Directory UMM :Data Elmu:jurnal:I:Insect Biochemistry and Molecular Biology:Vol30.Issue11.Nov2000:

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Cloning and functional expression of a Boophilus microplus

cathepsin L-like enzyme

Gaby Renard

a, b,*

_{, Jose´ F. Garcia}

1,a

_{, Felipe C. Cardoso}

_{, Marc F. Richter}

_{, Judy A.}

Sakanari

_{, Luiz S. Ozaki}

2,a

_{, Carlos Termignoni}

a, b

_{, Aoi Masuda}

a, c

a_{Centro de Biotecnologia do Estado do Rio Grande do Sul — UFRGS, C.P. 15005 — Campus do Vale, 91501-970 Porto Alegre RS, Brazil} b_{Departamento de Bioquı´mica, Universidade Federal do Rio Grande do Sul, Av. Bento Gonc¸alves 9500, C.P. 15005, 91501-970 Porto Alegre}

RS, Brazil

c_{Departamento de Biologia Molecular e Biotecnologia, Universidade Federal do Rio Grande do Sul, Av. Bento Gonc¸alves 9500, C.P. 15005,} 91501-970 Porto Alegre RS, Brazil

d_{Department of Biology, Sonoma State University, Rohnert Park, CA 94928-3609, USA}

Received 11 October 1999; received in revised form 1 March 2000; accepted 7 March 2000

Abstract

A cysteine proteinase gene homologous to cathepsins L genes was isolated from a B. microplus cDNA library. The precursor protein deduced from the nucleotide sequence contains 332 amino acid residues consisting of a signal sequence (pre-region), a pro-region and a mature proteinase. The DNA fragment coding for the proenzyme was cloned and expressed using the E. coli expression vector pMAL-p. The recombinant protein (MBP+PROCP) once activated is able to hydrolyze synthetic substrates as well as protein substrates like hemoglobin, vitellin and gelatin. Its optimal enzymatic activity on both fluorogenic and protein substrates was found to occur at an acidic pH. Expression of the proteinase gene was tested by RT-PCR with tick larvae RNA. Detection of amplified sequences indicates that the gene is expressed at this stage of the tick life cycle and the molecule is therefore potentially a target for chemotherapy or an immunogen in a vaccine.2000 Elsevier Science Ltd. All rights reserved.

Keywords: Boophilus microplus; Cysteine proteinase; Cathepsin L; Gene expression; cDNA

1. Introduction

The bovine tick Boophilus microplus is a blood suck-ing ectoparasite that causes severe production losses by its feeding activity and through transmission of other

Abbreviations: CP1, cysteine proteinase DNA fragment used as probe

for screening a B. microplus cDNA library; Bmcl1, cDNA which codes for the cathepsin L1 of B. microplus; BmCL1, enzyme encoded by

Bmcl1; MBP, maltose binding protein; E-64, L-trans-epoxysuccinyl

leucylamido (4-guanidino)-butane; MCA, 7-amido-4 methylcoumarin; DTT, dithiothreitol; PMSF, phenylmethanesulphonyl fluoride.

* Corresponding author. Tel.:+55-51-316-6078; fax:+ 55-51-319-1079.

E-mail address: gaby@dna.cbiot.ufrgs.br (G. Renard). 1 _{Present address, Departmento de Apoio, Produc¸a˜o e Sau´de}

Ani-mal, Universidade Estadual Paulista, Rua Clovis Pestana 793, 16050-680 Arac¸atuba, SP, Brasil

2 _{Present address: Department of Microbiology and Immunology,}

Virginia Commonwealth University, 1101E Marshall Street, Rich-mond, VA 23298-0678, USA

pathogens (Jongejan and Uilenberg, 1994). Current con-trol methods depend heavily on the use of acaricides. However, the accompanying problems of resistance to the acaricides (Solomon, 1983; Nolan et al., 1989; Gon-zales, 1995) and the presence of chemical residues in meat and milk show the need for alternative control methods (Kunz and Kemp, 1994). Since proteinases are involved in key functions in many organisms they could be potential targets to which novel antiparasite chemo-therapy and/or immunoprophylaxis could be directed. In fact, an antigen derived from B. microplus (Bm91) shown to be protective to cattle infestation was charac-terized as a carboxydipeptidase similar to mammalian angiotensin-converting enzymes (Riding et al., 1994; Jarmey et al., 1995). Other midgut derived enzymes with cathepsin L cysteine and aspartic proteinases activities were described in B. microplus (Mendiola et al., 1996). Midgut cysteine and serine proteinase activities have

also been detected in Haemaphysalis longicornis

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embryogenesis has been attributed to a cathepsin L-like proteinase (Fagotto, 1990). More recently, an aspartic proteinase precursor also involved in yolk degradation was isolated from B. microplus eggs (Logullo et al., 1998) and it was shown that cattle vaccinated with this protein were partially protected against tick infestation (Da Silva Vaz et al., 1998).

In the present study we report cloning, expression and characterization of a functional B. microplus cysteine proteinase. The papain superfamily of cysteine protein-ases represents the largest group of proteinprotein-ases described so far. They have been isolated from a wide range of sources including plant and animal tissues. Different physiological roles associated with parasitic life have been assigned to these proteinases. Potential functions include degradation of extracellular matrix for parasite penetration (Scholze and Tannich, 1994; Rhoads and Fetterer, 1996), involvement in molting processes (Richer et al., 1993) and hydrolysis of both host immunoglobulins (Carmona et al., 1993; Smith et al., 1993) and the C3 component of complement (Reed et al., 1989) as an escape mechanism from the host immune response. The availability of enough quantities of the active enzyme will enable studies on the protein struc-ture and of their role in tick physiology. This cysteine proteinase can also be tested as a putative immunogen for the development of a vaccine.

2. Materials and methods

Restriction endonucleases were supplied by Cbiot (Porto Alegre, Brazil), Gibco-BRL Life Technologies (Gaithersburg, USA) and Pharmacia Biotech Inc. (Sa˜o Paulo, Brazil), T4 DNA Ligase by Gibco-BRL Life Technologies and T4 DNA Polymerase by Cbiot. Amyl-ose resin was from New England BioLabs. Inc. (Beverly, USA). Other molecular biology grade chemical com-pounds were purchased from Merck (Rio de Janeiro, Brazil), Sigma (St. Louis, USA) and Pharmacia Biotech Inc. Chromogenic substrate H-D-Val-Phe-Lys-pNA was supplied by Chromogenix AB (Mo¨lndal, Sweden). Fluo-rogenic substrates N-Cbz-Phe-Arg-MCA, N-Cbz-Arg-Arg-MCA, N-t-Boc-Gly-Arg-N-Cbz-Arg-Arg-MCA, Gln-Ala-Arg-MCA, Val-Pro-Arg-MCA, t-Boc-Glu-Ala-Arg-MCA, t-Boc-Ile-Glu-Gly-Arg-MCA,

N-t-Boc-Glu-Lys-Lys-MCA and

N-t-Boc-Gln-Gly-Arg-MCA were purchased from Sigma.

2.1. Ticks

B. microplus ovipositing females, eggs and larvae

(Porto Alegre strain) were maintained in the laboratory at 28°C and 85% relative humidity and their parasitic life cycle was completed in calves, housed in individual

pens on slatted floors. Twenty day old larvae were

col-lected after egg hatching and kept at₂70°C until used.

2.2. PCR amplification and cloning of a DNA fragment representing a conserved region from a B. microplus cysteine proteinase gene

A DNA fragment was amplified by PCR using tick larvae cDNA and three oligonucleotides based on the sequences flanking the conserved residues of cysteine and asparagine present in the active site of cysteine pro-teinases (Eakin et al., 1990; Sakanari et al., 1989). RNA was obtained using a single-step purification procedure

(Chomczynski and Sacchi, 1987). Poly (A)+RNA was

purified by oligo(dT)-cellulose chromatography as

described in Sambrook et al. (1989). cDNA was

synthe-sized from Poly (A)+ RNA using the cDNA Synthesis

System Plus kit (Amersham). The oligonucleotides syn-thesized were a 33-mer corresponding to the region

flanking the cysteine residue, 59

ACAGAATTCCARGGGCARTGYGGGTCGTGYTGG-39 (primer 411) and two 33-mer corresponding to the

region flanking the asparagine residue: 59

TTAAAGCTTCCARCTRTTYTTGACRATCCARTA-39 (primer 413) and 59

-TTAAAGCTTCCAGGART-TYTTGACRATCCARTA-3₉ (primer 414). The primer

411 had a 5₉recognizing site for the restriction enzyme

EcoRI (underlined sequence) and the primers 413 and

414 a 3₉ recognizing site for HindIII (underlined

sequence). PCR conditions were as follows: the first step at 94°C for 5 min, followed by 45 cycles of 30 s at 94°C, 2 min at 25°C and 2 min at 72°C, followed by a final step at 72°C for 7 min. An approximately 500 bp long PCR product (referred to as CP1) was obtained and cloned into the pBluescript vector DNA digested with

EcoRI and HindIII restriction enzymes. 2.3. cDNA library screening

A cDNA library was made in λZAPII vector

(Stratagene, La Jolla, USA) using poly A+ RNA from

the tick B. microplus at different stages of its life cycle (larvae, nymphs, young females, adult males and semi-engorged adult females). Five thousand individual cDNA clones were screened on nitrocellulose mem-branes (Schleicher and Schu¨ll, USA) using the 500 bp long DNA fragment of B. microplus cysteine proteinase

(CP1) labeled with [a-32_{P]dATP by random priming}

(Sambrook et al., 1989). Hybridization was carried out

overnight at 65°C in hybridization buffer (6× SSC

(Sambrook et al., 1989) containing 5% (w/v) cow

non-fat dry milk, 200 µg/ml denatured salmon sperm DNA

and the CP1 probe at 107 _{c.p.m.). Filters were then}

washed for 30 min each in 6×SSC at room temperature

followed by 2×SSC, 1×SSC, 0.5×SSC and 0.2×SSC

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2.4. Nucleotide sequencing and sequence analysis

A clone hybridizing to the CP1 probe was selected and sequenced using M13 universal forward and reverse primers and subsequently gene specific primers.

Sequen-cing was performed using T7_SequencingTM _Kit

(Pharmacia). Analyses of nucleotide and deduced amino acid sequences were carried out using Genepro software program (Riverside Scientific Enterprises). Database searches were carried out using the FASTA e-mail server to search nonredundant set of databases (Pearson and Lipman, 1988). The putative signal peptide and the cor-responding cleavage point (Von Heijne, 1986) were identified using the SIGSEQ program (Rockefeller Uni-versity, 1989). The cloned gene was named Bmcl1.

2.5. Subcloning of Bmcl1 DNA fragment

corresponding to the proproteinase and genomic DNA amplifications

The following oligonucleotides were used: primer 1

(59-GGGAATTCAGCTCTCAAGAAATCCTACGCA-39)

corresponding to nucleotides 52-73 and primer 2 (59

-GGGAATTCCTGCCAAAAGTTGTCGAC-3₉)

corre-sponding to nucleotides 345–363 of Bmcl1 nucleotide

sequence, and primer 3 (5₉

-GGGAGCTCCTGTTA-GACGAGGGGGTA-3₉) corresponding to the inverse

complement of nucleotides 986–1003 in Bmcl1. Primers

1 and 2 have an additional 5₉ recognizing site for the

restriction enzyme EcoRI (underlined) and primer 3 has an additional 3₉ recognizing site for XhoI (underlined). PCR were performed using two primers (1+3 or 2+3) and Bmcl1 DNA as template. After denaturation at 94°C for 5 min amplification was performed with 30 cycles of 94°C for 30 s, 60°C for 2 min and 72°C for 2 min followed by a final extension at 72°C for 5 min. A 950 bp and a 660 bp DNA fragment were obtained. The first one was cloned in the pMOSBlue vector (Amersham). After digestion with the restriction enzyme EcoRI, the fragment was subcloned into the pMAL-p expression vector (New England Biolabs) to produce a pro BmCL1 fused with MBP (pMAL.PROCP). The reading frame was confirmed by sequencing.

The two pair of primers were also used when tick gen-omic DNA was used as template. Gengen-omic DNA was extracted from 2 g of 12 days old larvae ground in a mortar and pestle under liquid nitrogen. The grounded material was suspended in 20 ml of extraction buffer (0.15 M NaCl, 0.1 M EDTA pH 8); 1 ml of RNase (0.5

mg/ml), 100 µl of proteinase K (100 mg/ml) and 1 ml

of sarcosyl 10% were added and the mixture incubated

at 37°C for 2 h. Protein was extracted with

phenol/chloroform and the lysate was precipitated with ethanol.

2.6. RNA isolation and RT-PCR

Total RNA was isolated from 20 days old B.

microplus larvae using the TRIzolTM

reagent as

described by the manufacturer (Gibco-BRL Life

Technologies). Fiveµg of total RNA were submitted to

reverse transcription (RT) before PCR. The RT reaction was done at 37°C in the presence of oligo(dT) (Pharmacia Biotech Inc.) and M-MLV Reverse Tran-scriptase (Gibco-BRL Life Technologies) according to the manufacturer’s instructions. The PCR was performed

using 1 µl of the RT reaction, 10 pmol of each primer

(1+3 or 2+3) and 2.5 U Taq polymerase in a final volume

of 50µl. Samples were denatured for 5 min at 94°C and

amplification was achieved through 30 cycles of 30 s at 94°C, 30 s at 54°C and 30 s at 72°C. Following the last cycle a final extension (72°C, 10 min) was carried out. A parallel cDNA sample with actin-specific primers was also amplified (340 pb) as a positive control.

2.7. Expression of proBmCL1 in E. coli cells and its purification

After induction with 0.3 mM IPTG for 2 h at 37°C, the cells containing pMAL.PROCP were harvested and lysed by freezing and thawing and homogenized in a French Press. Purification of the fusion protein by amyl-ose affinity chromatography was performed according to the manufacturer’s instructions (New England Biolabs). The buffer utilized was 20 mM Tris-HCl, 200 mM NaCl,

1mM EGTA, 1 mM EDTA, 10 mM b-mercaptoethanol

and 1 mM PMSF, pH 7. A pool of the eluted fractions from the affinity chromatography containing the recom-binant BmCL1 proenzyme was applied onto a DEAE-Sepharose CL-6B column (Pharmacia Biotech Inc.) equilibrated with 20 mM Tris-HCl pH 7. The column was eluted with a linear gradient of 100–1000 mM NaCl in 20 mM Tris-HCl pH 7. Fractions were tested for activity using the fluorogenic substrate N-Cbz-Phe-Arg-MCA.

2.8. Polyacrylamide gels containing copolymerized substrates

PAGE was adapted from the method described by Heussen and Dowdle (1980) with the exception that the gel did not contain SDS. After the electrophoretic separ-ation (using 0.15–0.2 pmol protein per slot) in a 10% non-denaturing polyacrylamide gel containing copoly-merized substrates (0.05% gelatin, 0.05% hemoglobin, 0.05% tick vitellin, 0.05% bovine serum albumin or 0.05% ovalbumin), the gels were incubated in 0.1 M sodium acetate buffer (pH 3.5 and pH 5.5) containing 5 mM DTT for 16 h at 37°C. Subsequently, the gels were washed with water and stained in 5% methanol/10%

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acetic acid/water containing 0.1% Coommassie Brilliant Blue R-250.

2.9. Enzyme assays

The mature cysteine proteinase was generated by incubating the purified BmCL1 fusion proenzyme at 37°C for 1 h in 25 mM sodium acetate pH 3.5 and 5 mM DTT. Assays with the activated enzyme were per-formed with chromogenic (0.5 mM) and fluorogenic

(20–30 µM) substrates in a final volume of 100µl

con-taining 20 mM citric acid/54 mM sodium phosphate pH 5.5 and 5 mM DTT. Fluorogenic substrates assays were

done with 51.6 µg/ml (6.79 pmols) of enzyme except

for N-Cbz-Phe-Arg-MCA where 25.8 µg/ml was used.

For the chromogenic substrate, 40µg/ml (5.27 pmol) of

enzyme was used. Fluorogenic assays were monitored by continuous fluorimetry in a fmax Microplate Reader (Molecular Devices Corporation). The wavelength pair for emission and excitation was 320–430 nm. Enzyme-catalyzed release of p-nitroanilide with chromogenic substrates was monitored at 405 nm in a Spectra Max

250 Microtiter Plate Reader (Molecular Devices

Corporation). Optimum pH for enzyme activity was determined using citrate-sodium phosphate buffer in pH range of 2.5–7.5 and H-D-Val-Phe-Lys-pNA as sub-strate.

Active site titration was performed allowing the titrant (E-64) to bind to the enzyme in 20 mM citric acid/54 mM sodium phosphate buffer pH 5.5 containing 5 mM DTT for 15 min before measuring the residual activity

upon N-Cbz-Phe-Arg-MCA (30 µM). Protein

concen-tration was determined by the Bradford method

(Bradford, 1976) using bovine serum albumin as a stan-dard. Values of Kmand Kcatwere determined for selected substrates by fitting initial rates of peptide cleavage at various substrate concentrations to the Michaelis–

Menten equation in the Enzfitter program

(Leatherbarrow, R.J. 1987. Enzfitter. Elsevier Biosoft, Cambridge, England). Specific enzyme activity was

expressed as µmol/min/mg.

2.10. Inhibitor studies

The following known proteinase inhibitors were

tested: E-64 at 50 µM, leupeptin at 0.1 mM, PMSF at

1 mM, EDTA at 10 mM, pepstatin A at 1µM and

anti-pain at 0.1 µM. The inhibitory activity was determined

by measuring the residual enzyme activity upon 0.5 mM H-D-Val-Phe-Lys-pNA after preincubating the activated enzyme for 15 min with the inhibitor.

3. Results and discussion

3.1. Cloning of a DNA fragment corresponding to the conserved region of a cysteine proteinase gene and isolation of a full cDNA clone

Tick larvae cDNA and degenerate oligonucleotides (see Materials and methods) based upon sequences flanking the active site cysteine and asparagine amino acids (Sakanari et al., 1989; Eakin et al., 1990) were used to amplify by PCR a conserved sequence of a cyst-eine proteinase gene of the bovine tick B. microplus. A DNA fragment of approximately 500 bp was obtained and cloned into the pBluescript KS vector. Nucleotide sequence analysis of the cloned fragment revealed a con-served sequence present in other eukaryotic cysteine pro-teinases (results not shown). The cloned fragment, referred to as CP1, was used to probe a B. microplus

cDNA library in λZAPII. Screening of 5×103 _plaques

resulted in the detection of a cDNA clone with an insert of 1123 bp. The sequence is deposited in Genbank datab-ase under accession number AF227957 and is referred to as Bmcl1.

3.2. Sequence analysis of Bmcl1

The total nucleotide sequence of Bmcl1 and its deduced amino acid sequence are shown in Fig. 1. A presumed initial ATG codon (Kozak, 1991) is found 32

nucleotides downstream of the 5₉end. Three stop codons

(nucleotides 997, 1039, 1063), a 94 bp of non-coding region including a presumed polyadenylation signal (Proudfoot and Brownlee, 1976), AATAAA (nucleotides

1065–1070) are found at the 39 end. Within the cDNA

sequence a single long open reading frame is found which encodes a predicted prepropeptide of 332 amino acids (BmCL1). It presents a putative signal peptide (pre-region) formed by 18 amino acid residues, a pro-region containing 97 amino acid residues and a predicted mature enzyme with 217 amino acid residues. The calcu-lated molecular masses for the proprotein and mature enzyme are 34.51 kDa and 23.46 kDa, respectively. Similar molecular sizes are found for other cysteine pro-teinases; examples are: Dictyostelium discoideum (Pears et al., 1985), Fasciola sp. (Yamasaki and Aoki, 1993; Roche et al., 1997), Nephrops norvegicus (Le Boulay et al., 1995) and Toxocara canis (Loukas et al., 1998). Comparing the deduced amino acid sequence with

sequences in the GenBankTM _{we found that BmCL1 is}

most similar to the papain-family of proteinases. The pre-region is composed predominantly of hydrophobic residues characteristic of a signal peptide (Von Heijne, 1983). The putative cleavage point (Von Heijne, 1986) for the release of the signal peptide is located at Ser298 -Ser297

(Fig. 1). Either secreted or lysosomal enzymes of the papain family are synthesized with signal peptides

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Fig. 1. Nucleotide and the corresponding deduced amino acid sequence of B. microplus cysteine proteinase gene (Bmcl1). The regions used for the synthesis of primers 1, 2 and 3 are underlined. The arrows indicate the presumed cleavage sites for the pre and proenzyme, respectively. The asterisk indicates a potential glycosylation site. The conserved residues involved in catalysis are double underlined. In bold is the polyadenil-ation signal.

and have a propeptide at the N-terminus. To protect cells from the potentially disastrous consequences of uncon-trolled degradative activity, essentially all known cellular and bacterial proteolytic enzymes are synthesized as inactive precursors (or zymogens) (Carmona et al., 1996; Khan and James, 1998). Acidic limited proteolysis is necessary for activation of these zymogens (Mason et al., 1987; Rawlings and Barret, 1994). The potential cleavage site of the pro-region of the mature enzyme was

estimated to be between residues Ser21 _{and Leu}1 _(Fig.

1). Many members of the papain family contain a proline residue at position 2 in the mature enzyme. This is also observed in BmCL1. The proline may serve to prevent unwanted N-terminal proteolysis (Rawlings and Barret, 1994). Glycosylation with mannose 6-phosphate has been shown to be an important sorting signal for routing proteins into lysosomes. Glycosylation occurs in lysoso-mal proteinases, including mamlysoso-malian cathepsins B, L,

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S and H, and cathepsins L-like proteinases from proto-zoan parasites (Yamasaki and Aoki, 1993; Kirschke et al., 1993). N-glycosylation sites have been detected also in the pro-region sequence of papain (Vernet et al., 1990), cathepsin S (Kirschke, et al., 1993), cathepsin F (Wang et al., 1998) and in a cysteine proteinase of

Blat-tella germanica (Liu et al., 1996). In some cases it is

located in the C-terminal region of the propeptide (Padilla-Zu´n˜iga and Rojo-Domı´nguez, 1998). A single potential N-linked glycosylation motif was observed in the propeptide region of BmCL1 (Fig. 1). This implies that targeting via mannose-6-phosphate receptor would only be possible for the precursor and not for the mature enzyme.

Like in papain, the recombinant BmCL1 contains the

conserved Cys25, His159 and Asn175 (papain

numbering) residues involved in the catalysis and also the six cysteine residues involved in disulfide bond for-mation (Cys22_{, Cys}56_{, Cys}65_{, Cys}108_{, Cys}157_{and Cys}206_). The sequences around the Cys25_{, His}164_{and Asn}184 resi-dues are also well conserved in cysteine proteinases (Rawlings and Barret, 1994). Cysteine proteinases simi-lar to the mammalian cathepsins L and H contain in their propeptide the ERFNIN motif (Karrer et al., 1993) and a set of conserved amino acids (Ishidoh et al., 1987) not present in cathepsin B-like proteinases. Both character-istics are present in the propeptide region of BmCL1 and are represented by Glu273_{, Arg}269_{, Phe}265_{, Asn}262_, Ile258 _{and Asn}254_{, and by Asn}239_{, Phe}237_{, Gly}236_, Asp235_{, Leu}234_{, Leu}233_{, His}231_{, Glu}230 _{and Phe}229_, respectively. It was suggested that the ERFNIN motif serves to inhibit proteinase activity and that the removal of the propeptide converts the protein into its enzy-matically active form (Karrer et al., 1993). Another motif conserved among cysteine proteinases, including the cathepsin B-like proteinases, is the GCNGG motif. It is invariant with the exception of the central asparagine

residue. This motif is also present in BmCL1 (Gly64_to

Gly68_{) and has a glutamic acid residue in place of} aspara-gine. It was suggested that it has an important structural role (Karrer et al., 1993).

The proteinase sequences found in the GenBank and Swiss-Prot databases highly similar to the proform of BmCL1 were cathepsin L proteinases from several organisms. BmCL1 showed 61.68% identity with

Sarco-phaga peregrina cathepsin L (Q26636), 63.66% with Penaeus vannamei cathepsin L (Q27759), 58.17% with Homarus americanus cysteine protease (P25782),

57.65% with Nephrops norvegicus cathepsin L

(Q27708), 55.06% with Rattus norvegicus cathepsin L (P07154), 53.01% with Sus scrofa peptidase (Q28944) and 60.85% with Bombyx mori (Q26425). At the nucleo-tide level Bmcl1 showed 73.61% identity with

Haema-physalis longicornis cathepsin L-like cysteine proteinase

A mRNA (Ab020492), 67.41% with H. longicornis

cathepsin L-like cysteine proteinase B mRNA

(Ab020491) and 64.25% with P. vannamei cathepsin L mRNA (X99730).

3.3. RT-PCR and genomic DNA amplification

The forward primers 1 or 2 and the reverse primer 3 (see Materials and methods) were used in RT-PCR to analyze the expression of Bmcl1 in 20 day old larvae using total RNA as template. DNA fragments with the predicted size for the proenzyme (950 bp) and for the mature region (660 pb) transcripts were amplified (Fig. 2). Control RT-PCR amplifications using actin-specific primers confirmed sample integrity. The amplification of

Bmcl1 fragments and the CP1 clone from larvae cDNA

(used as probe in the library screening) indicate that this gene is active in this B. microplus developmental stage. Fragments with approximately 950 pb and 660 pb were also obtained when PCR was carried out using tick gen-omic DNA as template and primers 1+3 and 2+3 (Fig. 2). Since the PCR amplified fragments have the same size when cDNA or genomic DNA were used as tem-plate, it can be inferred that large introns are not present within the amplified portion of the Bmcl1 gene.

3.4. Expression of BmCL1 in E. coli

A DNA fragment of approximately 950 bp corre-sponding to the proprotein was amplified by PCR from

Bmcl1 and expressed in E. coli as MBP-fusion protein

with the pMAL-p vector. The expressed fusion protein, BmCL1, has an apparent molecular mass of 76 kDa.

Fig. 2. PCR fragments generated from Bmcl1, genomic DNA and larvae cDNA. Lanes 2 and 5 show the fragments amplified from

Bmcl1, lanes 3 and 6 the fragments amplified from genomic DNA, and

lanes 4, 7 and 8 the fragments amplified from larvae cDNA. The pri-mers used (see material and methods) were 1+3 for the pro-region (lanes 2, 3 and 4), 2+3 for the mature region (lanes 5, 6 and 7) and specific B. microplus actin primers for larvae cDNA control (lane 8). In lane 1 are the molecular weight markers (100 bp ladder, Pharmacia). Samples were submitted to electrophoresis on a 2% agarose gel stained with ethidium bromide.

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After a first purification step using amylose affinity chro-matography, the sample containing the fusion protein still showed some impurities when analyzed on SDS-PAGE. For further purification the sample was applied onto a DEAE Sepharose column. Using a linear gradient from 100–1000 mM NaCl the fusion protein was eluted in a pure form at 300 mM NaCl.

3.5. Enzyme activity assays

The fraction eluted at 300 mM NaCl from the DEAE Sepharose column containing the purified fusion protein, BmCL1, was assayed for its activity upon chromogenic, fluorogenic and protein substrates. On polyacrylamide gels containing copolymerized protein substrates the enzyme was able to hydrolyze gelatin, bovine hemoglo-bin and tick vittelin at pH 3.5 (Fig. 3). It also hydrolyzes gelatin at pH 5.5 but not bovine serum albumin, ovalbu-min, hemoglobin and vittelin (data not shown). Similar findings were described by Brady et al. (1999) for

Schis-tosoma mansoni cathepsin L when hemoglobin was used

as substrate. Controls done with total bacteria (XL1Blue) extracts with and without pMAL-p vector induced under the same conditions did not show any proteolytic activity when tested in polyacrylamide gels containing gelatin (data not shown). On the other hand, with the synthetic

substrate H-D-Val-Phe-Lys-pNA BmCL1 exhibited

activity over a wide pH range (pH 2.5 to 6.5) with

opti-Fig. 3. Substrate gel (zymogram) of the purified recombinant BmCL1. The purified enzyme was subjected to electrophoresis on a 10% polyacrylamide gel containing tick vittelin (1), bovine hemoglo-bin (2) and gelatin (3). The gel strips were incubated in 0.1 M sodium acetate buffer pH 3.5 for 16 hours at 37°C. After staining with Coom-assie Brilliant Blue the strips were destained with 10% acetic acid. Areas of proteolysis appear as clear regions in the gel.

Fig. 4. pH dependence of the B. microplus recombinant cysteine pro-teinase activity. Maximal proteolytic activity was taken as 100%. Sub-strate: H-D-Val-Phe-Lys-pNA. Values are the mean of two determi-nations.

mum activity at pH 5.5 (Fig. 4). No activity was observed at pH 7. Similar pH activity profiles were also shown with cysteine proteinases purified from Xenopus

laevis embryos (Yoshizaki et al., 1998), Blattella germ-anica yolk (Liu et al., 1996), rat liver lysosomes

(Kirschke et al., 1989) and human liver (Mason et al., 1986). This distinguishes them from cathepsin S which is active at neutral pH (Kirschke et al., 1993).

Like most members of the papain family (Rawlings and Barret, 1994; Kirschke et al., 1993; Wang et al., 1998) BmCL1 hydrolyzes substrates containing a bulky

hydrophobic residue in P2 and arginine or lysine in P1

(Table 1). Almost no hydrolysis is observed when a pro-line is in the P2 position (Table 1). This type of result was also shown for other cathepsins L (Mason et al., 1984; Bro¨mme et al., 1989) which also discriminate

Table 1

Activity of the recombinant cysteine proteinase upon chromogenic and fluorogenic peptide substratesa

Peptide Activity

(µmol/min/mg protein)

H-D-Val-Phe-Lys-pNA 0.39

N-Cbz-Phe-Arg-MCA 31.2

N-Cbz-Arg-Arg-MCA 0.43

N-t-Boc-Gly-Arg-Arg-MCA 0.096 N-t Boc-Gln-Ala-Arg-MCA 0.14 N-t-Boc-Val-Pro-Arg-MCA 0.002 N-t-Boc-Glu-Ala-Arg-MCA 0.22 N-t-Boc-Ile-Glu-Gly-Arg-MCA NH N-t-Boc-Glu-Lys-Lys-MCA NH N-t-Boc-Gln-Gly-Arg-MCA NH

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Table 2

Inhibition BmCL1 by various proteinase inhibitors. The purified B.

microplus recombinant cysteine proteinase was preincubated with the

indicated inhibitors and assayed for residual activity using H-D-Val-Phe-Lys-pNA as substrate. Assays were done in duplicate

Inhibitors Specificity Concentration Inhibition % (mM)

E-64 all cysteine proteinases 0.05 100 Leupeptin most cysteine and trypsin- 0.1 100

like serine proteinases

PMSF serine proteinases 1 0

EDTA Metalloproteinases 10 0.1 Pepstatin Asparticproteinases 0.001 2.37 Antipain trypsin-like serine and 0.1 100

some cysteine proteinases

Without – 0 83.3

DTT

against proline in P2 whereas cruzipain is able to

hydrolyze such substrates (Del Nery et al., 1997). BmCL1 was inhibited by E-64, leupeptin and antipain, well known cysteine proteinases inhibitors. In addition, its activity decreased 83.3% in the absence of DTT (5 mM) (Table 2).

Using active site titration, the active BmCL1 concen-tration in the preparation used to measure the kinetic data was calculated to be 0.024 pmol/µl. The total BmCL1 concentration of 0.67 pmol/µl was calculated based on its molecular mass and protein concentration by the method of Bradford (1976). Therefore, 3.5% of the total protein was active. The enzyme efficiently hydrolyses the cathepsin L- and cathepsin B-specific substrate N-Cbz-Phe-Arg-MCA. Moreover, it exhibits minimal activity upon N-Cbz-Arg-Arg-MCA, a diagnostic sub-strate for cathepsin B. The BmCL1 Kcat/Km relationship for N-Cbz-Phe-Arg-MCA hydrolysis was 90.76-fold higher than for the hydrolysis of N-Cbz-Arg-Arg-MCA (Table 3). This specificity, the motifs present in propep-tide and the pH profile reinforce the conclusion that the enzyme is a cathepsin L-like proteinase.

In this paper, we described the cloning, expression and characterization of a tick cysteine proteinase (BmCL1). It is the first report, to our knowledge, that a cysteine proteinase of the cattle tick B. microplus had its

nucleo-Table 3

Reaction kinetics of BmCL1 on chromogenic and fluorogenic pep-tide substratesa

Substrate KmµM Kcats21 Kcat/Km µM21_s21

H-D-Val-Phe-Lys-pNA 282 ND ND N-Cbz-Phe-Arg-MCA 18.8 11.18×102 ₅₉

N-Cbz-Arg-Arg-MCA 23.6 15.5 0.65

a_{ND, not determined.}

tide sequence determined, the corresponding protein expressed in an active form and its substrate specificity characterized. At least one cysteine proteinase has been shown to be effective when used as a vaccine. Actually, a cysteine proteinase confers some immunity in cattle against Fasciola hepatica (Dalton et al., 1996). Also, inhibitors of cysteine proteinases were able to interfere with important biological functions, for example, in

Try-panosoma cruzi (Engel et al., 1998a,b), Plasmodium fal-ciparum (Rosenthal, 1995) and Leishmania major

(Selzer et al., 1999). Therefore, the availability of recom-binant BmCL1 would be helpful on studies concerning its potential as a candidate for the development of new

immunoprophylatic or chemotherapeutic strategies,

alternatives that are being developed for other peptidases (Dalton et al., 1996; McKerrow et al., 1999).

Acknowledgements

We wish to thank Cbiot for the supply of Taq Poly-merase and restriction enzymes and Prof. He´lio M.M. Maia and Luis Paulo da Silva Braga for helpful assist-ance in the French Press use. We are also grateful to Dr. Jair Ribeiro Chagas for his guidance on fluorogenic peptide hydrolysis assays, to Daniel T. Passos for the RNA purifications used in the cDNA library construction and to Carlos Alexandre S. Ferreira for valuable dis-cussion. This work was supported by grants from CAPES, CNPq, PRONEX, PADCT and FAPERGS.

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(1)

and have a propeptide at the N-terminus. To protect cells

from the potentially disastrous consequences of

uncon-trolled degradative activity, essentially all known cellular

and bacterial proteolytic enzymes are synthesized as

inactive precursors (or zymogens) (Carmona et al., 1996;

Khan and James, 1998). Acidic limited proteolysis is

necessary for activation of these zymogens (Mason et

al., 1987; Rawlings and Barret, 1994). The potential

cleavage site of the pro-region of the mature enzyme was

estimated to be between residues Ser

_{and Leu}

_(Fig.

1). Many members of the papain family contain a proline

residue at position 2 in the mature enzyme. This is also

observed in BmCL1. The proline may serve to prevent

unwanted N-terminal proteolysis (Rawlings and Barret,

1994). Glycosylation with mannose 6-phosphate has

been shown to be an important sorting signal for routing

proteins into lysosomes. Glycosylation occurs in

lysoso-mal proteinases, including mamlysoso-malian cathepsins B, L,

(2)

S and H, and cathepsins L-like proteinases from

proto-zoan parasites (Yamasaki and Aoki, 1993; Kirschke et

al., 1993). N-glycosylation sites have been detected also

in the pro-region sequence of papain (Vernet et al.,

1990), cathepsin S (Kirschke, et al., 1993), cathepsin F

(Wang et al., 1998) and in a cysteine proteinase of

Blat-tella germanica (Liu et al., 1996). In some cases it is

located in the C-terminal region of the propeptide

(Padilla-Zu´n˜iga and Rojo-Domı´nguez, 1998). A single

potential N-linked glycosylation motif was observed in

the propeptide region of BmCL1 (Fig. 1). This implies

that targeting via mannose-6-phosphate receptor would

only be possible for the precursor and not for the

mature enzyme.

Like in papain, the recombinant BmCL1 contains the

conserved

Cys25,

His159

and

Asn175

(papain

numbering) residues involved in the catalysis and also

the six cysteine residues involved in disulfide bond

for-mation (Cys

_{, Cys}

108

_{, Cys}

157

_{and Cys}

206

_).

The sequences around the Cys

_{, His}

164

_{and Asn}

184

resi-dues are also well conserved in cysteine proteinases

(Rawlings and Barret, 1994). Cysteine proteinases

simi-lar to the mammalian cathepsins L and H contain in their

propeptide the ERFNIN motif (Karrer et al., 1993) and

a set of conserved amino acids (Ishidoh et al., 1987) not

present in cathepsin B-like proteinases. Both

character-istics are present in the propeptide region of BmCL1 and

are represented by Glu

273

_{, Arg}

269

_{, Phe}

265

_{, Asn}

262

_,

Ile

258

_{and Asn}

254

_{, and by Asn}

239

_{, Phe}

237

_{, Gly}

236

_,

Asp

235

_{, Leu}

234

_{, Leu}

233

_{, His}

231

_{, Glu}

230

_{and Phe}

229

_,

respectively. It was suggested that the ERFNIN motif

serves to inhibit proteinase activity and that the removal

of the propeptide converts the protein into its

enzy-matically active form (Karrer et al., 1993). Another motif

conserved among cysteine proteinases, including the

cathepsin B-like proteinases, is the GCNGG motif. It is

invariant with the exception of the central asparagine

residue. This motif is also present in BmCL1 (Gly

_to

Gly

_{) and has a glutamic acid residue in place of}

aspara-gine. It was suggested that it has an important structural

role (Karrer et al., 1993).

The proteinase sequences found in the GenBank and

Swiss-Prot databases highly similar to the proform of

BmCL1 were cathepsin L proteinases from several

organisms. BmCL1 showed 61.68% identity with

Sarco-phaga peregrina cathepsin L (Q26636), 63.66% with

Penaeus vannamei cathepsin L (Q27759), 58.17% with

Homarus

americanus

cysteine

protease

(P25782),

57.65%

with

Nephrops

norvegicus

cathepsin

L

(Q27708), 55.06% with Rattus norvegicus cathepsin L

(P07154), 53.01% with Sus scrofa peptidase (Q28944)

and 60.85% with Bombyx mori (Q26425). At the

nucleo-tide level Bmcl1 showed 73.61% identity with

Haema-physalis longicornis cathepsin L-like cysteine proteinase

A mRNA (Ab020492), 67.41% with H. longicornis

cathepsin

L-like

cysteine

proteinase

B

mRNA

(Ab020491) and 64.25% with P. vannamei cathepsin L

mRNA (X99730).

3.3. RT-PCR and genomic DNA amplification

The forward primers 1 or 2 and the reverse primer 3

(see Materials and methods) were used in RT-PCR to

analyze the expression of Bmcl1 in 20 day old larvae

using total RNA as template. DNA fragments with the

predicted size for the proenzyme (950 bp) and for the

mature region (660 pb) transcripts were amplified (Fig.

2). Control RT-PCR amplifications using actin-specific

primers confirmed sample integrity. The amplification of

Bmcl1 fragments and the CP1 clone from larvae cDNA

(used as probe in the library screening) indicate that this

gene is active in this B. microplus developmental stage.

Fragments with approximately 950 pb and 660 pb were

also obtained when PCR was carried out using tick

gen-omic DNA as template and primers 1

+

3 and 2

+

3 (Fig.

2). Since the PCR amplified fragments have the same

size when cDNA or genomic DNA were used as

tem-plate, it can be inferred that large introns are not present

within the amplified portion of the Bmcl1 gene.

3.4. Expression of BmCL1 in E. coli

A DNA fragment of approximately 950 bp

corre-sponding to the proprotein was amplified by PCR from

Bmcl1 and expressed in E. coli as MBP-fusion protein

with the pMAL-p vector. The expressed fusion protein,

BmCL1, has an apparent molecular mass of 76 kDa.

Fig. 2. PCR fragments generated from Bmcl1, genomic DNA and larvae cDNA. Lanes 2 and 5 show the fragments amplified from

Bmcl1, lanes 3 and 6 the fragments amplified from genomic DNA, and

(3)

After a first purification step using amylose affinity

chro-matography, the sample containing the fusion protein

still showed some impurities when analyzed on

SDS-PAGE. For further purification the sample was applied

onto a DEAE Sepharose column. Using a linear gradient

from 100–1000 mM NaCl the fusion protein was eluted

in a pure form at 300 mM NaCl.

3.5. Enzyme activity assays

The fraction eluted at 300 mM NaCl from the DEAE

Sepharose column containing the purified fusion protein,

BmCL1, was assayed for its activity upon chromogenic,

fluorogenic and protein substrates. On polyacrylamide

gels containing copolymerized protein substrates the

enzyme was able to hydrolyze gelatin, bovine

hemoglo-bin and tick vittelin at pH 3.5 (Fig. 3). It also hydrolyzes

gelatin at pH 5.5 but not bovine serum albumin,

ovalbu-min, hemoglobin and vittelin (data not shown). Similar

findings were described by Brady et al. (1999) for

Schis-tosoma mansoni cathepsin L when hemoglobin was used

as substrate. Controls done with total bacteria (XL1Blue)

extracts with and without pMAL-p vector induced under

the same conditions did not show any proteolytic activity

when tested in polyacrylamide gels containing gelatin

(data not shown). On the other hand, with the synthetic

substrate

H-D-Val-Phe-Lys-pNA

BmCL1

exhibited

activity over a wide pH range (pH 2.5 to 6.5) with

mum activity at pH 5.5 (Fig. 4). No activity was

observed at pH 7. Similar pH activity profiles were also

shown with cysteine proteinases purified from Xenopus

laevis embryos (Yoshizaki et al., 1998), Blattella

germ-anica yolk (Liu et al., 1996), rat liver lysosomes

(Kirschke et al., 1989) and human liver (Mason et al.,

1986). This distinguishes them from cathepsin S which

is active at neutral pH (Kirschke et al., 1993).

Like most members of the papain family (Rawlings

and Barret, 1994; Kirschke et al., 1993; Wang et al.,

1998) BmCL1 hydrolyzes substrates containing a bulky

hydrophobic residue in P

and arginine or lysine in P

(Table 1). Almost no hydrolysis is observed when a

pro-line is in the P

position (Table 1). This type of result

was also shown for other cathepsins L (Mason et al.,

1984; Bro¨mme et al., 1989) which also discriminate

Table 1

Activity of the recombinant cysteine proteinase upon chromogenic and fluorogenic peptide substratesa

Peptide Activity

(µmol/min/mg protein)

H-D-Val-Phe-Lys-pNA 0.39

N-Cbz-Phe-Arg-MCA 31.2

N-Cbz-Arg-Arg-MCA 0.43

N-t-Boc-Gly-Arg-Arg-MCA 0.096

N-t Boc-Gln-Ala-Arg-MCA 0.14

N-t-Boc-Val-Pro-Arg-MCA 0.002

N-t-Boc-Glu-Ala-Arg-MCA 0.22

N-t-Boc-Ile-Glu-Gly-Arg-MCA NH

N-t-Boc-Glu-Lys-Lys-MCA NH

N-t-Boc-Gln-Gly-Arg-MCA NH

(4)

Table 2

Inhibition BmCL1 by various proteinase inhibitors. The purified B.

microplus recombinant cysteine proteinase was preincubated with the

indicated inhibitors and assayed for residual activity using H-D-Val-Phe-Lys-pNA as substrate. Assays were done in duplicate

Inhibitors Specificity Concentration Inhibition % (mM)

E-64 all cysteine proteinases 0.05 100 Leupeptin most cysteine and trypsin- 0.1 100

like serine proteinases

PMSF serine proteinases 1 0

EDTA Metalloproteinases 10 0.1

Pepstatin Asparticproteinases 0.001 2.37 Antipain trypsin-like serine and 0.1 100

some cysteine proteinases

Without – 0 83.3

DTT

against proline in P

whereas cruzipain is able to

hydrolyze such substrates (Del Nery et al., 1997).

BmCL1 was inhibited by E-64, leupeptin and antipain,

well known cysteine proteinases inhibitors. In addition,

its activity decreased 83.3% in the absence of DTT (5

mM) (Table 2).

Using active site titration, the active BmCL1

concen-tration in the preparation used to measure the kinetic data

was calculated to be 0.024 pmol/

µ

l. The total BmCL1

concentration of 0.67 pmol/

µ

l was calculated based on

its molecular mass and protein concentration by the

method of Bradford (1976). Therefore, 3.5% of the total

protein was active. The enzyme efficiently hydrolyses

the cathepsin L- and cathepsin B-specific substrate

N-Cbz-Phe-Arg-MCA. Moreover, it exhibits minimal

activity upon N-Cbz-Arg-Arg-MCA, a diagnostic

sub-strate for cathepsin B. The BmCL1 K

cat

/K

relationship

for N-Cbz-Phe-Arg-MCA hydrolysis was 90.76-fold

higher than for the hydrolysis of N-Cbz-Arg-Arg-MCA

(Table 3). This specificity, the motifs present in

propep-tide and the pH profile reinforce the conclusion that the

enzyme is a cathepsin L-like proteinase.

In this paper, we described the cloning, expression and

characterization of a tick cysteine proteinase (BmCL1).

It is the first report, to our knowledge, that a cysteine

proteinase of the cattle tick B. microplus had its

nucleo-Table 3

Reaction kinetics of BmCL1 on chromogenic and fluorogenic pep-tide substratesa

Substrate KmµM Kcats21 Kcat/Km

µM21_s21

H-D-Val-Phe-Lys-pNA 282 ND ND

N-Cbz-Phe-Arg-MCA 18.8 11.18×102 ₅₉

N-Cbz-Arg-Arg-MCA 23.6 15.5 0.65

a_{ND, not determined.}

tide sequence determined, the corresponding protein

expressed in an active form and its substrate specificity

characterized. At least one cysteine proteinase has been

shown to be effective when used as a vaccine. Actually,

a cysteine proteinase confers some immunity in cattle

against Fasciola hepatica (Dalton et al., 1996). Also,

inhibitors of cysteine proteinases were able to interfere

with important biological functions, for example, in

Try-panosoma cruzi (Engel et al., 1998a,b), Plasmodium

fal-ciparum (Rosenthal, 1995) and Leishmania major

(Selzer et al., 1999). Therefore, the availability of

recom-binant BmCL1 would be helpful on studies concerning

its potential as a candidate for the development of new

immunoprophylatic

or

chemotherapeutic

strategies,

alternatives that are being developed for other peptidases

(Dalton et al., 1996; McKerrow et al., 1999).

Acknowledgements

We wish to thank Cbiot for the supply of Taq

Poly-merase and restriction enzymes and Prof. He´lio M.M.

Maia and Luis Paulo da Silva Braga for helpful

assist-ance in the French Press use. We are also grateful to

Dr. Jair Ribeiro Chagas for his guidance on fluorogenic

peptide hydrolysis assays, to Daniel T. Passos for the

RNA purifications used in the cDNA library construction

and to Carlos Alexandre S. Ferreira for valuable

dis-cussion. This work was supported by grants from

CAPES, CNPq, PRONEX, PADCT and FAPERGS.

References