purification and characterization of active recombinant human napsin a

Eur. J. Biochem. 267, 2573±2580 (2000) q FEBS 2000

Purification and characterization of active recombinant human napsin A

Vesna Schauer-Vukasinovic1, Daniel Bur1, Eric Kitas1, Daniel Schlatter1, Gerard RosseÂ 1,*, Hans-Werner Lahm2 andThomas Giller1

1F. Hoffmann-La Roche Ltd, Pharma Division, Preclinical Research, Basel, Switzerland; 2F. Hoffmann-La Roche Ltd, Pharma division,

Roche Genetics, Basel, Switzerland

Recombinant human napsin A expressed in human embryonic kidney 293 cells was purified to homogeneity by a

single-step procedure using part of napsin A propeptide as affinity ligand. N-Terminal amino-acid sequencing of

the purified enzyme identified the mature form of napsin A. Treatment of purified napsin A with

endoglycosidases F and H resulted in a decrease in its molecular mass from 39 kDa to < 37 kDa, confirming

that napsin A is glycosylated. The kinetic properties were analyzed by using two fluorogenic synthetic substrates

K(Dabsyl)-TSLLMAAPQ±Lucifer yellow (DS1) and K(Dabsyl)-TSVLMAAPQ±Lucifer yellow (DS3). The Km

values obtained were 1.7 mm and 6.2 mm, respectively. A substrate-specificity study using a napsin A-targeted

peptide library confirmed the preference of napsin A for hydrophobic residues at positions P1 and P1 0. Adjacent

positions, P2±P4 and P2 0±P4 0, appeared less restricted in distribution of amino acids. A pH optimum between 4.0

and 5.5 at room temperature was determined. The purified enzyme was fully active for more than 10 h at pH 5.0

and 6.0, while a half-life of 4 h was determined at pH 7.0 and 37 8C.

Keywords: aspartic proteinases; fluorescence resonance energy transfer; kinetics; pH optimum; propeptide.

Napsin A is a member of the family of human asparticproteinases, the nucleotide sequence of which has been reportedrecently by Tatnell et al. [1]. This enzyme is localizedpredominantly in kidney and lung [1,2], and this restrictedtissue localization may suggest a distinct physiological functioncompared with other aspartic proteinases such as cathepsin D,cathepsin E, pepsin, gastricsin, and renin.

Full-length sequences of human aspartic proteinases knownso far consist of a signal peptide, followed by a propeptideregion, and a catalytically active mature chain. The main role ofthe signal sequence is to correctly target the proteinase into thesecretory pathway through endoplasmic reticulum [3]. The pro-segment is involved in the folding of the protein and isresponsible for temporary inhibition of the proteinase in itszymogen form. Mature human aspartic proteinases share40±50% sequence identity (an exception is BACE [4] whichshows less than 30% sequence identity with human pepsinfamily members), the length being between 326 (pepsin) and

348 (cathepsin D) amino acids. Mature forms of severalmembers of this family, such as napsin A and napsin B[1], ASP1 and ASP2 (patent number EP848062-A2 andEP855444-A2), the later being identical with BACE [4],contain a C-terminal extension. Despite these similarities,there are significant differences between aspartic proteinaseswith regard to their cellular and subcellular localization as wellas their enzymatic properties. For example, pepsin, gastricsin,and renin are secretory enzymes while cathepsin D andcathepsin E are intracellular enzymes located in lysosomesand endoplasmic reticulum, respectively [5]. The pH optima ofthese enzymes range from very acidic for pepsin and gastricsin(about 2.0) to almost neutral for renin (from 5.3 to 7.0) [6,7].Differences between members of this family can also be seenwith regard to their N-terminal sequences. They share nocommon pattern in the first 10 amino acids of mature enzymeand therefore the exact N-terminal sequence of the matureactive form has to be determined experimentally.

Aspartic proteinases have been extensively studied becauseof their important physiological roles and association withdifferent pathological states [8,9]. As a result, a lot of effort hasbeen made to purify and obtain them in an active form. Severalmethods have been proposed for their purification, rangingfrom ion-exchange and size-exclusion techniques to a variety ofaffinity-chromatography approaches [10±15]. For example,affinity chromatography using immobilized synthetic inhibitorsas affinity ligands has been successfully used for purification ofcathepsin D, pepsin and renin [16,17]. Recently, cathepsin Dwas purified by using part of its propeptide as an affinity ligand[18]. This method is based on the fact that cathepsin D has ahigh affinity for its propeptide at neutral pH but can be releasedon lowering the pH [19].

Here we describe the purification of recombinant maturehuman napsin A by using part of its propeptide. Furthermore,the purified napsin A was characterized with respect to itsN-terminal sequence, glycosylation, and stability. To facilitatestudies of enzyme catalytic properties and substrate specificity,

Correspondence to T. Giller, F. Hoffmann-La Roche Ltd, Pharma Division,

Preclinical Research, Grenzacherstrasse 124, CH-4070 Basel, Switzerland.

Fax: 1 41 61 6882438, Tel.: 1 41 61 6888681,

E-mail: [email protected]

Abbreviations: HEK293 cells, human embryonic kidney 293 cells; EDANS,

5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid; Dabsyl,

4 0-dimethylaminoazobenzene-4 0-sulfonyl; Lucifer yellow,

N-(2-aminoethyl)-4-amino-3,6-disulfo-1,8-naphthalimide; FRET,

fluorescence resonance energy transfer; TPTU, O-(1,2-dihydro-2-oxo-1-

pyridyl)-N, N, N 0, N 0-tetramethyluronium hexafluoroborate; Pbf, 2,2,4,6,7-

pentamethyldihydrobenzofuran-5-sulfonyl; Trt, trityl; DIPEA,

di-isopropylethylamine; HATU, O-(7-azabenzotriazol-1-yl)-N, N, N 0, N 0-tetramethyluronium hexafluorophosphate; HOAt, 7-aza-1-

hydroxybenzotriazol.

Enzyme: napsin A (EC 3.4.23.-).

*Present address: Selectide Corporation, A Subsidiary of Hoechst Marion

Roussel, Inc., 1580 E. Hanley Blvd. Tucson, AR 85737, USA.

(Received 14 February 2000, accepted 2 March 2000)

which are important in the search for specific inhibitors andnatural substrates, it is necessary to have a sensitive assay forenzyme activity. One of the approaches developed for continu-ous monitoring of enzyme activity is to design fluorogenicsubstrates. We have used this strategy using combinatorialpeptide chemistry for enzymatic characterization of humannapsin A.

E X P E R I M E N T A L P R O C E D U R E S

Expression of napsin A in human embryonic kidney 293(HEK293) cells

Expression was performed as described previously [2]. In short,HEK293 cells were transfected with an expression vectorcontaining human napsin A cDNA and carrying the G418resistance gene using Lipofectamin (Life Technologies Inc,Gaithersburg, MD, USA) according to the manufacturer'sprotocol. Cells were grown in a humidified incubator in a 5%CO2 atmosphere at 37 8C. Cell lysates and pellet fractionswere prepared in the presence of 20 mm EDTA, 1 mm phenyl-methanesulfonyl fluoride (Sigma) and 1% Elugent (Calbiochem).

Synthesis of propeptide ligands EK191 and EK193

Both ligands (Fig. 1A) were synthesized using the continuous-flow solid-phase synthesis method on a PioneerTM PeptideSynthesizer, starting from Tenta Gel S RAM resin (3.0 g;0.25 mmol´g21 Rapp Polymere GmbH, TuÈbingen, Germany)

by the published method [20]. The base-labile Fmoc group wasused for a-amino protection. Side chains were protected withthe following protection groups: Arg(Pbf ), His(Trt), Gln(Trt),Asn(Trt) and Thr(But). Fmoc-amino acids or d-biotin (3.0equiv.) were activated with an equivalent amount of 2-[2-oxo-1(2H)-pyridyl]-1,1,3,3-tetramethyluronium tetrafluoroborate(TPTU) [21] and di-isopropylethylamine (DIPEA). Fmocdeprotection was achieved with 20% piperidine in dimethyl-formamide. Fmoc-Thr(But)-Leu-Ile-Arg(Pbf )-Ile-Pro-Leu-His(Trt)-Arg(Pbf )-Val-Gln(Trt)-Pro-Gly-Arg(Pbf )-Arg(Pbf )-Ile-Leu-Asn(Trt)-Leu-amide Tenta Gel S-resin (5.0 g) wasdivided, and the synthesis of EK191 and EK193 was continuedstarting from 2.25 g peptide±resin each. The two peptide±resins were each treated with a mixture (100 mL) of 90%trifluoroacetic acid, 2% ethanediol, 5% water, 3% tri-isopro-pylsilane for 5 h. The reaction mixtures were concentrated andpoured into diethyl ether. The precipitates were collected byfiltration and lyophilized from water/acetonitrile. Crude pep-tides (EK191, 600 mg and EK193, 750 mg) were purified bypreparative RP-HPLC (C18). Both compounds gave correctamino-acid analysis and correct molecular peaks on ion-sprayMS.

Purification of napsin A using propeptide ligands

A volume of 50 mL cell extract (0.4 mg´mL21 total protein)containing napsin A was mixed with 40 mL buffer A (20 mmHepes, 150 mm NaCl, 1% Triton, 10% glycerol, pH 7.4) and20 mg either biotinylated EK191 or EK193 peptide (Fig. 1) for

Fig. 1. Affinity purification of mature

napsin A. (A) Ligands utilized for affinity

purification of the napsin A (see text for details

on synthesis of ligands). (B) Schematic

representation of the purification procedure. The

active napsin A binds to the biotinylated ligand

in a pH-dependent manner (pH 7.5). Solution

containing napsin A±ligand complex is mixed

with streptavidin±agarose beads, resulting in

binding of napsin A to the beads through a

biotin±streptavidin interaction. All other cellular

proteins are washed away. Elution of active

napsin A is achieved under acidic conditions,

pH 3.7.

2574 V. Schauer-Vukasinovic et al. (Eur. J. Biochem. 267) q FEBS 2000

3 h at room temperature. After the incubation, 100 mLstreptavidin±agarose beads (0.2±0.3 mg streptavidin; LifeTechnologies) previously washed in buffer A were added tothe reaction mixture and incubated for 1 h at room temperature.Unbound proteins were removed by washing with 6 � 1 mLbuffer B (20 mm Hepes, 150 mm NaCl, 0.1% Triton, 10%glycerol, pH 7.4) and bound napsin A was eluted with5 � 40 mL buffer C (0.1 m sodium formate, 0.1% NonidetP40, 10% glycerol, pH 3.75). All fractions were analyzed bySDS/PAGE and Western blot. The fractions containing purifiednapsin A [< 16 mg as estimated using calculated A280

(1 mg´mL21) � 1.34] were pooled and the enzyme was storedin elution buffer (buffer C) at 4 8C. Under these conditions,purified enzyme was stable for more than 6 months.

Electrophoresis and Western-blot analysis

SDS/PAGE was by the method of Laemmli [22]. The detailedexperimental procedure for SDS/PAGE and Western-blotanalysis including the generation of anti-(napsin A) IgG hasbeen described elsewhere [2]. To determine N-linked glycosy-lation of recombinant napsin A, purified protein was incubatedfor 1 h at pH 5.5 with endoglycosidases F and H (Calbiochem-Novabiochem, La Jolla, CA, USA) as recommended by themanufacturer, and subsequently analyzed by Western blot.

N-Terminal amino-acid sequencing

The protein sample was concentrated and desalted on apoly(vinylidene difluoride) membrane (Prosorb; PE-Biosystems,Foster City, CA, USA) and washed with 200 mL water. Themembrane was transferred to a protein-sequencing system(Procise 494HT; PE-Biosystems), and the first 10 residues weredetermined by phenylthiohydantoin-amino-acid analysis, yield-ing a double sequence at a 1-pmol level.

Synthesis of fluorescence resonance energy transfer(FRET)-based substrates DS1, DS3, S1, S2 and EKN

The fluorogenic substrates K(Dabsyl)-TSLLMAAPQ±Luciferyellow (DS1), K(Dabsyl)-TSVLMAAPQ±Lucifer yellow(DS3), K(Dabsyl)-PQFFTEQ±Lucifer yellow (S1), K(Dab-syl)-PQLFTEQ±Lucifer yellow (S2) and K(Dabsyl)-KPKLG-APSPGDKDK-(EDANS) (EKN) were synthesized usingstandard solid-phase synthesis and purification procedures, asdescribed previously. They were dissolved in dimethylsulfoxide at 10 mm and stored at 4 8C until use.

Synthesis of napsin A-targeted peptide library

The FRET substrate combinatorial library was synthesized onPEGA1900 [copolymer of poly(ethylene glycol) with molecularmass of 1900 and acrylamide] resin via split synthesisfollowing standard Fmoc chemistry. First, Fmoc-Gly-OH andFmoc-Glu±Lucifer yellow were coupled successively to theamino PEGA1900 resin using O-(7-azabenzotriazol-1-yl)-N, N, N 0, N 0-tetramethyluronium hexafluorophosphate (HATU)/7-aza-1-hydroxybenzotriazol (HOAt) [23] in the presence ofDIPEA in N-methylpyrrolidone. Synthesis was performed on asemiautomated shaking-vessel machine. Then the resin wastransferred in a Manual Multiple/Library Synthesizer (MULTI-BLOCKw) and split into 19 portions. A different and singleFmoc-protected amino acid was coupled to each of the 19portions using the HATU/HOAt method in N-methylpyrrol-idone. Double coupling was performed using TPTU [21] in the

presence of DIPEA in N-methylpyrrolidone. Then the portionswere mixed together and washed thoroughly. After Fmocdeprotection the library was split again and the synthesis wasrepeated on the MULTIBLOCK to obtain Fmoc-X1-X2-L/F-M/F/Y-X3-X4-E(G-PEGA1900 resin)±Lucifer yellow. Finally, allthe portions were mixed together in a large reaction vessel, andBoc-Lys(Dabsyl)-OH was successively coupled on the semi-automated shaking-vessel machine using the HATU/HOAt andTPTU method. Before the enzymatic assay, side-chain-protect-ing groups of the desired library portion were removed withtrifluoroacetic acid/water/tri-isopropylsilane (95 : 3: 2, by vol.)for 2 h.

Screening of the napsin A-targeted peptide library

A part of the library (< 25 000 beads) was washed with100 mm sodium phosphate buffer, pH 7.0, containing 20 mmEDTA, 0.01% Nonidet P40, and 1% glycerol. Then 20 mL ofthe purified napsin A was added to the resin in a final volume of4 mL in the reaction buffer. After shaking for 18 h at roomtemperature, the resin was filtered off and the beads wereextensively washed with the incubation buffer and 100 mm Mesbuffer, pH 4.8. The first selection of 300 fluorescent beadsfound under the fluorescence microscope (Leica MZ12 equippedwith Leica 2 videosystem) were isolated and incubated a secondtime under the same conditions. Finally, 26 beads were isolatedand submitted to Edman sequencing (Procise 494HT sequencer).

Determination of kinetic parameters

The cleavage of the two fluorogenic substrates, DS1 and DS3,was monitored by the increase in fluorescence per minute usinga Fluostar (BMG LabTechnologies, Offenburg, Germany) and96-well plates (Dynex Technologies Inc, Chantilly, VA, USA)at room temperature. The reaction was performed in reactionbuffer containing 100 mm sodium acetate and 20 mm EDTA,pH 4.7. Typically, 30 mL reaction buffer was mixed with10 mL napsin A solution (diluted 1 : 50 in reaction buffer), andthe reaction was initiated by the addition of 10 mL substrate.The final substrate concentration was in the range 0.5±16 mm.The increase in fluorescence using excitation at 390 nm andemission at 538 nm was recorded as a function of time. Theinitial velocity defined as fluorescence intensity per unit time(Vt) was determined from the slope during the linear phase ofcleavage, normally 5±8 min. Initial velocities were convertedfrom fluorescence units (Vt) to concentration per unit time (vc)using the formula:

vc � V t�S�0/�I100 2 I0�where [S]0 is initial substrate concentration, and I0 and I100

are the fluorescence intensities at time 0 s and after 100%hydrolysis. The Km and Vmax values were derived from theLineweaver±Burk plot. Cleavage of the fluorogenic substrateswas examined by HPLC analysis (data not shown). MS analysisconfirmed that both substrates were cleaved at the Leu±Metbond. Inhibition experiments were performed at DS3 concen-trations 1 mm, 5 mm and 10 mm in the pH 4.7 assay buffer at25 8C. Pepstatin was diluted into the assay buffer just beforerate measurements were made with final concentrations of 6.2,12.5, 25.0 and 50.0 nm.

pH optimum and pH stability

The pH optimum of purified napsin A was determined assum-ing first-order kinetics using a substrate DS3 concentration far

q FEBS 2000 Properties of active recombinant napsin A (Eur. J. Biochem. 267) 2575

below the Km (0.5 mm). The assay was carried out at roomtemperature at pH ranging from 3.0 to 5.5 (100 mm sodiumacetate/20 mm EDTA) and 6.0±8.0 (100 mm sodium phos-phate/20 mm EDTA). Recombinant human napsin A was mixedwith the fluorogenic substrate at the appropriate pH, and theinitial rates of substrate hydrolysis were monitored as describedabove. The pH stability of active napsin A was determined in100 mm sodium acetate buffer, pH 5.0, and 100 mm sodiumphosphate buffer, pH 6.0 and 7.0, containing 20 mm EDTA.The enzyme was incubated in the appropriate buffer at 37 8C.At predetermined time points, samples were removed from theindividual incubation mixtures, and the activity of napsin A wasmeasured using the fluorogenic substrate assay as described above.

R E S U L T S A N D D I S C U S S I O N

Purification of napsin A

We have recently reported the expression of recombinanthuman napsin A in HEK293 cells [2]. It was found by Western-blot analysis that most of the napsin A was associated with thecell pellet, and only detergent treatment of the pellet resulted inefficient extraction of the enzyme into solution. Such apreparation was used as the starting material for the purificationof napsin A. The purification method described in this article isbased on the affinity of mature napsin A for its propeptide atneutral pH. This affinity binding is pH-dependent and can bedisrupted by lowering the pH, resulting in release of maturenapsin A in a pure form (Fig. 1B).

The first important issue was to determine the length of thepropeptide necessary for efficient binding of the enzyme.Wittlin et al. [18] reported that the first 25 amino acids of thecathepsin D 44-residue propeptide are sufficient for interactionwith mature or pseudo-cathepsin D. Recently the structure ofmature gastricsin with the N-terminal part of its propeptide waspublished [24]. The propeptide of napsin A consists of < 40amino acids as deduced from the primary sequence [1]. Basedon the analogy with cathepsin D, a tentative proN fragment fornapsin A was selected (TLIRIPLHRVQPGRRILNL) as anaffinity ligand. For purification studies, the 19-residue proNpeptide was synthesized and biotinylated at its N-terminus.Between the peptide and the biotin two spacers of differentlength, C14 and C14-(b-Ala)7, were inserted to yield the twoaffinity ligands EK191 and EK193, respectively (Fig. 1A).These two spacers were used to establish the optimal distancebetween biotin and the propeptide. After studying the structureof gastricsin with bound propeptide [24], we assumed thatbiotinylation of the proN peptide via a spacer would notinterfere with its binding to mature napsin A. The biotin moietyshould extend significantly from napsin A after binding of thepropeptide fragment to the mature enzyme. This should allowsmooth interaction between the biotin and streptavidin±agarose,the latter being used as the solid phase. Our experimentsshowed that both ligands successfully bound to napsin A, as thefinal elution with buffer C (pH 3.75) resulted in fractionscontaining . 95% pure napsin A, as judged by SDS/PAGE andWestern blot (Fig. 2; representative data using EK193 areshown). No napsin was detected in fractions eluted with bufferB. The band corresponding to purified napsin A has a molecularmass of < 39 kDa, which is in good agreement with ourpreviously reported value for the molecular mass of recombi-nant napsin A [2]. Interestingly, binding was abolished whenthe propeptide was first bound to streptavidin and thenincubated with cell extract containing napsin A. This is inagreement with the observations of Wittlin et al. [18], who

reported that preformation of cathepsin D propeptide±streptavidin complex dramatically decreased the binding capa-city of propeptide for cathepsin D. Although steric hindrance bystreptavidin was suggested as a possible explanation, this doesnot seem likely as no difference in binding was observed whena biotin spacer significantly longer than the C14 chain was used.Studies aimed to solve this problem and use the proposedmethod for large-scale purification of napsin A are currentlyunder investigation in our laboratory.

It should be added that the classical purification methodusing pepstatin affinity chromatography (published procedure:patent WO 98/22597) was applied but was not successful.Although napsin A did bind to the solid support under acidicconditions, efficient elution of the enzyme could not be achieved,even at pH 9.5.

N-Terminal sequence analysis

A fraction containing purified napsin A was used for N-terminalsequence analysis as described above. Figure 3 shows the

Fig. 2. SDS/PAGE and Western-blot analysis of the purification of the

mature napsin A. (A) SDS/PAGE (4±20% Tris/glycine) stained with

silver. (B) Western-blot analyses using a polyclonal rabbit anti-(napsin A)

IgG and a goat anti-rabbit antibody conjugated to horseradish peroxidase.

Lanes 1, standard proteins with molecular masses given in kDa; lanes 2, cell

extract from which mature napsin A was purified; lanes 3, pooled fractions

eluted with buffer B (20 mm Hepes, 150 mm NaCl, 0.1% Triton, 10%

glycerol, pH 7.4); lanes 4, eluate from streptavidin±agarose beads

containing homogeneous mature napsin A (< 39 kDa).


complete amino-acid sequence of human prepro-napsin A.Sequence deduced from the N-terminal protein determinationof purified human napsin A is highlighted in blue. The matureenzyme starts with Ser60, which corresponds to position 40 ofthe pig pepsinogen pro-segment [1]. This is close to the positionof activation cleavage sites found in other aspartic proteinasezymogens [25,26]. Therefore, the isolated protein representsmature enzyme in which the tentative signal peptide and thepropeptide (Fig. 3) have been removed. The mechanism of thisprocessing and maturation is currently under investigation inour laboratory. N-Terminal sequencing also detected a weakersignal corresponding to the sequence VXLXLXGFQ (X corre-sponds to amino acids not reliably determined). This sequencecan be assigned to the peptide VRLCLSGFQ found at position351 (equivalent to position 281b using pig pepsin numbering[1]). The calculated molecular mass of this C-terminal peptidewould correspond to < 8 kDa. It seems likely that this peptide,which cannot be observed on SDS/PAGE, represents a minordegradation product of purified enzyme. Detection by Westernblot was not possible as the epitope recognized by our anti-(napsin A) IgG is not located within the sequence of thispeptide (Fig. 3, red box).

Successful expression and purification of mature napsin Asuggest that pronapsin A is correctly processed to its matureform in HEK293 cells. In general, three mechanisms have beenreported for in vitro activation of aspartic proteinases. The firstmechanism, complete autoactivation, has been found for porcinepepsinogen [26]. The second mechanism is represented by fullyassisted activation of prorenin [27]. The third mechanism,described for cathepsin D, is a combination of partial auto-activation and enzyme-assisted activation yielding matureenzyme [28]. To test whether or not autoproteolysis is involvedin maturation of napsin A, a peptide K(Dabsyl)-KPKLGAP-SPGDKDK-(EDANS) (EKN) was designed and used as a

model substrate for napsin A. This model peptide comprisesamino acids found at the N-terminus of mature napsin A(underlined) preceded by six amino acids upstream of thissequence (bold). The assumption was that, if napsin A cancompletely remove its propeptide from zymogen to yieldmature enzyme, then this model peptide (EKN) should becleaved by mature napsin A between proline and serine. Such acleavage should increase the fluorescence signal. However, nocleavage of EKN was observed, indicating that napsin A isprobably not involved in the final autoactivation step. Althoughit can be argued that the whole protein may be needed forsuccessful cleavage, this does not seem probable consideringthat cleavage would need to take place between proline andserine. It seems more likely that another peptidase activitypresent in HEK293 cells carries out this process.

Deglycosylation of napsin A

Treatment of purified napsin A with endoglycosidases F and Hresulted in a decrease in molecular mass from 39 kDa to< 37 kDa (Fig. 4), the later corresponding to the theoreticallycalculated molecular mass of non-glycosylated mature napsinA. Endoglycosidases F and H selectively release N-linkedoligosaccharides such as high-mannose moieties from glyco-proteins. Therefore, this carbohydrate is attached to napsin A(similar results have been reported for cathepsin D [29]). A

Fig. 3. Deduced amino-acid sequence of human prepro-napsin A.

Numbers on the left indicate amino-acid position. The putative signal

peptide followed by the propeptide is highlighted in yellow. Sequences

deduced from the N-terminal sequence analysis are highlighted in blue.

An asterisk denotes the beginning of mature napsin A. The potential

N-glycosylation sites are underlined. The epitope sequence recognized by

the anti-(napsin A) IgG is highlighted in red.

Fig. 4. Deglycosylation of recombinant human napsin A. Purified

mature napsin A was treated with endoglycosidases H and F. Western

blot of endoH/F-treated sample using anti-(napsin A) IgG revealed two

bands corresponding to glycosylated (< 39 kDa, arrowhead) and deglyco-

sylated (< 37 kDa) mature napsin A (lane 2). A decrease in mass of the

mature enzyme indicates that napsin A is N-glycosylated. Lane 3 contains

purified napsin A. Lanes 4 and 5 contain endoglycosidase H and F,

respectively. The band of molecular mass < 34 kDa appearing in lanes 2

and 4 is the result of a non-specific interaction between antibody and

endoglycosidase H. The molecular-mass standards are shown in lanes 1 and 6.

Table 1. Kinetic parameters for the hydrolysis of fluorogenic substrates. Where applicable, values are mean ^ SD (n � 3). An asterisk denotes cleavage

site between Leu and Met, as confirmed by HPLC and MS analysis.

Substrate Km (mm) Vmax (mm´min21) Vmax/Km (min21)

K(Dabsyl)-TSLL*MAAPQ-Lucifer yellow 1.70 �^ 0.18 0.020 �^ 0.004 0�.01

K(Dabsyl)-TSVL*MAAPQ-Lucifer yellow 6.20 �^ 0.70 0.20 �^ 0.02 0�.03


second band at < 34 kDa results from non-specific interactionsbetween the immunoreactive serum and endoglycosidase H(Fig. 4, lane 4). Human napsin A has three putative N-linkedoligosaccharide attachment sites as deduced from the amino-acid sequence (Fig. 3). The first glycosylation site, Asn90(Asn26 in pig pepsinogen), is in an identical position with thatfound in cathepsin E [30]. The second site, Asn133 (Asn67in pig pepsinogen), is also present in human cathepsin Dand renin. The third putative site, Asn336 (Asn268 in pig

pepsinogen), however, is unique to napsin A. It can be assumedthat napsin A is glycosylated in vivo as it contains a putativesignal peptide, which could be involved in translocation tothe endoplasmic reticulum via the signal-recognition particleroute. This route is also responsible for processing of theN-glycosylation sites.

Kinetic and substrate-specificity studies

To identify substrates that could be cleaved by napsin A, wescreened a readily available in-house combinatorial peptidelibrary using a FRET-based approach. By this method, twocleavable substrates, K(Dabsyl)-TSLLMAAPQ±Lucifer yellow(DS1) and K(Dabsyl)-TSVLMAAPQ±Lucifer yellow (DS3),containing a fluorescent donor, Lucifer yellow, and a quenchingacceptor, Dabsyl, have been identified. In our assay, activenapsin A cleaved both peptide substrates between Leu and Met(as confirmed by HPLC and MS analysis; data not shown),thereby releasing the fluorophore (Lucifer yellow) from itsquencher Dabsyl. Kinetic parameters for cleavage of the twosubstrates are presented in Table 1. Analysis of cleavage sites inthese two as well as several other peptide substrates from thelibrary (data not shown) revealed that napsin A has a preferencefor hydrophobic residues at positions P1 and P1 0.

To characterize further the substrate specificity of napsin Aat and around cleavage site, a specific peptide librarygenerally described as K(Dabsyl)X1-X2-L/F-M/F/Y-X3-X4-E(G-PEGA1900-resin)±Lucifer yellow was designed. Nineteennatural l-amino acids were chosen for positions X1, X2, X3 andX4 in the library. Cys was not included because of possibleoxidation and disulfide-bond formation. Position P1 wasoccupied by Leu or Phe, while position P1 0 included Met,Phe or Tyr. Our intention was to direct the site of cleavagebetween these fixed positions containing hydrophobic residues,and analyze the preference of napsin A for a hydrophobic pairand the influence of adjacent positions on substrate cleavage.The theoretical complexity of the library was therefore19 � 19 � 2 � 3 � 19 � 19 � 781 926 individual peptides.A subset of the peptide library representing < 25 000 beadswas incubated with the purified recombinant napsin A as above.A total of 26 bright fluorescent beads were discovered and

Table 2. Distribution of the amino acids at different positions relative to

the cleavage sites on the substrates of napsin A. The numbers were

calculated based on the sequences of 26 bead-bound FRET peptides

processed between P1 and P1 0 at the directed Leu/Phe±Met/Phe/ Tyr

cleavage site.

Amino

Position relative to cleavage site

acid P3 P2 P1 P1 0 P2 0 P3 0

A 1 1

D 1 1 4

E 1 6 3 7

F 2 8 23 1

G

H 1 1

I 4 2 2 1

K 1

L 2 18 2 1

M 2 1 3

N 1 1 2 2

P 10 2

Q 1 7 3 1

R 1 4 1

S 3 1 1

T 1 2 4 2

V 1 1 1

W

Y 1 2

Total 26 26 26 26 26 26

Fig. 5. Inhibition of napsin A by pepstatin.

Fluorescence measurements were carried out in

the pH 4.7 assay buffer at 25 8C. Rates were

derived from the initial 5±8 min of the reaction.

Each data point represents the mean of three

velocity (V) determinations; the SD of the

measured rates was always less than 10%.

Pepstatin was diluted into the assay buffer just

before rate measurements were made. The data

were taken at three concentrations of substrate

DS3 (indicated on plot) and are shown in the

form of a Dixon plot; the point of intersection

yields Ki 2 nm. Inset: Cornish±Bowden plot

[36].


collected (Table 2). Edman sequencing analysis showed thatLeu and Phe are the best tolerated at position P1, although Leuis the better preferred. Phe is found almost exclusively inposition P1 0 under our experimental conditions. P2, P2 0 and P3 0seem to tolerate a broad variety of residues, while in subsite P3,Pro is the most prominent amino acid in the bead-boundsubstrate. We infer from the distribution of the amino acids that,similarly to cathepsin D, napsin A is cleaved primarily at theposition before the Phe in peptide substrates. However, incontrast with the dominant cleavage between a doublet of Pheresidues [31], napsin A seems to favour Leu at P1. To confirmthis observation further, two peptides, K(Dabsyl)-PQFFTEQ±Lucifer yellow (S1) and K(Dabsyl)-PQLFTEQ±Lucifer yellow(S2), were synthesized. The sequences of the two peptidesmirror the amino-acid preference of napsin A, as deduced fromTable 2. On the basis of HPLC and MS analysis (data notshown), both substrates were cleaved by napsin A in solutionbetween Phe-Phe and Leu-Phe, respectively. The low solubilityof both substrates prevented measurement of Km and kcat,respectively. However, based on initial velocities at low sub-strate concentrations, it can be concluded that S2 is cleavedmore efficiently than S1. In view of these data, napsin A is aregular member of the aspartic proteinase family, as a similarpreference for cleavage between hydrophobic residues has beenreported for cathepsin D, cathepsin E and renin [32±34].

The activity of napsin A was inhibited by pepstatin, a generalaspartic proteinase inhibitor [35], with an inhibition constant(Ki) of 2 nm (Fig. 5). When the data were plotted by the methodof Cornish-Bowden [36] (Fig. 5, inset), parallel lines wereobtained, showing that pepstatin is a competitive inhibitor. Itshould be added that the absence of N-linked oligosaccharidesdid not alter the catalytic properties of the enzyme.

pH optimum and pH stability

The pH optimum of purified napsin A has been determined tobe between 4.0 and 5.5, as can be seen from Fig. 6. A rapiddecrease in activity was measured at more acidic conditions,while more basic conditions are better tolerated, 40% activitybeing left at pH 7.0.

The pH-stability study was performed at pH 5.0 (lysosomalpH), pH 6.0 and pH 7.0 (cytosolic pH). Napsin A was fullyactive at pH 5.0 and 6.0 for more than 10 h as judged byretention of catalytic activity. The stability at pH 7.0 was lower,with a half-life of < 4 h. This is significantly different for

cathepsin D, which is known to be stable but inactive at pH 7.0[29]. In contrast, human renin, which circulates in plasma,shows a broad plateau of stability between pH 6.0 and 10.0with a half-life longer than 20 min [37]. These data indicatethat napsin A may function in acidic as well as neutral com-partments. However, at present there is not enough evidence toconfirm that these assumptions are valid in vivo.

Conclusions

To gain a better understanding of the properties of humannapsin A, we purified and characterized it. Recombinant humannapsin A has been successfully expressed in HEK293 cells andpurified to greater than 95% homogeneity when part of itspropeptide was used as an affinity ligand. Noting the complex-ity of the lysate from which napsin A was isolated, this resultshows that our affinity ligand specifically recognizes thisenzyme. Furthermore, this method provides the means forexclusive isolation of < 39-kDa mature napsin A without itsproform (the propeptide of mature enzyme is removed, which isa common feature of active aspartic proteinases [28,30]). Ourstudy using a limited napsin-targeted peptide library shows thatnapsin A prefers Leu and Phe at P1 and P1 0, respectively, andthat adjacent positions do not seem to be restrictive inaccommodating a variety of amino acids. Purified napsin Awas characterized with regard to pH optimum and pH stability.Taken together, these studies show that napsin A shares simi-larity with lysosomal proteinases (pH optimum 4.7). However,the long half-life at pH 7.0 associated with stable activitysuggests the possibility of a non-lysosomal localization. Thelatter assumption is in accord with results from immunohisto-chemical studies using healthy kidney tissue, which indicatedthe localization of napsin A to be disperse and possiblymembrane associated [2]. Further studies on napsin A shouldclarify its subcellular localization. At present, the physiologicalrole of napsin A is not known. Restricted localization in thekidney and lung may suggest a function different from`housekeeping'. Recently, Chuman et al. [38] reported thatnapsin A may have a role as a marker for primary lungadenocarcinoma and in processing of pro-surfactant proteins.Obviously, further clarification of the physiological andpathological relevance of napsin A is important.

A C K N O W L E D G E M E N T S

We would like to thank Rana Gardiner for excellent technical assistance.

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purification and characterization of active recombinant human napsin a

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