytic activity under any test condition(Fig. 4).As most lysosomal hydrolases are not active as zym-ogens, we determined the SC activity in homogenatesof HT1080 cells and HT1080-Scpep1 cells (data notshown). Although the latter mainly show Scpep1 in itsprocessed form, we could not detect any differences inacid SC activity. To exclude cell line and vector-specific effects of His-tagged Scpep1, we assayedChase (h) 0 455PNGase F –+ –+MEF3518320101∗∗Lane 1 2 3 4Fig. 3. Glycosylation of Scpep1. MEFs were pulse labelled andchased for 4 h. Scpep1 was immunoprecipitated from the lysates,treated with PNGase F and separated by SDS-PAGE. The filledarrowheads point to the fully glycosylated forms of Scpep1 withthe number of their N-glycans, and the open arrowheads to degly-cosylated forms of the 35 kDa processed form and the 18 kDa pro-cessed form. Nonspecific signals are marked with asterisks (*) atchase time 0.K. Kollmann et al. Functional characterization of lysosomal Scpep1FEBS Journal 276 (2009) 1356–1369 ª 2009 The Authors Journal compilation ª 2009 FEBS 1359HT1080 cells that had been transiently transfected withan untagged variant of Scpep1, His-tagged Scpep1 orthe mock vector (pcDNA3.1-Hygro) (Fig. 5A), as wellas COS-7 cells transfected with Scpep1-His6 and thepCI-neo mock vector (Fig. 5B). Although largeamounts of processed Scpep1 were detected in thehomogenates, no differences in acid SC activity could bemeasured regardless of the cell line and substrate used.Scpep1-gt miceIn order to obtain an insight into Scpep1 function,we generated a gene trap mouse model. A blast ofScpep1 cDNA within the BayGenomics (San Fran-cisco, CA, USA) database identified the ES cell lineRST426 as a Scpep1-gt cell line. The gene trap vectorused by BayGenomics contains a splice acceptor siteupstream of a b-galactosidase ⁄ neomycin phosphotrans-ferase (geo) fusion gene (Fig. S2), which inserted intointron 7 of the Scpep1 gene as confirmed by genomicsequencing. Hence, the downstream exons 8–13 weredeleted from the gene trap transcript and werereplaced by the promoterless geo cassette. Mostimportantly, two amino acids, Asp371 and His431, ofthe putative catalytic triad for SC activity wereexcluded from the resulting fusion product.As gene trapping does not consistently result in theinactivation of a gene, we checked for Scpep1 mRNAwith a 3¢-specific probe by northern blot analyses(Fig. S3) and for Scpep1 protein by western blotting(Fig. 5A). In Scpep1-gt mice, the 35 kDa Scpep1 signalwas absent from virtually all tissues tested. However,an antibody against the geo moiety of the gene trapfusion product detected a 200 kDa protein in thetissues of Scpep1-gt mice, corresponding to the 35 kDaScpep1 expression pattern in wild-type mice,confirming the calculated size for the Scpep1-geofusion protein of about 200 kDa (Fig. 6A).Subcellular fractionation of mouse liver after TritonWR-1339 (tyloxapol) injection, including differentialcentrifugation steps followed by a discontinuoussucrose gradient, enables the isolation of a fraction(F2) which is  50-fold enriched in lysosomal markerenzymes such as b-hexosaminidase. Western blot ana-lysis of each fraction of the lysosomal purification fromwild-type mice showed co-fractionation of the pro-cessed 35 kDa Scpep1 and 18 kDa Scpep1 with lyso-somal proteins such as cathepsin D (Ctsd) andlysosomal associated membrane protein 1 (Lamp1) infraction F2 (Fig. 6B). Western blot analyses from sub-cellular fractions derived from Scpep1-gt mice failed todetect Scpep1 in fraction F2 (Fig. 6C). In contrast, thegeo antibody revealed a specific  200 kDa signal inthe microsomal fraction P, indicating that the Scpep1-geo fusion product was retained in the endoplasmicreticulum and ⁄ or in the Golgi (Fig. 6C).Phenotype of Scpep1-gt miceGenotyping of 350 offspring from heterozygous bree-dings showed the expected Mendelian frequency with23.6% homozygous Scpep1-gt mice, indicating thatScpep1 is not essential for correct embryonic develop-ment. Homozygous Scpep1-gt mice and wild-type miceshowed comparable sizes and weight developments, aswell as fertility and mortality (data not shown). Deter-minations of blood (full blood count), serum (e.g.aspartate aminotransferase, c-glutamyl transferase)and urine parameters showed no pathological findings.The activities of several lysosomal hydrolases werenormal in various tissues and lysosomes from liver andkidney of Scpep1-gt mice, and were inconspicuous withregard to their distribution in a Percoll gradient, indi-cating that the density and size of the lysosomes wereunaltered (data not shown).The following organs were regularly examined histo-logically using semithin sections: liver, lung, kidney,spleen, pancreas, retina, cornea, and spinal cord; insome instances, the inner ear (cochlea) and cerebellarcortex were also investigated. Ultrastructural examina-tion was performed on liver and spinal cord of twowild-type and two Scpep-gt mice. We were unable tofind any consistent differences between wild-type andScpep-gt mice. In particular, there was no evidence oflysosomal storage in any of the numerous cell typesinspected.3.5 4.5 5.5 6.5 7.5 ytivitca CS .ceps(U·mg–1)Fig. 4. SC activity determination. C-terminally His-tagged 55 kDaScpep1 precursor (h) was purified from stably expressing HT1080cells and incubated at different pH values ranging from 3.5 to 8.5with FA-Phe-Phe as SC substrate. Yeast CPY (d) served as a posi-tive control and BSA (D) as a negative control.Functional characterization of lysosomal Scpep1 K. Kollmann et al.1360 FEBS Journal 276 (2009) 1356–1369 ª 2009 The Authors Journal compilation ª 2009 FEBSSC activity in Scpep1-gt miceHomogenates from various tissues and lysosome-enriched fractions from liver (F2) of control mice andScpep1-gt mice showed equal levels of acid SC activity(data not shown). We further separated F2 fractionsfrom control and Scpep1-gt mice by gel filtration andtested each fraction for Scpep1 and Ctsa by westernblot analysis and acid SC activity. The Ctsa elutionprofiles were similar in both F2 fractions ranging from1. CBZ-Leu-Phe CBZ-Gly-Leuα1pepcS-α-GAPDH0801THkcom +1pepcS +ASubstrate*553575503725kDa376siH-1pepcS +0801THkcom +1pepcS +6siH-1pepcS +0801THkcom +1pepcS +6siH-1pepcS +0801THkcom +1pepcS +6siH-1pepcS + CBZ-Leu-Phe CBZ-Gly-LeuBα1pepcS-α-GAPDH7-SOCkcom +6siH-1pepcS +7-SOCkcom +6siH-1pepcS +7-SOCkcom +6siH-1pepcS +Substrate7-SOCkcom +6siH-1pepcS +*553575503725kDa37Specific activity of acid carboxypeptidases (U·mg–1)Specific activity of acid carboxypeptidases (U·mg–1)Fig. 5. Expression and acid SC activity of Scpep1 in COS-7 and HT1080 cells. HT1080 (A) and COS (B) cells were transfected with eitherthe appropriate mock construct (pCI-neo for COS-7; pcDNA3.1-Hgyro for HT1080), Scpep1 or Scpep-His6, as indicated, and assayed for acidSC activity using various artificial substrates. The columns represent the mean of three technical replicates for each cell line. Scpep1 expres-sion was monitored by western blot analysis using 100 lg of the cell homogenates, and glyceraldehyde 3-phosphate dehydrogenase(GAPDH) served as loading control. Nonspecific signals are marked with asterisks (*).K. Kollmann et al. Functional characterization of lysosomal Scpep1FEBS Journal 276 (2009) 1356–1369 ª 2009 The Authors Journal compilation ª 2009 FEBS 1361fraction 16 to 18 (Fig. 7), whereas Scpep1 signals weresolely detectable in fractions 15–17 from control mice(Fig. 7). However, the lysosomal SC activity distribu-tion was roughly identical in both elution profiles,regardless of the presence or absence of Scpep1(Fig. 7). Thus, Scpep1 did not show proteolytic activitytowards common lysosomal SC type C and Dsubstrates.DiscussionTo date, four putative lysosomal SCs have beenidentified, but proteolytic activity has only beenproven for Ctsa and the distantly related prolyl-carboxypeptidase [19,20]. The third putative SC,carboxypeptidase vitellogenic-like (Cpvl), has beenreported to be a lysosomal SC restricted toT W t g T W t g T W t g T W t g T W t g T W t g T W t g T W t g T W t g T W t g 1 p e p c S l o r t n o c T W t g α α 1 p e p c S - α α -geo kDa 25 20 100 75 50 37 15 α α -GAPDH 34 250 150 0 8 0 1 T H F E M A α α -Scpep1 α α -Ctsd α α -Lamp1 kDa -37 -25 - 150 - 100 -20 -20 -50 -37 -25 α α -geo -150 -50 -75 N E M L P S F1 F2 F3 F4 B C Wild-type mice Fraction α α -Scpep1 α α -geo α α -Lamp1 -100 - 150 -37 -25 -20 -50 -75 N E M L P S F1 F2 F3 F4 Scpep1-gt mice Fraction Testis Liver Kidney Intestine Stomach Bladder Brain Lung Heart Spleen Fig. 6. Differential western blot analysis ofScpep1 expression in tissues from wild-type(WT) and Scpep1-gt mice. (A) Protein fromvarious tissue extracts (200 lg per lane) andcell lysates (HT1080 and MEF, 50 lg perlane) were separated by SDS-PAGE, blottedonto poly(vinylidene difluoride) membraneand probed with the antibodies as indicated.Tyloxapol-filled lysosomes from mouse liverof control mice (B) and Scpep1-gt mice (C)were separated by differential centrifugation(corresponding to fractions N–S). Fraction L(light mitochondria) was loaded under asucrose gradient (F1–F4), resulting in acodistribution of Scpep1 with the lysosomalmarker proteins cathepsin D (Ctsd) andLamp1 in F2, as shown by western blotanalysis after SDS-PAGE loaded with250 lg for each fraction of the differentialcentrifugation (N–S) and 50 lg of F1–F4 ofthe sucrose gradient. The blot membranewas additionally probed with antibodiesagainst neomycin phosphotransferase(a-geo). E, postnuclear fraction; F1–F4,sucrose gradient fractions 1–4; L, lightmitochondria fraction; M, heavymitochondria fraction; N, nuclear fraction;P, microsomal fraction; S, cytosolic fraction.Functional characterization of lysosomal Scpep1 K. Kollmann et al.1362 FEBS Journal 276 (2009) 1356–1369 ª 2009 The Authors Journal compilation ª 2009 FEBSmacrophages [21]. Recently, it has been demonstratedthat Cpvl localizes to the endoplasmic reticulumrather than to lysosomes, and hence a role in majorhistocompatibility complex loading has been sug-gested [22], but proof for the enzymatic activity ofCpvl has not yet been served. In this study, wefocused on the characterization of the fourthputative lysosomal SC, Scpep1, with regard to itslocalization, processing, glycosylation and presumedSC activity.Molecular forms of Scpep1Our antisera raised against the 55 kDa Scpep1precursor confirmed the lysosomal localization ofendogenous Scpep1 by immunofluorescence andco-fractionation, as postulated previously by ourgroup and others [7,11]. Tissue-specific expressionanalyses were performed according to a recent study[11], with highest Scpep1 levels found in visceralorgans such as the liver and kidney. Western blotanalyses of homogenates from HT1080-Scpep1 cellsand from a 50-fold lysosome-enriched fractionrevealed the presence of the expected 35 kDa pro-cessed fragment and an as yet unknown 18 kDa C-terminal fragment, in contrast with a recent publica-tion [11] in which the processing of the Scpep1 pre-cursor to a C-terminal 35 kDa fragment and aputative, but undetected, N-terminal 16 kDa peptidewas postulated. The maturation from a zymogen intoa two-chain form bears resemblance to a number ofother SCs, such as barley SC [23] and lysosomalCtsa, in particular. Ctsa is synthesized as a 54 kDaprecursor and further processed into N-terminal32 kDa and C-terminal 20 kDa polypeptides [24].However, although both subunits of Ctsa are linkedto each other by disulfide bonds to form the 54 kDamonomer [24], the two-chain form of Scpep1 doesnot form disulfide bridges. Moreover, although Ctsadimerizes and, together with b-galactosidase andsialidase, forms a large multienzyme complex [25],Scpep1 from mouse liver elutes at  50 kDa in gelfiltration assays (Fig. 1B), as predicted for the mono-mer, and in this regard resembles the yeast SCsCPY [26] and KEX1 [27], which are also active asmonomers.The maturation of lysosomal hydrolases from azymogen is essential for their functional activation,and hence must be tightly regulated in terms of pro-tecting the cell against self-digestion. We could notmimic autoprocessing, as described in vitro for lyso-somal cathepsin B, D or L [13,14,28]. The matura-tion of the Scpep1 precursor into the 35 kDafragment is a multistep process (Fig. 2A), which isprevented by the addition of the serine proteinaseinhibitor AEBSF (Fig. 2B), as well as by the NH4Cl-mediated uncoupling of MPR-dependent lysosomaltransport (Fig. 2C), suggesting that Scpep1 process-ing is mediated by a lysosomal serine proteinase.Previously, we have demonstrated that partially puri-fied mouse Scpep1 derived from BHK cells binds onimmobilized MPR46 and MPR300 and is internalizedby I-cell fibroblasts in an M6P-dependent manner[7]. Limited deglycosylation by PNGase F digestdemonstrated that all putative N-glycosylation sitesare occupied in MEFs. Sleat et al. [29] identifiedM6P sites on 92 MPR-binding proteins derived fromhuman and mouse brain, which were both of lyso-somal function or unknown function. Although atotal of 135 M6P sites were identified in 69 proteins,M6P sites on Scpep1 escaped the analysis [29]. Mostprobably, these sites were missed because of the sizeof tryptic peptides containing the N -glycosylationsites, which range from 30 to 62 amino acids, andthus may exceed the preset mass range of the MSanalysis [29].13 16 17 18 1914 15α-Ctsaα-Ctsaα-Scpep1α-Scpep1F2 WTF2 gtBA13 14 15 16 17 18 1901234FractionSpec. SC activity (mU·mg–1)Fig. 7. SC activity profiling and western blot analyses of Scpep1and Ctsa after gel filtration of lysosome-enriched fractions. F2 frac-tions derived from wild-type mice (s) and Scpep1-gt mice () wereseparated on an FPLC Superdex 200 10 ⁄ 300 GL column in 20 mMMes, pH 4.5, 150 mM NaCl. The collected fractions were assayedfor SC activity (A) and analysed by western blot analyses (B), usingthe Scpep1 antiserum to detect the 35 kDa fragment and an anti-Ctsa rat IgG2B to detect the 32 kDa heavy chain of Ctsa.K. Kollmann et al. Functional characterization of lysosomal Scpep1FEBS Journal 276 (2009) 1356–1369 ª 2009 The Authors Journal compilation ª 2009 FEBS 1363The lysosomal localization, processing and conserva-tion of critical domains are shared features of Scpep1and Ctsa. However, under conditions adapted to SCssuch as Ctsa or CPY, we failed to demonstrate acidSC activity of Scpep1 in any approach addressed sofar, regardless of which molecular form of Scpep1 orsubstrate was assayed. Because of its zymogen status,it is reasonable that the purified Scpep1 precursor doesnot exhibit SC activity; however, surprisingly, homo-genates of HT1080 and COS-7 cells, which highlyexpressed and subsequently processed mouse Scpep1 ina tagged or untagged version, did not show anyelevated acid SC activity. The lack of additional SCactivity on Scpep1 overexpression may be ascribed toan unknown limiting factor of Scpep1 activation. Asan example, lysosomal sulfatases are modified in theendoplasmic reticulum by the formylglycine-generatingenzyme, which has been shown to be an essential andlimiting factor for sulfatase activity [30,31]. Further-more, it has been reported that the activation of thecathepsin D precursor is accelerated when it is com-plexed with prosaposin [32].The Scpep1-gt mouse did not exhibit an obviousphenotype and did not show any lysosomal storage,although we confirmed the loss of the lysosomal35 kDa mature form of Scpep1. Despite the deletionof the entire C-terminus, including two critical aminoacids of the putative catalytic triad, we were unable toshow reduced acid SC activity in Scpep1-deficientmice.We would like to point out that the computationalmodelling of Scpep1 also predicts a Ctsa- (1ivyA)and CPY-like (1cpy_) folding (http://swissmodel.expasy.org/SWISS-MODEL.html; Fig. 8). In addition,the alignment of Scpep1 with several SCs frommouse, Saccharomyces cerevisiae and Trypanoso-ma cruzei identifies a highly conserved substratebinding site (I), as well as three conserved catalyticregions (II–IV), each embedding one amino acid ofthe catalytic triad, and hence strongly favouringacidic SC activity (Fig. 9).Consequently, the apparent lack of in vitro SCactivity of Scpep1 in its processed form must beascribed either to the selection of an inappropriatesubstrate or to a nonproteolytic function of Scpep1.It is worth mentioning that another study failed todemonstrate proteolytic activity of Scpep1 for Ctsasubstrates such as endothelin-1 and, in combinationwith immunohistological studies, suggests a functionin the homeostasis of the renal and reproductiveCtsa (1ivyA) Scpep1 (Model: 1ivyA)CPY (1cpy_) Scpep1 (Model: 1cpy_ )Fig. 8. Predicted three-dimensional struc-ture of Scpep1. Scpep1 was homologymodelled with Ctsa and CPY as templatesto predict its three-dimensional structureusing theSWISSMODEL alignmentmode (http://swissmodel.expasy.org//SWISS-MODEL.html).Functional characterization of lysosomal Scpep1 K. Kollmann et al.1364 FEBS Journal 276 (2009) 1356–1369 ª 2009 The Authors Journal compilation ª 2009 FEBSsystems [11]. A recent gene target Ctsa mouse model(CtsaS190A), in which the catalytic serine residue wassubstituted but the protective protein function waspreserved [33], does not develop a secondary galacto-sialidosis like the ‘classic’ Ctsa-deficient mouse [34].In CtsaS190Amice, neither Scpep1 nor any otherpredicted lysosomal SC efficiently compensates forthe loss of in vitro SC activity, resulting in residualactivities of 5–10% in visceral organs [33], althoughScpep1 and, moreover, Ctsa and Scpep1 areFig. 9. Multiple alignment of the primary amino acid sequences of SCs. SCs were aligned according to the CLUSTALW algorithm. Sequencesare highlighted in grey for two or three homologous sequences or in black for amino acids conserved in all four sequences. The substratebinding site (I) is defined by the line. The catalytic domains embedding the amino acids of the catalytic triad (*) are marked as II–IV. Saccha-romyces cerevisiae CPY (GI:115901); Trypanosoma cruzei SCP (GI:35181448); mouse Ctsa (GI:84042523); mouse Scpep1 (GI:13436038);mouse vitellogenic-like carboxypeptidase VLCP (GI:187952735).K. Kollmann et al. Functional characterization of lysosomal Scpep1FEBS Journal 276 (2009) 1356–1369 ª 2009 The Authors Journal compilation ª 2009 FEBS 1365[...]... 3 010 –30 21 10 Chen J, Streb JW, Maltby KM, Kitchen CM & Miano JM (20 01) Cloning of a novel retinoid-inducible serine carboxypeptidase from vascular smooth muscle cells J Biol Chem 276, 3 417 5–3 418 1 11 Lee TH, Streb JW, Georger MA & Miano JM (2006) Tissue expression of the novel serine carboxypeptidase Scpep1 J Histochem Cytochem 54, 7 01 711 12 Jung G, Ueno H & Hayashi R (19 98) Proton-relay system of carboxypeptidase. .. FEBS Journal 276 (2009) 13 56 13 69 ª 2009 The Authors Journal compilation ª 2009 FEBS 13 67 Functional characterization of lysosomal Scpep1 13 14 15 16 17 18 19 20 21 22 23 24 K Kollmann et al studies on mutagenic replacement of his 397 J Biochem 12 4, 446–450 Rozman J, Stojan J, Kuhelj R, Turk V & Turk B (19 99) Autocatalytic processing of recombinant human procathepsin B is a bimolecular process FEBS... & Sandhoff K (19 82) Lysosomal enzyme precursors in human fibroblasts Activation of cathepsin D precursor in vitro and activity of beta-hexosaminidase A precursor towards ganglioside GM2 Eur J Biochem 12 5, 317 – 3 21 Leake DS & Peters TJ (19 81) Proteolytic degradation of low density lipoproteins by arterial smooth muscle cells: the role of individual cathepsins Biochim Biophys Acta 664, 10 8 11 6 Wendland... Calpha-formylglycine-generating enzyme J Biol Chem 280, 14 900 14 910 Wattiaux R, Wibo M & Baudhuin P (19 63) [Effect of the injection of Triton WR 13 39 on the hepatic lysosomes of the rat] Arch Int Physiol Biochim 71, 14 0 14 2 Gieselmann V, Pohlmann R, Hasilik A & von Figura K (19 83) Biosynthesis and transport of cathepsin D in cultured human fibroblasts J Cell Biol 97, 1 5 Taylor S & Tappel AL (19 73) Lysosomal peptidase... & Fincher GB (19 88) The A- and B-chains of carboxypeptidase I from germinated barley originate from a single precursor polypeptide J Biol Chem 263, 11 106 11 110 Galjart NJ, Gillemans N, Harris A, van der Horst GT, Verheijen FW, Galjaard H & d’Azzo A (19 88) Expression of cDNA encoding the human ‘protective protein’ associated with lysosomal beta-galactosidase and 13 68 25 26 27 28 29 30 31 32 33 34 35... available: Fig S1 Lysosomal localization of endogenous Scpep1 by immunofluorescence Fig S2 Disruption of the Scpep1 gene (A) Insertion of the b-geo gene trap cassette into the Scpep1 gene (B) Southern blot (C) Multiplex-PCR analysis of genomic DNA derived from three F2 mice Fig S3 Multitissue northern blot analysis with RNA derived from wild-type (+ ⁄ +) mouse tissues, Scpep1gt () ⁄ )) tissues and heterozygous... were determined according to [ 41] for CBZ substrates and [42] for FA substrates Cloning, transfection and expression of Scpep1 Generation of the Scpep1-gt mouse model The Scpep1 cDNA was subcloned into the pcDNA3 .1 ⁄ Hygro(+) vector and pCI-neo (Promega GmbH, Mannheim, Germany) by add-on PCR, as described previously [36] HT1080 cells and COS-7 cells were transfected with Fugene6 reagent (Roche GmbH, Mannheim,... Robinson D & Galjaard H (19 82) Molecular defect in combined betagalactosidase and neuraminidase deficiency in man Proc Natl Acad Sci USA 79, 4535–4539 Jung G, Ueno H & Hayashi R (19 99) Carboxypeptidase Y: structural basis for protein sorting and catalytic triad J Biochem 12 6, 1 6 Latchinian-Sadek L & Thomas DY (19 93) Expression, purification, and characterization of the yeast KEX1 gene product, a polypeptide... al (19 95) Mouse model for the lysosomal disorder galactosialidosis and correction of the phenotype with overexpressing erythroid precursor cells Genes Dev 9, 2623–2634 Pohlmann R, Boeker MW & von Figura K (19 95) The two mannose 6-phosphate receptors transport distinct complements of lysosomal proteins J Biol Chem 270, 27 311 –27 318 Deuschl F, Kollmann K, von Figura K & Lubke T ¨ (2006) Molecular characterization. .. function of lysosomal SCs, it could be insightful to crossbreed Scpep1-deficient mice with CtsaS190A mice to investigate the overlap and distinct functions of Scpep1 and Ctsa Purification of Scpep1-His6 from stably expressing HT1080 cells Materials and methods Deglycosylation by PNGase F Cell lines and cell culture Cell lysates of HT1080-Scpep1 were subjected to PNGase F (Roche) treatment as described previously . preset mass range of the MSanalysis [29]. 13 16 17 18 19 14 15 α-Ctsaα-Ctsaα-Scpep1α-Scpep1F2 WTF2 gtBA 13 14 15 16 17 18 19 0 1 234FractionSpec M5535 18 kDakDa0801TH-0801THsiH -1 pepcS08 01 TH-0801THsiH -1 pepcS0801TH-080 1 THsiH-1pepcSALane 1 2 3 4 5 6 7 8 9 10 11 12 Fig. 1. Molecular forms of Scpep1. (A)
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