Protein Expression and PuriWcation 45 (2006) 157–167

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High yield synthesis and characterization of phosphorylated recombinant human procathepsin D expressed in mammalian cells

Marina Démoz a, Roberta Castino a, Carlo Follo a, Andrej Hasilik b, Bonnie F. Sloane c,d, Ciro Isidoro a,¤

a Dipartimento di Scienze Mediche, Università “A. Avogadro,” via Solaroli 17, 28100 Novara, Italyb Institut für Physiologische Chemie, Philipps-Universität, Marburg D-35033, Germany

c Department of Pharmacology, Wayne State University, Detroit, MI 48201, USAd Barbara Ann Karmanos Cancer Institute, Wayne State University, Detroit, MI 48201, USA

Received 18 April 2005, and in revised form 6 July 2005Available online 19 August 2005

Abstract

We used a vaccinia virus expression system for the production of recombinant human cathepsin D (CD), a lysosomal proteaseimplicated in various patho-physiological processes including cancer, neurodegeneration, and development. The recombinant pro-tein was successfully expressed in various human and non-human cells. It was correctly synthesized as a glycosylated 53 kDa precur-sor (proCDrec) that reacted with a polyclonal antibody against residues 7–21 of the propeptide sequence. In contrast to the control, incells infected with the recombinant virus proCDrec was largely secreted into the culture medium, although it contained high-mannoseoligosaccharides with uncovered mannose-6-phosphate residues. Intracellular proCDrec was processed into the 48 kDa intermediatesingle-chain and the 31 plus 13 kDa double-chain forms, however, the processing was slower than in normal cells. A method basedon Pepstatin A-aYnity chromatography allowed to isolate the recombinant protein from the medium of infected cells. Based on itslatency in activity assay at acid pH and on its reactivity with antibodies speciWc for the N-terminus, the puriWed protein was judgedto be in the inactive precursor form. During incubation at acid pH the puriWed proCDrec underwent autocatalytic processing andacquired pepstatin A-sensitive enzyme activity, as expected for correctly folded proCD. Antiserum raised in rabbits against proCDrec

speciWcally reacted with human, but not with mouse proCD under non-denaturing conditions. We conclude that our vaccinia virus-directed proCDrec displays structural and functional features resembling those of native human proCD. This system can therefore beexploited for the synthesis of large quantities of human proCD, allowing further studies on the structure and function of this interest-ing protein. 2005 Elsevier Inc. All rights reserved.

Keywords: Lysosomes; Vaccinia virus; Protein folding; Conformational epitopes

1

Cathepsin D (CD) is an aspartic endopeptidase nor-mally located in endosomal and lysosomal organellesand ubiquitously expressed in mammalian cells [1].While in lysosomes CD is mainly involved in the degra-dation of (auto) phagocytosed proteins, within endo-

* Corresponding author. Fax: +39 0321 620421.E-mail address: isidoro@med.unipmn.it (C. Isidoro).

1 Abbreviations used: CD, cathepsin; M6P, mannose-6-phosphate;Pst, pepstatin A; BrdU, 5-bromodeoxyuridine; TK, thymidine kinase.

1046-5928/$ - see front matter 2005 Elsevier Inc. All rights reserved.doi:10.1016/j.pep.2005.07.024

somes this protease aVects the limited proteolysis ofvarious substrates (such as pro-hormones and pro-enzymes), which results in their biological activation [2].CD-mediated proteolysis has been shown to be impor-tant in various patho-physiological conditions includingtissue homeostasis and organ development [3], neurode-generative diseases [4–7], ageing [8,9], and cancer [10–12].In certain conditions, CD is translocated into the cytosolwhere it exerts a pro-apoptotic activity [13,14]. This activ-ity has been demonstrated directly by microinjecting CD

158 M. Démoz et al. / Protein Expression and PuriWcation 45 (2006) 157–167

into cultured cells [15]. We have recently demonstratedthat CD secreted from the rat mammary gland can act atphysiological pH in the extracellular environment toprocess the prolactin hormone [16].

CD is synthesized in the endoplasmic reticulum as a53 kDa pro-enzyme that becomes activated after reach-ing the endosomal–lysosomal compartment. In vitro,proCD is capable of autocatalytic acid-dependentremoval of 26 residues, which leads to a 51 kDa single-chain known as pseudocathepsin D [17]. In cells, theprocessing proceeds through the removal of the entireprosequence of 44 residues and formation of 48 kDa sin-gle-chain processing intermediate. In the acidic pre-lyso-somal and lysosomal compartments the intermediateform is eventually converted to a double-chain form thatis composed of 31 kDa (large) and 13 kDa (small) chains[18,19]. Transport of proCD from the Golgi complex todistal compartments is mediated by receptors speciWc formannose-6-phosphate (M6P) groups [20], by interactionwith prosaposin [21,22] or other unknown molecules.The domain for interaction with prosaposin is located atthe N-terminus of proCD and pseudoCD, within theregion comprising the residues Ile27 and Glu44 [22].ProCD also contains a domain, so far not yet identiWed,for interaction with ceramide [23]. Prosaposin and cera-mide have been shown to promote the autocatalyticactivation of proCD [22,23]. The structures of crystal-lized mature form of bovine and human CD have beendescribed [24–26]. A 3D structure of human proCD isnot yet available, and this limits our knowledge in regardto the mechanisms of proCD transport and pro-peptideremoval at acid pH. Human recombinant CD has beenproduced in Escherichia coli [27,28] and in insect cellsinfected with recombinant baculovirus [29,22]. Theserecombinant proteins, however, were not subjected toproper post-translational modiWcation of N-linked sug-ars [29] and therefore cannot be considered as surrogatesof authentic human CD. We document here the Wrst pro-duction of recombinant human proCD obtained inmammalian cells by using a vaccinia virus system anddescribe its puriWcation, and biochemical and structuralcharacteristics. We demonstrate that our system is usefulfor producing large quantities of recombinant humanproCD with the characteristics of the normal nativeprotein.

Materials and methods

Reagents and chemicals

Vaccinia virus strain was purchased from ATCC(Rockville, MD, USA); pepstatin A (Pst) and 5-bromo-deoxyuridine (BrdU) were purchased from Sigma (St.Louis, MO); Endo-N-acetylglucosaminidase H (EH) andalkaline phosphatase (AP) were purchased from Boeh-

ringer Mannheim; [35S]methionine and 32P were pur-chased from Amersham (Aylesbury, UK); Zeocin wasobtained from Invitrogen (CA, USA). The monoclonalanti-tubulin antibody, and the Texas Red- and theFITC-conjugated secondary antibodies were purchasedfrom Sigma (St. Louis, MO). The following antiseraraised in rabbits immunized with mature CD puriWedfrom human placenta (�h-mCD; [11]) or with matureCD puriWed from rat liver (�rat-mCD; [30]) or with puri-Wed proCDrec (see below) were produced in Dr. Isidoro’slaboratory. The rabbit antiserum against the His7-Gly21synthetic peptide was obtained from Genosys Biotech-nologies (Cambridge, UK).

Cell lines and cell cultures

The following cell lines obtained from ATCC (Rock-ville, MD, USA) were used: CV-1 African green monkeykidney cells; BSC1 monkey kidney cells; TK¡143Bhuman sarcoma cell line lacking the thymidine kinase(TK) gene; HeLa, human cervix carcinoma; HepG2,human hepatocarcinoma; MCF7, human breast carci-noma; NIHOVCAR3, human ovarian cancer; RBL, ratbasophilic leukaemia; L929 mouse Wbrosarcoma cells.Cells were cultivated in MEM containing 10% bovinefetal serum at 37 °C in a humidiWed incubator saturatedwith 95% air and 5% CO2.

Viral infection and plasmid transfection

The manipulation of the vaccinia virus and the infec-tion with the recombinant virus are detailed in the text.Transfection of L929 and of RBL cells with thepcDNA3.1 expression vector (pcDNA3.1ZEO (¡), Invit-rogen, CA, USA) carrying the cDNA for CD [31] wasperformed using Lipofectamine 2000 as instructed bythe manufacturer (Invitrogen, CA, USA). L929 clonesstably expressing CD were selected after 30 days of cul-ture in the presence of the antibiotics zeocin (0.5 �g/ml).

Analysis of proCD processing in infected cells

Metabolic labeling with the radioactive precursors[35S]methionine or [32P]phosphate was performed inappropriate deWcient medium. Radioactively labeled CDwas isolated by immunoprecipitation and denaturedimmunoprecipitates were incubated with EH or AP asdescribed previously [32,33]. Complete removal of N-linked high-mannose-type sugars and of monoesterphosphates by EH or AP treatment, respectively, wasascertained by performing extensive hydrolysis (i.e., bydoubling the enzyme concentration and the time of incu-bation). EH treatment of 32P-labeled CD immunoprecip-itates conWrmed that the [32P]phosphate was associatedwith high-mannose-type sugars (not shown and [33]).Proteins were separated by SDS–PAGE and revealed by

M. Démoz et al. / Protein Expression and PuriWcation 45 (2006) 157–167 159

standard procedures of Xuorography (for [35S]methio-nine- or 32P-labeled CD) or Western blotting. Cellhomogenates for Western blotting analysis were pre-pared in 0.5% sodium-deoxycholate by freeze–thawing(3 cycles) and sonication. A polyclonal antiserum raisedagainst mature CD (�h-mCD) was used as primary anti-body [11]. After incubation with horseradish peroxidase-conjugated secondary antibody, immunocomplexes wererevealed by ECL-chemiluminescence (Perkin-Elmer)according to manufacturer’s instructions.

Chromatography puriWcation of recombinant proCD

Medium from BSC1 cells was clariWed by centrifuga-tion (10 min at 12,000g) and applied to a Pst-Sepharose(Sigma–Aldrich, Deisenhofen, Germany) column(1.5 £ 2.5 cm diameter). Immediately prior to the appli-cation the medium was mixed with the binding buVer(0.7 M NaCl, 0.2% Triton X-100, 0.2 M formic acidadjusted to pH 3.5 with NaOH). The column waswashed with 5 volumes 0.4 M NaCl, 0.1% Triton X-100,0.1 M sodium formate, pH 3.5, and one volume each0.4 M NaCl, 10 mM sodium formate, pH 3.5, and 0.4 MNaCl sodium acetate, pH 5.0. The enzyme was elutedwith 0.4 M NaCl, 20 mM Tris–HCl, pH 8.3. Chromatog-raphy was performed at 4 °C. The eluted proCDrec wasfurther applied to a 7 £ 35 mm Bio-Rad UNO-Q1 anionexchange FPLC column and from this eluted with a lin-ear gradient at a Xow rate of 0.5 ml/min using 2 mMTris–HCl (pH 8.3) and 150 mM Tris–HCl (pH 8.9) as ini-tial and Wnal buVers, respectively. The fractions werepooled and concentrated for subsequent analyses. Thepresence of CD was estimated in aliquots by SDS–PAGE stained with silver-nitrate reagent [34] or byWestern blotting. In the latter case, a polyclonal rabbitantiserum raised against mature CD [11] or against theN-terminus of CD (obtained from Genosys Biotechnol-ogies, Cambridge, UK) was used as speciWed in the text.

CD enzyme assay

CD activity was assayed at pH 3.65 using an aminom-ethylcoumarine (AMCA)-conjugated hemoglobin assubstrate (a kind gift of Dr. LoeZer, University of Mar-burg) [35]. An aliquot (1–20 �l) of puriWed proCDrec wasincubated for up to 30 min at 37 °C in a formate–acetatebuVer (12.5 mM, pH 3.65), 0.2�g hemoglobin, 0.05AMCA-hemoglobin in 40 �l Wnal volume. The assay wasrun on a multiwell plate and Xuorescence was read in aspectroXuorometer at 365 and 460 nm excitation andemission wavelengths, respectively. Before running theassay some samples were incubated at 37 °C for 15 minin 50 mM formate–acetate buVer at pH 3.65 to generatepseudo-CD by autoproteolysis. To prove that substratewas hydrolyzed by CD, the inhibitor Pst was added toparallel samples.

Production and characterization of anti-proCDrec antiserum

A rabbit was immunized with four cycles of injectionwith 200 �g of puriWed proCDrec that was emulsiWedwith Freund’s complete (Wrst injection) or incomplete(following injections) adjuvant. Peripheral blood wastaken from the ear vein before (pre-immune serum) andafter immunization (�proCDrec). The use and care of theanimal was approved by the University Commission forAnimal Care following the criteria of the ItalianNational Research Council. The speciWcity of this antise-rum toward proCD and processed CD molecules, eithernon-denatured or denatured, was checked by Westernblotting, dot blotting or immunoXuorescence. For West-ern blotting analysis cell homogenates and culture mediawere denatured in Laemmli buVer, fractionated by SDS–PAGE, and electro-transferred onto a nitrocelluloseWlter. For dot-blot analysis under non-reducing condi-tion, the conditioned media of mouse or human cellswere directly transferred onto nitrocellulose Wlter(0.45 �m pore size) using a dot-blot apparatus (Bio-dot,Bio-Rad). Prior to further manipulation the Wlters weretemporally stained with Ponceau red (0.2% solution in180 mM trichloroacetic acid for 5 min) to reveal theamount of protein blotted. The Wlters were then pro-cessed for immunostaining following a standard proce-dure. For immunoXuorescence studies the cells wereseeded on sterile glass coverslips, then Wxed in methanol,and processed for immunoXuorescence staining of CD[36]. To this end the cover-slip was incubated Wrst with arabbit anti-CD polyclonal antiserum (either �rat-mCDor �proCDrec, as indicated) and a monoclonal anti-tubu-lin antibody, then with Texas red-conjugated goat anti-rabbit IgG (to reveal CD) and a FITC-conjugated goatanti-mouse IgG (to reveal tubulin). Both the primaryand secondary antibodies were diluted (1:200) in phos-phate buVer containing 2.5% bovine serum albumin and0.1% Triton X-100. Images were captured under a confo-cal Xuorescence microscope.

Results

Generation of the recombinant vaccinia virus carrying the CD cDNA

To express human cathepsin D (CD) at a high rate inmammalian cells we generated a recombinant vacciniavirus. To this end the full-length CD cDNA comprisingthe ATG codon was inserted downstream of a syntheticvaccinia late promoter into the vector pMJ601 [37]which also contains the E. coli LacZ gene. This expres-sion cassette is Xanked by segments of the vaccinia thy-midine kinase (TK) gene that allows homologousrecombination with the wild-type virus (Fig. 1, inset).

160 M. Démoz et al. / Protein Expression and PuriWcation 45 (2006) 157–167

The pMJ601/CD was transfected into CV-1 cells previ-ously infected with the WR strain of the vaccinia virus(with this strain >95% of the infectious virus remainscell-associated), and the cell homogenates were furtherused to infect TK¡143B cells that are deWcient of TKgene. The TK selection method is based on the inser-tional inactivation of the TK gene. In the presence ofactive TK, added BrdU is phosphorylated and incorpo-rated into viral DNA, where it causes lethal mutations. IfTK¡ cells are used, then TK¡ (recombinant) viruseswill replicate normally in the presence of BrdU, whereasTK+ virus will not. Because the product of a singlecross-over is still TK+, only double cross-over will beselected. This selection has been combined with �-galac-tosidase screening (based on the co-insertion of theE. coli LacZ gene under the control of a separate vac-cinia virus promoter into the vaccinia virus genomealong with the CD gene). The combination of TK selec-tion and �-galactosidase screening allowed us to discrim-inate TK¡ recombinant (blue plaques) fromspontaneous TK¡ mutants (white plaques). The generalscheme of the method is outlined in Fig. 1. Severalrounds of plaque puriWcation were used to ensure theabsence of residual wild-type virus. Recombinant virusplaques were picked and used for rounds of ampliWca-tion in HeLaS3 cells and Wnally used to infect mamma-lian cells.

Expression of recombinant proCD in human and non-human cells

Heterologous human proCD was expressed in vari-ous human and non-human cells by infection with therecombinant vaccinia virus RecVVpMJ/CD at a multi-plicity of 3 plaque-forming units (p.f.u.) per cell. After24 h of infection, homogenates and media from infectedcultures and from control cultures were analyzed by

Western blotting for the expression of CD. The poly-clonal �-human CD antiserum (�h-mCD; [11]) recog-nized several molecular species of the expected size[10,11] of both the endogenous and the heterologousCDs (Fig. 2). As shown in Fig. 2C in infected cells, highlevels of immature forms of CD, corresponding to the53 kDa precursor and the 48 kDa intermediate single-chain, accumulated. In media from non-infected culturesproCD was not detectable (Fig. 2B), while in media frominfected cultures this molecule was present in amountslargely suYcient to be detected by Western blotting(Fig. 2D). From this experiment, we conclude that ourrecombinant vaccinia virus is an eYcient system allow-ing high production of human CD in mammalian cells.

Transport and maturation of recombinant CD werefurther analyzed in infected BSC1 cells. To evaluate side-eVects of viral infection on the synthesis and transport ofendogenous CD, we prepared a recombinant vacciniavirus carrying the empty pMJ601 vector (RecVVpMJ).BSC1 cells were infected with either RecVVpMJ or Rec-VVpMJ/CD viruses. Several clones were isolated andcharacterized for the synthesis and maturation ofproCD. Cells and media were analyzed »24 h after theviral infection. The immunoblot in Fig. 3 shows in con-trol non-infected BSC1 cells (lane 1) the presence of the53 kDa proCD and of the 34 kDa LM, the latter formrepresenting more than 75% of the total intracellularCD. The 48 kDa intermediate form is not detectable. NoproCD could be evidenced in medium by Western blot-ting. Thus, in BSC1 cells under basal conditions proCDis eYciently segregated into the endosomal–lysosomalcompartment and it is fully processed into the double-chain mature form. In cells infected with the RecVVpMJempty virus only the LM form was detectable, and inamounts reduced with respect to control non-infectedparental cells (Fig. 3, compare lanes 1 and 2). This verylikely reXects the inhibition of host protein synthesis as a

Fig. 1. Scheme of method for the generation of recombinant vaccinia virus bearing the cathepsin D cDNA. Inset shows the generation of the plasmidpMJ/CD (see the text for detailed explanation).

M. Démoz et al. / Protein Expression and PuriWcation 45 (2006) 157–167 161

ognition of the heterologous recombinant CD. Five

consequence of the viral infection as well as the turnoverof CD stored in lysosomes. Thus, at the time of maximallate gene expression, in infected cells synthesis of endog-enous protein was impaired and this facilitated the rec-

Fig. 3. Expression of CD in BSC1 cells infected with the recombinantvaccinia virus bearing or not bearing the CD cDNA. BSC1 cells wereinfected with the control empty vector recVVpMJ (lane 2) or with rec-VVpMJ/CD encoding for human CD (lanes 3–7). Parental non-infected (lane 1) and infected cells were analyzed by Western blottingfor the expression of CD in homogenates (A) and media (B). FivediVerent clones infected with recVVpMJ/CD were analyzed (lanes 3–7). Cells were plated at the same cell density. Western blotting analysiswas performed 24 h post-infection. Equal amount of cell proteins andequal amount of culture media were analyzed. A representative blot(out of two with analogous patterns) is shown. The intensity of the CDbands was quantiWed by densitometry using the apparatus VersaDoc(Bio-Rad) equipped with the software QuantityOne 4.5 (Bio-Rad). P,precursor; I, intermediate single-chain; LM and SM, large and smallmature chains of the double-chain form, respectively.

clones were isolated from BSC1 cultures infected withthe recombinant pMJ/CD vaccinia virus. Western blot-ting analysis (Fig. 3, lanes 3–7) revealed that hCD wasexpressed at very high levels. In these cells the intermedi-ate form, and the proCD and LM forms were detectedon the gel. Densitometric analysis of the Western blotindicated that in these clones more than 55% of the cell-associated immunoreactive CD was in the precursorform, while »15% was represented by the intermediateform and »30% by the mature double-chain form. Thislatter form likely also includes some residual endoge-nous CD. Apparently the rate of synthesis is many foldshigher than the rate of transport and maturation. West-ern blotting analysis of culture media revealed nosecreted CD in the control or recVVpMJ-infected cul-tures (Fig. 3B, lanes 1 and 2). In contrast, a strong bandcorresponding to proCDrec was evident in media ofBSC1 cells infected with recVVpMJ/CD virus (Fig. 3B,lanes 3–7). By densitometry, the amount of secretedproCD represented about 30–40% of the total CD accu-mulated during the infectious period. Further, theamount of proCD (P) and of mature CD (LM) accumu-lated in high expressor clones (lanes 5–7) was 2.4- to 3.0-fold and 1.4- to 1.6-fold, respectively, the level of the cor-responding proteins accumulated in low expressorclones (lanes 3–4), and the amount of proCD secreted byhigh expressor clones (Fig. 3B, lanes 5–7) was about 1.5-fold higher than that secreted by low expressor clones(Fig. 3B, lanes 3–4). Taken together these observationsindicate that proCDrec was eYciently expressed in BSC1cells, but only a small portion of it was further processedinto mature forms, while a major portion of it accumu-lated within the cell and the medium. Ammonium

Fig. 2. Expression of recombinant human CD in mammalian cells. The following types of human (MCF7, breast cancer; HepG2, hepatocarcinoma;HeLa, cervix cancer) and non-human (BSC1, simian kidney epithelial) cells were infected with the recombinant VVpMJ/CD. Parental non-infected(A, cell homogenates; B, media) and infected cultures (C, cell homogenates; D, media) were then analyzed by Western blotting for the expression ofhuman CD. Cells were seeded at the same cell density and harvested 24 h post-infection. Equal amount of cell proteins and equal volume of culturemedia were analyzed. The polyclonal antiserum raised against human mature CD (�h-mCD) recognized the following CD molecular forms (molecu-lar weights are indicated): P, precursor; I, intermediate; LM and SM, large and small mature chains of the double-chain form, respectively. In media,no additional immunoreactive bands were revealed beside the precursor. The experiment was repeated two times with similar results. Note that theproportion of LM and SM peptides diVer among cell lines, likely due to the diVerent stability of the latter.

162 M. Démoz et al. / Protein Expression and PuriWcation 45 (2006) 157–167

chloride, a lysosomotropic weak base known to stimu-late the secretion of lysosomal pro-enzymes [18], did notenhance the rate of secretion of proCDrec in infectedBSC1 cells (not shown). The latter results consistentlysuggest that the export of proCDrec from trans-Golginetwork in highly expressing infected cells could be satu-rated. In the following experiments we further character-ized the structural and functional features ofrecombinant CD.

Analysis of the processing of CD-associated oligosaccharides

Newly synthesized human CD bears two N-linkedhigh-mannose-type saccharides. In the Golgi complexthese carbohydrate side chains become modiWed withone or two terminal M6P that ultimately prevent forma-tion of complex-type oligosaccharides [38]. We checkedthat N-glycosylation, mannose trimming, and synthesisof the M6P group occurred in proCDrec expressed inBSC1 cells. In a Wrst set of experiments cells wereinfected with recVVpMJ601/CD and 24 h later (whenviral gene expression reached the maximum) cells wereincubated for a further 24 h in medium containing[35S]methionine to label newly synthesized proteins.Under these conditions synthesis of endogenous CD wasshown to be depressed, whereas synthesis of recCDreached the maximum (see Fig. 3). CD was then immu-noprecipitated from media and cell extracts, and sub-jected to extensive endoglycosidase H (EH) digestion[33]. This treatment is known to remove selectively high-mannose- and hybrid-type sugars from the glycoprotein,so that its apparent molecular weight is diminished indenaturing gel electrophoresis. This experiment revealedthat: (i) intracellular proCD (almost totally representedby the recombinant protein, see Fig. 3) only bears high-mannose-type, EH-sensitive sugars (Fig. 4A); (ii) aminor portion of secreted proCDrec appears to be par-tially resistant to EH digestion (indicated by the arrowin Fig. 4A). The apparent molecular weight of EH-degly-cosylated CD forms corresponds to that of CDs isolatedfrom infected cells labeled with [35S]methionine in thepresence of tunicamycin, a toxin that prevents N-glyco-sylation (not shown).

In a second set of experiments recVVpMJ601/CD-infected BSC1 cells were incubated for 20 h with 32Pinorganic phosphate to examine the formation of thecovered and uncovered M6P groups [32,33]. By treating‘in vitro’ the 32P-labeled immunoprecipitates with alka-line phosphatase (AP), which hydrolyzes phosphomono-ester- but not phosphodiester-groups, we discriminatedthe M6P moieties still covered by N-acetyl-glucosamine(that is resistant to AP) from the uncovered forms (thatlose the phosphate) [33]. Immunoprecipitation from cellextract and medium essentially isolated recombinantproCD, as under these experimental conditions there

was little endogenous proCD (see Fig. 3). Immunopre-cipitates were incubated ‘in vitro’ with or without AP,then fractionated by SDS–PAGE, and the polypeptideswere visualized by Xuorography. The data shown inFig. 4B indicate that: (i) CDrec-associated oligosaccha-ridic chains are phosphorylated (Fig. 4B, lane 1); (ii)about 20% of 32P-labeled proCD is still in the ‘covered,’AP-resistant form (Fig. 4B, lane 2); (iii) the majority ofsecreted proCDrec bears ‘uncovered,’ AP-sensitive, M6Pgroups (Fig. 4B, lower panel, compare lanes 1 and 2).

PuriWcation and enzymatic characterization of recombinant proCD

As an indication of correct folding of the protein, wechecked if the active site of CDrec was functional. Theantibiotic Pst shows high aYnity for the active site ofCD and can be used either as its inhibitor or as its sub-strate for solid-phase chromatography. Pst Wts into andbinds to the active site of CD only if the protein is cor-rectly folded. We therefore attempted to purify proCDrec

from the medium of infected cultures by aYnity chroma-tography on a Pst–agarose column (see the Materialsand methods for details). For this purpose we infectedBSC1 cells cultured at high density for 48 h. This methodpermitted us to concentrate high amounts of secretedproCDrec in the medium. To favor the interaction ofproCDrec with Pst, just prior entering the column themedium was mixed with the binding buVer at pH 3.5 (seeMaterials and methods). To verify the purity and theidentity of the protein recovered after chromatography,

Fig. 4. Analysis of the processing of proCDrec-associated sugars. Rec-VVpMJ/CD-infected BSC1 cells were labeled with either [35S]methio-nine (A) or 32P-inorganic phosphate (B) and CD immunoprecipitatedfrom cell (C) and medium (M) extracts was analyzed by gel electro-phoresis and Xuorography after endoglycosidase H (EH, Fig. 4A) oralkaline phosphatase (AP, Fig. 4B) treatment. In (A) the reduction ofmolecular size demonstrates almost complete removal of sugars (lane2, the arrow points to secreted molecules partially resistant to EH,likely bearing one complex-type sugar). In (B) reduction of intensity ofthe bands demonstrates the removal of monoester phosphates by AP(lane 2). The following CD forms were visible: P, precursor, LM, largechain of the mature double-chain. A representative gel (out of three) isshown.

M. Démoz et al. / Protein Expression and PuriWcation 45 (2006) 157–167 163

eluates were analyzed by gel electrophoresis followed bysilver staining of the gel and Western blotting. Consider-ing the sensitivity of these methods and on the basis ofthe results shown in Figs. 5A and B the isolated proC-Drec was assumed >90% pure. As to the yield of puriWca-tion, about 1.2 mg of proCDrec could be recovered from1 liter of culture medium of 100 £ 106 infected cells. Asan additional way to identify the recombinant protein asauthentic human proCD we performed a Western blot-ting analysis of the puriWed protein using a polyclonalantiserum raised against a synthetic peptide correspond-ing to the sequence His7 through Gly21 [22], based onthe cDNA sequence [39]. The result shown in Fig. 5CconWrmed the identity of the puriWed protein as humanproCD and, indirectly, proved that the stretch of aminoacids at the N-terminus of proCDrec upstream of theautocatalytic cleavage site was intact.

Like other aspartic proteases (e.g., pepsinogen),proCD undergoes autoactivation at acid pH, i.e., a pep-tide (residues 1–26) at the N-terminus is removed andthe protein acquires enzymatic activity. In vitro thisreaction takes place within a few minutes of exposure ofproCD to pH 3.65, is Pst-sensitive [40], and gives rise tothe so-called ‘pseudo-CD’ [28]. We veriWed the ability ofrecombinant human proCD to undergo pH-dependentautoactivation and to acquire enzymatic activity. To thisend, we assayed over time the activity of recombinantCD at pH 3.65 using aminomethylcoumarin-conjugatedhemoglobin as substrate (see Materials and methods fordetails). PuriWed proCDrec and pseudo-CDrec (the latterwas obtained by a 15 min pre-incubation of proCDrec atacid pH) were incubated with the substrate in theabsence or the presence of Pst and generation of Xuores-

cence was continuously monitored for up to 30 min. Theassay revealed that in samples containing proCDrec

hydrolysis of the substrate and generation of Xuores-cence increased with the time of incubation (Fig. 6).When the assay was performed in the presence of Pst,proCDrec did not acquire enzymatic activity conWrmingthat the activation process requires a proteolytic event

Fig. 6. Recombinant proCD undergoes self-activation at acid pH.PuriWed proCDrec was pre-incubated or not at acid pH for 15 min andthen further incubated at pH 3.65 in the presence of a Xuorochromicsubstrate to evaluate the enzymatic activity. The experiment revealsthat proCDrec acquires enzyme activity upon incubation at acid pH.As expected, CD activity is inhibited in the presence of Pst.

Fig. 5. IdentiWcation of puriWed proCDrec in chromatographic fractions. ProCDrec was puriWed by Pst-aYnity chromatography from the medium ofrecVVpMJ/CD-infected BSC1cells. CD was eluted, and analyzed by SDS–PAGE and silver staining (A) and Western blotting (B) of the gel. Loadingof samples as follow: Std, molecular weight standard proteins; lane 1, aliquot of medium from infected culture prior to puriWcation; lane 2, aliquot ofmedium from infected culture after Pst-aYnity chromatography; lanes 3–6, washes; lanes 7–14, aliquot form elution fractions. Western blotting(in B) showed higher sensitivity than silver staining and revealed the presence of proCDrec also in fractions 12, 13, and 14. In (C) is shown the Westernblotting of puriWed proCDrec (aliquot of the pool of fractions 7–13) performed with a polyclonal antiserum that recognizes the amino acids 7–21 ofthe N-terminus. Representative data of several trials of puriWcation are shown.

164 M. Démoz et al. / Protein Expression and PuriWcation 45 (2006) 157–167

mediated by the protein itself. This experiment demon-strated that at acid pH proCDrec underwent the typicalautoproteolytic removal of the inhibitory peptide asexpected for authentic human CD [28] and conWrmedthat the active site was folded correctly. In a separateexperiment, the time- and acid-dependent conversion ofproCDrec into the pseudo-form was shown to be associ-ated with the reduction in size of approximately 2 kDa,as estimated by SDS-gel electrophoresis (not shown).The kinetics of pseudo-CDrec activity are also shown inFig. 6. It should be noted that pseudo-CDrec showed nolatency and immediately started to hydrolyze the sub-strate. On the contrary, the fact that Pst-aYnity puriWedproCDrec became active only upon incubation at acidpH further indicates that the puriWcation methodallowed to isolate the protein structurally intact, as inde-pendently conWrmed by Western blotting with the anti-body speciWc for the N-terminus (Fig. 5C).

SpeciWcity of the antiserum raised against proCDrec

All the above observations suggest that the recombi-nant human proCD expressed in mammalian cells isstructurally and functionally identical to native humanCD. We Wnally checked whether this molecule could beexploited for the production of antibodies speciWcallydirected toward conformational epitopes present in non-denatured proCD. PuriWed proCDrec was used to immu-nize a rabbit, and the speciWcity of the immune serumtoward human and rodent CD molecular forms, eitherdenatured or non-denatured, was analyzed by Westernblotting, dot-blotting, and immunoXuorescence. HumanCD was ectopically expressed either in rat (RBL) ormouse (L929) cells by transfection with the vectorpcDNA3.1 carrying the speciWc cDNA. The images inFig. 7A clearly show the speciWcity of the antiserumtoward human CD. The upper panels show endogenousCD as revealed by an anti-rat CD antiserum [30] thatcross-reacts also with human and mouse CD. The lowerpanels show immunoXuorescence staining of CD per-formed with the anti-proCDrec antiserum in cultures ofrat (RBL) and of mouse (L929) cells that have been tran-siently transfected with a plasmid encoding human CD.Non-transfected cells in the same Weld do not show posi-tive immunostaining for CD and serve as internal con-trol (Fig. 7A, lower panels). For comparison,immunoXuorescence staining of CD in human ovariancarcionoma (NIHOVCAR3) cells is included. Thus, inmethanol-Wxed cells the anti-rat CD antiserum was ableto reveal the endogenous CD in all cell lines tested,whereas the �proCDrec antiserum speciWcally reactedonly with human, not rat or mouse, CD. It is remarkablethat in denatured cell homogenates analyzed by Westernblotting the anti-proCDrec antiserum recognizes themature form, but not the precursor form, of rat andmouse CD (not shown). We further investigated the

ability of this antiserum to speciWcally recognize epi-topes present in the propiece sequence of proCD byimmunoblotting analysis of serum-free culture mediaunder denaturing or non-denaturing conditions. For thisexperiment, we used the concentrated medium of MCF7cells (which are known to secrete large amounts ofproCD), and the culture medium from L929 wild-type(L929wt) and from a clone of L929 (L92910hCD) stablytransfected and over-expressing human CD. To increaseaccumulation of the protein in the medium, secretion ofproCD was stimulated by incubating the cells for 24 hwith 10 mM ammonium chloride [18]. Denatured mediawere subjected to SDS–PAGE and Western blottinganalysis. The results of this experiment (shown inFig. 7B) conWrm that the anti-proCDrec antiserum pref-erentially reacts with human proCD. The same Wlter wasstripped and re-probed with an anti-rat CD antiserum todemonstrate that mouse proCD was in fact present inthe secretion of ammonium chloride-treated L929 cells(not shown). We also performed a dot-blotting analysisof non-denatured conditioned media directly applied tothe nitrocellulose Wlter (see Materials and methods). Theresults of such analyses (shown in Fig. 7C) demonstratethat the antiserum raised against aYnity-puriWed proC-Drec reacts against non-denatured CD of human origin,but not of mouse origin. This suggests that the antiserumcontains antibodies against conformational epitopespresent only in human proCD.

Discussion

We describe a system for production of large quanti-ties of recombinant human proCD that is normallyfolded (since autoactivable and able to bind to Pst) andnormally glycosylated (since sensitive to both endogly-cosidase H and alkaline phosphatase). This contrastswith the recombinant human proCD that has beenobtained in E. coli [27] and Spodoptera frugiperda cells[22,29]. The former accumulated in inclusion bodiesand was folded ‘in vitro’ with a low yield. The enzymesynthesized in the insect cells folded normally,however, its carbohydrate side chains contained shortnon-phosphorylated oligosaccharides. Peptide process-ing, N-glycosylation, trimming, and phosphorylationof high-mannose-type oligosaccharides in proCDresult from the activity of enzymes that speciWcally rec-ognize a structural feature that is common and uniqueto soluble lysosomal proteins [41]. By employing amodiWed vaccinia virus, we have assessed the produc-tion of recombinant human proCD in mammalian cellsthat provide the convenient cellular environment toensure proper co- and post-translational processing ofectopic pre-proCD. The vaccinia virus expression sys-tem has been previously shown useful for the produc-tion of recombinant lysosomal cathepsin B in

M. Démoz et al. / Protein Expression and PuriWcation 45 (2006) 157–167 165

mammalian cells [42]. Here, we show that this systemhas a high capacity and Wdelity for the production oflarge quantities of human proCDrec with structural andfunctional features of the normally processed precursorbearing uncovered phosphate residues. Infection ofmammalian cells with the recombinant vaccinia viruswas so highly productive that the lysosomal segrega-tion appeared to be saturated, thus favoring secretionof proCDrec. This precursor was released into themedium of recVVpMJ601/CD-infected BSC1 cells inan uncovered phosphorylated form and could be

bound to a Pst–agarose column at acidic pH. After anelution, it was able to undergo a Pst-sensitive auto-acti-vation and it acquired enzymatic activity when incu-bated at an acidic pH. The activation was accompaniedby a reduction in the apparent size of proCDrec byapproximately 2 kDa (not shown), indicating the for-mation of pseudo-cathepsin D and conWrming that theprecursor was correctly folded [17,22,28,40]. This pre-cursor was used to immunize a rabbit. The antiserumraised against Pst-puriWed human proCDrec recognizedthe mature form of denatured CD from rat and mouse

Fig. 7. SpeciWcity of the antiserum raised against proCDrec. The immune antiserum (�proCDrec) raised in a rabbit was tested for its speciWcity towardnon-denatured or denatured CD from rat, mouse, and human cells. (A) ImmunoXuorescence staining of CD in cultures of human ovarian carcinoma(NIHOVCAR3), of rat (RBL), and of mouse (L929) cells. In the upper panels, wild-type cultures were processed for immunoXuorescence stainingusing as primary antibody an anti-rat CD that cross-reacts with both human and mouse CD. In the lower panels, RBL and L929 cells transientlytransfected with a plasmid encoding human CD and wild-type NIHOVCAR3 cells were processed for immunoXuorescence staining with the �proC-Drec polyclonal antiserum. Representative Welds are shown (red color, CD; green color, tubulin). Empty vector transfectants were negative for CDimmunostaining (not shown). (B) Western blotting of concentrated conditioned media from human breast cancer cells (MCF7) or from mouse not-transfected (L929wt) or humanCD-stably transfected (L92910hCD) cells. In parallel samples accumulation of proCD in media was favored by incubat-ing the cells with ammonium chloride (NH4Cl). Equal volume of culture media was loaded. A representative Western blot (out of three) is shown.The experiment reveals that �proCDrec preferentially recognizes human proCD under denaturing conditions. (C) Dot-immunoblotting (top panel)under non-denaturing conditions of media from human and mouse cells (see explanation in B). In this experiment, a twofold volume of culturemedium from L929wt cells was loaded with respect to the volume of culture medium from MCF7 or L92910hCD cells, as indicated by Ponceau redstaining of the Wlter (bottom panel). The experiment (repeated three times in double with analogous results) conWrms the ability of �proCDrec to spe-ciWcally recognize human proCD under non-denaturing condition. ImmunoXuorescence and immunoblot staining with pre-immune serum were neg-ative (not shown). (For interpretation of the references to color in this Wgure legend, the reader is referred to the web version of this paper.)

166 M. Démoz et al. / Protein Expression and PuriWcation 45 (2006) 157–167

species, besides human; this is indeed not surprising,considering the high homology of amino acidsequences among the three CDs [39,43,44]. The antise-rum, however, speciWcally recognized human proCD,but not rat or mouse proCD, regardless if the proteinswere denatured or not. This property is likely linked tosome diVerences in the amino acid sequence of the CDpro-peptides of these species. These observations indi-cate that the proCDrec produced with the vaccinia virusexpression system provides a unique immunogen forthe production of antibodies speciWcally directedtoward conformational epitopes expressed on the pro-sequence of human proCD. In addition, the availabilityof large quantities of proCDrec identical to nativeproCD oVers an opportunity for structural studies ofits 3D conformation, for identiWcation of domainsinteracting with peptides (such as prosaposin) or lipids(such as ceramide) potentially involved in the matura-tion and ‘alternative’ transport of proCD, and for com-prehension of its acid-dependent auto-activation, aswell as for designing drugs inhibiting its active site.

Acknowledgments

This work was supported by grants from the Con-sorzio Interuniversitario per le Biotecnologie (CIB,Trieste, Italy), the Consiglio Nazionale delle Ricerche(target project Biotechnology, contract No.9900386PF.49 to C.I.), the Università del Piemonte Ori-entale, National Institutes of Health (grants CA 36481and CA 56586), the Duetsche Forshungsgemeinschaft(grant Ha 915/7-1).

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