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Comparative Biochemistry and Physiology, Part B 149 (2008) 599 – 607 www.elsevier.com/locate/cbpb
BYC, an atypical aspartic endopeptidase from Rhipicephalus (Boophilus) microplus eggs Maria Clara L. Nascimento-Silva a , Alexandre T. Leal b , Sirlei Daffre c , Luiz Juliano d , Itabajara da Silva Vaz Jr. b , Gabriela de O. Paiva-Silva a , Pedro L. Oliveira a , Marcos Henrique F. Sorgine a,⁎ a
Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil b Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil c Departamento de Parasitologia, Instituto de Ciencias Biomedicas, Universidade de Sao Paulo, Sao Paulo, Brazil d Departamento de Biofisica, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil Received 24 August 2007; received in revised form 13 December 2007; accepted 14 December 2007 Available online 28 December 2007
Abstract An aspartic endopeptidase was purified in our laboratory from Rhipicephalus (Boophilus) microplus eggs [Logullo, C., Vaz, I.S., Sorgine, M.H., Paiva-Silva, G.O., Faria, F.S., Zingali, R.B., De Lima, M.F., Abreu, L., Oliveira, E.F., Alves, E.W., Masuda, H., Gonzales, J.C., Masuda, A., and Oliveira, P.L., 1998. Isolation of an aspartic proteinase precursor from the egg of a hard tick, Rhipicephalus (Boophilus) microplus. Parasitology 116, 525–532]. Boophilus yolk cathepsin (BYC) was tested as component of a protective vaccine against the tick, inducing a significant immune response in cattle [da Silva, V.I., Jr., Logullo, C., Sorgine, M., Velloso, F.F., Rosa de Lima, M.F., Gonzales, J.C., Masuda, H., Oliveira, P.L., and Masuda, A., 1998. Immunization of bovines with an aspartic proteinase precursor isolated from Rhipicephalus (Boophilus) microplus eggs. Vet. Immunol. Immunopathol. 66, 331–341]. In this work, BYC was cloned and its primary sequence showed high similarity with other aspartic endopeptidases. In spite of this similarity, BYC sequence shows many important differences in relation to other aspartic peptidases, the most important being the lack of the second catalytic Asp residue, considered to be essential for the catalysis of this class of endopeptidases. When we determined BYC cleavage specificity by LC-MS, we found out that it presents a preference for hydrophobic residues in P1 and P1' in accordance to most aspartic endopeptidases. Also, when analyzed by circular dicroism, BYC presented high β sheet content, also a characteristic of aspartic endopeptidases. On the other hand, although both native and recombinant BYC are catalytically active, they present a very low specific activity, what seems to indicate that this peptidase will digest its natural substrate, vitellin, very slowly. We speculate that such a slow Vn degradative process might constitute an important strategy to preserve egg protein content to the hatching larvae. © 2007 Elsevier Inc. All rights reserved. Keywords: Rhipicephalus microplus; Aspartic endopeptidases; Catalytic mechanism; Vitellin degradation
1. Introduction The hard tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) is a bovine ectoparasite responsible for great econom-
⁎ Corresponding author. Universidade Federal do Rio de Janeiro, Centro de Ciências da Saúde, Instituto de Bioquímica Médica, Bloco D-Sala SS05-Ilha do Fundão, RJ-CEP: 21.941-590 Brazil. Tel.: +55 21 25626751. E-mail address:
[email protected] (M.H.F. Sorgine). 1096-4959/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2007.12.007
ical losses in tropical and subtropical areas of the globe, such as Central and South America and Australia. Besides the damage caused by blood feeding, R. microplus is also the vector of several lethal cattle diseases, such as babesiosis and anaplasmosis (Jongejan and Uilenberg, 2004). Endopeptidases form a large protein functional group and account for almost 2% of all sequenced genes in eukaryotic genomes (Barrett et al., 1998). The knowledge about aspartic endopeptidases catalytic mechanism is very limited when compared to other endopeptidases. The exact mechanism by which the
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hydrolysis reaction takes place is not completely understood, but it is known that, in the catalytic pocket of aspartic endopeptidases, two aspartic acid residues are responsible for binding water molecules, essential for the cleavage of the peptide-bond. Structural characterization of retropepsins, the aspartic endopeptidases of retroviruses, provided support to the concept that the two aspartic acid residues are essential for peptide-bond hydrolysis by aspartic endopeptidases. Since retropepsins have only one Asp residue in each polypeptide chain, this enzyme only works as a homodimer (Lapatto et al., 1989). In this work, the cDNA coding for BYC was cloned by RTPCR and the analysis of its deduced amino acid sequence confirmed great similarity with other aspartic endopeptidases. However, BYC sequence showed a unique feature, which was the lack of the highly conserved second Asp residue found in the active site of this class of endopeptidases. We have tried to define molecular aspects of the protein catalysis for this unique enzyme and speculate about the consequence of the lack of one Asp catalytic residue to the enzyme role in yolk degradation and tick development.
(120 × 2.4 cm) equilibrated with 0.15 M NaCl, 10 mM Tris–HCl buffer, pH 7.2. Protein purity was monitored by SDS-PAGE. 2.5. cDNA cloning and analysis
Eggs were homogenized in a Potter-Elvehjem tissue grinder in 20 mM Tris–HCl buffer, pH 7.4, with soybean trypsin inhibitor (20 μM), leupeptin (10 μM), and benzamidine (20 μM). Egg homogenates were centrifuged at 45,000 g for 60 min at 4 °C. The floating lipids and the pellet were discarded, and the crude egg extract supernatant was used for protein isolation.
Total RNA from fat bodies of engorged female ticks was purified using TRIzol reagent (Invitrogen Inc.). Five micrograms of total RNA were reverse transcribed using the ‘Superscript preamplification system’ (Invitrogen Inc). A degenerated primer (CGNAARGAYCGNATHATHATG) based on BYC aminoterminal sequence (Logullo et al., 1998) was used together with a NotI-(dT)18 primer (Amersham Pharmacia Biotech) to amplify BYC cDNA in a 35 cycles PCR reaction (94 °C for denaturation, 55 °C for annealing and 70 °C for extension) using Pfx DNA polymerase. The PCR product was analyzed in 1% agarose gel and extracted using “Concert Rapid Gel Extraction System” (Life Technologies Inc.). This product was cloned into a pT7Blue-3 vector using the Perfectly Blunt TM cloning kit (Novagen Inc.), according to the manufacturer's instructions. DNA sequencing was performed using the dideoxy method at the Molecular Genetics Instrumentation Facility of the University of Georgia. Several clones produced in different PCR and cloning reactions were sequenced. The obtained sequences were subjected to similarity searches in non-redundant sequence databases such as NCBI (www.ncbi.nlm.nih.gov/BLAST). Multiple alignments were made using ClustalW (www.expasy.org) (Thompson et al., 1994) and T-coffee, version 1.4(http://www.ch.embnet.org/ software/TCoffee.htmL) (Notredame et al., 2000). Physicochemical and structural features were analyzed using the following programs: Prot-Param (www.expasy.org), for general parameters (Guruprasad et al., 1990); SignalP 3.0 (http://www. cbs.dtu.dk/services/SignalP/) for signal peptide cleavage site identification; PROSITE search (www.expasy.org) for secondary structure prediction (Bairoch et al., 1997); SwissModel First Approach Mode (http://www.expasy.org/spdbv/) for secondary structure comparison and alignment and SwissModel First Approach Mode (http://www.expasy.org/spdbv/) for tertiary structure modeling (Peitsch and Tschopp, 1995; Guex and Peitsch, 1997; Schwede et al., 2003).
2.3. Electrophoresis
2.6. Recombinant BYC
Polyacrylamide gels (10%) were run in the presence of SDS at a constant current of 20 mA. Gels were stained with Coomassie Blue G according to the method of Neuhoff et al., 1988. and destained with deionized water.
The amino acid sequence of the BYC clone obtained in this work was incomplete when compared to the sequence determined by Logullo et al., 1998, since the six N-terminal amino acids were absent. In order to express recombinant BYC, the full coding sequence was restored by PCR and sub-cloned into a pET32b (Novagen) vector, producing a recombinant protein fused with histidine tagged thioredoxin protein (rBYC-trx) (Leal et al., 2006). Recombinant BYC (rBYC-trx) was expressed in inclusion bodies. The inclusion bodies were purified as described by Marks et al., 2001. Briefly, the induction was made using 1 mM IPTG in 250 mL of LB medium for 6 h at 37 °C. Cultures were centrifuged at 3000 ×g for 10 min at 4 °C, the cell pellet was incubated in KTE (50 mM Tris pH 8, 100 mM KCl, 1 mM
2. Materials and methods 2.1. Animals R. (B.) microplus of Porto Alegre strain, were reared on calves obtained from a tick-free area and maintained at the Faculdade de Veterinaria of the Universidade Federal do Rio Grande do Sul, Brazil. Engorged adult females were kept in Petri dishes at 28 C and 85% relative humidity. Eggs were collected at the first day after oviposition and were frozen at − 70 C until use. 2.2. Egg homogenates
2.4. Native BYC purification BYC was purified as previously described (Logullo et al., 1998). Briefly, first-day egg homogenate was applied to an anion exchange DEAE-Toyopearl (20 × 3 cm) column equilibrated with 10 mM Tris–HCl, pH 8.4 and eluted with a NaCl gradient (0 to 0.3 M NaCl) at a 1 mL/min flow. Fractions containing BYC (identified by SDS-PAGE) were concentrated in a Speed-Vac system (Savant) and applied to a Sephacryl S-200 column
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EDTA) with 5 μg/mL DNAase for 10 min and sonicated on ice. After that, the preparation was centrifuged at 15,000 ×g for 15 min at 4 °C and washed with KTE. This procedure was repeated four times. The obtained inclusion bodies were washed twice in 20 mM Tris pH 8.5, 2.5 mM EDTA, 5 mM imidazole, 1% Triton X-100, centrifuged at 27,000 ×g for 10 min at 4 °C and solubilized in 15 mL 0.3% N-laurosyl sarcosine with 50 mM CAPS, pH 11, and 1 mM DTT for 30 min at room temperature. After that, the preparation was centrifuged at 11,000 ×g for 15 min at 4 °C and the supernatant was collected and dialyzed at 4 °C against 0.15 M NaCl, 20 mM Tris–HCl, pH 8.0, using three refolding steps, 4 h per step: 1) 0.1 mM DTT 2) buffer only 3) with 0.2 mM oxidized glutathione and 1 mM reduced glutathione. Protein purity was monitored by SDS-PAGE. 2.7. Proteolytic activity To determine peptidase activity, both native and recombinant BYC were incubated in 0.1 M sodium acetate buffer, pH 3.5, at 37 °C with its substrates. Abz-peptidyl-EDDnp fluorogenic substrates (6 μM) were incubated with 0.5 μM of the enzyme and the reactions were monitored after 12 h by measuring the fluorescence (excitation at 320 nm wavelength, emission at 420 nm) with a Cary Eclipse spectrofluorometer.
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2.8. Hemoglobin hydrolysis and BYCs cleavage specificity analysis by LC-MS To follow hydrolysis of hemoglobin, 20 μg of native BYC and 20 μg of bovine hemoglobin were incubated in 0.1 M sodium acetate buffer pH 3.5, at 37 °C for different times. Reactions were stopped by the addition of 3 μl of SDS sample buffer to the 15 μl of the reaction mixture and the samples were immediately boiled for 3 min. Hemoglobin hydrolysis was observed by SDS-PAGE. For analysis of cleavage specificity, 50 μg of native BYC and 65 μg of bovine hemoglobin were incubated in 0.1 M sodium acetate buffer pH 3.5, at 37 °C for 12 h in a 50 μl reaction. This reaction was diluted 10 times in 0.05% TFA and fractioned in a C18 capillary HPLC column equilibrated in 5% acetonitrile, 0.05% TFA and eluted by a 5–80% acetonitrile gradient. This column was coupled to a Finnigan LCQ Duo ion Trap mass spectrometer. Data was obtained with 3 s intervals in a 150–2000 m/z range. The sequenced fragments were analyzed by Turbo Sequest (Thermo Finnigan) software, using bovine proteins database. 2.9. Circular dichroism CD spectra were made at room temperature in a Jasco J-715 spectropolarimeter, between 190–260 nm, using a 1 mm cuvette
Fig. 1. BYC cloning. BYC complete nucleotide and amino acid sequence. The box marks the region corresponding to the primer used for PCR. The shaded region corresponds to the pre-pro-region. The asterisk corresponds to the cleavage site of the pre-pro-BYC, predicted using SignalP, which matches the amino terminal determined by Edmann degradation (Logullo et al., 1998).
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and 0.05 mg/mL of BYC, in 0.10 M sodium acetate buffer, pH 3.5. 2.10. Fluorogenic synthetic peptides All fluorogenic peptides used in this work were synthesized as previously described (Hirata et al., 1991). 3. Results 3.1. BYC cloning The amino-terminal sequence, obtained by Edman degradation of native BYC (Logullo et al., 1998), was used to construct a degenerated primer (CGNAARGAYCGNATHATHATG) that, together with a NotI-(dT) 18 primer, was used to clone BYC from tick fat body RNA (GenBank accession no. AY966003). The obtained cDNA was Blasted against B. microplus sequences available at The Institute for Genomic Research (TIGR) Gene Index Project (http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/ gimain.pl?gudb=b_microplus) and a sequence named BEACL02 was identified as identical to BYC, containing not only its coding sequence but also its 5'UTR. When translated, the obtained sequences encodes a protein of 375 amino acids (41,844.6 Da) with a pI of 5.38 (Fig. 1). When this cDNA sequence was used to perform a search in NCBI Blastp database, some of the closest obtained identities were with aspartic
endopeptidases from Schistossoma mansoni (GenBank accession no. SMU60995) and Aedes aegypti (GenBank accession no. H477377) (34% and 35%, respectively), which were aligned with BYC in Fig. 2. 3.2. BYC sequence analysis When BYC sequence was compared with other aspartic endopeptidases, we noticed some important differences in the amino-acids of its active site (Fig. 2). The substrate binding S2 sub-site, in the carboxy-end of BYC amino acid sequence was not preserved, as well as the nearby conserved cysteine pair. But the most important change was the absence of the second aspartic acid residue, highly conserved in this enzyme class. As previously described, aspartic endopeptidases have two aspartic residues, responsible for binding the water molecules in its active site, leading to substrate hydrolysis (Barrett et al., 1998). 3.3. Secondary and tertiary structure prediction BYC amino acid sequence was analyzed using Predictprotein (Columbia University) and SWISS-MODEL software for secondary structure prediction. The higher identity matches in SWISS-MODEL template database were with rennin, from Mus musculus (code 1smrA, 32% identity) and Homo sapiens (code 1hrnA, 33% identity). The predicted secondary structure
Fig. 2. Alignment of BYC amino acid sequence with Aedes aegypti (GenBank accession no. H477377) and Schistossoma mansoni (GenBank accession no. SMU60995) aspartic endopeptidases. The structural and functional domains are assigned: Conserved loops are shaded, catalytic aspartic residues are in gray boxes marked with an arrow; S2 substrate subsite is assigned with a dark star; S3 substrate subsite is assigned with a white star and Cys residues with arrowheads.
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Fig. 3. Circular Dichroism of BYC. BYC presented a β-sheet typical profile, with a negative peak at 216 nm.
showed a predominance of β-sheet, which was in accordance with the circular dichroism spectra of native BYC, which displayed a typical β-sheet profile, with a negative peak at 216 nm (Fig. 3). Using the alignments with M. musculus and H. sapiens rennins we obtained a tertiary structure model for BYC, using the “first approach” mode of Swiss-Model. Besides, another alignment was done, using T-Coffee software, having human recombinant renin (PDB- 2Ren) as template. From this second alignment, another BYC model was made, using the “Alignment Interface” mode of Swiss-Model. Both models were analyzed on RasTop and SPDBV software and the main residues were located in nearly identical positions. The comparison of BYC model with renin structure showed a serine residue in BYC sequence that would be in the same position occupied by one of the rennin catalytic asp residues, suggesting a role for this amino acid in BYC catalytic mechanism (Fig. 4).
Fig. 5. Bovine hemoglobin hydrolysis by BYC. SDS PAGE (20%) of BYC incubated with hemoglobin for 12 h. 1-BYC and hemoglobin without incubation, 2-acidic medium pH 3.5, 3-acidic medium in the presence of pepstatin A and 4-neutral medium.
3.4. Determination of cleavage specificity by LC-MS Native BYC activity was tested against hemoglobin, a classic substrate for aspartic endopeptidases (Fig. 5). When incubated in acidic medium for 12 h, purified native BYC was able to hydrolyze hemoglobin. However, when incubated in neutral medium or in presence of pepstatin A, an aspartic endopeptidase inhibitor, proteolysis was totally abolished. Considering that BYC is an active aspartic endopeptidase that lacks an important residue in its catalytic site, we wanted to determine its cleavage specificity. BYC (20 μg) was incubated
Fig. 4. BYC Structure prediction. (A) The prediction was made using first approach mode, SWISS-MODEL. Human recombinant renin PDB — 2ren_ two catalytic Asp-residues are shown in (B) and BYC residues that align in the active region are shown in (C).
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Fig. 6. Clevage specificity analysis. (A) Cleavage sites identificed by “⁎” in α and β chains of bovine hemoglobin by overnight 15 h incubations with BYC were determined by mass spectrometry LC-MS (B) Cleavage profile of the fluorogenic peptides by BYC after a 20 h incubation, pH 3,5 and 37 °C. Results are indicated as means ± S.E.M. of three independent experiments.
with hemoglobin (20 μg) for 15 h. The reaction mixture was analyzed in a HPLC capillary C18 column coupled to a mass spectrometer (LC-MS). The proteolysis products were partially sequenced and analyzed using a bovine protein database. It was possible to identify 17 different BYC cleavage sites in hemoglobin sequence (Fig. 6A).
LARNF and AIAFFSRQ); eight peptides presented a lower cleavage rate (LERMFLSP, AEALERMF, SHSLLVTL, LVTLASHL, TAFWGKV, LGRLLVVY, TQRFFESF and GRLLVVYP) and the other four were not susceptible at all to proteolysis by BYC (LDKFLANV, STVLTSKY, FLANVSTV and LQADFQKV) (Fig. 6B).
3.5. Fluorogenic synthetic peptides
3.6. Recombinant BYC activity
Based on the cleavage profile obtained by the LC-MS analysis, 14 synthetic peptides with 8 amino acids were made (from P4 to P4'). In these peptides, P1 and P1' were the probable cleavage site determined by bovine hemoglobin proteolysis (Fig. 6A). One more peptide was synthesized, using a consensus hydrolysis sequence for aspartic endopeptidases (peptide A, AIAFFSRQ). All the synthetic peptides were incubated with native BYC for 48 h and the reaction medium fluorescence was registered at different times. When the fluorescence values obtained were analyzed, three peptides were more efficiently cleaved than the others, reaching the maximum of fluorescence after 20 h of incubation (HSLLVTLA, VVV-
Bacteria containing pET-32b/BYC (Leal et al., 2006) were grown in LB medium with IPTG. The recombinant BYC, fused with histidine tagged thioredoxin (Trx) was expressed in inclusion bodies, which were purified to obtain rBYC-Trx. After refolding, the extraction product presented the expected molecular mass for rBYC-Trx (50 kDa ) when observed by SDS PAGE (Fig. 7A). Recombinant BYC activity was measured using the synthetic peptide A, consensus for aspartic endopeptidases, in the same conditions as the native protein. Recombinant BYC was able to hydrolyze peptide A and its activity was abolished in the presence of pepstatin A, a specific aspartic endopeptidase
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Fig. 7. Recombinant BYC is proteolytic active (A) SDS PAGE (10%) of recombinant BYC 1-Molecular mass marker and 2-purified inclusion bodies. The arrow indicates BYC. (B) Synthetic peptide A fluorescence when incubated with recombinant BYC in the absence (1) or presence (2) of pepstatin A. Results are representative of three independent experiments.
inhibitor (Fig. 7B). Bacterial extracts containing only PET32b plasmid showed no activity under these conditions (data not shown). 4. Discussion 4.1. An atypical aspartic endopeptidase BYC amino terminal sequence (Logullo et al., 1998) was used to design a degenerated primer to clone its cDNA by RT-PCR (Fig. 1). The first amino-acids and the 5'UTR of the gene were further obtained by blasting the obtained sequence against the Boophilus microplus EST database at the TIGR Gene Index Project. BYC cDNA sequence (Fig. 1) was compared to other aspartic endopeptidases. BLASTp analysis showed great sequence identities (about 35%) with arthropods endopeptidases, and with those of Schistossoma mansoni (34%). Despite overall sequence similarity, BYC showed important differences towards these typical aspartic endopeptidases (Fig. 2). The most important is the absence of the second Asp residue from the catalytic site. Since present knowledge about the reaction mechanism of aspartic endopeptidases establishes that two conserved Asp residues must bind the water molecules responsible for hydrolysis of substrate peptide bond, we wonder how BYC could preserve proteolytic activity even in the lack of this residue of aspartic acid. A few reports on aspartic endopeptidases that also lack important residues in their active sites can be found in the literature and include mammal PSPs, cockroach proteins and an aspartic peptidase from Plasmodium falciparum. The Pregnancy Specific Proteins (PSPs) are found in placenta of ungulate mammals and are described as inactive members of the aspartic endopeptidase family (Xie et al., 1991). PSPs present several modifications in its catalytic site, among which the most relevant is the absence of the second Asp residue, a feature that is also observed in BYC sequence. These proteins
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are proposed to have no catalytic activity, in spite of being capable of binding peptides (up to 7 amino acids) to its active site (Butler et al., 1982). Lma-p54 is a surface protein present in adult Leucophaea maderae and proposed to be closely related to aspartic endopeptidases. However, some amino-acid substitutions at the active site seem to result in the absence of enzymatic activity. Proteolysis assays performed in conditions close to those observed in the natural secretions suggested that Lma-p54 is not active on the insect body surface (Cornette et al., 1991). The most similar protein to Lma-p54 is the cockroach allergen Bla g 2, of B. germanica (Arruda et al., 1995) (48.5% amino acid Identity). The structure of Bla g 2, obtained using recombinant enzyme expressed in Pichia pastoris revealed that this protein has a bilobal fold with a large cleft located between the two domains, as expected for aspartic endopeptidases. However, four critical amino-acid substitutions in the active site and in the flap region cause important modifications in the orientation of the aspartate residues important to its catalytic mechanism (Gustchina et al., 2005). No enzymatic activity was measured using milk-clotting and hemoglobin assays but a residual proteolytic activity was found at high protein concentrations (approximately 4 mM), suggesting that proteolysis may not be the primary function of this allergen. (Wünschmann et al., 2005). Despite a very low activity, both Bla g 2 and PSPs are capable of biding ligands to its active site (Butler et al., 1982, Wünschmann et al., 2005) and their role as a ligand-binding or carrier molecules are under investigation. Another aspartic endopeptidase presenting a modification in its catalytic site has been characterized. HAP (Histo-aspartic endopeptidase) is an aspartic endopeptidase located in the digestive vacuole of Plasmodium falciparum that presents a His residue in the place of the first Asp catalytic residue (Berry et al., 1999). This substitution allows the enzyme to be active, although it results in a change of the enzyme optimum pH towards more neutral instead of acidic ones. This data shows that other amino-acids can also bind the water molecule, performing role of at least one of the aspartic acid residues in hydrolysis of substrate peptide bonds. Nevertheless, it is important to notice that, in the case of BYC, a change in the enzyme optimum activity pH is not observed, being its activity maximum at acidic pHs, similar to all the other “classical” aspartic endopeptidases (Fig. 5). An alternative explanation would be that BYC, as well as HIV retropepidase, works as a dimmer, using only one aspartic acid residue from each subunit to break the peptide bond. Nevertheless, all molecular weight determinations, by native PAGE or gel exclusion chromatography, showed no evidence of such fact and, indeed, indicate that BYC works as a monomer (Logullo et al., 1998). Comparison of BYC amino acid sequence with other aspartic endopeptidases shows that the region around the first Asp residue is highly conserved, including the Gln residue of the S3 sub-site, important in substrate binding. In contrast, the region near the position of the missing Asp residue of BYC is also different from other aspartic endopeptidases since a sequence similar to the whole S2 sub-site is not found in the tick peptidase.
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Several studies with vertebrate and fungi aspartic endopeptidases suggested that S2 and S3 sub-sites are both essential for peptidase-substrate interaction (Dunn et al., 1986; Dunn et al., 1987 and Wong et al., 1997). Vertebrate cathepsins D present eight conserved loops, some of them involved in maturation and processing of precursor polypeptides. Loop 3 is responsible for a cleavage that leads to dimer formation, stabilizing the tertiary structure of the enzyme (Yonezawa et al., 1988). Some enzymes do not have this loop but, instead, present other cleavage site or never form a dimer. BYC and the peptidases that alignment better with it did not present loop 3 (Fig. 2). Secondary structure prediction using BYC amino acid sequence indicates a high β sheet content, a known characteristic of aspartic endopeptidases (Davies, 1990). This prediction was in agreement with circular dichroism spectra of native BYC (Fig. 3). When BYC was compared to other aspartic endopeptidases that already have its tertiary structure determined (SWISS-PROT database), it presented higher similarity with rennins. Based on the structure of human rennin, a model for BYC tertiary structure was created, making it possible to compare the different amino acid positions in the active site region (Fig. 4). The residue that better fits the second Asp position, in this model, was a serine. The lack of a cysteine pair in this region could lead to a modified conformation in comparison to other aspartic endopeptidases, suggesting that only a complete resolution of BYC structure, together with a site-directed mutagenesis study, could answer this question. 4.2. BYC activity, cleavage specificity and importance to the tick larvae Brindley et al., 1997, studied carefully the cleavage specificity of the cathepsin D — like aspartyl endopeptidases of S. mansoni and S. japonicum. Using human hemoglobin as substrate they found 15 and 13 cleavages sites, respectively. Both enzymes showed preference for hydrophobic residues in P1 and P1', specially leucine and phenylalanine. BYC cleavage specificity analysis by LC-MS identified seventeen cleavage sites in bovine hemoglobin (Fig. 6A). Ten of these sites were in hemoglobin α chain and seven in β chain. Among them, thirteen had leucine or phenylalanine in P1 and six in P1'. This profile is very similar to the one exhibited by S. mansoni cathepsin D when assayed against human hemoglobin (Brindley et al., 1997). Furthermore, BYC shares six of the thirteen human cathepsin D cleavage sites in bovine hemoglobin (Fruitier et al., 1998). The cleavage profile of synthetic peptides by BYC confirmed the enzyme preference for hydrophobic and aromatic residues in P1 and P1' (Fig. 6B). These results showed that BYC displays a typical cathepsin D-like cleavage primary specificity, although lacking an important residue in its active site. To exclude the possibility that BYC previously demonstrated activity (Logullo et al., 1998) was due to a minor contamination of the preparation by another egg aspartic endopeptidase, such as THAP (Sorgine et al., 2000), we expressed the protein in E. coli system (Leal et al., 2006). After purification, activity was tested using synthetic peptide A, showing that the recombinant enzyme is proteolytically active (Fig. 7) and shows the same
cleavage specificity of the native enzyme. For these reasons, we believe that BYC, as described here, might constitute a novel aspartic endopeptidase with a unique catalytic mechanism. Further studies, particularly using single point mutations, are still necessary to better elucidate its action mechanism. Vitellins (Vns) are the major storage proteins of arthropod eggs and are degraded during embryogenesis to provide raw materials/substrates for embryo development (Postlethwait and Giorgi, 1985). BYC is associated with yolk degradation in R. microplus (Abreu et al., 2004). Logullo et al., 2002, showed that only one third of total Vn is degraded during the embryogenesis of R. microplus – which takes about three weeks from oviposition to hatching – suggesting that this is a very slow and controlled process. Besides BYC, two other yolk endopeptidases from R. (B.) microplus eggs have already been described: a cysteine endopeptidase VTDCE (vitellin degrading cysteine endopeptidase) (Seixas et al., 2003) and another aspartic endopeptidases, THAP (Tick Heme-binding Aspartic endopeptidase) (Sorgine et al., 2000). If the activity of these peptidases were not tightly controlled, Vn would probably be degraded much faster than it actually occurs in vivo. Therefore, the mechanisms by which each one of these enzymes is regulated are not only extremely important for a better understanding of embryogenesis in the tick, but also represents a very interesting model to increase our understanding on substrate-enzyme interaction and cleavage specificity. One important issue about BYC activity is that significant levels of activity can only be detected after long incubations in high enzyme/substrate molar ratios, sometimes close to 1:1, indicating that the enzyme possesses a very low specific activity. It seems much likely that this low specific activity be due to the lack of the second Asp catalytic residue. BYC is the most abundant endopeptidase in R. microplus eggs. When determined by densitometry of SDS gels, this protein represents approximately 6% of egg total protein content (data not shown). Considering that Vn may constitute up to 90% of the egg protein content (Logullo et al., 2002), one may conclude that the physiological molar ratio of BYC to Vn in the egg is of approximately 0.6 to 1, which is probably one of the highest enzyme to substrate ratios ever described. In this way, if BYC had a high specific activity, Vn would be degraded too soon, a fact that cannot happen since by the time of hatching only 40% of the original Vn content has been consumed and the rest of this protein is necessary to sustain the larvae before it finds a new host (a process that can take a few months) (Logullo et al., 2002). In this way, we believe that the presence of an endopeptidase with high specificity and low activity might constitute an important strategy developed by the tick to spare egg energetic resources for the first periods of larvae existence. Acknowledgements We are grateful to Dr. Misao Onuma for cooperation in cloning experiments and S R Cassia for technical assistance. This work was supported by grants from HHMI, PRONEX and CNPq.
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