Two Cathepsins B Are Responsible for the Yolk Protein Hydrolysis in Culex quinquefasciatus

Despite the established role of Culex quinquefasciatus as a vector of various neurotropic viruses, such as the Rift Valley and West Nile viruses, as well as lymphatic filariasis, little is known regarding the organism’s reproductive physiology. As in other oviparous animals, vitellogenin, the most important source of nutrients for the embryo development, is digested by intracellular proteases. Using mass spectrometry, we have identified two cathepsin B homologues partially purified by self-proteolysis of Cx. quinquefasciatus total egg extract. The transcriptional profile of these two cathepsin B homologues was determined by quantitative RT-PCR, and the enzymatic activity associated with the peptidase was determined in ovaries after female engorgement. According to the VectorBase (vectorbase.org) annotation, both cathepsin B homologues shared approximately 66% identity in their amino acid sequences. The two cathepsin B genes are expressed simultaneously in the fat body of the vitellogenic females, and enzymatic activity was detected within the ovaries, suggesting an extra-ovarian origin. Similar to the transcriptional profile of vitellogenin, cathepsin B transcripts were shown to accumulate post-blood meal and reached their highest expression at 36 h PBM. However, while vitellogenin expression decreased drastically at 48 h PBM, the expression of the cathepsins increased until 84 h PBM, at which time the females of our colony were ready for oviposition. The similarity between their transcriptional profiles strongly suggests a role for the cathepsin B homologues in vitellin degradation.


Introduction
Culex quinquefasciatus (Diptera: Culicidae) is a cosmopolitan mosquito that is highly anthropophilic and completely adapted to urban conditions. This mosquito is a competent vector of neurotrophic viruses such as the St. Louis and Japanese encephalitis viruses, the eastern and western equine encephalomyelitis viruses and the Rift Valley and West Nile viruses [1][2][3][4]. Moreover, Cx. quinquefasciatus is the most important Brazilian vector of Wuchereria bancrofti, the lymphatic filariasis agent [5].
Similar to other oviparous animals, Cx. quinquefasciatus generates and stores within the oocytes the nutrients needed for the embryonic development. Nutrient reserves are synthesised in the maternal fat body, a tissue analogous in function to the vertebrate liver. The primary source of amino acids and lipids for embryonic development is vitellogenin (Vg), a glycosylated phospholipoprotein that is secreted into the haemolymph and then incorporated via receptor mediated endocytosis by the developing ovarian follicles [6] and stored into the yolk platelets as vitellin [7].
Haematophagous mosquitoes of the Aedes, Anopheles and Culex genera share multiple biochemical, morphological, developmental and behavioural characteristics. However, Cx. quinquefasciatus diverges from mosquitoes of other genera in the fine structure of their salivary glands, saliva composition [26,27], cellular and biochemical mechanisms governing blood digestion and haem detoxification [28,29] and their response to odorants and biting behaviour [30].
In the following study, we build on our initial description of the morphofunctional aspects of Cx. quinquefasciatus oogenesis and identify two cathepsin B proteinases, which are present in the Cx. quinquefasciatus eggs, expressed in the female fat bodies following a blood meal and are involved in promoting yolk protein degradation.

Ethics Statement
The protocols used in this work were approved by the Animal Experimentation Ethics Committee of the Institute of Biomedical Sciences (University of São Paulo, São Paulo, Brazil-process number CEAU 103/2012).
All extracts were immediately used or stored at -20°C until needed.

Ovary extract
Ovaries of adult females 96 and 120 hours post blood meal (PBM) were processed as described above for white eggs.

Determination of cathepsin B activity
Total protein from egg extract (40 ng) was incubated for 30 min at 27°C with 20 μM of Z-Arg-Arg-NHMec (benzyloxycarbonylarginyl-arginine 4-methylcoumarin-7-amide) in 10 mM sodium acetate buffer pH 5.0 at a final volume of 200 μl. The fluorescent product was detected using a Fluoroskan Ascent Microplate Fluorometer (Thermo Scientific, USA) with an excitation wavelength of 360 nm and emission of 450 nm. Control samples were established by adding 50 μM E-64 (N-[N-(L-3-trans-carboxyirane-2-carbonyl)-L-leucyl]-agmatine), a specific inhibitor of cysteine proteases [33]. A standard curve was obtained by measuring the fluorescence of 0.8 nM 7amino-4-methyl Coumarin solution (AMC). The cathepsin proteolytic activities were expressed in units (U) defined as the amount of enzyme that hydrolyses 1 μmol of substrate per minute.

Enzyme partial purification
Total egg extract was incubated at 27°C for 18 h in 10 mM sodium acetate buffer pH 5.0 supplemented with 0.5 mM phenylthiocarbamide. The degradation profile was analysed by SDS-PAGE and the enzymatic activity of the proteolytic product was determined as described above.

Mass spectrometry analyses
Protein bands of interest were excised from the gel and in-gel digestion was performed as previously described [36]. Following desalting with Zip-Tip (Millipore, USA), tryptic fragments were analysed using a nano-HPLC-MS/MS (LCQDuo ion trap mass spectrometer; Finnigan, Thermo Fisher Scientific Inc. Waltham, Massachusetts, USA) through a 120 min gradient from 5% to 56% acetonitrile in 0.2% formic acid. The digestion spectra were analysed with the Sequest program (Thermo Fisher Scientific Inc.) using a NCBI (National Center for Biotechnology Information, http://blast.ncbi.nlm.nih.gov/blast) non-redundant database of Culex and confirmed with the specific vector database (https://www.vectorbase.org/). Peptides were validated using protein probability 1 x 10 -7 , dCN 0.05, and Xcorr values of 1.5, 2.2, and 2.7 for singly, doubly or triply charged peptides, respectively. Proteins with a minimum of two validated peptides were considered.

RNA extraction
Total RNA was extracted using Trizol (Life Technologies, USA) from dissected fat bodies of adult females fed on sucrose (SUC) and at 12, 24, 36, 48, 60, 72, and 84 hours post blood meal (PBM). Alternatively, whole adult females 24 hours PBM were dissected at different times and total RNA was extracted as described above. In all samples, residual genomic DNA was removed by incubation with DNase I, Amp Grade (Invitrogen, USA) and RNA integrity was evaluated by agarose gel electrophoresis. RNA was quantified using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, USA).

RT-PCR
Reverse transcription (RT) was carried out with 2.2 μg of total RNA primed with 500 ng of Oligo dT (Life Technologies, USA) using the SuperScript II first-strand synthesis system (Life Technologies, USA) according to the manufacturer's instructions.
PCR was performed with 1 μl of complementary DNA (cDNA) as template and 0.4 μM of each primer (Table 1). Reactions were performed in a T3 Biometra (Biometra, Germany) thermocycler as follows: 2 min at 94°C, followed by 40 cycles of 94°C for 20 s, 57°C for 20 s, and 72°C for 40 s. The amplified products were resolved on 1.0% agarose gels, stained with Gel Red (Uniscience, Brazil), and visualised using an Image Quant 300 (GE Life Sciences, USA).

Quantitative RT-PCR
Quantitative RT-PCR (qRT-PCR) was performed using a StepOne Real-Time PCR System (Applied Biosystems, USA) in 96-well optical reaction plates (Applied Biosystems, USA). One microliter of cDNA template, 0.6 μM of each specific primer (Table 1), and 1 μl of Maxima SYBR Green (Thermo Scientific, USA) were used for each reaction. The thermal cycling program was: 10 min at 95°C, followed by 40 cycles of 95°C for 15 s, 57°C for 1 min, and 60°C for 1 min. The threshold cycle was normalised according to the rp49 ribosomal protein expression, and relative gene expression was calculated by the 2 -ΔΔC T method [38], using expression data generated from adult females fed on sucrose as a reference condition. All depicted data correspond to three independent biological samples analysed in triplicate.

Determination of cathepsin B activity during vitellogenesis
Three pairs of ovaries dissected from females fed on sucrose (SUC) and at 12, 24, 36, 48, 60, 72, and 84 hours post blood meal (PBM) were homogenised in 60 μl of PBS in the absence of protease inhibitors. Enzymatic activity was measured using 10 μl of homogenate as described above.

Statistical analysis
All experiments were repeated three times and the data were analysed using one-way or twoway analysis of variance (ANOVA) and the Tukey's HSD (Honestly Significant Difference) test with confidence intervals of 95% with the GraphPad InStat 3.02 software package (GraphPad Software Inc., USA). Experimental values were obtained from three independent assays and expressed as the means ± standard error.

Results and Discussion
Enzymatic activity profile of cathepsin B The activity of cathepsin B has been previously characterised by hydrolysis of the specific synthetic substrate, Z-Arg-Arg-NHMec [43], at an acidic pH in ovaries and eggs of Diptera such as Drosophila melanogaster [8], Musca domestica [9,10] and Aedes aegypti [11]. For all of these insects, cathepsin B has been shown to play a role in the degradation of yolk during embryogenesis.
Based on these data, we confirmed cathepsin B activity in total extract from dark eggs (approximately 24 h after oviposition) of Cx. quinquefasciatus with Z-Arg-Arg-NHMec at an optimum pH level between 5.0 and 5.5 (Fig. 1), similar to the conditions described previously [8][9][10][11]. Based on these data, all future experiments were conducted at pH 5.0 at 27°C. Self-proteolysis The SDS-PAGE profile of the dark eggs' total extract ( Fig. 2A) revealed a drastic decrease in band number compared to extracts from vitellogenic ovaries or white eggs (S1 Fig.). These results confirm recent data [44], which describes advanced embryonic development at 24 h after oviposition.
Only one protein band was observed at approximately 30 kDa (the approximate mass of the dipteran cathepsin B homologues previously described [9][10][11]; Fig. 2A, arrow) within egg extracts that were incubated without protease inhibitors for 18 hours at pH 5.0 and 27°C [10] compared to the total egg extract prior to incubation ( Fig. 2A).
Together, these results suggested that at least one active protease remains within the egg extract (Fig. 2B). The residual enzymatic activity was shown to be inhibited by E-64 and CA-074, a specific inhibitor of cysteine proteases [33] and cathepsin B [34], respectively, but not by any other enzyme inhibitors tested, including PMSF (serine proteases), EDTA (metalloproteases) and pepstatin (aspartyl proteases; Fig. 2B).
Mass spectrometry identifies cathepsin B peptides The 30 kDa band ( Fig. 2A) was excised from the gel and submitted for in gel reduction, alkylation and trypsinisation. The resulting tryptic peptides were individually analysed by mass spectrometry. Sequest analysis [45] of MS/MS data revealed that the peptides corresponding to the protein band shared identity with two cathepsins B of Cx. quinquefasciatus deposited in the VectorBase database with codes CPIJ015761 and CPIJ015762 (S1 Table). In this work, we will refer to CPIJ015761 as CatB1 and CPIJ015762 as CatB2.
The protein sequences of CatB1 and CatB2 identified by peptide analysis were found to display high alignment similarity with a lysosomal cathepsin B of Homo sapiens (AAH10240.1) [43] and with a cathepsin B of Ae. aegypti (AAEL007585) [11,12] (S2 Fig.; S2 Table). This analysis also identified the catalytic dyad, which consists of a cysteine and histidine residue and is specific to cysteine proteases [43].
The alignment of the deduced amino acid sequences of CatB1 (342 aa) and CatB2 (353 aa) revealed approximately 66% shared similarity (S2 Fig.; S2 Table).  Enzymatic activity analysis of the total extract of Cx. quinquefasciatus dark eggs. A: 12% SDS-PAGE of the total extract (TE) before or after incubation at 27°C for 18 h without protease inhibitors (WI) or in the presence of specific inhibitors of various proteases: 50 mM E-64 (cysteine proteases); 1 mM CA-074 (cathepsin B); 1 mM PMSF (serine proteases); 2 mM EDTA (metalloproteases); 10 mM pepstatin (aspartyl proteases); WI: without inhibitor. A total of 2 μg of protein was loaded into each lane, and the molecular weight (MW) is shown in kiloDaltons. The arrow points to the approximately 30 kDa band, which was analysed by mass spectrometry. B: The proteolytic activity, calculated at pH 5.0, of the total extract from Cx. quinquefasciatus dark eggs prior to and after incubation in the presence or not of the same specific protease inhibitors. Proteolytic activity is expressed in units (U) defined as the amount of enzyme that hydrolyses 1 μmol of substrate per minute. Each column represents the mean expression ± standard error of three independent biological experiments; asterisks denote statistically significant divergences compared to the standard condition (total extract) as determined by one-way ANOVA (*: p < 0.001; **: p < 0.0001) and the Tukey's HSD (Honestly Significant Difference) test.
The nucleotide sequence analysis of the two Cx. quinquefasciatus cathepsins B genes obtained from the VectorBase database showed 73% shared identity, with the exception of noncoding regions within the 3' UTR region (S3 Fig.), which allowed for the design and synthesis of specific reverse primers for each gene (Table 1). Oligonucleotide specificity was confirmed by sequencing PCR products and by the dissociation curve analysis generated by real-time PCR, which revealed a single peak for each cathepsin B transcript (data not shown).
Expression profiling of cathepsins B1 and B2 Previous work, performed in several insect species, has highlighted the role of a single acting vitellolytic cathepsin B in yolk protein degradation. However, it remains unclear whether multiple peptidases may contribute to targeted protein degradation within the oocyte. In support of potential enzyme cooperativity, Price and co-workers [12] reported the e transcription of four genes of cathepsin B within the fat body of Ae. aegypti female following a blood meal; however, the enzymatic activities were not analysed. In contrast, Snigirevskaya and colleagues [46] reported only a single cathepsin B at the yolk granule periphery in the oocytes of the same mosquito. As vitellogenin, this enzyme is synthesised by the vitellogenic fat body as a 44 kDa precursor protein, which is then secreted into the haemolymph and internalised by the developing oocytes. Following fertilisation, it is activated by the acidification of the granule to produce an active enzyme of 33 kDa [11,46].
These findings led us to investigate whether CatB1 and CatB2 of Cx. quinquefasciatus are co-expressed. For this purpose, Cx. quinquefasciatus females 24 hours PBM were individually tested by quantitative relative real-time PCR and revealed that CatB1 and CatB2 were simultaneously expressed in each mosquito analysed (Fig. 3). Based on the finding that the cathepsins B1 and B2 of Cx. quinquefasciatus were expressed simultaneously and reside within the same phylogenetic tree constructed from the amino acid sequences of cathepsins B of Cx. quinquefasciatus and Ae. aegypti [11,12,47], we hypothesise that they are likely the product of a successful gene duplication [48,49] (S4 Fig.).
In addition to simultaneous expression, both enzymes were shown to be upregulated post blood meal (Fig. 4), with peak expression occurring at 36 h PBM, similar to vitellogenin (Fig. 4). Interestingly, while the expression of vitellogenin showed a sharp decrease at 60 h PBM, CatB1 and CatB2 expression remained high until 84 h PBM, which coincides with female entry into oviposition (Patricia S. Yogi, unpublished data). The amount of cDNA was confirmed using the RP49 gene, which was also used as housekeeping control gene for the qRT-PCR (S5 Fig.).
Enzyme activity profiling of cathepsins B during vitellogenesis  (12,24,36,60,72,84). Each column represents the mean expression ± standard error of three independent biological replicates; asterisks denote data with a statistically significant differences compared to the standard condition (SUC), as determined by one-way ANOVA (*: p < 0.05; **: p <0.001; ***: p < 0.001) and the Tukey's HSD (Honestly Significant Difference) test. doi:10.1371/journal.pone.0118736.g004 While the transcription of both cathepsins B was shown to be upregulated at 12 h PBM ( Fig. 4A and 4B), the enzymatic activity was only detectable at 24 h PBM and continuously increased until reaching a plateau at 60 h PBM (Fig. 5). We hypothesise that this observation may be explained by cessation of incorporation, as this time is synonymous with endochorion secretion by the follicular cells [50].

Conclusions
In this paper, we describe the transcriptional profile of two cathepsin B homologues, which are expressed simultaneously in the fat body of Cx. quinquefasciatus vitellogenic females from 12 h PBM.
Both cathepsins were identified by mass spectrometry from the total extract of Cx. quinquefasciatus eggs after self-proteolysis. While in silico analysis suggested that both sequences harbour catalytic residues that may contribute to the observed enzymatic activity, it was not possible to determine whether both enzymes are simultaneously activated.
Finally, the transcriptional profile of the two cathepsin B genes is similar to that of vitellogenin, which supports the notion that the two enzymes likely cooperate in vitellin degradation. Enzymatic activity of cathepsin B was measured in a single ovary of adult females fed on sucrose (SUC) and every 12 h PBM (12,24,36,60,72,84). U/ovary: Units per ovary, where unit is defined as the amount of enzyme that hydrolyses 1 μmol of substrate per minute. Each column represents the mean expression ± standard error of three independent biological replicates; asterisks denote data with a statistically significant differences compared to the standard condition (SUC), as determined by one-way ANOVA (*: p < 0.05; **: p < 0.001) and Tukey's HSD (Honestly Significant Difference) test.  [43], Ae. aegypti (AAEL007585; VectorBase) [11,12] and Cx. quinquefasciatus (CatB1: CPIJ015761 and CatB2: CPIJ015762; VectorBase) were compared. Box region: signal peptides; black background indicate identical amino acids; asterisks indicate catalytic site residues: cysteine (C) and histidine (H). (TIF)  Table. The physicochemical properties of the peptide sequences of Cx. quinquefasciatus cathepsins B. Listed are each of the peptides detected by mass spectrometry of the 30 kDa band and the percentage of similarity observed between each peptide and the protein sequences of each cathepsin B (CPIJ015761 and CPIJ015762) using BLASTp. The bolded amino acid (H) represents the catalytic histidine. The cross correlation (Xcorr) function was used to assess the quality of peptide spectra matches. The Delta Correlation (DeltaCN) represents the difference between the normalised Xcorrs of the primary and secondary matches.