SmCB2, a novel tegumental cathepsin B from adult Schistosoma mansoni

SmCB2, a novel tegumental cathepsin B from adult Schistosomamansoni�

Conor R. Caffrey a,*, Jason P. Salter a, Kimberley D. Lucas a, Dustin Khiem a,Ivy Hsieh a, Kee-Chong Lim a, Andreas Ruppel b, James H. McKerrow a,

Mohammed Sajid a

a Tropical Disease Research Unit, Department of Pathology, Box 0511, University of California San Francisco, San Francisco, CA 94143, USAb Abteilung fur Tropenhygiene und offentliches Gesundheitswesen, Hygiene Institut, Ruprecht-Karls-Universitat Heidelberg, Im Neuenheimer Feld 324,

Heidelberg 69120, Germany

Received 17 October 2001; received in revised form 22 January 2002; accepted 25 January 2002

Abstract

Papain-like cysteine endopeptidases have been recognized as potential targets for chemotherapy and serodiagnostic reagents in

infections with the human parasitic helminth Schistosoma . A novel cathepsin B endopeptidase from adult S. mansoni has been

isolated and characterized. The enzyme is termed SmCB2 to distinguish it from the first recorded schistosome cathepsin B, SmCB1,

also known as Sm31. A rapid and convenient protocol involving anion exchange and affinity chromatography is described for the

isolation of SmCB1 and SmCB2 from the same parasite starting material. SmCB2 has been functionally expressed in and purified

from Pichia pastoris . Both native and recombinant SmCB2 migrate similarly (33 kDa) by SDS-PAGE. Both display strict acidic pH

activity profiles and similar Km and kcat for dipeptidyl amidomethylcoumarin substrates. We conclude that the recombinant enzyme

is properly folded. The S2 subsite specificity of recombinant SmCB2 exhibits the preferences Phe�/Leu�/Val�/Arg. By

immunoblotting with anti-SmCB2 IgG, a 33 kDa protein was identified in soluble extracts of male schistosomes. By

immunohistochemistry, SmCB2 was localized in the tegumental tubercles and parenchyma of males with less product being

visualized in the parenchyma of females. The enzyme may be lysosomal and function at the host parasite-interface. # 2002 Elsevier

Science B.V. All rights reserved.

Keywords: Schistosoma ; Cysteine endopeptidase; SmCB2; Recombinant expression; Chemotherapy; Diagnosis

1. Introduction

Helminth endoparasites of the genus Schistosoma are

a major public health concern in 74 tropical and sub-

tropical countries. Over 200 million people are infected

with 20 million people being severely debilitated by the

disease schistosomiasis [1]. Adult worms live in the

mesenteric veins and portal vein leading to the liver and

feed on blood. Schistosomiasis is associated with severe

morbidity mainly arising from immunopathological

reactions to parasite eggs in various tissues, particularly

in the liver, intestinal tract and/or bladder.

The characterization and isolation of schistosome

proteolytic enzymes (peptidases) is driven in large part

by their potential as chemotherapeutic targets. Indeed,

targeting peptidases with small-molecule inhibitors has

been shown to inhibit skin penetration by larval

schistosomes [2], and decrease worm burden and egg

production by adult Schistosoma [3]. In addition,

schistosome endopeptidases have a demonstrated value

as selective diagnostic markers of disease [4�/6].

Most studies concerning peptidases of the adult

schistosome have dealt with cysteine endopeptidases

Abbreviations: AEC, aminoethyl carbazole; BCIP, 5-bromo-4-

chloro-3-indolyl-1-phosphate; EST, expressed sequence tag; NBT,

nitro blue tetrazolium; NMec, 7-amido-4-methylcoumarin; PVDF,

polyvinylidene difluoride; TBS-T, Tris-buffered saline-Tween 20

solution; YPD, yeast extract-peptone-dextrose; Z, benzyloxycarbonyl.�

Note: Nucleotide sequence data reported in this paper have been

submitted to the GenBankTM, EMBL and DDBJ databases with the

accession number AJ312106.

* Corresponding author. Tel.: �1-415-514-3052; fax: �1-415-514-

3165.

E-mail address: [email protected] (C.R. Caffrey).

Molecular & Biochemical Parasitology 121 (2002) 49�/61

www.parasitology-online.com

0166-6851/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved.

PII: S 0 1 6 6 - 6 8 5 1 ( 0 2 ) 0 0 0 2 2 - 1

mailto:[email protected]

related to papain (Clan CA, family C1). Genes encoding

a cathepsin B (aka Sm31) [7], two cathepsin Ls (SmCL1

and SmCL2) [8,9], and an exopeptidase, cathepsin C

[10], have been characterized. These enzymes have been

functionally expressed in different recombinant systems

[11�/15]. The open reading frames (ORF) of all of the

above cysteine peptidases encode pre-pro-proteins ana-

logous to their respective mammalian orthologs and

homology modeling has been applied to predict three

dimensional structures [16�/18].

Perhaps the best clues to a biological function of these

peptidases have come from immunolocalization experi-

ments with specific antibodies. Thus, Sm31 [19], SmCL1

[14,20] and SmCL2 [20], being localized in the schisto-

some gut, are considered to act as alimentary endopep-

tidases. A function in schistosome reproduction has also

been proposed for SmCL2 due to its presence in the

female reproductive tract [9]. Finally, a cathepsin C-like

activity has also been localized in the gut by fluorescent

histochemistry [21].

In this report, we have characterized a cDNA

sequence encoding a novel cathepsin B endopeptidase,

termed SmCB2. For the sake of clarity and the establish-

ment of a systematic nomenclature, we rename Sm31,

the first cathepsin B gene described [7], as SmCB1. This

nomenclature is comparable, therefore, with that used

for the schistosome cathepsin L endopeptidases, SmCL1

and SmCL2. We describe a technique to simultaneously

isolate SmCB1 and SmCB2 from parasite extracts.

Further, SmCB2 was functionally expressed in Pichia

pastoris and isolated, and mono-specific antibodies

raised to localize the endopeptidase in adult parasite

tissues. Interestingly, the enzyme was preferentially

expressed in male worms and was localized in the

tegument in addition to the parenchyma. Both native

and recombinant SmCB2, along with native SmCB1 and

mammalian cathepsins B and L, were compared and

contrasted in their respective abilities to degrade a range

of dipeptidyl substrates.

2. Materials and methods

2.1. Parasites

S. mansoni (Puerto Rican isolate) was maintained by

cycling through the vector snail Biomphalaria glabrata

and NMRI mice or Syrian Golden hamsters. Adult

worms were obtained by portal perfusion of mice.

Specific techniques for the maintenance of the schisto-

some life cycle and collection of parasites have been

described previously [22].

2.2. Purification of SmCB1 and SmCB2 from parasite

extracts

Adult S. mansoni worms (400�/600 pairs) weresonicated on ice in 2�/3 ml 20 mM sodium phosphate

buffer, pH 6.0 and the homogenate centrifuged for 20

min at 10 000 g . The supernatant (soluble worm extract)

was sterile-filtered (0.2 mm; Millipore, Bedford, MA)

and then cycled three times over a 3 ml bed-volume of

DE-52 anion exchanger (Whatman, Maidstone, UK)

that had been pre-equilibrated with the same buffer. The

material not adsorbed to the column (wash fraction) andcontaining SmCB2 was cycled three times over a second

3 ml bed-volume of DE-52 pre-equilibrated with the

same buffer, and then made 2 mM with respect to DTT

(Sigma, St. Louis, MO). The wash fraction was mixed

end-over-end with a 6.5 ml slurry of Ahx-Sepharose 4B

(Sigma) that had been coupled to the reversible cathe-

psin B inhibitor H-Gly-Phe-Gly-semicarbazone (Ba-

chem, Torrance, CA) as described previously [23]. Theaffinity matrix had been pre-equilibrated with 50 mM

tri-sodium citrate, pH 5.0, 1 mM di-sodium-EDTA, 2

mM DTT. Isolation of SmCB2 was then continued as

described previously for the cathepsin B-like enzyme of

Schistosoma japonicum (SjCB1) [24].

In contrast to SmCB2, SmCB1 is adsorbed to the first

DE-52 column under the conditions used. After removal

of the wash fraction, the column was washed with a 10-fold bed-volume of equilibration buffer containing 100

mM NaCl. Then, material containing SmCB1 was

eluted with equilibration buffer containing 300 mM

NaCl. The eluate, usually 6�/8 ml, was concentrated to

1�/2 ml by ultrafiltration over a YM10 membrane

(Millipore). The solution was then made 2 mM with

respect to DTT and mixed with a 3.5 ml slurry of the

cathepsin B-affinity matrix for isolation of SmCB1 aspreviously described [24]. For both SmCB1 and SmCB2,

purification was monitored by SDS-PAGE [25] through

12.5% polyacrylamide gels and silver staining [26].

Endopeptidase activity was monitored by hydrolysis of

the fluorogenic peptidyl substrate benzyloxycarbonyl-

phenylalanyl-arginine-7-amido-4-methylcoumarin [27]

(see Section 2.10). Protein concentrations were deter-

mined with the micro-adaptation of the procedure ofBradford [28].

2.3. N-terminal sequencing

Purified native SmCB1, SmCB2 or recombinant

SmCB2 (see Section 2.6) was subjected to SDS-PAGE

and transferred to PVDF membranes (Immobilon-P;

Millipore) for 45 min at 25 V using the procedures and

apparatus supplied by Invitrogen. Protein bands werevisualized in 0.1% Ponceau S (Sigma) and destained in

water. Sequencing was performed using the Edman

degradation technique by Dr. Armin Bosserhoff of the

C.R. Caffrey et al. / Molecular & Biochemical Parasitology 121 (2002) 49�/6150

ZMBH, Ruprecht-Karls Universitat, Heidelberg and

Dr. Ralph Reid at the UCSF Biomolecular Resource

Center.

2.4. Sequencing of SmCB2 cDNA

Poly A�/mRNA was isolated (Amersham Pharmacia

Biotech Inc., Piscataway, NJ) from 6-week-old S.

mansoni worms of mixed sex and converted to single-

stranded cDNA for subsequent polymerase chain reac-

tions (PCR) using AMV Reverse Transcriptase as

described by the manufacturer (Life Technologies,

Inc., Rockville, MD).To obtain sufficient SmCB2 cDNA for sequencing, a

semi-nested PCR was performed using two degenerate

forward primers derived from the N-terminal sequence

of native SmCB2 (see Fig. 2). The outer and inner

forward primers were 5?-CCNAARTCNTTYGAYGC-

NAG-3? and 5?-GAYGCNAGNGTNGARTGGCC-3?,respectively, and the reverse primer was a (dT)18

oligonucleotide (Roche Molecular Biochemicals, India-napolis, IN). The first PCR (100 ml final volume)

contained 50 ng single-stranded cDNA, 10 mM Tris�/

HCl, pH 8.85, 25 mM potassium chloride, 5 mM

ammonium sulphate, 2 mM magnesium sulphate, 0.2

mM dNTP, 0.1 nmol of the outer forward and the

reverse primers and 1.0 U Pwo polymerase (Roche). The

reaction was performed in a Perkin Elmer Thermal

Cycler with 35 cycles of 94, 48 and 72 8C, each for 1 min.On completion of the first PCR, 1 ml of the reaction

product was added as template for the second reaction

(performed as described above) with the inner forward

primer and the same reverse primer. PCR products were

resolved in 1% agarose gels containing 0.5 mg ml�1

ethidium bromide, and a band of approximately 1.0 kb

(based on the expected size of the mature catalytic

domain plus a 3?-UTR sequence) excised and purifiedusing silica beads (BIO 101, Vista, CA). Purified DNA

was submitted directly for sequencing using the inner

forward degenerate primer (UCSF Biomolecular Re-

source Facility or Sequetech Corp., Mountain View,

CA). From the sequence obtained, additional forward

and reverse primers were designed and used to sequence

the entire 1.0 kb PCR product. To allow for sequencing

of the SmCB2 ORF upstream of the N-terminus of thecatalytic domain, 5?-RACE was performed according to

the manufacturer’s instructions (Clontech, Palo Alto,

CA). Sequencing of this section of the ORF was

completed using a forward primer to the N-terminal

signal sequence and a reverse primer derived from the

catalytic domain. The overall sequencing strategy en-

sured that the entire SmCB2 ORF was sequenced in

both directions. The putative cleavage point between thesignal and pro-peptides was identified using the SignalP

V1.1 World Wide Web Prediction Server at the Center

for Biological Sequence Analysis, Denmark (http://

genome.cbs.dtu.dk/services/SignalP). Networks were

trained on sequences from eukaryotes and the software

is based on the algorithm of von Heijne [29]. A multiple

sequence alignment of the primary sequence of SmCB2with SmCB1 ([7]; accession number AAA29865), SjCB1

([30]; P43157) and human cathepsin B ([31]; P07858) was

developed using the CLUSTAL W program [32] avail-

able at the UCSF Sequence Analysis Resource Facility.

2.5. Design of a vector construct for expression of

SmCB2 in P. pastoris

The nucleotide sequence encoding the pro-domain ofSmCB2 was included in the expression vector construct

as it was found previously for cysteine-class cathepsins

that the pro-region is necessary for proper protein

folding [33]. Pro-SmCB2 was amplified by PCR from

single stranded cDNA derived from adult worms

(above). PCR primers were: forward, 5?- ATACTCGA-

GAAAAGAGATGCTAGACGACATAAACGTATG-

3? which incorporated an Xho I (Roche) site immedi-ately 5? of the P. pastoris Kex 2 endopeptidase recogni-

tion site (both sites are underlined); and reverse 5?-AATGCGGCCGCCTAGTTTTTTATTTTTGGTAT-

TCCAGCAT-3?, which incorporated a transcription

termination codon immediately 3? of a Not I site (both

sites are underlined). Amplification reactions were as

described in Section 2.4 except that the annealing

temperature was 55 8C. The approximately 1.2 kbPCR product, purified from agarose gels, was restriction

digested overnight at 37 8C with Xho I and Not I, re-

purified and ligated into the expression vector pPIC ZaA (Invitrogen Corp., San Diego, CA) that had been

similarly restriction digested and purified. The resulting

construct places the pro-SmCB2 gene downstream and

in frame with the a-mating factor of P. pastoris , thereby

targeting SmCB2 for secretion. Sufficient recombinantplasmid was produced in and purified from E. coli

(DH5a strain; Life Technologies, Inc., Rockville, MD)

for transformation of P. pastoris (see below).

2.6. Expression and purification of recombinant SmCB2

Procedures to express SmCB2 were as detailed by the

manufacturer (Invitrogen). Briefly, to allow for homo-logous incorporation of the recombinant plasmid into

the AOX 1 locus of the P. pastoris genome, the pro-

SmCB2-pPIC Za A plasmid construct (10 mg) was

linearized with Sac I overnight at 37 8C and purified.

Electrocompetent X33 strain of P. pastoris (Invitrogen)

was electroporated at 1.5 kV and 129 ohms in electro-

poration cuvettes (2 mm gap; BTX, San Diego, CA).

Pichia colonies growing at 30 8C under zeocin-selection(100 mg ml�1; Invitrogen) on yeast extract-peptone-

dextrose (YPD)-agar plates were picked for expansion

first in 10 ml and then in 500 ml of YPD medium

C.R. Caffrey et al. / Molecular & Biochemical Parasitology 121 (2002) 49�/61 51

http://genome.cbs.dtu.dk/services/SignalP

http://genome.cbs.dtu.dk/services/SignalP

containing the antibiotic. Expression of recombinant

protein was induced by incubation of P. pastoris for 24�/

48 h at 30 8C in buffered minimal medium con-

taining 1% methanol as the sole carbon source. En-dopeptidase activity in the medium (1�/5 ml) was

monitored by hydrolysis of Z-Phe-Arg-NMec (see

Section 2.10).

Media (200�/300 ml) from P. pastoris that had been

induced were lyophilized and stored in a desiccator at

4 8C until use. Lyophilized samples were resuspended in

50 mM sodium acetate, pH 4.5 (10% of the original

volume) and complete equilibration with this buffer wasaccomplished using PD10 columns (Amersham Phar-

macia Biotech Inc.). The solution was loaded onto a

Mono S HR 5/5 cation exchange column, which had

been pre-equilibrated in 50 mM sodium acetate, pH 4.5,

and protein resolved by use of a 0�/1 M NaCl gradient.

Endopeptidase activity associated with SmCB2 eluted at

500 mM NaCl. Fractions (1 ml) were stored at �/80 8Cuntil use. Purification was monitored by SDS-PAGEthrough 4�/12% polyacrylamide gels (Invitrogen). After

electrophoresis, gels were stained in 40% methanol, 10%

acetic acid containing 0.5% Coomassie Brilliant Blue R-

250 (BIO-RAD, Hercules, CA) and destained in the

same solution, but omitting Coomassie Blue.

2.7. Production of antiserum to recombinant SmCB2 and

purification of IgG

Antiserum was raised in a New Zealand white rabbit

by Covance, Richmond, CA. Prior to the first injection

of enzyme, pre-immune serum was collected and stored

at �/20 8C. SmCB2 (200 mg) was deglycosylated with

500 U PNGase F for 4 h at 37 8C as described by the

manufacturer (New England Biolabs, Beverly, MA) and

resolved by SDS-PAGE. The Coomassie stained band

was excised and mixed with an approximately equalvolume of adjuvant. Freund’s Complete Adjuvant was

used for the primary injection, whereas Freund’s In-

complete Adjuvant was used for the second and third

injections on days 18 and 36, respectively. The rabbit

was exsanguinated at 54 days and serum stored at

�/20 8C. IgG was isolated from both pre-immune and

hyper-immune sera using Protein G-Sepharose (1 ml) as

described [34] and stored at �/20 8C.

2.8. Immunoblotting

Adult male (100) and female (40) S. mansoni worms

were sonicated over an ice bath in 0.5 ml 50 mM sodium

phosphate buffer, pH 7.0. After centrifugation at 9000 g

for 5 min at 4 8C, the supernatant was either used

immediately or stored at �/70 8C. Worm supernatant(100 mg) or pure recombinant SmCB2 (5 mg) were

resolved by SDS-PAGE (4�/12% gradient gels) and

transferred to PVDF membrane as described above.

Following blocking of the membrane for 2 h at room

temperature in TBS-T (100 mM Tris�/HCl, 100 mM

NaCl, pH 8.0, 0.05% Tween 20), the membrane was

incubated for 1 h with preimmune or anti-SmCB2 IgG

at a dilution of 1:3000 in TBS-T. The membrane was

then washed 5�/5 min in TBS-T and incubated for 1 h

in TBS-T containing alkaline phosphatase-conjugated

goat anti-rabbit IgG diluted 1:4000 (2.5 mg; Life

Technologies). After the same washing steps, blots

were developed in 100 mM Tris�/HCl, 100 mM NaCl,

5 mM MgCl2, pH 9.0 containing 0.33% nitro blue

tetrazolium (NBT; Promega, Madison, WI) and 0.66%

5-bromo-4-chloro-3-indolyl-1-phosphate (BCIP; Pro-

mega). Development was stopped by incubating the

membrane in TBS containing 0.005 M di-sodium

EDTA.

2.9. Immunohistochemistry

Adult S. mansoni worms were washed once in PBS

and fixed in 2% paraformaldehyde/0.1% glutaraldehyde/

PBS for 2�/4 h at room temperature. The schistosome

material was allowed to stand in PBS overnight at 4 8C.

The embedding and immunolabeling procedures have

been previously described [35]. Briefly, parasites were

dehydrated through a graded series of acetone solutions,

embedded in JB-4 (Polyscience, Warrington, PA) and

sectioned at 2 mm with a Sorvall JB-4 microtome.

Sections were placed onto glass slides, air-dried over-

night and incubated in 0.25% trypsin (Zymed, South

San Francisco, CA) at 37 8C for 10 min. Sections were

washed 3�/5 min in wash buffer (PBS containing 0.05%

Tween-20 and 0.1% BSA). Potential endogenous perox-

idase activity was quenched by incubating sections for

10 min at room temperature in 1% H2O2/0.1% NaN3 in

the blocking buffer that is part of the Vectastain Elite

ABC Kit (Vector Labs, Burlingame, CA). Sections were

incubated in blocking buffer alone for a further 30 min

and then incubated overnight at 4 8C with a 1:100

dilution (10 mg) of purified rabbit pre-immune or anti-

SmCB2 IgG in blocking buffer. After the above series of

washes, sections were incubated for 30 min at room

temperature with a 1:200 dilution of biotinylated goat

anti-rabbit IgG (Vectastain Elite ABC Kit). After

washing, sections were incubated for 30 min in the

Vectastain ABC Reagent (horseradish peroxidase H

conjugated to avidin DH), washed again and incubated

for 5 min in AEC (aminoethyl carbazole) Single Solu-

tion (Zymed). Following a further series of washes, the

sections were stained with Gill’s Hematoxylin solution

No. 3 (Sigma), washed in water and air-dried. Sections

were mounted in Aquamount (Lerner Labs, Pittsburgh,

PA).


2.10. Endopeptidase activity measured with fluorogenic

substrates: pH optimum and S2 subsite specificity

Routine assays used Z-Phe-Arg-NMec and initialrates of activity were measured at room temperature in

black 96-well microtiter plates using a Labsystems

Fluoroskan II fluorometer at excitation and emission

wavelengths of 350 and 460 nm, respectively. To activate

enzyme activity, either native SmCB1 or SmCB2 (both

0.5 nM), or recombinant SmCB2 (5 nM) was preincu-

bated for 5 min in 100 ml 100 mM sodium citrate, pH

5.5, containing 2 mM DTT. Substrate (100 ml; 40 mM),in the same buffer, was added and the reaction

continued for 5 min. Control reactions contained buffer

in place of enzyme. Activity was expressed either as

nmol NMec released min�1 mg�1 (purification of

native enzymes) or mol NMec released min�1 mol

enzyme�1 (recombinant SmCB2) using a standard curve

of free NMec prepared in ethanol and further diluted in

water. The concentration of active enzyme used inassays was determined by active site titration with E-

64 (Sigma), a stoichiometric irreversible inhibitor of

papain-like cysteine endopeptidases [36].

The pH optimum of native and recombinant SmCB2

was measured with Z-Phe-Arg-NMec, suitable for the

assay of both cathepsins B and L, and Z-Arg-Arg-

NMec, the cathepsin B-selective substrate [27]. A 100

mM citrate�/phosphate buffer over the pH range of 3.0�/

8.0 was used. All buffers contained 300 mM NaCl to

minimize variations in ionic strength. Otherwise assays

were performed as above. To probe the S2 subsite

specificity of SmCB2 (nomenclature of Schechter and

Berger [37]), four substrates varying in the P2 amino acid

(Phe, Leu, Val or Arg) were used (Z-Leu-Arg-NMec and

Z-Val-Arg-NMec were kindly provided by Dr. Dieter

Bromme, Mount Sinai School of Medicine, New York).Given that the choice of substrate can itself influence pH

activity profiles [38], the initial rates of hydrolysis were

measured between pH 4.0 and 7.0.

2.11. Determination of Km and kcat values with NMec

substrates

For determination of the Michaelis constant, Km, a

range of substrate concentrations between 10 and 300mM (solubility limit), and between 50 and 1400 mM was

employed for Z-Phe-Arg-NMec and Z-Arg-Arg-NMec,

respectively. Otherwise, the design of the assays was as

described above. Km and Vmax values were measured by

the direct liner plot method [39]. The catalytic rate

constant kcat was determined from Vmax/e , where e

represents the active enzyme concentration, as measured

by titration with E-64. For comparative purposes underour experimental conditions, kinetic constants for

bovine spleen cathepsin B (Sigma) and rat-liver cathe-

psin L (kindly provided by Dr. Heidrun Kirschke,

Institut fur Physiologische Chemie, Martin-Luther Uni-

versitat, Halle, Germany) were also determined.

3. Results

3.1. Isolation of a novel cathepsin B from schistosome

extracts

Two distinct cysteine endopeptidases were isolatedfrom extracts of adult S. mansoni by DE-52 anion-

exchange chromatography followed by H-Gly-Phe-Gly-

semicarbazone affinity chromatography. N-terminal

sequencing of the purified enzymes identified the activity

adsorbed to the DE-52 matrix as the previously

characterized SmCB1 [7] and the activity not adsorbed

as novel. The latter enzyme was eventually termed

SmCB2 on confirmation by nucleotide sequencing ofits cathepsin B-like characteristics. For both enzymes, a

single protein species at 33 kDa was resolved by SDS-

PAGE and silver-staining (Fig. 1, lane 3; only data for

SmCB2 shown). Both enzymes are minor worm proteins

as the relevant molecular mass areas of soluble worm

extract (Fig. 1, lane 1) and the DE-52 wash fraction

(from the second DE-52 column; lane 2) were relatively

poorly stained. With Z-Phe-Arg-NMec as substrate, thespecific activities of purified native SmCB2 and SmCB1

were 22 1719/6272 and 20 5239/2892 nmol NMec re-

Fig. 1. Purification of SmCB2 from schistosome extracts. Acidic

soluble extracts of adult S. mansoni were subjected to DE-52 ion

exchange and Ahx-Sepharose 4B-Gly-Phe-Gly-semicarbazone affinity

chromatographies as described in the text. SDS-PAGE in 12.5% gels

followed by silver staining were used to analyze purification. Lane 1,

starting material (30 mg); lane 2, material not adsorbed to the DE-52

columns (wash fraction; 20 mg); lane 3, purified SmCB2 (3 mg).

Molecular masses are indicated on the right.


leased min�1 mg�1, respectively, compared to the

endopeptidolytic activity of soluble worm extract, which

was 1789/97 nmol NMec released min�1 mg�1 (average

from three purifications). Thus, the fold purification ofSmCB2 and SmCB1 was 142 and 115, respectively. Final

activity yields of both enzymes were between 3 and 8%

of the starting material.

3.2. Confirmation that SmCB2 is a novel schistosome

cathepsin B

The first 13 N-terminal amino acid residues of native

SmCB2 were sequenced (Fig. 2, underlined). Thisfacilitated the design of a semi-nested PCR using

degenerate forward primers (based on the amino acid

sequence with the dashed line in Fig. 2) and a (dT)18

reverse primer to isolate the appropriate DNA sequence

from adult S. mansoni single-stranded cDNA. A 1.0 kb

product was amplified. Sequencing of this product

identified a novel endopeptidase distinct from SmCB1

(see below). 5?-RACE amplified the remaining SmCB2sequence upstream of the N-terminus of the mature

catalytic domain. The ORF of SmCB2 is made up of

1041 nucleotides with a 285 base 3?-UTR and encodes a

pre-pro-protein of 347 amino acids with a calculated

molecular mass of 39 017 Da (Fig. 2). A predicted

hydrophobic leader sequence of 23 amino acids is

upstream of a typical cathepsin B pro-peptide of 70

residues. The 254 amino acid residue catalytic domain ofSmCB2 is 49, 51 and 52% identical, and 57, 59 and 61%

similar to SmCB1, SjCB1 and human cathepsin B,

respectively (Fig. 3). SmCB2 possesses the conserved

active site residues Cys29, His200, and Asn220 (number-

ing system as depicted in Fig. 3) of papain-like cysteine

endopeptidases. In addition, SmCB2 contains all those

residues of the papain superfamily endopeptidases that

interact with the main chain of the bound substrate,namely, Gln23, Gly73, Gly74, and Trp222 [40]. All 12

cysteine residues that form the 6 intramolecular disulfide

bridges within the catalytic domain are conserved

between the schistosome and human enzymes [41].

Characteristic of the cathepsin B group, SmCB2 has

an occluding loop (residues 108 through 119), which is

responsible for dipeptidyl peptidase activity [41]. Two

potential Asn-linked glycosylation sites are present inthe pro-peptide and catalytic domain, at positions p27

and 95, respectively.

3.3. Expression of recombinant active SmCB2 in P.

pastoris

SmCB2 was expressed by P. pastoris as an active

endopeptidase. Yields of purified enzyme were in therange of 40�/60 mg l�1 of culture medium, and on a

weight basis, tests with the stoichiometric irreversible

inhibitor E-64 indicated that the enzyme was 50% active.

By SDS-PAGE, the media from induced P. pastoris

cultures contained a predominant protein of 33 kDa and

a second band at 43 kDa (Fig. 4, lane 1). A single

purification step with Mono S cation exchange chroma-tography was sufficient to isolate SmCB2 as judged by

overloading SDS gels with column fractions containing

the greatest hydrolytic activity against Z-Phe-Arg-NMec

(Fig. 4, lane 2). The purified enzyme migrated with an

apparent mass of 33 kDa, but on treatment with the

glycosidase PNGase F, the molecular mass was approxi-

mately 31 kDa (data not shown), thus indicating that

the recombinant protein is N-glycosylated. N-terminalsequencing of the recombinant enzyme yielded G Y I S

D E, i.e. most of the pro-domain had been removed to

within six amino acids of the N-terminus of the catalytic

domain.

3.4. Identification of SmCB2 in adult schistosome

extracts

By immunoblotting, rabbit anti-SmCB2 IgG reactedwith its recombinant target (Fig. 5, lane 2), whereas

preimmune IgG did not (lane 4). When tested with

parasite extracts of male worms, the antibody reacted

with bands of 33 and 40 kDa (Fig. 5, lane 1). Preimmune

IgG did not react (lane 3). No reaction was visible with

female worm extracts (not shown), and this was in spite

of varying conditions of protein loading and antibody

dilutions.

3.5. Immunolocalization of SmCB2 in adult male and

female S. mansoni

Immunohistochemical studies with anti-SmCB2 IgG

localized the enzyme in tissues of both sexes, but the

reaction was more marked in sections of male worms. In

males, reactivity was detected in the dorsal and lateraltubercles of the tegument; otherwise the tegument was

not stained (Fig. 6A). Labeling was distributed in a

punctate manner throughout the parenchyma below the

muscle layers. Labeling was not observed in the testes

(not shown), gastrodermis or gut lumen. In females, less

labeling was evident compared to males and was found

in the parenchyma below the muscle layers (Fig. 6C).

Also, the reaction was occasionally found in theparenchyma between the vitellaria. No reaction was

detected in the tegument, vitellaria, gastrodermis or in

the gut lumen. For both sexes, no reaction was observed

with pre-immune IgG (Fig. 6B and D).

3.6. Acidic pH optima, S2 subsite specificity and kinetics

of substrate hydrolysis

Both native and recombinant SmCB2 degraded Z-

Phe-Arg-NMec, the cathepsin L/B substrate and Z-Arg-

Arg-NMec, the cathepsin B-selective substrate [27]. For


Fig. 2. Nucleotide and deduced amino acid sequence of SmCB2. The N-terminal sequence obtained from affinity purified native SmCB2 is

underlined and the dashed underline identifies the sequence used to design degenerate primers for semi-nested PCR (see text for details). The start of

the pro- and catalytic domains is indicated by an arrow. Potential N-glycosylation sites are boxed and the active site Cys29, His200, and Asn220

(numbering system used in Fig. 4) are indicated in bold face.


both forms of SmCB2, the pH optima for hydrolysis of

Z-Phe-Arg-NMec (Fig. 7A) and Z-Arg-Arg-NMec (Fig.

7B) were 5.0 and 5.5, respectively. For native SmCB1,

the pH optima for hydrolysis of Z-Phe-Arg-NMec and

Z-Arg-Arg-NMec were 4.5 and 5.5, respectively (not

shown). At pH values at and above 6.5 with either

Fig. 3. Multiple sequence alignment of SmCB2 with other cathepsin B enzymes. Alignments were made with SmCB1 ([7]; accession number

AAA29865), SjCB1 ([30]; P43157) and human cathepsin B ([31]; P07858). Above the sequences, numbers prefixed with a ‘‘p’’ refer to the pro-domain

of the enzyme. Below the sequences, identities across all four sequences are indicated by asterisks, one or more conservative substitutions are

indicated by colons and, where non-conservative changes are present, points are used. The catalytic triad residues (Cys29, His200 and Asn220) are

indicated in bold face. Those residues making up the S2 specificity subsite [41] are indicated with � symbols in bold face.


substrate, no hydrolysis was detected for SmCB2 or

SmCB1.

For papain-like endopeptidases, such as SmCB2, the

S2 subsite of the active site cleft defines the primary

specificity towards peptidyl substrates. Four dipeptidyl

NMec substrates, each varying in the P2 amino acid

(Phe, Leu, Val or Arg), were employed to probe the S2

specificity of recombinant SmCB2. At optimal pH, the

substrate with Phe at P2 was degraded 3-fold quicker

than those in which P2 was Leu or Val (Fig. 8). Arg at P2

was least preferred (see also kinetic results below).

Both native and recombinant SmCB2 had similar Km

and kcat values when tested with Z-Phe-Arg-NMec or Z-

Arg-Arg-NMec (Table 1). SmCB2 had a greater speci-

ficity constant (kcat/Km values) for Z-Phe-Arg-NMec

over Z-Arg-Arg-NMec. The preference for the substrate

with Phe at P2 was 69- and 80-fold for native and

recombinant SmCB2, respectively. These values are

significantly greater than those recorded for native

SmCB1 (12-fold) and bovine spleen cathepsin B (5-

fold; Table 1). The kinetic constants measured here for

mammalian cathepsins B and L are consistent with

those obtained by others [27,42].

4. Discussion

We have isolated, functionally expressed and char-

acterized a novel cathepsin B endopeptidase from the

parasitic helminth S. mansoni . The enzyme was tissue-

localized in sections of adult worms. We have termed the

endopeptidase SmCB2, as it is the second cathepsin B ofS. mansoni for which a gene has been identified, the first

being Sm31 [7]. For this report, we have consolidated

the nomenclature regarding schistosome cathepsin Bs,

analogous to that of schistosome cathepsin L enzymes

[14,18,20]. Thus, we now describe Sm31 as SmCB1.

SmCB2 was originally purified from acidic, soluble

worm extracts by a combination of DE-52 anion-

exchange and inhibitor-affinity chromatographies. Thepurification scheme is quicker and involves less handling

compared to previous purifications of schistosome

cathepsins [43�/45]. It is especially convenient as the

Fig. 4. Recombinant expression and purification of active SmCB2.

Concentrated induced culture medium (see text) from P. pastoris

transformed with pro-SmCB2-pPIC Za A was equilibrated with 50

mM sodium acetate buffer, pH 4.5 and loaded onto a Mono S HR 5-5

cation exchange column. Protein was eluted in a 0�/1 M NaCl gradient

and fractions containing highest endopeptidolytic activity (with Z-Phe-

Arg-NMec as substrate) were pooled. A 4�/12% gradient SDS gel

stained with Coomassie Blue is shown. Lane 1, concentrated Pichia

culture medium (10 mg); lane 2, purified SmCB2 pool (70 mg).

Molecular masses are indicated on the right.

Fig. 5. Reaction of monospecific IgG with recombinant SmCB2 and

adult male S. mansoni extracts. Worm supernatant (100 mg; lanes 1 and

3) or pure recombinant SmCB2 (5 mg; lanes 2 and 4) were resolved by

SDS-PAGE (4�/12% gradient gels) and transferred to PVDF mem-

brane. Blots were probed with IgG purified from either a rabbit serum

raised to deglycosylated recombinant SmCB2 (lanes 1 and 2) or pre-

immune serum (lanes 3 and 4). Note the reaction in worm extracts of

protein species of 35 and 43 kDa (lane 1), presumed to represent the

fully processed and zymogen forms of SmCB2, respectively.


same batch of starting material can be used to purify

both SmCB1 and SmCB2. Though sufficient native

enzyme could be purified for limited biochemical

analyses, to perform more extensive analyses, including

crystallography, it is necessary to have a recombinant

source of SmCB2. Accordingly, the information gained

from the N-terminal sequence of purified native SmCB2

facilitated the design of a semi-nested PCR strategy to

amplify, sequence and eventually clone SmCB2 for

expression in P. pastoris .

Sequencing of the ORF of SmCB2 was accomplished

directly from gel-purified PCR products. No ambigu-

ities arose regarding nucleotide identities during the

course of sequencing indicating that the PCR product

contained a single DNA sequence. The sequence of

SmCB2 is clearly related to other cathepsin B enzymes

from both schistosomes and mammals. Most notable is

the presence in SmCB2 of a section encoding the

occluding loop (residues 108�/119) which restricts access

of potential substrates to the S? subsites of the active site

cleft and confers the peptidyl dipeptidase activity unique

to cathepsin B among the cysteine-class cathepsins [41].

Using the entire SmCB2 ORF, a standard nucleotide-

nucleotide BLAST (blastn) analysis limited to the S.

mansoni EST database returned 14 partial but exactly

matching sequences. This would suggest that, thus far,

no isoforms of SmCB2 exist. Also, as the ESTs

identified were derived from both male and female S.

mansoni cDNA libraries, the analysis confirms the

presence by immunohistochemistry of SmCB2 in both

sexes.

The expression and purification of active recombinant

SmCB2 using P. pastoris was straightforward. SmCB2

was the major protein secreted by P. pastoris with an

Fig. 6. Immunolocalization of SmCB2 in adult male and female S. mansoni . Experiments using JB-4-embedded sections of male (A and B) and

female (C and D) worms were performed with IgG purified from a rabbit antiserum to deglycosylated recombinant SmCB2 (A and C) or preimmune

serum (B and D). Secondary antibody was biotinylated goat anti-rabbit IgG which was followed by incubation with horseradish peroxidase H

conjugated to avidin DH. Development of the reaction causes precipitation of the red substrate aminoethyl carbazole at those sites where SmCB2 is

present. Sections were counter-stained with Gill’s Hematoxylin solution No. 3. Reaction product was seen in the parenchyma (p) and tegumental

tubercles (tl) of male worms, and in the parenchyma of females. Note the greater presence of SmCB2 in male worms. g, gastrodermis; gl, gut lumen;

m, muscle; v, vitellaria. Magnification �250.


apparent molecular mass of 33 kDa. The presence of a

second protein at 43 kDa might represent an unpro-

cessed pro-form of the enzyme. Recombinant SmCB2

was isolated by cation exchange chromatography over a

Mono S column and N-terminal sequencing confirmed

that the enzyme had been processed to within six amino

acids of the start of the mature domain. Accordingly,

autoactivation procedures used by others to process the

related cathepsins F [46] and V [47], which were secreted

as unprocessed pro-enzymes in the same expression

system, were not required.

The presence of six extra amino acids belonging to the

pro-region influenced neither the pH activity nor kinetic

profiles of recombinant SmCB2 compared to the native

enzyme. The results suggest that in all likelihood, the

recombinant enzyme had been correctly folded with the

proper active site topography and charge-distribution.

Consistent with most papain-like endopeptidases,both native and recombinant SmCB2 had a greater

preference for Z-Phe-Arg-NMec over Z-Arg-Arg-

NMec. The preferences of 69-fold (native) and 80-fold

(recombinant) were greater than those recorded for

either bovine cathepsin B (5-fold) or SmCB1 (12-fold).

This might be in part due to the fact that SmCB2 differs

from these respective enzymes by three and four residues

of the seven that make up the S2 specificity subsite ([41];see residues highlighted by a �/ symbol in Fig. 3). In

contrast to mammalian cathepsin B, all of the schisto-

some enzymes shared similar low specificity constants

(kcat/Km) for Z-Arg-Arg-NMec. This is mainly due to

the influence of kcat rather than affinity for the

substrate. When tested with four peptidyl substrates

with Phe, Leu, Val or Arg at P2, SmCB2 had a 3-fold

preference for the bulky aromatic Phe over either of thebranched aliphatics Leu and Val. In contrast, mamma-

lian cathepsin B has 5- and 10-fold preference for

phenylalanine over leucine and valine, respectively, at

P2 [46]. This would suggest that SmCB2 has a less

restricted S2 pocket than the mammalian enzyme.

Immunoblotting and immunohistochemical experi-

ments using anti-SmCB2 IgG demonstrated that though

the enzyme is expressed in adult worms of both sexes, itspresence is significantly greater in males. The reaction of

a 33 kDa protein by immunoblotting extracts of male

worms is consistent with the molecular mass of the

native and mature recombinant enzymes. The 40 kDa

protein also identified in the blots might be the pro-form

of SmCB2. Pro-forms of cathepsins have been pre-

viously identified in immunoblots of schistosome ex-

tracts [24].Some inferences can be made as to the biology of

SmCB2 in S. mansoni based on the results of immuno-

histochemical experiments. In male and female sections,

SmCB2 was localized predominantly in the parench-

Fig. 7. pH optima of native and recombinant SmCB2 with dipeptidyl

NMec substrates. Enzymes were pre-incubated in buffer containing

DTT for 5 min as described in the text. Substrate was added to a final

concentration of 20 mM and the reaction continued at room

temperature for 5 min. Data is plotted as a percentage of that activity

at optimal pH. Results for Z-Phe-Arg-NMec and Z-Arg-Arg-NMec

are depicted in A and B, respectively. Open and solid symbols refer to

the use of native and recombinant SmCB2, respectively. Assays were

performed twice each in triplicate. Standard deviations of individual

values from the mean were always less than 5%.

Fig. 8. S2 subsite specificity of recombinant SmCB2. Substrates of the

form Z-Xxx-Arg-NMec (where Xxx refers to Phe, Leu, Val or Arg)

were tested at a final concentration of 20 mM as described in the text.

Initial rates of activity were measured over the pH range 4.0�/8.0. The

experiment was performed twice each in duplicate and the results of

one experiment are presented. Phe, closed circles; Leu, closed squares;

Val, open circles; Arg, open squares.


ymous tissues. The antibody reactivity was not evenly

distributed, but was found in discrete areas suggesting a

localization of action. The enzyme may well be present

in the lysosomes or another acidified compartment as

suggested by the strict acidic pH profile of the enzyme

recorded here using dipeptidyl substrates. Of particularnote is the absence of SmCB2 from the gastrodermis and

lumen of the parasite gut. Accordingly, the enzyme is

unlikely to participate directly in the digestion of

ingested nutrients, a function that has been postulated

for the gut-localized endopeptidases SmCB1 [3,7,24],

SmCL1 [14] and SmCL2 [20]. Interestingly, the dorsal

and lateral tubercles of the male were heavily stained,

suggesting that SmCB2 functions at the host�/parasiteinterface. The enzyme may participate in the turnover of

tegumental proteins, the degradation of endocytosed

proteins and/or operate in a protective capacity.

Acknowledgements

Supported by a TDRU grant (AI35707) from the

NIAID and the Sandler Family Foundation.

References

[1] WHO. Schistosomiasis. Fact Sheet no. 115 (http://www.who.int/

inf-fs/en/fact115.html); 1996.

[2] Lim KC, Sun E, Bahgat M, et al. Blockage of skin invasion by

schistosome cercariae by serine protease inhibitors. Am J Trop

Med Hyg 1999;60:487�/92.

[3] Wasilewski MM, Lim KC, Phillips J, McKerrow JH. Cysteine

protease inhibitors block schistosome hemoglobin degradation in

vitro and decrease worm burden and egg production in vivo. Mol

Biochem Parasitol 1996;81:179�/89.

[4] Ruppel A, Shi YE, Wei DX, Diesfeld HJ. Sera of Schistosoma

japonicum -infected patients cross-react with diagnostic 31/32 kD

proteins of S. mansoni . Clin Exp Immunol 1987;69:291�/8.

[5] Li YL, Idris MA, Corachan M, Han JJ, Kirschfink M, Ruppel A.

Circulating antigens in schistosomiasis: detection of 31/32-kDa

proteins in sera from patients infected with Schistosoma japoni-

cum , S. mansoni , S. haematobium , or S. intercalatum . Parasitol

Res 1996;82:14�/8.

[6] Grogan J, Rotmans P, Ghoneim H, Deelder A, Yazdanbakhsh

M, Klinkert M-Q. Recognition of Schistosoma mansoni cathe-

psins B and L by human IgG1 and IgG4 antibodies. Parasite

Immunol 1997;19:215�/20.

[7] Klinkert M-Q, Felleisen R, Link G, Ruppel A, Beck E. Primary

structures of Sm31/32 diagnostic proteins of Schistosoma mansoni

and their identification as proteases. Mol Biochem Parasitol

1989;33:113�/22.

[8] Smith AM, Dalton JP, Clough KA, et al. Adult Schistosoma

mansoni express cathepsin L proteinase activity. Mol Biochem

Parasitol 1994;67:11�/9.

[9] Michel A, Ghoneim H, Resto M, Klinkert M-Q, Kunz W.

Sequence, characterization and localization of a cysteine protei-

nase cathepsin L in Schistosoma mansoni . Mol Biochem Parasitol

1995;73:7�/18.

[10] Hola-Jamriska L, Tort JF, Dalton JP, et al. Cathepsin C from

Schistosoma japonicum -cDNA encoding the preproenzyme

and its phylogenetic relationships. Eur J Biochem

1998;255:527�/34.

[11] Gotz B, Felleisen R, Shaw E, Klinkert M-Q. Expression of an

active cathepsin B-like protein Sm31 from Schistosoma mansoni in

insect cells. Trop Med Parasitol 1992;43:282�/4.

[12] Gotz B, Klinkert M-Q. Expression and characterization of a

cathepsin B-like enzyme (Sm31) and a proposed ‘haemoglobinase’

(Sm32) from Schistosoma mansoni . Biochem J 1993;290:801�/6.

[13] Lipps G, Fullkrug R, Beck E. Cathepsin B of Schistosoma

mansoni . Purification and activation of the recombinant proen-

zyme secreted by Saccharomyces cerevisiae . J Biol Chem

1996;271:1717�/25.

[14] Brady CP, Dowd AJ, Brindley PJ, Ryan T, Day SR, Dalton JP.

Recombinant expression and localization of Schistosoma mansoni

cathepsin L1 support its role in the degradation of host

hemoglobin. Infect Immun 1999;67:368�/74.

[15] Hola-Jamriska L, King LT, Dalton JP, Mann VH, Aaskov JG,

Brindley PJ. Functional expression of dipeptidyl peptidase I

(Cathepsin C) of the oriental blood fluke Schistosoma japonicum

in Trichoplusia ni insect cells. Protein Expr Purif 2000;19:384�/92.

[16] Klinkert M-Q, Cioli D, Shaw E, Turk V, Bode W, Butler R.

Sequence and structure similarities of cathepsin B from the

parasite Schistosoma mansoni . FEBS Lett 1994;351:397�/400.

[17] Butler R, Michel A, Kunz W, Klinkert M-Q. Sequence of

Schistosoma mansoni cathepsin C and its structural comparison

with papain and cathepsins B and L of the parasite. Prot Pept Lett

1995;2:313�/20.

Table 1

Comparison of kinetic values for native and recombinant SmCB2, native SmCB1, bovine-spleen cathepsin B and rat-liver cathepsin L

Enzyme Substrate Difference in kcat/Km values (-fold)

Z-Phe-Arg-NMec Z-Arg-Arg-NMec

Km (mM) kcat (s�1) kcat/Km (mM�1 s�1) Km (mM) kcat (s�1) kcat/Km (mM�1 s�1)

SmCB2 0.056 35 625 1.0 9 9 69

rSmCB2 0.063 70 1111 0.65 9 14 80

SmCB1 0.055 11 196 1.4 23 17 12

Cat Ba 0.25 122 488 1.3 121 93 5

Cat L 0.008 43 5326 n.h.b

a Assays were performed as described in the text, except for bovine cathepsin B (assays at pH 6.0 with Z-Arg-Arg-NMec) and SmCB1 (assays at pH

4.5 with Z-Phe Arg-NMec). For each enzyme, experiments were performed twice, each in duplicate. The results of one experiment for each enzyme

are shown here.b n.h., not hydrolyzed.


http://www.who.int/inf-fs/en/fact115.html

http://www.who.int/inf-fs/en/fact115.html

[18] Brady CP, Brinkworth RI, Dalton JP, Dowd AJ, Verity CK,

Brindley PJ. Molecular modeling and substrate specificity of

discrete cruzipain-like and cathepsin L-like cysteine proteinases of

the human blood fluke Schistosoma mansoni . Arch Biochem

Biophys 2000;380:46�/55.

[19] Ruppel A, Breternitz U, Burger R. Diagnostic Mr 31,000

Schistosoma mansoni proteins: requirement of infection, but not

of immunization, and use of the ‘miniblot’ technique for the

production of monoclonal antibodies. J Helminthol 1987;61:11�/

9.

[20] Bogitsh BJ, Dalton JP, Brady CP, Brindley PJ. Gut-associated

immunolocalization of the Schistosoma mansoni cysteine pro-

teases, SmCL1 and SmCL2. J Parasitol 2001;87:237�/41.

[21] Bogitsh BJ, Dresden MH. Fluorescent histochemistry of acid

proteases in adult Schistosoma mansoni and S. japonicum . J

Parasitol 1983;69:106�/10.

[22] Ruppel A, Shi YE, Moloney NA. Schistosoma mansoni and S.

japonicum : comparison of levels of ultraviolet irradiation for

vaccination of mice with cercariae. Parasitology 1990;101:23�/6.

[23] Rich DH, Brown MA, Barrett AJ. Purification of cathepsin B by

a new form of affinity chromatography. Biochem J 1986;235:731�/

4.

[24] Caffrey CR, Ruppel A. Affinity isolation and characterization of

the cathepsin B-like proteinase Sj31 from Schistosoma japonicum .

J Parasitol 1997;83:1112�/8.

[25] Laemmli UK. Cleavage of structural proteins during the assembly

of the head of bacteriophage T4. Nature 1970;227:680�/5.

[26] Merril CR. Gel-staining techniques. Methods Enzymol

1990;182:477�/88.

[27] Barrett AJ, Kirschke H. Cathepsin B, Cathepsin H, and

Cathepsin L. Methods Enzymol 1981;80:535�/61.

[28] Bradford MM. A rapid and sensitive method for the quantitation

of microgram quantities of protein utilizing the principle of

protein-dye binding. Anal Biochem 1976;72:248�/54.

[29] Nielsen H, Brunak S, von Heijne G. Machine learning approaches

to the prediction of signal peptides and other protein sorting

signals. Prot Eng 1999;12:3�/9.

[30] Merckelbach A, Hasse S, Dell R, Eschlbeck A, Ruppel A. cDNA

sequences of Schistosoma japonicum coding for two cathepsin B-

like proteins and Sj32. Trop Med Parasitol 1994;45:193�/8.

[31] Chan SJ, San Segundo B, McCormick MB, Steiner DF. Nucleo-

tide and predicted amino acid sequences of cloned human and

mouse preprocathepsin B cDNAs. Proc Natl Acad Sci USA

1986;83:7721�/5.

[32] Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: Improv-

ing the sensitivity of progressive multiple sequence alignment

through sequence weighting, positions-specific gap penalties and

weight matrix choice. Nucleic Acids Res 1994;22:4673�/80.

[33] Tao K, Stearns N, Dong J, Wu Q, Sahagian GG. The proregion

of cathepsin L is required for proper folding, stability, and ER

exit. Arch Biochem Biophys 1994;311:19�/27.

[34] Harlow E, Lane D. Using Antibodies. A Laboratory Manual.

New York: Cold Spring Harbor Press, 1999.

[35] Yezzi MJ, Hsieh IE, Caughey GH. Mast cell and neutrophil

expression of dog mast cell protease-3. A novel tryptase-related

serine protease. J Immunol 1994;152:3064�/72.

[36] Barrett AJ, Kembhavi AA, Brown MA, et al. L-trans -Epoxysuc-

cinyl-leucylamido(4-guanidino)butane (E-64) and its analogues as

inhibitors of cysteine proteinases including cathepsins B, H and L.

Biochem J 1982;201:89�/98.

[37] Schechter I, Berger A. On the size of the active site in proteases. I.

Papain. Biochem Biophys Res Commun 1967;27:157�/62.

[38] Khouri HE, Plouffe C, Hasnain S, Hirama T, Storer AC, Menard

R. A model to explain the pH-dependent specificity of cathepsin

B-catalyzed hydrolyses. Biochem J 1991;275:751�/7.

[39] Eisenthal R, Cornish-Bowden A. The direct linear plot. A new

graphical procedure for estimating enzyme kinetic parameters.

Biochem J 1974;139:715�/20.

[40] Turk B, Turk D, Turk V. Lysosomal cysteine proteases: more

than scavengers. Biochim Biophys Acta 2000;1477:98�/111.

[41] Musil D, Zucic D, Turk D, et al. The refined 2.15 A X-ray crystal

structure of human liver cathepsin B: the structural basis for its

specificity. EMBO J 1991;10:2321�/30.

[42] Hasnain S, Hirama T, Tam A, Mort JS. Characterization of

recombinant rat cathepsin B and nonglycosylated mutants

expressed in yeast. New insights into the pH dependence of

cathepsin B-catalyzed hydrolyses. J Biol Chem 1992;267:4713�/21.

[43] Chappell CL, Dresden MH. Purification of cysteine proteinases

from adult Schistosoma mansoni . Arch Biochem Biophys

1987;256:560�/8.

[44] Lindquist RN, Senft AW, Petitt M, McKerrow J. Schistosoma

mansoni : purification and characterization of the major

acidic proteinase from adult worms. Exp Parasitol 1986;61:398�/

404.

[45] Ghoneim H, Klinkert MQ. Biochemical properties of purified

cathepsin B from Schistosoma mansoni . Int J Parasitol

1995;25:1515�/9.

[46] Wang B, Shi GP, Yao PM, Li Z, Chapman HA, Bromme D.

Human cathepsin F. Molecular cloning, functional expression,

tissue localization, and enzymatic characterization. J Biol Chem

1998;273:32000�/8.

[47] Bromme D, Li Z, Barnes M, Mehler E. Human cathepsin V.

Functional expression, tissue localization, electrostatic surface

potential, enzymatic characterization, and chromosomal localiza-

tion. Biochemistry 1999;38:2377�/85.


SmCB2, a novel tegumental cathepsin B from adult Schistosoma mansoni

Documents

Transcript of SmCB2, a novel tegumental cathepsin B from adult Schistosoma mansoni