Cathepsin L proteases of the parasitic copepod, Lepeophtheirus salmonis

Aquaculture 356–357 (2012) 264–271

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Cathepsin L proteases of the parasitic copepod, Lepeophtheirus salmonis Elaine McCarthy a,⁎, Eleanor Cunningham a, Lorraine Copley b, David Jackson b, David Johnston c, John P. Dalton d, Grace Mulcahy a a

School of Agriculture, Food Science and Veterinary Medicine, Veterinary Sciences Building, University College Dublin, Belfield, Dublin 4, Ireland Marine Institute, Rinville, Oranmore, Co. Galway, Ireland Biomedical Parasitology Division, Department of Zoology, Natural History Museum, Cromwell Road, London, UK d Institute of Parasitology, McGill University, Montreal, Canada b c

a r t i c l e

i n f o

Article history: Received 7 July 2010 Received in revised form 23 April 2012 Accepted 5 May 2012 Available online 12 May 2012 Keywords: L. salmonis Protease Cathepsin L Ectoparasite

a b s t r a c t The salmon louse, Lepeophtheirus salmonis, is a parasitic copepod that feeds on the mucus, skin and blood of salmonids. We describe the identification of two complete L. salmonis cathepsin L-like gene sequences and their molecular characterisation. L. salmonis cathepsin L1 (LsCL1), is 978 base pairs in length, encoding a protein of 325 amino acid residues while L. salmonis cathepsin L2 (LsCL2) is 1149 base pairs in length, encoding a protein of 382 amino acid residues. The predicted molecular weights of LsCL1 and LsCL2 are 35,964 Da and 42,150 Da respectively. The two proteases share only 25% identity in the primary sequences; however, the catalytic triad of cysteine, histidine and asparagine is highly conserved for both. Biochemical analysis of L. salmonis extracts revealed that cathepsin L has an optimum activity at pH 6.5, at 15 °C and remains stable at this temperature. Cathepsin L activity is present in all of the parasite life stages assayed, with the chalimus life stage extract exhibiting the most activity. Cathepsin L activity was also observed in the secretory/excretory products possibly indicating a role for this protease in immunoevasion and establishment of the parasite on the host. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Lepeophtheirus salmonis infection is a parasitic infection that causes major economic loss in the salmonid farming industry worldwide (Costello, 2009). During infection, the copepodid stage attaches to the host and molts into the first of four chalimus stages, all of which are physically attached to their host by a frontal filament The fourth chalimus stage molts into the first of two pre-adult stages which are free moving on the host with the exception of a short time during the molt when a temporary frontal filament is formed. The second preadult stage molts into free moving adults which undergo no further molting. All developmental stages of the parasite feed on fish mucus and skin, while the pre-adult and adult stages can also ingest blood. While heavy infestations rarely occur in wild populations, sea lice infection can have a devastating effect on farmed salmon if left untreated (Schram, 1993). While the literature relating to sea lice is ever expanding and our knowledge of the parasites' biology and ecology is growing, more research is required to identify the molecules employed by these parasites to establish and maintain infection. Cysteine proteases are members of the papain-like proteases classed as the C1 family (Bromme, 2001). All papain-like cysteine proteases consist of a signal peptide, a pro-peptide and a mature proteolytically active enzyme. Signal peptides are responsible for translocation into the endoplasmic reticulum during ribosomal protein expression. Pro⁎ Corresponding author. Tel.: + 1 353 1 7166138; fax: + 1 353 1 7166185. E-mail address: [email protected] (E. McCarthy). 0044-8486/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2012.05.007

peptides of variable length function as a scaffold for protein folding of the catalytic domain, as a chaperone for the transport of the propeptide to the endosomal lysosomal compartment and as a high affinity reversible inhibitor preventing the premature activation of the catalytic domain (Lecaille et al., 2002). Several cathepsin-type cysteine proteases have been detected in non-lysosomal regions of eukaryotic cells and embryos, leading to speculation that the enzymes function in reactions outside lysosomes and may accumulate in different organelles (Butler et al., 2001). Cathepsin L cysteine proteases belong to the C1 family and are categorised into the Clan CA (Rawlings and Barrett, 1994; Yamaji et al., 2009). This class of protease has long been implicated in critical parasitic functions. In the parasitic ciliate Ichthyophthirius multifiliis, it has been suggested that cathepsin L may play a role in invasion of the fish host epidermis (Jousson et al., 2007). Cathepsin L-like proteases from the helminth parasites Fasciola hepatica and Schistosoma mansoni have been shown to be involved in activities such as invasion of tissues, feeding, immune evasion and egg shell formation (Brady et al., 2000; Dvorak et al., 2009; Smooker et al., 2000). Similarly, in the Chinese liver fluke, Clonorchis sinensis, cathepsin L facilitates the invasion of the cercariae into the intermediate fish host by secretion of the enzyme through the tegument of parasite thus penetrating the skin and migrating to the host muscle (Li et al., 2009). In crustaceans, cathepsin L has been purified from the gastrointestinal juice of the American lobster, Homarus americanus (Laycock et al., 1989). Additionally, cathepsin L cDNA has been cloned from the digestive glands, the hepatopancreas, of H. americanus (Laycock et al., 1991) and from the shrimps Litopenaeus

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vannamei (Le Boulay et al., 1996) and Metapenaeus ensis (Hu and Leung, 2004). More recent studies have demonstrated that shrimp cathepsin L has a role in intracellular digestion within the vacuoles of cells and is secreted extracellularly into the lumen of the hepatopancreas and stomach (Hu and Leung, 2007). To date, there has been limited characterisation of the proteases of L. salmonis. A number of L. salmonis trypsin and trypsin-like serine proteases have been identified and localised to the gut, involved in digestion (Johnson et al., 2002; Kvamme et al., 2004, 2005) and to the egg, involved in yolk degradation (Skern-Mauritzen et al., 2009). Our group has identified and biochemically characterised an L. salmonis cathepsin B-like cysteine protease (Cunningham et al., 2010) and localised it to the gut of the parasite (unpublished data), but there is little characterisation on other cysteine proteases of the parasite. Three reportedly complete L. salmonis cathepsin L-like proteins have been deposited in GenBank (GenBank accession nos. ADD24149, ADD24160 and EF490928), and a single putative incomplete cysteine protease nucleotide sequences was deposited in GenBank following an EST sequencing project (GenBank accession no. EF490847), but no further analysis on these sequences has been published. The present paper details two complete cathepsin L gene sequences and their molecular characterisation. We also show that cathepsin L activity is expressed in the different L. salmonis life stages and have biochemically characterised the activity in adult stages. 2. Materials and methods 2.1. Chemicals and parasites The peptide substrate Z-Phe-Arg-NHMec was purchased from Bachem Biosciences (Germany). Protease inhibitors used included L-3-trans-(propylcarbamyl)oxirane-2-carbonyl)-L-isoleucyl-L-proline (CA074) which inhibits cathepsin B activity and trans-epoxysuccinyl-Lleucylamido(4-guanidino)butane (E64) (Sigma St. Louis, Mo.) which inhibits both cathepsin L and B activity. Adult L. salmonis were collected from infected salmon found on established fish farms in the West of Ireland. Female adult lice were separated and stored in RNAlater® (Ambion®, Applied Biosystems, Austin, TX 78744‐1832) at −20 °C and subsequently used for library construction. Various life stages (chalimus I–IV, preadult male I–II, preadult female I–II, adult male, adult female and egg strings) were collected from infected fish and stored at −20 °C. Nauplius stages were hatched from egg strings as per Pietrak and Opitz (2004) and stored at −20 °C. Life stages of parasites processed into extracts were subsequently used for biochemical characterisation as per Section 2.5. 2.2. Construction of L. salmonis cDNA library Approximately 120 adult female sea lice were homogenised under RNase-free conditions. Total RNA was isolated using RNeasy® Protect Midi Kit (Qiagen, CA 91355, USA) according to the manufacturer's instructions. mRNA was subsequently prepared with an Oligotex mRNA Midi kit (Qiagen). Poly-A RNA (6 μg) was used for cDNA library construction using a ZAP cDNA synthesis kit (Stratagene, CA 92037, USA). cDNA was digested with Xho I and resulting cDNA fragments were size fractionated on a Sepharose column and ligated into the Uni-ZAP vector. Following ligation, the whole library was packaged using Gigapack® III Gold packaging extract (Stratagene) according to the manufacturer's protocol. 2.3. L. salmonis cDNA library sequencing In vivo mass excision was performed on the library according to manufacturer's recommendations. Plasmids were purified from resulting white colonies and glycerol stocks prepared. Over 1900 expressed

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sequence tags (ESTs) were sequenced commercially in one direction using the M13 reverse primer (5′-CATGGTCATAGCTGTTTCC-3′). Vector sequences were trimmed using the Sequencher program and BLAST analysis was performed on the resulting sequences against the GenBank database. The partial L. salmonis cathepsin L2 gene was identified by this method. 2.4. PCR amplification and sequencing of L. salmonis cathepsin L1 and L2 genes and phylogenetic analysis Analysis of the GenBank database revealed four L. salmonis cathepsin L sequences in the public databases (GenBank accession nos. EF490928, EF490847, ADD24149 and ADD24160). L. salmonis cathepsin L1 was so named as it was most similar to the previously defined copepod crustacean Caligus clemensi cathepsin L1 (GenBank accession no. ACO15375). L. salmonis cathepsin L2 was named to continue this numerical sequence. Based on alignments carried out on the GenBank accession no. EF490928, it was apparent that this sequence was incomplete. In order to complete this L. salmonis cathepsin L1, (LsCL1), rapid amplification of cDNA ends (RACE) was performed (First choice® RLM-RACE kit, Ambicon). Gene specific RACE reverse primers (outer primer: 5′-GAAATATTGTCCCTCTACAGAG-3′; inner primer: 5′AACCTTTGGCTAATCCAGT-3′) were designed based on the nucleotide sequence available and used to amplify the 5′ end of the gene, thus confirming a nucleotide insertion in the published sequence. In order to confirm a deletion present in the published sequenced, the 3′ end of the gene was amplified using the forward primer (5′-CTTATTGCTAATAAAGGTATAGCTAC-3′) and reverse primer (5′-TTAAATGACGGGATATGAAGCCAGTG-3′). The resultant cDNA fragments were ligated into the pGem-T Easy® vector (Promega, Madison, WI 53711, USA) for sequencing. A partial sequence for LsCL2 was obtained following the sequencing of the L. salmonis cDNA library (Section 2.3). RACE was used to amplify the missing 5′ end of LsCL2 (outer primer: 5′-ACCAGTCACAGATGAGGCAT-3′; inner primer: 5′‐GGCACCCTTACATCC-3′). cDNA fragments amplified by this method were sequenced and found to be identical to the published sequence (GenBank accession no. EF490847) albeit missing its 5′ nucleotide sequence. Using the sequence data above, a full length LsCL2 gene was amplified from L. salmonis adult female cDNA using the forward primer (5′ATGAAAATTCATTTCAAGATG-3′) and the reverse primer (5′TTACTCAAATGTGGCTCC-3′) and ligated into the pGem-T Easy® vector for sequencing. Each PCR reaction contained 1X PCR Buffer, 2.5 mM MgCl2, 0.2 mM dNTP, 1 U DNA taq polymerase, 10 pmol primer and cDNA template (0.1 μg/μl) cycled as follows: initial denaturation of 1 cycle of 95 °C for 3 min, 35 cycles of denaturation at 95 °C for 1 min, primer annealing at 50 °C for 1 min, extension at 72 °C for 1 min and a final extension of 1 cycle of 72 °C for 7 min. The ExPASy Molecular Biological Server (http://ca.expasy.org/) (Gasteiger et al., 2005) provided the tools by which to analyse the secondary structure of the encoded LsCL1 and LsCL2 proteins. The program Prot Param available on ExPASy was used to predict the molecular weight and theoretical pI of the elucidated L. salmonis cathepsin L protein sequences. Glycosylation sites were predicted using the NetNGly 1.0 program available on ExPASy. The prediction of signal peptide cleavage sites was carried out using the SignalP program available on ExPASy. Multiple alignments were performed on the UniProt Server (http://www.uniprot.org/) using the ClustalW algorithm (Thompson et al., 1994). Positioning of relevant cathepsin L conserved sites in Fig. 1 were determined following an alignment of the L. salmonis cathepsin L sequences (GenBank accession numbers: HM439291, HM439290, ADD24160 and ADD24149) and the closest matches of the elucidated LsCL1 sequence (C. clemensi cathepsin L1 precursor, C. clemensi cathepsin L precursor, Caligus rogercresseyi cathepsin L precursor and L. vannamei cathepsin L available at GenBank accession

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Fig. 1. Clustal 2.0.10 multiple sequence alignment comparing LsCL1 (GenBank accession no. HM439291), LsCL2 (GenBank accession no. HM439920), L. salmonis cathepsin L-like proteinase, termed LsCL-like (GenBank accession no. ADD24149) and L. salmonis cathepsin L, termed LsCL (GenBank accession number. ADD24160). Conserved residues are denoted by *. The signature sequences for cathepsin L “ERFNIN”, “GNFD” and “GCNGG” are highlighted in blue and are shown above the alignment. The highly conserved catalytic triad, Cys-131, His-270, Asn-292 (LsC1 numbering), is highlighted in red. The S2 subsite for determining substrate specificity is indicated above the alignment. The cleavage site predicted by SignalP in LsCL1 is denoted by “▼” and in LsCL2 is denoted by “■” above the alignment. The beginning of the mature peptide for LsCL1 and LsCL2 is indicated by “ ” above the alignment.

numbers: ACO15375; ACO14903; ACO10357 and CAA74241 respectively) and the closest matches of the elucidated LsCL2 sequence (C. clemensi cathepsin K, Tetrahymena thermophila cysteine protease and I. multifiliis cathepsin L cysteine protease ICP1 available at GenBank accession numbers: ACO15154; XP_001022356 and ABH06549 respectively) identified using the BLAST server available at NCBI (http://blast.ncbi.nlm.nih.gov/). Alignment of the above sequences allowed identification of the mature peptide, conserved catalytic site residues, cathepsin L motifs and the substrate binding site as per the published sequence analysis on the I. multifiliis cathepsin L cysteine protease ICP1 (Jousson et al., 2007; Karrer et al., 1993). Trees were created using the Neighbour Joining algorithm (Saitou and Nei, 1987). Sequence data were subjected to phylogenetic

analysis by a maximum likelihood tree estimation using the Jukes Cantor substitution model (Felsenstein, 1981) available at CLC DNA Workbench 5 (CLC bio©, Aarhus, Denmark). 2.5. L. salmonis extracts and secretory/excretory products (SEPs) Various life stages (nauplius I–II, chalimus I–IV, preadult male I–II, preadult female I–II, adult male, adult female and egg strings) of L. salmonis, collected from infected fish or hatched from egg strings (nauplius), were homogenized in PBS using a mortar and pestle. The samples were then sonicated on ice for two 30 second pulses at 10 W and frozen at − 80 °C for 15 min. The samples were allowed to defrost on ice and the sonication and freezing cycle repeated twice. They were then centrifuged at 4 °C for 20 min and the protein

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concentration of the supernatants was determined using the BCA protein assay. Supernatants were stored at −20 °C. Secretory/excretory products are those compounds produced by the parasite secreted onto the surface of the host that have been postulated to be involved with feeding or immune evasion (Fast et al., 2004). SEPs were prepared as previously described by Fast et al. (2004) with some amendments. Briefly, approximately two adult lice/ml were incubated in sterile seawater with 0.1 mM dopamine (Sigma-Aldrich) at 10 °C for 1 h. Lice were removed and the solution centrifuged at 3500 ×g for 5 min to remove debris. The protein concentration of SEPs was determined by BCA protein assay (Biorad). 2.6. Cathepsin L fluorometric assay of L. salmonis homogenates Cathepsin L activity was measured using the fluorogenic peptide substrate Z-Phe-Arg-NHMec, (Bachem Biosciences, Germany). Assays were carried out in duplicate in 0.1 mM Tris–HCl pH, 6.5, the presence of 20 μM substrate and 1 mM diothiothreithrol (DTT) in a 96-well plate and repeated in triplicate. The substrate and enzyme were incubated for 1 h, the reaction was stopped with 1.7 M acetic acid and the release of the fluorescent moiety (NHMec) was measured at excitation 370 nm and emission of 440 nm. Enzyme activity for each extract was as calculated in nmol mg− 1 min− 1 and triplicates of experiments are represented by error bars. The inhibitor for cathepsin B, CA074, and the general cysteine protease inhibitor, E64, were used to determine specific cathepsin L activity. Twenty μM of the inhibitor was added to selected wells with sample and incubated for 1 h before addition of the substrate. All assays were carried out at room temperature unless specified otherwise. The pH profile of cathepsin L was determined using several buffers, namely Clarks and Lubs (pH 1–2), glycine–HCl (pH 2.2–3), Mc Ilvaine (pH 3–7.5), Clarks and Lubs (pH 7.5–9.5) (McIlvaine, 1921). Temperature stability studies were performed at 20 °C and 45 °C over a period of 24 h. Samples were taken at hourly intervals and stored at −20 °C until all samples were collected. The temperature activity profile was assessed using a temperature range between 4 °C and 80 °C. 3. Results 3.1. Identification of L. salmonis cathepsin L1 and L2 genes EST sequencing of the cDNA library revealed a partial transcript for a putative cathepsin L, LsCL2. RACE of the 5′ end allowed completion of the full sequence of the LsCL2 gene, comprising 1149 base pairs (GenBank accession no. HM439290), encoding a predicted protein of 382 amino acid residues. The molecular weight of LsCL2 is 42,151 Da, with a theoretical pI of 5.45 as predicted by the Prot Param program available on ExPASy (Gasteiger et al., 2005). According to NetNGly 1.0 predictions, there are two glycosylation sites present at positions 166 and 233 respectively. The SignalP server predicts that the cleavage site of the signal peptide lies between positions 24/25 (AAG/TS). The beginning of the mature LsCL2 peptide, as determined by multiple alignment analysis (alignment not shown, see Section 2.4 for details), lies at position 125. Comparison of this sequence to the database at NCBI identified a partial cathepsin L from L. salmonis, GenBank accession no. EF490847, with 94% identity to LsCL2. A second cathepsin L-like gene sequence was identified in the NCBI database, (GenBank accession no. EF490928) that differed to LsCL2, which we term LsCL1. Although described as a full-length coding sequence, our analysis revealed that both an insertion and subsequent deletion of a single nucleotide yielded a frame shift in the denoted amino acid sequence, therefore the coding sequence described for GenBank accession no. EF490928 is incomplete. Primers designed based on the published sequence were used to amplify the complete L. salmonis cathepsin L-like gene. The resulting sequence, LsCL1, is 978 base pairs in length (GenBank accession no. HM439291), encoding a

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protein of 325 amino acid residues. The predicted molecular weight of LsCL1 is 35,964 Da, with a theoretical pI of 5.73. NetNGly 1.0 predicts one glycosylation site at position 112. SignalP predicts the site of cleavage of the signal peptide between 19/20 (LSG/EW). The beginning of the mature LsCL1 peptide, as determined by multiple alignment analysis (not shown), lies at amino acid position 108. 3.2. Sequence and phylogenetic analysis of L. salmonis cathepsins L1 and L2 Fig. 1 shows an alignment of LsCL1, LsCL2, L. salmonis cathepsin L-like proteinase and L. salmonis cathepsin L (GenBank accession nos. HM439291, HM439290, ADD24149 and ADD24160 respectively). The amino acid sequences for LsCL1 and LsCL2 are 25% identical. LsCL2 contains a N-terminal extension of 15 residues and a C-terminal extension of 30 residues compared to LsCL1. The ERFNIN sequence characterised by Karrer et al. (1993) is present but slightly different in LsCL1 (IRFNIN), while an ERFNIN-like sequence is also observed in LsCL2 (LKFNIN). However, an additional cathepsin L signature, GNFD, as characterised by Vernet et al. (1995) is maintained in LsCL2, while a GNFD-like sequence is present in LsCL1 (ENYD). The cathepsin L signature sequence, GCNGG, as described by Rawlings and Barrett (1994) is present in LsCL1 (GCNGG), while a GCNGG-like sequence is observed in LsCL2 (GGSGG). The catalytic triad of cysteine, histidine and asparagine is highly conserved for both LsCL1 and LsCL2. The active site cysteine is embedded in a highly conserved peptide sequence, CGSCWAFS (Lecaille et al., 2002). Only a small number of cysteine proteases have amino acid replacements in this region and while LsCL1 conforms to the predicted motif, LsCL2 has a valine residue substituted for the conserved alanine. The active site histidine residue is found adjacent to small amino acid residues such as glycine, found in LsCL1, or alanine, found in LsCL2, followed by 4 aliphatic hydrophobic residues such as isoleucine, leucine or valine (loosely retained in LsCL1 and not retained in LsCL2), followed by a glycine residue (present in LsCL1 & LsCL2) (Bromme, 2001; Lecaille et al., 2002). The active site asparagine residue is embedded in the asparagine, serine, tryptophan (Asn-Ser-Trp) motif (Bromme, 2001), found to be conserved in both LsCL1 and LsCL2 sequences. The residues occupying the S2 subsite differ in LsCL1 and LsCL2, namely leucine and threonine respectively. The L. salmonis cathepsin L-like proteinase (GenBank accession no. ADD24149) is a complete protein sequence of 380 amino acids and is more similar to LsCL2, sharing 42% identity, compared to 24% identity with LsCL1. Like LsCL2, it possesses the cathepsin L signature ERFNINlike sequence, a GNFD sequence and a GCNGG-like sequence. The L. salmonis cathepsin L (GenBank accession no. ADD24160) is also a complete protein sequence of 334 amino acids and is more similar to LsCL1, sharing 53% identity, compared to 29% identity with LsCL2. It is the only L. salmonis cathepsin L homologue analysed that possesses an intact ERFNIN signature sequence. L. salmonis cathepsin L has a GNFDlike and a GCNGG-like sequence. The catalytic triad is conserved for both L. salmonis cathepsin L-like amino acid sequences, while LsCL2 and the cathepsin L-like proteinase share a leucine residue in the S2 subsite. A phylogenetic tree (Fig. 2) was prepared following an alignment of the L. salmonis cathepsin L protein sequences with their closest matches from GenBank. LsCL1 is so named as it is most similar (64% identity) to a previously defined cathepsin L1 sequence from the copepod crustacean C. clemensi (GenBank accession no. ACO15375). This close relationship between LsCL1 and its homolog in C. clemensi is illustrated in the phylogenetic tree. A separate branch within this clade contains L. salmonis cathepsin L, closely positioned with the crustacean cathepsin L precursor sequence from C. clemensi (GenBank accession no. ACO14903) and a cathepsin L sequence from C. rogercresseyi (GenBank accession no. ACO10357). L. salmonis CL2 forms a separate clade in the tree, comprised of two branches. On one branch, LsCL2 is positioned with its closest matches, cathepsin K from C. clemensi (GenBank accession no. ACO15154) and L. salmonis cathepsin L-like protease (GenBank

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A

958 C. clemensi CK 776 L. salmonis CL-like

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100 LsCL2

80 60 40 20 0 1

2

3

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T. thermophila CP

6

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998 I. multifiliis

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0 0

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Fig. 2. A phylogenetic tree was constructed from the ClustalW alignment of L. salmonis cathepsin L-like protease (GenBank accession no. ADD24149), L. salmonis cathepsin L (GenBank accession no. ADD24160), LsCL1 (GenBank accession no. HM439291) and its 4 closest matches following a BLAST search of the GenBank database, Caligus clemensi cathepsin L1 (CL1) precursor (GenBank accession no. ACO15375), Caligus clemensi cathepsin L (CL) precursor (GenBank accession no. ACO14903), Caligus rogercresseyi cathepsin L (CL) precursor (GenBank accession no. ACO10357) and Litopenaeus vannamei cathepsin L (CL) (GenBank accession no. CAA74241). Also included in the tree are LsCL2 (GenBank accession no. HM439290) and its 3 closest relevant matches and following a BLAST search of the GenBank database, Caligus clemensi cathepsin K (CK) (GenBank accession no. ACO15154), Tetrahymena thermophila papain family cysteine protease (CP) (GenBank accession no. XP_001022365) and Ichthyophthirius multifiliis cathepsin L cysteine protease ICP1 (CP1) (GenBank accession no. ABH06549). The tree shows the relationship between the L. salmonis cathepsin L sequences. Bootstrap values on 1000 replicates are indicated at each branch point.

accession no. ADD24149). The cysteine proteases of the protozoan species I. multifiliis (GenBank accession no. ABH06549) and T. thermophila (GenBank accession no. XP_001022365) fall on a separate branch.

Relative activity (%)

0.1

20 15

80 60 40 20 0 0

2

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6

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10 12 14 16 18 20 22 24

Time (hours) Fig. 3. (A) pH profile of L. salmonis cathepsin L activity in adult female lice extracts. The optimum pH was determined to be pH 6.5. (B) Temperature profile of L. salmonis cathepsin L activity measured over a range of 4–60 °C. The optimal activity was recorded at 15 °C. (C) Stability of L. salmonis cathepsin L over time at the 15 °C and 20 °C. The enzyme demonstrates increased activity at 15 °C compared to 20 °C and retains the increased activity for longer (24 h).

3.3. Biochemical characterisation of cathepsin L activity in adult female L. salmonis

3.4. Cathepsin L activity in different L. salmonis life stages

Biochemical analysis revealed that extracts of adult female L. salmonis contain cathepsin L activity with a pH optimum for activity at pH 6.5 (Fig. 3A). In the pH range of 1.5–5.5, enzyme activity remains low, increasing dramatically at pH 6.0 and decreases markedly above 6.5. The optimum temperature for activity of cathepsin L occurs at 15 °C, decreasing rapidly at temperatures over 40 °C (Fig. 3B). However, approximately 45% activity remains at a temperature of 10 °C. The activity of the enzyme at both 15 °C and 20 °C is relatively stable, with maximum activity at 15 °C occurring after 3 h (Fig. 3C).

The chalimus stage exhibits the most cathepsin L activity (6.35 ± 0.515 nmol mg − 1 min − 1), decreasing with progression to the preadult male (3.83±0.59 nmol mg− 1 min− 1) and female (1.137± 0.163 nmol mg− 1 min− 1) life stages and decreasing even further following the progression to the adult male (0.34±0.09 nmol mg− 1 min− 1) and female (0.94±0.032 nmol mg− 1 min− 1) life stages. Lower activity was observed in the nauplius life stage (0.65±0.035 nmol mg− 1 min− 1). Cathepsin L activity was also observed in the secretory/excretory products (5.58±0.523 nmol mg− 1 min− 1) (Fig. 4).

Units activity mg/ml/min

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7.5

5.0

2.5

0.0 AM

AF

PAM

PAF

CH

NP

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Life stages Fig. 4. Cathepsin L activity in the different life stages of L. salmonis; adult male (AM), adult female (AF), preadult male (PAM), preadult female (PAF), chalimus (CH), nauplius (NP) and secretory/excretory products (SEP). Enzyme activity was found in all extracts examined, with the highest activity recorded in the chalimus extract.

4. Discussion Two complete cathepsin L sequences from L. salmonis have been elucidated and termed LsCL1 and LsCL2. Both L. salmonis cathepsin L proteases possess a signal sequence allowing for targeting of the propeptide to various locations for storage and are synthesised as inactive precursors, activated by proteolytic removal of the N-terminal propeptide. LsCL1 and LsCL2 have catalytic domains of 217 and 257 amino acids respectively which fall within the expected size range for papain-like cysteine proteases (214–260 amino acids) (Bromme, 2001). Analysis of the LsCL1 and LsCL2 sequences reveals that they represent two individual cathepsin L preproenzymes, sharing only 25% identity with each other. LsCL2 is a longer peptide than LsCL1 possessing a 15 amino acid N-terminal extension in a similar manner to the differing lengths displayed by S. mansoni cathepsins L1, L2 and L3 (Dvorak et al., 2009). LsCL2 also possesses a 30 amino acid C-terminal extension that is not present in LsCL1. The presence of both the N and C-terminal extensions would suggest that the LsCL1 sequence represents a cathepsin F-like cysteine protease; however, analysis of the propeptide reveals that LsCL2 does not contain the ERFNAQ signature sequence evident in cathepsin F sequences from other species (Pinlaor et al., 2009). As the C-terminal extension is well conserved in the closest matches of LsCL2, it would appear it is a feature of this cathepsin L subtype in L. salmonis. Cathepsin L from all species demonstrates the highest conservation in the mature domain of the polypeptide that represents the catalytically active protease (Bromme, 2001). However, the relatedness of the different subfamilies of cysteine proteases (cathepsin B-like, cathepsin L-like and cathepsin F-like) is reflected in the conserved motifs in the pro region (Lecaille et al., 2002). LsCL1 and LsCL2 show all the characteristics of the peptidase subfamily C1A, namely the conserved ERFNIN, GNFD and GCNGG motifs. These motifs were slightly different in LsCL1 and LsCL2, although sufficient conservation is retained to determine the cysteine protease sequences from L. salmonis as cathepsin L enzymes. The functions of these conserved motifs remain unclear but it is thought that the ERFNIN motif functions as an auto-inhibitory domain and the GCNGG motif contains a cysteine residue involved in formation of a disulphide bridge and is therefore believed to be structurally important. The GNFD motif plays a role in processing and folding of C1A proteases (Jousson et al., 2007). Cathepsin L proteases have primary substrate preference at the S2 subsite, whereby the P2 residue of the peptide substrate fits into the protease S2 subsite forming a pocket determining substrate specificity (Jousson et al., 2007). L. salmonis cathepsins L1 and L2 have different residues at their S2 subsite, with different properties suggesting different capabilities. A similar phenomenon has also been observed in the parasitic ciliate I. multifiliis where Icp1 and Icp2 have charged (Asp) and uncharged (Ser) residues respectively. This structural

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diversity of the S2 subsite may imply a potential broadening the range of metabolically relevant substrates. This paper identifies four potential cathepsin L homologues in the copepod sea lice species. A thorough examination of the EST database available at NCBI may reveal more than those discussed here, however, this analysis is beyond the scope of this paper. Analysis of the alignment of LsCL1 and LsCL2 demonstrates that all four can be classified as cathepsin L proteases as all have intact or slightly modified cathepsin L motifs. It is apparent upon analysis of the phylogenetic tree that four cathepsin L homologues exist for the sea lice species indicated by the separate branching of cathepsin L sequences within the two distinct clades that are formed in the tree. Cathepsin L activity was observed in all the parasite life stages and also in the secretory/excretory products, pointing to potential roles in many aspects of the biology of this parasite. However, although cathepsin L activity has been measured in parasite extracts, it is not possible definitively to attribute this activity to one of the four cathepsin L genes described in this paper. Due to its activity in the parasitic life stages analysed, it is plausible to suggest that L. salmonis cathepsin L is involved in extracellular digestion of host mucus, skin and blood in the parasite. This is supported by the identification of a single cysteine peptidase gene associated with abdominal growth using EST sequencing and microarray analysis (Eichner et al., 2008). Eichner et al. (2008) also identified an L. salmonis cysteine peptidase similar to the trophozoite cysteine proteinase from Plasmodium falciparum malaria parasite that is associated with the degradation of haemoglobin. The authors suggest the L. salmonis cysteine peptidase may play a similar role following ingestion of blood from the salmon host (Eichner et al., 2008). The American lobster (H. americanus) is a crustacean that has cathepsin L activity present in the gastrointestinal juice, indicating that the protease can be secreted out of a cell and has the ability to digest food extracellularly (Laycock et al., 1989). Similarly, in the shrimp M. ensis, cathepsin L has been identified in the hepatopancreas indicating that it is involved in digestion also (Hu and Leung, 2007). In the helminth parasite S. mansoni, discrete functions for cathepsin L1 and cathepsin L2 have been suggested whereby SmCL1 was localised to the gastrodermal cells while SmCL2 was localised to the reproductive system of male and female worms (Brady et al., 2000). An additional S. mansoni cathepsin L, SmCL3, has been described that was localised to the gastrodermis of both male and female worms, but unlike SmCL1, is not present in the parasite excretory/secretory (ES) extract (Dvorak et al., 2009). It has been suggested that the production of multiple cathepsin L proteases with overlapping specificity could digest the host macromolecules more efficiently (Stack et al., 2008) and this may be the case in L. salmonis, however further research is required to substantiate this claim. The physiological pH for salmon skin and mucus is near neutral (Shephard, 1994) therefore, based on its pH profile, native L. salmonis cathepsin L has the ability to cleave mucus as a substrate at this pH. This observation provides a potential role for cathepsin L not only in the digestive process within the parasite, but also in infection and establishment of the parasite on the host. Most mammalian cathepsins have an acidic pH optimum, allowing activity intracellularly within the lysosomal compartment. In contrast, many parasitic cysteine proteases, including the parasitic ciliate of fish I. multifiliis, are most active at neutral pH (Jousson et al., 2007; Sajid and McKerrow, 2002). Indeed, L. salmonis cathepsin L shows little activity at acidic pHs. The neutral pH optimum of cathepsin L is in accordance with the extracellular activity observed for these proteases in nutrition, tissue and cell invasion, ex/encystment, hatching and immunoevasion (Sajid and McKerrow, 2002). The presence of cathepsin L activity in the SEP extract is interesting as this activity has also been described in the excretory/secretory extracts of many parasites including the helminths F. hepatica (Dowd et al., 1994), S. mansoni (Dalton et al., 1996), C. sinensis (Li et al., 2009)

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and several arthropod parasites. It is thought that these secretions aid feeding and immunoevasion. The SEP extract of L. salmonis has not been well characterised to date, although trypsin and trypsin-like serine proteases (Firth et al., 2000; Ross et al., 2000) and the prostanoid prostaglandin E2 have been identified in secretions of L. salmonis (Fast et al., 2004; Fast et al., 2007). The research of Firth et al. (2000) describes an absence of cysteine protease activity in sea lice infected salmon mucus however, non‐detection may be due to the cysteine protease activity not being sufficiently high to visualise by zymography. It is also of note that as SEP cathepsin L activity assays were carried out in a low salt buffer, it is plausible to suggest that if exposed to a high saline buffer, more similar to the environment of the SEP, a higher level of cathepsin L activity may be observed. However, the identification of cathepsin L activity in the SEP of L. salmonis implicates the involvement of this protease in vital functions such as immunoevasion and establishment of the parasite on the host. Cathepsin L activity in the non-feeding nauplius stages indicates a different function of the protease in this instance. It is possible that it is involved in the molting process of the sea lice occurring in the transition from nauplius I to nauplius II. This hypothesis is supported by the high level of cathepsin L activity in the chalimus life stages which along with its feeding activity, goes through multiple molts during its development on the host. In contrast, a lower level of activity is observed in the preadult stages which also undergo molting during their development. It is conceivable that the enzyme activity observed in the preadult lice homogenate is predominantly cathepsin L used during the molting process, while a portion of the digestive function of the enzyme is excreted into the SEP fraction for external digestion of host tissue once the parasite is mobile. This is supported by lower levels of cathepsin L activity observed in the non-molting adult homogenates. Similarly, cysteine protease activity has previously been implicated in the regulation of molting in the crustacean shrimp Artemia franciscana (Warner and Matheson, 1998). It is worth noting that enzyme activity levels observed in the preadult and chalimus stages may also be related to the amount of gut tissue relative to other sea lice tissues present in the sample. The temperature profile of L. salmonis cathepsin L demonstrates that the enzyme is active over a wide temperature profile. Over 20% activity is retained at 4 °C, with enzyme activity increasing as the temperature increases, to a maximum (100%) relative activity at 15 °C. Furthermore, native L. salmonis cathepsin L retains over 60% of its activity after 24 h at 15 °C demonstrating its capacity to remain functional over long periods of time at this temperature, perhaps carrying out vital roles in the parasite. Although cathepsin L activity was demonstrated between 20 and 40 °C, this is clearly not within the physiological capabilities of the parasite in vivo. The optimum temperature for L. salmonis has not been fully elucidated however it is believed that this species probably requires temperatures of 4 °C or higher to complete its life cycle successfully (Boxaspen, 2006; Boxaspen & Næss, 2000). The capacity for cathepsin L to remain active over a wide range of temperatures is reflected in the fact that sea lice are tolerant of temperature variations in vivo and this is supported by numerous studies that have shown that temperature fluctuation does not affect sea lice abundance on hosts (Revie et al., 2002; Saksida et al., 2007; Yatabe et al., 2011). 5. Conclusion This paper characterises two complete cathepsin L sequences (LsCL1 and LsCL2) from the ectoparasite, L. salmonis. Searches of the GenBank database resulted in the identification of two additional complete cathepsin L and cathepsin L-like proteases from L. salmonis. The characterisation and clarification of the roles of proteases, such as LsCL1 and LsCL2, of L. salmonis will provide a better understanding of the host parasite interaction. Many of the leading vaccine candidates against metazoan parasites of man and animals are enzymes. Hence, this characterisation is a prerequisite to the ultimate goal of designing

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