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Identification and characterization of a cathepsin D homologue from lampreys (Lampetra japonica)
Developmental and Comparative Immunology 49 (2015) 149–156
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Developmental and Comparative Immunology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / d c i
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Identification and characterization of a cathepsin D homologue from lampreys (Lampetra japonica) Rong Xiao a,b,1, Zhilin Zhang a,b,1, Hongyan Wang a,b, Yinglun Han a,b, Meng Gou a,b, Bowen Li a,b, Dandan Duan a,b, Jihong Wang a,b, Xin Liu a,b, Qingwei Li a,b,* a b
School of Life Sciences, Liaoning Normal University, Dalian 116081, China Lamprey Research Center, Liaoning Normal University, Dalian 116081, China
A R T I C L E
I N F O
Article history: Received 9 July 2014 Revised 28 October 2014 Accepted 28 October 2014 Available online 31 October 2014 Keywords: Lamprey Buccal gland Cathepsin D Immune defense
A B S T R A C T
Cathepsin D (EC 3.4.23.5) is a lysosomal aspartic proteinase of the pepsin superfamily which participates in various digestive processes within the cell. In the present study, the full length cDNA of a novel cathepsin D homologue was cloned from the buccal glands of lampreys (Lampetra japonica) for the first time, including a 124-bp 5′ terminal untranslated region (5′-UTR), a 1194-bp open reading frame encoding 397 amino acids, and a 472-bp 3′-UTR. Lamprey cathepsin D is composed of a signal peptide (Met 1-Ala 20), a propeptide domain (Leu 21-Ala 48) and a mature domain (Glu 76-Val 397), and has a conserved bilobal structure. Cathepsin D was widely distributed in the buccal glands, immune bodies, hearts, intestines, kidneys, livers, and gills of lampreys. After challenging with Escherichia coli or Staphylococcus aureus, the expression level of lamprey cathepsin D in the buccal gland was 8.5-fold or 6.5-fold higher than that in the PBS group. In addition, lamprey cathepsin D stimulated with Escherichia coli was also up-regulated in the hearts, kidneys, and intestines. As for the Staphylococcus aureus challenged group, the expression level of lamprey cathepsin D was found increased in the intestines. The above results revealed that lamprey cathepsin D may play key roles in immune response to exogenous pathogen and could serve as a potential antibacterial agent in the near future. In addition, lamprey cathepsin D was subcloned into pcDNA 3.1 vector and expressed in the human embryonic kidney 293 cells. The recombinant lamprey cathepsin D could degrade hemoglobin, fibrinogen, and serum albumin which are the major components in the blood, suggested that lamprey cathepsin D may also act as a digestive enzyme during the adaptation to a blood-feeding lifestyle. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Cathepsin D is an intracellular aspartic protease of the pepsin superfamily which is synthesized in rough endoplasmic reticulum with a signal peptide, a propeptide and a mature peptide (Zaidi et al., 2008). After the signal peptide was removed, cathepsin D with a propeptide and a mature peptide reaches its targeted intracellular
Abbreviations: BCA, bicinchoninic acid; BLAST, basic local alignment search tool; DTT, dithiothreitol; E. coli, Escherichia coli; ESTs, expressed sequence tags; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HEK 293 cell, human embryonic kidney 293 cell; IrCD, Ixodes ricinus cathepsin D; L. japonica, Lampetra japonica; PBS, phosphate buffered saline; RACE, rapid amplification of cDNA ends; S. aureus, Staphylococcus aureus; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TiCaD, Triatoma infestans cathepsin D; UTR, terminal untranslated region. * Corresponding author. School of Life Sciences, Lamprey Research Center, Liaoning Normal University, Dalian 116081, China. Tel.: +86 411 8582 7799; fax: +86 411 8582 7799. E-mail address: [email protected] (Q. Li). 1 These authors contributed equally. http://dx.doi.org/10.1016/j.dci.2014.10.014 0145-305X/© 2014 Elsevier Ltd. All rights reserved.
vesicular structures (lysosomes, endosomes, and phagosomes), where it could be activated by endopeptidases (Laurent-Matha et al., 2006). Usually, cathepsin D is widely distributed in most eukaryotic cells, and plays key roles in the lysosomal digestive process (Fusek and Veˇtvicˇka, 2005; Luca et al., 2009). Recently, a great number of studies have demonstrated that cathepsin D participates in the regulation of apoptosis, activation of polypeptide hormones, growth factors, enzymatic precursors, etc. (Benes et al., 2008). In addition, the expression pattern of cathepsin D usually changed during many pathological processes, such as Alzheimer’s disease, atherosclerosis and cancer (Benes et al., 2008). Thus, cathepsin D might also act as a biomarker for the prevention and detection of diseases in the near future (Vetvicka and Fusek, 2012). Lately, several studies have focused on cathepsin D from the blood feeding parasites which was proved to be related to the blood digestion process due to its ability to degrade hemoglobin (Brinkworth et al., 2001). In 2012, three cathepsin D (Ixodes ricinus cathepsin D, IrCD) forms were identified from the gut tissue of ticks (Ixodes ricinus). Among the three paralogs, IrCD1 with a shortened propeptide region and a unique posttranslational modification was recombinant and also proved to act
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as a hemoglobinase which facilitates the blood digestion process of ticks (Sojka et al., 2012). At the same time, two native cathepsin D proteins (Triatoma infestans cathepsin D, TiCaD and TiCaD2) purified from the lumen of the small intestine of the hematophagous bug (Triatoma infestans) were characterized as proteases targeting the ingested blood (Balczun et al., 2012). Moreover, cathepsin D might also act as an immune defender in fishes and parasites. During the infection process with pathogenic microorganisms, the expression level of cathepsin D was found increased in the various tissues of grass carp (Ctenopharyngodon idella), channel catfish (Ictalurus punctatus), half-smooth tongue sole (Cynoglossus semilaevis), hard tick (Haemaphysalis longicornis and Ixodes ricinus), and triatomine (Rhodnius prolixus) (Boldbaatar et al., 2006; Borges et al., 2006; Chen et al., 2011; Dong et al., 2012; Feng et al., 2011; Franta et al., 2010; Horn et al., 2009; Jeffers and Roe, 2008; Mu et al., 2013). In addition, cathepsin D is also involved in egg development and tissue invasion process (Fialho et al., 2005; Follo et al., 2013; Williamson et al., 2003). So far, several cathepsin D homologues have been identified and characterized in a variety of tissues from bugs, fishes, hookworms, mites, nematodes, schistosomes, and ticks (Balczun et al., 2012; Bartley et al., 2012; Chen et al., 2011; Dong et al., 2012; Feng et al., 2011; Fragoso et al., 2009; Mu et al., 2013; Sojka et al., 2012; Verity et al., 1999; Williamson et al., 2002, 2003). The jawless lampreys (Lampetra japonica, L. japonica) are one of the most primitive vertebrates still alive, which are considered as ideal animal models to study vertebrate evolution, embryo development and the origin of the adaptive immune system due to their unique position during the long-term evolution process (Chang et al., 2014; Forey and Janvier, 1993; Nikitina et al., 2009). In addition, lampreys are also well known for their blood sucking habit which may bring harmful effects to marine fishes (Lennon, 1954). In 2007, Xiao et al. have reported the fibrinogenolytic properties of buccal gland secretion which may help lampreys counteract the blood coagulation for the first time (Xiao et al., 2007). In recent years, a variety of bioactive proteins and peptides have been identified in the buccal glands of lampreys, which were proved to be involved in counteracting the hemostasis, nociceptive, and immune responses of host fishes (Chi et al., 2009; Gao et al., 2005; Ito et al., 2007; Liu et al., 2009; Sun et al., 2008, 2010a, 2010b; Wang et al., 2010; Wong et al., 2012; Xiao et al., 2007, 2012; Xue et al., 2011). In contrast to the extensive studies of cathepsin D from the other vertebrates and invertebrates, little work has been done on cathepsin D from lampreys. In the present study, a cathepsin D homologue was found in the buccal glands of lampreys for the first time. And the connection of this enzyme with immune defense in response to bacterial stimulation and digestion process of lampreys was also investigated. 2. Materials and methods 2.1. Animals Adult lampreys (L. japonica) at spawning migration stage were obtained in December 2011 in Tong River, a branch of Songhua River in Heilongjiang province of China. The handling of lampreys was approved by the Animal Welfare and Research Ethics Committee of the Institute of Dalian Medical University (Permit Number: SYXK2004—0029). 2.2. Cloning of a cathepsin D homologue from lampreys In the previous studies, the cDNA libraries from the buccal gland, liver, and leukocyte cell of lampreys (L. japonica) have been constructed, respectively (Gao et al., 2005; Zhang et al., 2010; Zhu et al., 2008). Based on the analysis of the expressed sequence tags (ESTs)
of the buccal gland cDNA library from the lampreys, two cDNA sequences which are homologous to cathepsin D were identified by using the Basic Local Alignment Search Tool X (BLASTX) in the National Center for Biotechnology Information (NCBI, http:// www.ncbi.nlm.nih.gov/). All PCR primers were designed based on the sequences in the lamprey buccal gland cDNA library which are homologous to cathepsin D (Supplementary Table S1). Total RNA was isolated from the buccal gland of lampreys (L. japonica) according to the manufacturer’s protocol (TaKaRa, Dalian, China) and treated with the RNase-free DNase I (TaKaRa, Dalian, China) to remove genomic DNA contamination. First-strand cDNA was synthesized from the total RNA with PrimeScriptTM RT-PCR Kit (TaKaRa, Dalian, China) and stored at −80 °C. To obtain the full length cDNA of lamprey cathepsin D, rapid amplification of 5′ cDNA ends (5′-RACE) and 3′RACE was performed with the 5′ Full RACE Core Set Kit (TaKaRa, Dalian, China) and 3′ Full RACE Core Set Kit (TaKaRa, Dalian, China), respectively. All the PCR amplifications were carried out by using LA Taq DNA polymerase (TaKaRa, Dalian, China) and cloned into a pMD19-T Simple Vector for sequence confirmation. 2.3. Sequence analysis The full length cDNA sequence of lamprey cathepsin D was spliced by Sequencher 4.2 software and the deduced amino acids were analyzed with DNAMAN V6 and DNASTAR 5.0 software, respectively. The primary structure was analyzed by ProtParam (http:// www.expasy.org/). A three-dimensional structure model of lamprey cathepsin D was constructed using the Phyre software (version 0.2) with the X-ray structure of pepsin-like acid proteases (d3psga_) as a template, and visualized using the UCSF Chimera program package (Kelley and Sternberg, 2009; Pettersen et al., 2004). 2.4. Sequence alignment and phylogenetic tree construction Additional 29 cathepsin D sequences from the other species were obtained from ExPASy (http://www.expasy.ch/tools/blast). The multiple sequence alignments of cathepsin D were performed by ClustalX 1.83 software using default settings (Thompson et al., 1997). A neighbor-joining tree was constructed by MEGA 4.1 software based on the pair-wise deletion of gaps/missing data and a p-distance matrix of an amino acid model with 1000 bootstrapped replicates (Tamura et al., 2007). 2.5. Real-time quantitative PCR analysis The lampreys with an average body weight of 200 g were kept in aquaria at 16 ± 2 °C for two weeks before challenging. The Escherichia coli (E. coli) or Staphylococcus aureus (S. aureus) was cultured at 37 °C to logarithmic growth in LB liquid medium. The cells were then inactivated with formalin (He et al., 2005). The inactivated bacteria cells were resuspended after centrifugation to approximately 2 × 108 CFU/ml in phosphate buffered saline (PBS, pH 7.2). The lampreys were randomly divided into three groups (ten lampreys in each group) and intraperitoneally injected with PBS (0.1 ml), inactivated E. coli (0.1 ml) and S. aureus (0.1 ml) at 8-day intervals, respectively. After the fourth injection, the buccal glands, gills, hearts, livers, intestines, kidneys, and immune bodies of the lampreys were respectively dissected for RNA extraction as described above. Three tissues were pooled together as one sample (three lampreys per pool, three pools per tissue). The expression level of lamprey cathepsin D was assayed by using TaKaRa SYBR® PrimeScriptTM RT-PCR Kit with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control (Kawano-Yamashita et al., 2007; Pfaffl, 2001). Each reaction contained 1 × SYBR Premix Ex Taq, forward primer (10 μM), reverse primer (10 μM) and cDNA (100 ng/μl) with a final volume of 25 μl. The amplification was carried out on a TaKaRa PCR Thermal
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Cycler Dice Real Time System with the parameters as follows: 95 °C for 10 s, followed by 45 cycles of 5 s at 95 °C, 30 s at 60 °C, 30 s at 72 °C, and a final extension step at 72 °C for 10 min. Specific primers for lamprey cathepsin D and lamprey GAPDH were designed based on the sequences in the lamprey buccal gland cDNA library (Supplementary Table S1). Each sample was performed in triplicate and the data were analyzed with the SAS Proprietary Software Release 8.02. Student’s t-tests were used to determine statistical significance (* < 0.05; ** < 0.01) (Livak and Schmittgen, 2001). 2.6. Expression and purification of lamprey cathepsin D Lamprey cathepsin D without the signal peptide (Leu 21-Val 397) was amplified and subcloned into pcDNA 3.1 vector with a HisFlag tag. The protein expression was induced in the human embryonic kidney 293 (HEK 293) cells for 48 h. The cells were digested by 0.25% trypsin (Hyclone, USA), collected by centrifugation, and washed in PBS. After centrifugation, the cells were resuspended in 50 mM Tris–HCl (pH 8.0) containing 500 mM NaCl, 0.1% Triton X-100, 1 mM dithiothreitol (DTT), and 5% glycerol, and then sonicated on ice for 10 min. The soluble supernatant was collected through centrifugation and subjected to an ANTI-FLAG® M2 affinity resin column (Sigma, USA) equilibrated with binding buffer (50 mM Tris–HCl, 300 mM NaCl, 0.1% Triton X-100, 1 mM DTT, and 5% glycerol, pH 8.0). After washing the column with wash buffer (50 mM Tris–HCl, 500 mM NaCl, 1 mM DTT, and 5% glycerol, pH 8.0), the recombinant protein was collected in elution buffer containing 50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 150 μg/μl Flag peptide (Sigma, USA), 1 mM DTT and 10% glycerol. The concentration of recombinant lamprey cathepsin D was measured using a Bicinchoninic Acid (BCA) Protein Assay kit (Pierce, USA). The purified recombinant lamprey cathepsin D (0.2 μg) was analyzed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie brilliant blue. 2.7. Identification of recombinant lamprey cathepsin D The protein bands in the gel were excised from the gel and destained in acetonitrile containing 50 mM ammonium bicarbonate. Subsequently, the samples were dehydrated by acetonitrile for 10 min. After reductive alkylation by dithiothreitol (10 mM) and iodoacetamide (50 mM) for 2 h, the samples were digested with trypsin (25 mM, Promega) in-gel overnight. The peptide mixtures were analyzed by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF/TOF) mass spectrometry (Bruker, USA).
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3. Result 3.1. A cathepsin D homologue was found in the buccal glands of lampreys A full length cDNA (1790 bp) of lamprey cathepsin D homologue was obtained from the cDNA library of the buccal glands of lampreys (Fig. 1), including a 124-bp 5′ terminal untranslated region (5′-UTR), a 1194-bp encoding region and a 472-bp 3′ terminal untranslated region (3′-UTR). A polyadenylation signal sequence, AATAAAA, was located at 16 bp upstream of the poly (A) tail. The open reading frame of lamprey cathepsin D was 1194 bp encoding a protein with 397 amino acid residues. According to the ProtParam program predication, the predicted molecular mass weight and the theoretical isoelectric point of lamprey cathepsin D were 42, 772 Da and 6.23, respectively. Similar to the other cathepsin D proteins, lamprey cathepsin D also contains a signal peptide (Supplementary Fig. S1A, orange, Met 1-Ala 20), a propeptide domain (Supplementary Fig. S1A, marine, Leu 21-Ala 48) and a mature domain (Supplementary Fig. S1A, green, Glu 76-Val 397). According to the analysis of the amino acid sequence, one N-glycosylation site (Supplementary Fig. S1A, yellow, Asn 62) and two catalytic motifs (Supplementary Fig. S1A, red, Asp 95-Ser 98 and Asp 282-Thr 285) were found in lamprey cathepsin D. As shown in Supplementary Fig. S1A, the active site flap of lamprey cathepsin D was AIQYGTGSLSG, in which the specific substrates or inhibitors could be enclosed. The nucleotide sequence of lamprey cathepsin D has been submitted to the GenBank database (accession number: KM051432). Spatial homology model of lamprey cathepsin D showed a completely conserved bilobal structure with two catalytic motifs (Supplementary Fig. S1B, red, DTGS and DTGT) on each side of the active site flap (Supplementary Fig. S1B, purple, AIQYGTGSLSG) which is related to substrate binding. A “β-loop” (Supplementary Fig. S1, pink, Cys316-Gly345) and a “polyproline loop” (Supplementary Fig. S1, blue, Gly359-Pro379) were found in the proximity of the active site flap. Conserved Lys 318 and Lys 344 (Supplementary Fig. S1, black) were located at the “β-loop”, while repeated proline residues were found in the “polyproline loop” region of lamprey cathepsin D. Different from human cathepsin D, lamprey cathepsin D only has two conserved lysine residues, but lacks a Lys 203 residue which is important for mammal phosphotransferase recognition (Steet et al., 2005). 3.2. Lamprey cathepsin D shares high homology with the cathepsin D from the other species
2.8. Recombinant lamprey cathepsin D activity assay The recombinant lamprey cathepsin D (0.1 μg/μl, final concentration) which was dissolved in 50 mM Tris–HCl containing 150 mM NaCl (pH 8.0) was added into 100 mM formic acid for 3 h at 25 °C for auto-activation (pH of the mixture, 3.5). The recombinant lamprey cathepsin D in 50 mM Tris–HCl containing 150 mM NaCl (0.1 μg/ μl, final concentration) was incubated with bovine hemoglobin (7 mg/ml, final concentration, Sigma, USA), fibrinogen (2 mg/ml, final concentration, Sigma, USA), and bovine serum albumin (0.8 mg/ ml or 2 mg/ml, final concentration, Solarbio, China), respectively. Hemoglobin, fibrinogen and bovine serum albumin were dissolved in 100 mM formic acid, respectively. Each mixture (pH 3.5) was incubated at 25 °C for 20 h. The reaction was terminated by addition of 4% (w/v) SDS in 100 mM Tris–HCl buffer (pH 6.8) containing 200 mM DTT, 0.2% bromophenol blue and 20% glycerol. Hemoglobin, fibrinogen, and bovine serum albumin which were dissolved in 100 mM formic acid were also incubated at 25 °C for 20 h, respectively. Aliquots were taken for 12% SDS-PAGE and stained with Coomassie brilliant blue.
Multiple sequence alignments of cathepsin D indicated that lamprey cathepsin D is a very conservative gene and shares high homology with cathepsin D in the other species (Fig. 2). Lamprey cathepsin D shows about 67–72% identities with the cathepsin D from teleosts, amphibians, reptiles, birds, and mammals. While it only has 52–59% sequence similarities with the cathepsin D from the invertebrates, including echinoderms, arthropods, and mollusks (Supplementary Table S2). According to the sequence analysis, the catalytic motifs (DTGS and DTGT) were completely conserved among all of the cathepsin D sequences. In addition, the active site flap of lamprey cathepsin D (AIQYGTGSLSG) shares high similarity with that from the other organisms. In order to study the phylogenetic relationship between lamprey cathepsin D and the other cathepsin D, a phylogenetic tree was constructed through the neighbor-joining method (Supplementary Fig. S2). Phylogeny of 30 cathepsin D sequences indicated that all cathepsin D homologues used in this study were classified into two clusters: vertebrate cluster and invertebrate cluster. Vertebrate cluster includes cathepsin D from birds, reptiles, agnathans, mammals,
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teleosts and amphibians; the second includes cathepsin D from mammals; and the third includes those from reptiles, birds and agnathans, respectively (Supplementary Fig. S2). Phylogenetic analysis indicated that lamprey cathepsin D was clustered as the out group of cathepsin D from reptiles and birds. 3.3. The expression pattern of lamprey cathepsin D Real-time quantitative PCR was used to investigate the relative expression pattern of cathepsin D in the various tissues of lampreys (L. japonica). Lamprey GAPDH was used as an internal control. In the control group, the expression level of lamprey cathepsin D was a little higher in the buccal glands, immune bodies, and hearts than that in the intestines, kidneys, livers, and gills (Fig. 3). After stimulation with E. coli or S. aureus by intraperitoneal injection, the expression level of lamprey cathepsin D in the buccal gland was 8.5fold or 6.5-fold higher than that in the PBS group. In addition, lamprey cathepsin D stimulated with E. coli was also up-regulated in the hearts, kidneys, and intestines of lampreys (Fig. 3, P < 0.05). As for the lampreys treated with S. aureus, the expression level of lamprey cathepsin D was also increased in the intestines. Compared with the E. coli stimulated group, lamprey cathepsin D was not significantly expressed in the S. aureus treated group. 3.4. The recombinant lamprey cathepsin D could degrade hemoglobin, fibrinogen, and serum albumin
Fig. 1. The nucleotide and deduced amino acid sequences of lamprey cathepsin D. The nucleotide (upper line) and amino acid (lower line) sequences are numbered from the guanine and the initial methionine, respectively. The stop codon is marked with an asterisk and indicated in red. A signal peptide splice site is located between Ala 20 and Leu 21 labeled with a purple arrow. One N-glycosylation site and two catalytic residues are indicated in green and blue, respectively. A polyadenylation signal sequence, AATAAAA, is located at 16 bp upstream of the poly (A) tail marked with yellow. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
amphibians and teleosts; invertebrate cluster is composed of cathepsin D from mollusks and arthropods. Except the cathepsin D from Ictalurus punctatus, vertebrate cluster can be further classified into three subgroups, the first one contains cathepsin D from
Lamprey cathepsin D was subcloned into pcDNA 3.1 vector and expressed as a His-Flag-tagged fusion protein in HEK 293 cells. As shown in Supplementary Fig. S3A, there are mainly two protein bands detected on 12% SDS-PAGE. According to the MALDI-TOF/ TOF analysis, the peptides generated in gel digestion (66 kDa) were the fragments (ISSIQSIVPALEIANAHR, TLNDELEIIEGMKFDR, KPLVIIAEDVDGEALSTLVLNR, and LVQDVANNTNEEAGDGTTTATVLAR) of heat shock protein 60 (GI:306890); while the peptides generated in gel digestion (43 kDa) were the fragments (NVFSFYLNR and YYKGELSYVPVTR) of lamprey cathepsin D, suggesting that lamprey cathepsin D was expressed successfully. After auto-activation, the recombinant lamprey cathepsin D migrated as a single band on 12% SDS-PAGE (Supplementary Fig. S3B), suggesting that the recombinant lamprey cathepsin D was able to degrade heat shock protein 60 in acidic conditions. Recently, several reports have shown that cathepsin D from the bloodsuckers could degrade hemoglobin in acidic conditions (Brinkworth et al., 2001; Sojka et al., 2012; Williamson et al., 2002). To investigate the potential roles of lamprey cathepsin D in their buccal glands, the recombinant lamprey cathepsin D was incubated with hemoglobin in acidic conditions for 20 h. 12% SDS-PAGE showed that the hemoglobin was degraded in the presence of the recombinant lamprey cathepsin D (Supplementary Fig. S3C). And several degradation fragments could be detected on the gel. According to the analysis of gray scan of the hemoglobin, 50% hemoglobin was digested by lamprey cathepsin D (data not shown). When the recombinant lamprey cathepsin D was incubated with fibrinogen at 25 °C for 8 h, the Aα and Bβ chains of fibrinogen were digested completely. And the γ chain was degraded partly. As the incubation time extended to 20 h, the γ chain was also hydrolyzed completely (Supplementary Fig. S3D). In addition, bovine serum albumin was also hydrolyzed in the presence of the recombinant lamprey cathepsin D, and several degradation fragments had also been detected on the gel (Supplementary Fig. S3E). 4. Discussion Parasitic phase lampreys are eel like animals which usually attack the host fishes to suck the flesh and blood. Like the other
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Fig. 2. Multiple sequence alignments of lamprey cathepsin D with cathepsin D from the other species by using ClustalX 1.83. The accession numbers of the amino acid sequences extracted from the EXPASY database are as follows: Homo sapiens, NP_001900.1; Mus musculus, NP_034113.1; Gallus gallus, NP_990508.1; Pelodiscus sinensis, XP_006134990.1; Xenopus laevis, BAC57431.1; Danio rerio, NP_571785.1; Lampetra japonica, KM051432; Aedes aegypti, XP_001657556.1; Pinctada margaritifera, AFE48185.1; Apostichopus japonicus, AEG79714.1. Dashes represent gaps inserted into the alignment. Identical residues are indicated by asterisks. Strong and weak homologous residues are indicated in colons (:) and dots (.), respectively. The catalytic motifs and aspartyl protease active site flap are covered with the transparent box and gray box, respectively.
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Fig. 3. The mRNA expression level of lamprey cathepsin D after stimulation with E. coli and S. aureus, respectively. Real time quantitative RT-PCR was performed to determine the lamprey cathepsin D mRNA levels in various lamprey tissues. Total RNAs were extracted from buccal glands, hearts, kidneys, intestines, immune bodies, livers and gills of lampreys collected after stimulation with PBS, E. coli, and S. aureus, respectively. Lamprey GAPDH served as an internal control to calibrate the cDNA template for all the samples. The significant differences of lamprey cathepsin D expression between the challenged groups and the blank group are indicated by asterisks (*: P < 0.05; **: P < 0.01).
bloodsuckers, such as triatomas, bugs, ticks, leeches, and vampire bats (Basanova et al., 2002; Zavalova et al., 2002), the buccal gland secretion of lampreys also contains several regulators related to the anticoagulation, analgesia, and immune defense (Chi et al., 2009; Gao et al., 2005; Ito et al., 2007; Liu et al., 2009; Sun et al., 2008, 2010a, 2010b; Wang et al., 2010; Wong et al., 2012; Xiao et al., 2007, 2012; Xue et al., 2011). In this study, the full-length cDNA of a cathepsin D homologue was obtained from the buccal gland of lampreys (L. japonica) for the first time. Sequence analysis indicated that lamprey cathepsin D is a highly conserved gene and also contains a typical structure including a signal peptide, a propeptide, and a mature domain which should be activated by a series of proteinases. According to the spatial structure analysis, lamprey cathepsin D showed a conserved bilobal structure with two catalytic motifs on each side of the active site flap. In addition, the catalytic motifs (DTGS and DTGT) and the active site flap (AIQYGTGSLSG) of lamprey cathepsin D, which are typical characteristics of aspartic proteases, were highly conserved. Thus, the novel cathepsin D homologue found in the buccal gland of lampreys may be considered as a digestive enzyme which is essential for the process of blood intake. In 1995, Fortenberry et al. reported that glycosylation was not necessary for the folding and expression of cathepsin D, but was essential for the new synthetized cathepsin D to anchor to the lysosome (Fortenberry et al., 1995). In addition, Lys 203, and the “β-loop” which contained Lys 267 and Lys 293 in the human cathepsin D were proved to help cathepsin D target the lysosome through the mannose 6-phosphate pathway (Baranski et al., 1991). Based on the sequence analysis, a N-glycosylation site (Asn 62) and two conserved lysines (Lys318 and Lys 344) were also found in the lamprey cathepsin D, suggested that these residues are important in lamprey cathepsin D location. Lamprey cathepsin D was widely expressed in the buccal glands, immune bodies, hearts, intestines, kidneys, livers, and gills of
lampreys based on real-time quantitative PCR analysis. And the strongest expression was found in the buccal glands, which suggested that lamprey cathepsin D may be related to the feeding habit of lampreys. Recently, a great number of studies have shown that several cathepsin D proteins from the blood suckers prefer to degrade hemoglobin which is the main source of amino acids for the hematophagous parasites (Brinkworth et al., 2001; Sojka et al., 2012; Verity et al., 1999; Williamson et al., 2002). After auto-catalytical activation, the mature form of cathepsin D could degrade the substrates such as hemoglobin in acidic conditions (Brinkworth et al., 2001; Sojka et al., 2012; Williamson et al., 2002). In the present study, lamprey cathepsin D was successfully expressed in the HEK 293 cells. In acidic conditions, the recombinant protein could also hydrolyze hemoglobin, fibrinogen, and serum albumin which are the major components present in blood. As lampreys feed on the blood and flesh of host fishes, their buccal glands may also provide cathepsin D as a digestive enzyme. Previous studies have indicated that several cathepsin D proteins from parasites and fishes could also participate in the immune defense process against microbial pathogens (Boldbaatar et al., 2006; Borges et al., 2006; Buarque et al., 2013; Chen et al., 2011; Dong et al., 2012; Franta et al., 2010; Horn et al., 2009; Jeffers and Roe, 2008; Mu et al., 2013). And the expression level of cathepsin D was also found increased in the various tissues of grass carp (Ctenopharyngodon idella), channel catfish (Ictalurus punctatus), half-smooth tongue sole (Cynoglossus semilaevis), hard tick (Haemaphysalis longicornis and Ixodes ricinus), and triatomine (Rhodnius prolixus) during the infection with pathogenic microorganisms (Boldbaatar et al., 2006; Borges et al., 2006; Chen et al., 2011; Dong et al., 2012; Franta et al., 2010; Horn et al., 2009; Jeffers and Roe, 2008; Mu et al., 2013). After intraperitoneal injection with E. coli or S. aureus, the expression level of lamprey cathepsin D in the buccal gland was 8.5-fold or 6.5-fold higher than that in the PBS group (control), which indicated that lamprey cathepsin D may act as an effective immune defender. Usually, the microbial pathogens are widely distributed in the marine environment where the parasitic phase lampreys lived. And the surface of the host fishes may also carry some specific bacteria. Except the fleshes and blood of host fishes, lampreys might also suck the microbial pathogens with their unique buccal funnel during the feeding process. Thus, the cathepsin D secreted from the buccal gland would act as the first barrier to protect lampreys from infection by the bacteria effectively. Interestingly, lamprey cathepsin D was more sensitive to gram negative bacteria (such as E. coli) than gram positive bacteria (such as S. aureus) based on real-time quantitative PCR analysis. In 2006, the activity of cathepsin D in the midgut of Rhodnius prolixus infected with Trypanosoma cruzi was higher than that in the uninfected group (Borges et al., 2006). Compared with the PBS group, the increase in lamprey cathepsin D expression was also found in the intestines after stimulation with E. coli or S. aureus, suggesting that the intestines of lampreys might be the other organs important for exogenous pathogen clearance. Several studies have indicated that cathepsin family proteases have bactericidal abilities such as cathepsin-D, -L, -B or -G (Thorne et al., 1976). In 2009, Eugenio Carrasco-Marín et al. put forward that cathepsin D might kill Listeria monocytogenes or Streptococcus pneumonia through degradation of listeriolysin O or pneumolysin at the same undecapeptide sequence between the Trp 491 and Trp 492 residues or between Trp 435 and Trp 436 residues (Carrasco-Marín et al., 2009). In addition, the activated cathepsin D was proved to be able to induce macrophage apoptosis through down-regulation of Mcl-1, which enhances the antimicrobial effect of macrophage during late phase killing of pneumococci (Bewley et al., 2011). However, little work has been done on the antibacterial activity and mechanisms of lamprey cathepsin D, and it needs further studies to elucidate.
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5. Conclusions This report represents the characterization of a novel cathepsin D homologue obtained from the buccal gland of lampreys for the first time. And expression pattern analysis showed that lamprey cathepsin D was related to immune defense and might act as a potential antibacterial agent in the near future. In addition, the recombinant lamprey cathepsin D was related to the digestive process of lampreys due to its ability to degrade hemoglobin, fibrinogen, and serum albumin.
Acknowledgements This work was supported by grants from The National Natural Science Foundation of China (No. 31301880), China Postdoctoral Science Foundation (No. 2013M541246), New Teacher of Specialized Research Foundation for the Doctoral Program of Higher Education of China (No. 20112136120002), Scientific Research Fund of Liaoning Provincial Education Department (No. L2011187), and the Scientific and Technological Research Projects of Dalian (No. 2011J21DW014).
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Developmental and Comparative Immunology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / d c i
Short communication
Identification and characterization of a cathepsin D homologue from lampreys (Lampetra japonica) Rong Xiao a,b,1, Zhilin Zhang a,b,1, Hongyan Wang a,b, Yinglun Han a,b, Meng Gou a,b, Bowen Li a,b, Dandan Duan a,b, Jihong Wang a,b, Xin Liu a,b, Qingwei Li a,b,* a b
School of Life Sciences, Liaoning Normal University, Dalian 116081, China Lamprey Research Center, Liaoning Normal University, Dalian 116081, China
A R T I C L E
I N F O
Article history: Received 9 July 2014 Revised 28 October 2014 Accepted 28 October 2014 Available online 31 October 2014 Keywords: Lamprey Buccal gland Cathepsin D Immune defense
A B S T R A C T
Cathepsin D (EC 3.4.23.5) is a lysosomal aspartic proteinase of the pepsin superfamily which participates in various digestive processes within the cell. In the present study, the full length cDNA of a novel cathepsin D homologue was cloned from the buccal glands of lampreys (Lampetra japonica) for the first time, including a 124-bp 5′ terminal untranslated region (5′-UTR), a 1194-bp open reading frame encoding 397 amino acids, and a 472-bp 3′-UTR. Lamprey cathepsin D is composed of a signal peptide (Met 1-Ala 20), a propeptide domain (Leu 21-Ala 48) and a mature domain (Glu 76-Val 397), and has a conserved bilobal structure. Cathepsin D was widely distributed in the buccal glands, immune bodies, hearts, intestines, kidneys, livers, and gills of lampreys. After challenging with Escherichia coli or Staphylococcus aureus, the expression level of lamprey cathepsin D in the buccal gland was 8.5-fold or 6.5-fold higher than that in the PBS group. In addition, lamprey cathepsin D stimulated with Escherichia coli was also up-regulated in the hearts, kidneys, and intestines. As for the Staphylococcus aureus challenged group, the expression level of lamprey cathepsin D was found increased in the intestines. The above results revealed that lamprey cathepsin D may play key roles in immune response to exogenous pathogen and could serve as a potential antibacterial agent in the near future. In addition, lamprey cathepsin D was subcloned into pcDNA 3.1 vector and expressed in the human embryonic kidney 293 cells. The recombinant lamprey cathepsin D could degrade hemoglobin, fibrinogen, and serum albumin which are the major components in the blood, suggested that lamprey cathepsin D may also act as a digestive enzyme during the adaptation to a blood-feeding lifestyle. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Cathepsin D is an intracellular aspartic protease of the pepsin superfamily which is synthesized in rough endoplasmic reticulum with a signal peptide, a propeptide and a mature peptide (Zaidi et al., 2008). After the signal peptide was removed, cathepsin D with a propeptide and a mature peptide reaches its targeted intracellular
Abbreviations: BCA, bicinchoninic acid; BLAST, basic local alignment search tool; DTT, dithiothreitol; E. coli, Escherichia coli; ESTs, expressed sequence tags; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HEK 293 cell, human embryonic kidney 293 cell; IrCD, Ixodes ricinus cathepsin D; L. japonica, Lampetra japonica; PBS, phosphate buffered saline; RACE, rapid amplification of cDNA ends; S. aureus, Staphylococcus aureus; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TiCaD, Triatoma infestans cathepsin D; UTR, terminal untranslated region. * Corresponding author. School of Life Sciences, Lamprey Research Center, Liaoning Normal University, Dalian 116081, China. Tel.: +86 411 8582 7799; fax: +86 411 8582 7799. E-mail address: [email protected] (Q. Li). 1 These authors contributed equally. http://dx.doi.org/10.1016/j.dci.2014.10.014 0145-305X/© 2014 Elsevier Ltd. All rights reserved.
vesicular structures (lysosomes, endosomes, and phagosomes), where it could be activated by endopeptidases (Laurent-Matha et al., 2006). Usually, cathepsin D is widely distributed in most eukaryotic cells, and plays key roles in the lysosomal digestive process (Fusek and Veˇtvicˇka, 2005; Luca et al., 2009). Recently, a great number of studies have demonstrated that cathepsin D participates in the regulation of apoptosis, activation of polypeptide hormones, growth factors, enzymatic precursors, etc. (Benes et al., 2008). In addition, the expression pattern of cathepsin D usually changed during many pathological processes, such as Alzheimer’s disease, atherosclerosis and cancer (Benes et al., 2008). Thus, cathepsin D might also act as a biomarker for the prevention and detection of diseases in the near future (Vetvicka and Fusek, 2012). Lately, several studies have focused on cathepsin D from the blood feeding parasites which was proved to be related to the blood digestion process due to its ability to degrade hemoglobin (Brinkworth et al., 2001). In 2012, three cathepsin D (Ixodes ricinus cathepsin D, IrCD) forms were identified from the gut tissue of ticks (Ixodes ricinus). Among the three paralogs, IrCD1 with a shortened propeptide region and a unique posttranslational modification was recombinant and also proved to act
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as a hemoglobinase which facilitates the blood digestion process of ticks (Sojka et al., 2012). At the same time, two native cathepsin D proteins (Triatoma infestans cathepsin D, TiCaD and TiCaD2) purified from the lumen of the small intestine of the hematophagous bug (Triatoma infestans) were characterized as proteases targeting the ingested blood (Balczun et al., 2012). Moreover, cathepsin D might also act as an immune defender in fishes and parasites. During the infection process with pathogenic microorganisms, the expression level of cathepsin D was found increased in the various tissues of grass carp (Ctenopharyngodon idella), channel catfish (Ictalurus punctatus), half-smooth tongue sole (Cynoglossus semilaevis), hard tick (Haemaphysalis longicornis and Ixodes ricinus), and triatomine (Rhodnius prolixus) (Boldbaatar et al., 2006; Borges et al., 2006; Chen et al., 2011; Dong et al., 2012; Feng et al., 2011; Franta et al., 2010; Horn et al., 2009; Jeffers and Roe, 2008; Mu et al., 2013). In addition, cathepsin D is also involved in egg development and tissue invasion process (Fialho et al., 2005; Follo et al., 2013; Williamson et al., 2003). So far, several cathepsin D homologues have been identified and characterized in a variety of tissues from bugs, fishes, hookworms, mites, nematodes, schistosomes, and ticks (Balczun et al., 2012; Bartley et al., 2012; Chen et al., 2011; Dong et al., 2012; Feng et al., 2011; Fragoso et al., 2009; Mu et al., 2013; Sojka et al., 2012; Verity et al., 1999; Williamson et al., 2002, 2003). The jawless lampreys (Lampetra japonica, L. japonica) are one of the most primitive vertebrates still alive, which are considered as ideal animal models to study vertebrate evolution, embryo development and the origin of the adaptive immune system due to their unique position during the long-term evolution process (Chang et al., 2014; Forey and Janvier, 1993; Nikitina et al., 2009). In addition, lampreys are also well known for their blood sucking habit which may bring harmful effects to marine fishes (Lennon, 1954). In 2007, Xiao et al. have reported the fibrinogenolytic properties of buccal gland secretion which may help lampreys counteract the blood coagulation for the first time (Xiao et al., 2007). In recent years, a variety of bioactive proteins and peptides have been identified in the buccal glands of lampreys, which were proved to be involved in counteracting the hemostasis, nociceptive, and immune responses of host fishes (Chi et al., 2009; Gao et al., 2005; Ito et al., 2007; Liu et al., 2009; Sun et al., 2008, 2010a, 2010b; Wang et al., 2010; Wong et al., 2012; Xiao et al., 2007, 2012; Xue et al., 2011). In contrast to the extensive studies of cathepsin D from the other vertebrates and invertebrates, little work has been done on cathepsin D from lampreys. In the present study, a cathepsin D homologue was found in the buccal glands of lampreys for the first time. And the connection of this enzyme with immune defense in response to bacterial stimulation and digestion process of lampreys was also investigated. 2. Materials and methods 2.1. Animals Adult lampreys (L. japonica) at spawning migration stage were obtained in December 2011 in Tong River, a branch of Songhua River in Heilongjiang province of China. The handling of lampreys was approved by the Animal Welfare and Research Ethics Committee of the Institute of Dalian Medical University (Permit Number: SYXK2004—0029). 2.2. Cloning of a cathepsin D homologue from lampreys In the previous studies, the cDNA libraries from the buccal gland, liver, and leukocyte cell of lampreys (L. japonica) have been constructed, respectively (Gao et al., 2005; Zhang et al., 2010; Zhu et al., 2008). Based on the analysis of the expressed sequence tags (ESTs)
of the buccal gland cDNA library from the lampreys, two cDNA sequences which are homologous to cathepsin D were identified by using the Basic Local Alignment Search Tool X (BLASTX) in the National Center for Biotechnology Information (NCBI, http:// www.ncbi.nlm.nih.gov/). All PCR primers were designed based on the sequences in the lamprey buccal gland cDNA library which are homologous to cathepsin D (Supplementary Table S1). Total RNA was isolated from the buccal gland of lampreys (L. japonica) according to the manufacturer’s protocol (TaKaRa, Dalian, China) and treated with the RNase-free DNase I (TaKaRa, Dalian, China) to remove genomic DNA contamination. First-strand cDNA was synthesized from the total RNA with PrimeScriptTM RT-PCR Kit (TaKaRa, Dalian, China) and stored at −80 °C. To obtain the full length cDNA of lamprey cathepsin D, rapid amplification of 5′ cDNA ends (5′-RACE) and 3′RACE was performed with the 5′ Full RACE Core Set Kit (TaKaRa, Dalian, China) and 3′ Full RACE Core Set Kit (TaKaRa, Dalian, China), respectively. All the PCR amplifications were carried out by using LA Taq DNA polymerase (TaKaRa, Dalian, China) and cloned into a pMD19-T Simple Vector for sequence confirmation. 2.3. Sequence analysis The full length cDNA sequence of lamprey cathepsin D was spliced by Sequencher 4.2 software and the deduced amino acids were analyzed with DNAMAN V6 and DNASTAR 5.0 software, respectively. The primary structure was analyzed by ProtParam (http:// www.expasy.org/). A three-dimensional structure model of lamprey cathepsin D was constructed using the Phyre software (version 0.2) with the X-ray structure of pepsin-like acid proteases (d3psga_) as a template, and visualized using the UCSF Chimera program package (Kelley and Sternberg, 2009; Pettersen et al., 2004). 2.4. Sequence alignment and phylogenetic tree construction Additional 29 cathepsin D sequences from the other species were obtained from ExPASy (http://www.expasy.ch/tools/blast). The multiple sequence alignments of cathepsin D were performed by ClustalX 1.83 software using default settings (Thompson et al., 1997). A neighbor-joining tree was constructed by MEGA 4.1 software based on the pair-wise deletion of gaps/missing data and a p-distance matrix of an amino acid model with 1000 bootstrapped replicates (Tamura et al., 2007). 2.5. Real-time quantitative PCR analysis The lampreys with an average body weight of 200 g were kept in aquaria at 16 ± 2 °C for two weeks before challenging. The Escherichia coli (E. coli) or Staphylococcus aureus (S. aureus) was cultured at 37 °C to logarithmic growth in LB liquid medium. The cells were then inactivated with formalin (He et al., 2005). The inactivated bacteria cells were resuspended after centrifugation to approximately 2 × 108 CFU/ml in phosphate buffered saline (PBS, pH 7.2). The lampreys were randomly divided into three groups (ten lampreys in each group) and intraperitoneally injected with PBS (0.1 ml), inactivated E. coli (0.1 ml) and S. aureus (0.1 ml) at 8-day intervals, respectively. After the fourth injection, the buccal glands, gills, hearts, livers, intestines, kidneys, and immune bodies of the lampreys were respectively dissected for RNA extraction as described above. Three tissues were pooled together as one sample (three lampreys per pool, three pools per tissue). The expression level of lamprey cathepsin D was assayed by using TaKaRa SYBR® PrimeScriptTM RT-PCR Kit with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control (Kawano-Yamashita et al., 2007; Pfaffl, 2001). Each reaction contained 1 × SYBR Premix Ex Taq, forward primer (10 μM), reverse primer (10 μM) and cDNA (100 ng/μl) with a final volume of 25 μl. The amplification was carried out on a TaKaRa PCR Thermal
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Cycler Dice Real Time System with the parameters as follows: 95 °C for 10 s, followed by 45 cycles of 5 s at 95 °C, 30 s at 60 °C, 30 s at 72 °C, and a final extension step at 72 °C for 10 min. Specific primers for lamprey cathepsin D and lamprey GAPDH were designed based on the sequences in the lamprey buccal gland cDNA library (Supplementary Table S1). Each sample was performed in triplicate and the data were analyzed with the SAS Proprietary Software Release 8.02. Student’s t-tests were used to determine statistical significance (* < 0.05; ** < 0.01) (Livak and Schmittgen, 2001). 2.6. Expression and purification of lamprey cathepsin D Lamprey cathepsin D without the signal peptide (Leu 21-Val 397) was amplified and subcloned into pcDNA 3.1 vector with a HisFlag tag. The protein expression was induced in the human embryonic kidney 293 (HEK 293) cells for 48 h. The cells were digested by 0.25% trypsin (Hyclone, USA), collected by centrifugation, and washed in PBS. After centrifugation, the cells were resuspended in 50 mM Tris–HCl (pH 8.0) containing 500 mM NaCl, 0.1% Triton X-100, 1 mM dithiothreitol (DTT), and 5% glycerol, and then sonicated on ice for 10 min. The soluble supernatant was collected through centrifugation and subjected to an ANTI-FLAG® M2 affinity resin column (Sigma, USA) equilibrated with binding buffer (50 mM Tris–HCl, 300 mM NaCl, 0.1% Triton X-100, 1 mM DTT, and 5% glycerol, pH 8.0). After washing the column with wash buffer (50 mM Tris–HCl, 500 mM NaCl, 1 mM DTT, and 5% glycerol, pH 8.0), the recombinant protein was collected in elution buffer containing 50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 150 μg/μl Flag peptide (Sigma, USA), 1 mM DTT and 10% glycerol. The concentration of recombinant lamprey cathepsin D was measured using a Bicinchoninic Acid (BCA) Protein Assay kit (Pierce, USA). The purified recombinant lamprey cathepsin D (0.2 μg) was analyzed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie brilliant blue. 2.7. Identification of recombinant lamprey cathepsin D The protein bands in the gel were excised from the gel and destained in acetonitrile containing 50 mM ammonium bicarbonate. Subsequently, the samples were dehydrated by acetonitrile for 10 min. After reductive alkylation by dithiothreitol (10 mM) and iodoacetamide (50 mM) for 2 h, the samples were digested with trypsin (25 mM, Promega) in-gel overnight. The peptide mixtures were analyzed by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF/TOF) mass spectrometry (Bruker, USA).
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3. Result 3.1. A cathepsin D homologue was found in the buccal glands of lampreys A full length cDNA (1790 bp) of lamprey cathepsin D homologue was obtained from the cDNA library of the buccal glands of lampreys (Fig. 1), including a 124-bp 5′ terminal untranslated region (5′-UTR), a 1194-bp encoding region and a 472-bp 3′ terminal untranslated region (3′-UTR). A polyadenylation signal sequence, AATAAAA, was located at 16 bp upstream of the poly (A) tail. The open reading frame of lamprey cathepsin D was 1194 bp encoding a protein with 397 amino acid residues. According to the ProtParam program predication, the predicted molecular mass weight and the theoretical isoelectric point of lamprey cathepsin D were 42, 772 Da and 6.23, respectively. Similar to the other cathepsin D proteins, lamprey cathepsin D also contains a signal peptide (Supplementary Fig. S1A, orange, Met 1-Ala 20), a propeptide domain (Supplementary Fig. S1A, marine, Leu 21-Ala 48) and a mature domain (Supplementary Fig. S1A, green, Glu 76-Val 397). According to the analysis of the amino acid sequence, one N-glycosylation site (Supplementary Fig. S1A, yellow, Asn 62) and two catalytic motifs (Supplementary Fig. S1A, red, Asp 95-Ser 98 and Asp 282-Thr 285) were found in lamprey cathepsin D. As shown in Supplementary Fig. S1A, the active site flap of lamprey cathepsin D was AIQYGTGSLSG, in which the specific substrates or inhibitors could be enclosed. The nucleotide sequence of lamprey cathepsin D has been submitted to the GenBank database (accession number: KM051432). Spatial homology model of lamprey cathepsin D showed a completely conserved bilobal structure with two catalytic motifs (Supplementary Fig. S1B, red, DTGS and DTGT) on each side of the active site flap (Supplementary Fig. S1B, purple, AIQYGTGSLSG) which is related to substrate binding. A “β-loop” (Supplementary Fig. S1, pink, Cys316-Gly345) and a “polyproline loop” (Supplementary Fig. S1, blue, Gly359-Pro379) were found in the proximity of the active site flap. Conserved Lys 318 and Lys 344 (Supplementary Fig. S1, black) were located at the “β-loop”, while repeated proline residues were found in the “polyproline loop” region of lamprey cathepsin D. Different from human cathepsin D, lamprey cathepsin D only has two conserved lysine residues, but lacks a Lys 203 residue which is important for mammal phosphotransferase recognition (Steet et al., 2005). 3.2. Lamprey cathepsin D shares high homology with the cathepsin D from the other species
2.8. Recombinant lamprey cathepsin D activity assay The recombinant lamprey cathepsin D (0.1 μg/μl, final concentration) which was dissolved in 50 mM Tris–HCl containing 150 mM NaCl (pH 8.0) was added into 100 mM formic acid for 3 h at 25 °C for auto-activation (pH of the mixture, 3.5). The recombinant lamprey cathepsin D in 50 mM Tris–HCl containing 150 mM NaCl (0.1 μg/ μl, final concentration) was incubated with bovine hemoglobin (7 mg/ml, final concentration, Sigma, USA), fibrinogen (2 mg/ml, final concentration, Sigma, USA), and bovine serum albumin (0.8 mg/ ml or 2 mg/ml, final concentration, Solarbio, China), respectively. Hemoglobin, fibrinogen and bovine serum albumin were dissolved in 100 mM formic acid, respectively. Each mixture (pH 3.5) was incubated at 25 °C for 20 h. The reaction was terminated by addition of 4% (w/v) SDS in 100 mM Tris–HCl buffer (pH 6.8) containing 200 mM DTT, 0.2% bromophenol blue and 20% glycerol. Hemoglobin, fibrinogen, and bovine serum albumin which were dissolved in 100 mM formic acid were also incubated at 25 °C for 20 h, respectively. Aliquots were taken for 12% SDS-PAGE and stained with Coomassie brilliant blue.
Multiple sequence alignments of cathepsin D indicated that lamprey cathepsin D is a very conservative gene and shares high homology with cathepsin D in the other species (Fig. 2). Lamprey cathepsin D shows about 67–72% identities with the cathepsin D from teleosts, amphibians, reptiles, birds, and mammals. While it only has 52–59% sequence similarities with the cathepsin D from the invertebrates, including echinoderms, arthropods, and mollusks (Supplementary Table S2). According to the sequence analysis, the catalytic motifs (DTGS and DTGT) were completely conserved among all of the cathepsin D sequences. In addition, the active site flap of lamprey cathepsin D (AIQYGTGSLSG) shares high similarity with that from the other organisms. In order to study the phylogenetic relationship between lamprey cathepsin D and the other cathepsin D, a phylogenetic tree was constructed through the neighbor-joining method (Supplementary Fig. S2). Phylogeny of 30 cathepsin D sequences indicated that all cathepsin D homologues used in this study were classified into two clusters: vertebrate cluster and invertebrate cluster. Vertebrate cluster includes cathepsin D from birds, reptiles, agnathans, mammals,
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teleosts and amphibians; the second includes cathepsin D from mammals; and the third includes those from reptiles, birds and agnathans, respectively (Supplementary Fig. S2). Phylogenetic analysis indicated that lamprey cathepsin D was clustered as the out group of cathepsin D from reptiles and birds. 3.3. The expression pattern of lamprey cathepsin D Real-time quantitative PCR was used to investigate the relative expression pattern of cathepsin D in the various tissues of lampreys (L. japonica). Lamprey GAPDH was used as an internal control. In the control group, the expression level of lamprey cathepsin D was a little higher in the buccal glands, immune bodies, and hearts than that in the intestines, kidneys, livers, and gills (Fig. 3). After stimulation with E. coli or S. aureus by intraperitoneal injection, the expression level of lamprey cathepsin D in the buccal gland was 8.5fold or 6.5-fold higher than that in the PBS group. In addition, lamprey cathepsin D stimulated with E. coli was also up-regulated in the hearts, kidneys, and intestines of lampreys (Fig. 3, P < 0.05). As for the lampreys treated with S. aureus, the expression level of lamprey cathepsin D was also increased in the intestines. Compared with the E. coli stimulated group, lamprey cathepsin D was not significantly expressed in the S. aureus treated group. 3.4. The recombinant lamprey cathepsin D could degrade hemoglobin, fibrinogen, and serum albumin
Fig. 1. The nucleotide and deduced amino acid sequences of lamprey cathepsin D. The nucleotide (upper line) and amino acid (lower line) sequences are numbered from the guanine and the initial methionine, respectively. The stop codon is marked with an asterisk and indicated in red. A signal peptide splice site is located between Ala 20 and Leu 21 labeled with a purple arrow. One N-glycosylation site and two catalytic residues are indicated in green and blue, respectively. A polyadenylation signal sequence, AATAAAA, is located at 16 bp upstream of the poly (A) tail marked with yellow. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
amphibians and teleosts; invertebrate cluster is composed of cathepsin D from mollusks and arthropods. Except the cathepsin D from Ictalurus punctatus, vertebrate cluster can be further classified into three subgroups, the first one contains cathepsin D from
Lamprey cathepsin D was subcloned into pcDNA 3.1 vector and expressed as a His-Flag-tagged fusion protein in HEK 293 cells. As shown in Supplementary Fig. S3A, there are mainly two protein bands detected on 12% SDS-PAGE. According to the MALDI-TOF/ TOF analysis, the peptides generated in gel digestion (66 kDa) were the fragments (ISSIQSIVPALEIANAHR, TLNDELEIIEGMKFDR, KPLVIIAEDVDGEALSTLVLNR, and LVQDVANNTNEEAGDGTTTATVLAR) of heat shock protein 60 (GI:306890); while the peptides generated in gel digestion (43 kDa) were the fragments (NVFSFYLNR and YYKGELSYVPVTR) of lamprey cathepsin D, suggesting that lamprey cathepsin D was expressed successfully. After auto-activation, the recombinant lamprey cathepsin D migrated as a single band on 12% SDS-PAGE (Supplementary Fig. S3B), suggesting that the recombinant lamprey cathepsin D was able to degrade heat shock protein 60 in acidic conditions. Recently, several reports have shown that cathepsin D from the bloodsuckers could degrade hemoglobin in acidic conditions (Brinkworth et al., 2001; Sojka et al., 2012; Williamson et al., 2002). To investigate the potential roles of lamprey cathepsin D in their buccal glands, the recombinant lamprey cathepsin D was incubated with hemoglobin in acidic conditions for 20 h. 12% SDS-PAGE showed that the hemoglobin was degraded in the presence of the recombinant lamprey cathepsin D (Supplementary Fig. S3C). And several degradation fragments could be detected on the gel. According to the analysis of gray scan of the hemoglobin, 50% hemoglobin was digested by lamprey cathepsin D (data not shown). When the recombinant lamprey cathepsin D was incubated with fibrinogen at 25 °C for 8 h, the Aα and Bβ chains of fibrinogen were digested completely. And the γ chain was degraded partly. As the incubation time extended to 20 h, the γ chain was also hydrolyzed completely (Supplementary Fig. S3D). In addition, bovine serum albumin was also hydrolyzed in the presence of the recombinant lamprey cathepsin D, and several degradation fragments had also been detected on the gel (Supplementary Fig. S3E). 4. Discussion Parasitic phase lampreys are eel like animals which usually attack the host fishes to suck the flesh and blood. Like the other
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Fig. 2. Multiple sequence alignments of lamprey cathepsin D with cathepsin D from the other species by using ClustalX 1.83. The accession numbers of the amino acid sequences extracted from the EXPASY database are as follows: Homo sapiens, NP_001900.1; Mus musculus, NP_034113.1; Gallus gallus, NP_990508.1; Pelodiscus sinensis, XP_006134990.1; Xenopus laevis, BAC57431.1; Danio rerio, NP_571785.1; Lampetra japonica, KM051432; Aedes aegypti, XP_001657556.1; Pinctada margaritifera, AFE48185.1; Apostichopus japonicus, AEG79714.1. Dashes represent gaps inserted into the alignment. Identical residues are indicated by asterisks. Strong and weak homologous residues are indicated in colons (:) and dots (.), respectively. The catalytic motifs and aspartyl protease active site flap are covered with the transparent box and gray box, respectively.
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Fig. 3. The mRNA expression level of lamprey cathepsin D after stimulation with E. coli and S. aureus, respectively. Real time quantitative RT-PCR was performed to determine the lamprey cathepsin D mRNA levels in various lamprey tissues. Total RNAs were extracted from buccal glands, hearts, kidneys, intestines, immune bodies, livers and gills of lampreys collected after stimulation with PBS, E. coli, and S. aureus, respectively. Lamprey GAPDH served as an internal control to calibrate the cDNA template for all the samples. The significant differences of lamprey cathepsin D expression between the challenged groups and the blank group are indicated by asterisks (*: P < 0.05; **: P < 0.01).
bloodsuckers, such as triatomas, bugs, ticks, leeches, and vampire bats (Basanova et al., 2002; Zavalova et al., 2002), the buccal gland secretion of lampreys also contains several regulators related to the anticoagulation, analgesia, and immune defense (Chi et al., 2009; Gao et al., 2005; Ito et al., 2007; Liu et al., 2009; Sun et al., 2008, 2010a, 2010b; Wang et al., 2010; Wong et al., 2012; Xiao et al., 2007, 2012; Xue et al., 2011). In this study, the full-length cDNA of a cathepsin D homologue was obtained from the buccal gland of lampreys (L. japonica) for the first time. Sequence analysis indicated that lamprey cathepsin D is a highly conserved gene and also contains a typical structure including a signal peptide, a propeptide, and a mature domain which should be activated by a series of proteinases. According to the spatial structure analysis, lamprey cathepsin D showed a conserved bilobal structure with two catalytic motifs on each side of the active site flap. In addition, the catalytic motifs (DTGS and DTGT) and the active site flap (AIQYGTGSLSG) of lamprey cathepsin D, which are typical characteristics of aspartic proteases, were highly conserved. Thus, the novel cathepsin D homologue found in the buccal gland of lampreys may be considered as a digestive enzyme which is essential for the process of blood intake. In 1995, Fortenberry et al. reported that glycosylation was not necessary for the folding and expression of cathepsin D, but was essential for the new synthetized cathepsin D to anchor to the lysosome (Fortenberry et al., 1995). In addition, Lys 203, and the “β-loop” which contained Lys 267 and Lys 293 in the human cathepsin D were proved to help cathepsin D target the lysosome through the mannose 6-phosphate pathway (Baranski et al., 1991). Based on the sequence analysis, a N-glycosylation site (Asn 62) and two conserved lysines (Lys318 and Lys 344) were also found in the lamprey cathepsin D, suggested that these residues are important in lamprey cathepsin D location. Lamprey cathepsin D was widely expressed in the buccal glands, immune bodies, hearts, intestines, kidneys, livers, and gills of
lampreys based on real-time quantitative PCR analysis. And the strongest expression was found in the buccal glands, which suggested that lamprey cathepsin D may be related to the feeding habit of lampreys. Recently, a great number of studies have shown that several cathepsin D proteins from the blood suckers prefer to degrade hemoglobin which is the main source of amino acids for the hematophagous parasites (Brinkworth et al., 2001; Sojka et al., 2012; Verity et al., 1999; Williamson et al., 2002). After auto-catalytical activation, the mature form of cathepsin D could degrade the substrates such as hemoglobin in acidic conditions (Brinkworth et al., 2001; Sojka et al., 2012; Williamson et al., 2002). In the present study, lamprey cathepsin D was successfully expressed in the HEK 293 cells. In acidic conditions, the recombinant protein could also hydrolyze hemoglobin, fibrinogen, and serum albumin which are the major components present in blood. As lampreys feed on the blood and flesh of host fishes, their buccal glands may also provide cathepsin D as a digestive enzyme. Previous studies have indicated that several cathepsin D proteins from parasites and fishes could also participate in the immune defense process against microbial pathogens (Boldbaatar et al., 2006; Borges et al., 2006; Buarque et al., 2013; Chen et al., 2011; Dong et al., 2012; Franta et al., 2010; Horn et al., 2009; Jeffers and Roe, 2008; Mu et al., 2013). And the expression level of cathepsin D was also found increased in the various tissues of grass carp (Ctenopharyngodon idella), channel catfish (Ictalurus punctatus), half-smooth tongue sole (Cynoglossus semilaevis), hard tick (Haemaphysalis longicornis and Ixodes ricinus), and triatomine (Rhodnius prolixus) during the infection with pathogenic microorganisms (Boldbaatar et al., 2006; Borges et al., 2006; Chen et al., 2011; Dong et al., 2012; Franta et al., 2010; Horn et al., 2009; Jeffers and Roe, 2008; Mu et al., 2013). After intraperitoneal injection with E. coli or S. aureus, the expression level of lamprey cathepsin D in the buccal gland was 8.5-fold or 6.5-fold higher than that in the PBS group (control), which indicated that lamprey cathepsin D may act as an effective immune defender. Usually, the microbial pathogens are widely distributed in the marine environment where the parasitic phase lampreys lived. And the surface of the host fishes may also carry some specific bacteria. Except the fleshes and blood of host fishes, lampreys might also suck the microbial pathogens with their unique buccal funnel during the feeding process. Thus, the cathepsin D secreted from the buccal gland would act as the first barrier to protect lampreys from infection by the bacteria effectively. Interestingly, lamprey cathepsin D was more sensitive to gram negative bacteria (such as E. coli) than gram positive bacteria (such as S. aureus) based on real-time quantitative PCR analysis. In 2006, the activity of cathepsin D in the midgut of Rhodnius prolixus infected with Trypanosoma cruzi was higher than that in the uninfected group (Borges et al., 2006). Compared with the PBS group, the increase in lamprey cathepsin D expression was also found in the intestines after stimulation with E. coli or S. aureus, suggesting that the intestines of lampreys might be the other organs important for exogenous pathogen clearance. Several studies have indicated that cathepsin family proteases have bactericidal abilities such as cathepsin-D, -L, -B or -G (Thorne et al., 1976). In 2009, Eugenio Carrasco-Marín et al. put forward that cathepsin D might kill Listeria monocytogenes or Streptococcus pneumonia through degradation of listeriolysin O or pneumolysin at the same undecapeptide sequence between the Trp 491 and Trp 492 residues or between Trp 435 and Trp 436 residues (Carrasco-Marín et al., 2009). In addition, the activated cathepsin D was proved to be able to induce macrophage apoptosis through down-regulation of Mcl-1, which enhances the antimicrobial effect of macrophage during late phase killing of pneumococci (Bewley et al., 2011). However, little work has been done on the antibacterial activity and mechanisms of lamprey cathepsin D, and it needs further studies to elucidate.
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5. Conclusions This report represents the characterization of a novel cathepsin D homologue obtained from the buccal gland of lampreys for the first time. And expression pattern analysis showed that lamprey cathepsin D was related to immune defense and might act as a potential antibacterial agent in the near future. In addition, the recombinant lamprey cathepsin D was related to the digestive process of lampreys due to its ability to degrade hemoglobin, fibrinogen, and serum albumin.
Acknowledgements This work was supported by grants from The National Natural Science Foundation of China (No. 31301880), China Postdoctoral Science Foundation (No. 2013M541246), New Teacher of Specialized Research Foundation for the Doctoral Program of Higher Education of China (No. 20112136120002), Scientific Research Fund of Liaoning Provincial Education Department (No. L2011187), and the Scientific and Technological Research Projects of Dalian (No. 2011J21DW014).
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