Characterization of Two Novel Cysteine Proteases in the Free-Living Flatworm Macrostomum Lignano

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Characterization of Two Novel Cysteine Proteases in the Free-Living Flatworm Macrostomum lignano

Phillip Zanet, Parasitology, McGill University, Montreal Submitted July 2013

A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Master of Science (MSC).

Table of Contents:

Acknowledgements...... 4 Abstract...... 5 Abbreviations...... 7 List of Tables...... 8 List of Figures...... 8

CHAPTER 1 – Introduction ...... 12 Introduction ...... 13 Parasitic Trematodes ...... 14 Free-Living Planarians ...... 15 Feeding in Planarians ...... 17 Macrostomum Lignano ...... 18 Phytoplankton: Nitzschia Curvilineata ...... 20 Peptidases ...... 21 Cathepsins ...... 23

CHAPTER 2 – Characterization of the Free-Living Macrostomum lignano Cathepsin L1 ...... 27 1. Introduction ...... 28 2. Materials and Methods ...... 29 2.1 Gene Identification and Phylogeny ...... 30 2.2 Gene Editing ...... 31 2.3 Cloning ...... 31 2.4 Recombinant Protein Production, Purification and Quantification ...... 32 2.5 Colony Blot ...... 32 2.6 Antibodies Production and Purification ...... 33 2.7 Kinetics and Substrate Specificities of MlCL1 ...... 34 2.8 pH Profile of MlCL1 ...... 34 2.9 Activity of MlCL1 at Different Temperatures ...... 35

2.10 Activity of MlCL1 at Different Salinities ...... 35 2.11 Stability of MlCL1 at Different Times ...... 36 2.12 Stability of MlCL1 at Different Temperatures ...... 36 2.13 Stability of MlCL1 at Different Salinities ...... 37 2.14 Inhibition of MlCL1 by E-64 and CAA0225 ...... 37 3. Results ...... 37 3.1 Phylogeny ...... 37 3.2 Cloning and Colony Blot ...... 38 3.3 Protein Purification and Quantification...... 40 3.4 Determining Kinetics and Substrate Specificities ...... 41 3.5 pH Profile of MlCL1 ...... 42 3.6 Determining the Activity of MlCL1 at Different Temperatures and Salinities ...43 3.7 Determining the Stability of MlCL1 at Different Times, Temperatures and Salinities ...... 45 3.8 Inhibition of MlCL1 by E-64 and CAA0225...... 47

CHAPTER 3 – Characterization of the Free-Living Macrostomum lignano Cathepsin B2 ...... 49 1. Introduction ...... 50 2. Materials and Methods ...... 51 2.1 Gene Identification and Phylogeny ...... 51 2.2 Gene Editing ...... 52 2.3 Cloning ...... 53 2.4 Colony Blot ...... 54 2.5 Recombinant Protein Production, Purification and Quantification...... 55 2.6 Antibodies Production and Purification ...... 55 2.7 Kinetics and Substrate Specificities of MlCB2 ...... 56 2.8 pH Profile of MlCB2 ...... 56 2.9 Activity of MlCB2 at Different Temperatures ...... 57 2.10 Activity of MlCB2 at Different Salinities ...... 57 2.11 Stability of MlCB2 at Different Times ...... 57

2.12 Stability of MlCB2 at Different Temperatures ...... 58 2.13 Stability of MlCB2 at Different Salinities ...... 58 2.14 Inhibition of MlCB2 by E-64 and CA-074 …...... 59 3. Results ...... 60 3.1 Phylogeny ...... 60 3.2 Cloning and Colony Blot ...... 61 3.3 Protein Purification and Quantification...... 62 3.4 Antibodies Production and Purification...... 64 3.5 Determining Kinetics and Substrate Specificities ...... 64 3.6 pH Profile of MlCB2 ...... 64 3.7 Determining the Activity of MlCB2 at Different Temperatures and Salinities ...65 3.8 Determining the Stability of MlCB2 at Different Times, Temperatures and Salinities ...... 66 3.9 Digestion of Natural Substrates by MlCB2...... 66 3.10 Inhibition of MlCB2 by E-64 and CA-074…...... 70

CHAPTER 4- Discussion and Conclusion ...... 72

4. Discussion ...... 72

5. Conclusion ...... 81

Bibliography ...... 83

Acknowledgements

This thesis is the result of support and contributions from several individuals who brought forth their unique expertise and assistance. I would like to first acknowledge all the members of the Institute of Parasitology at the MacDonald Campus of McGill University. I wish to thank gracefully and admirably my supervisor Dr. John P. Dalton for giving me a chance to hone my scientific abilities and entrusting me with such a difficult project. I would like to also give further thanks to my past and current labmates, faculty and staff who have lent me their time, patience and wisdom to my project, including Dr. Robin Beech, Dr. Petra Rorhbach, Dr.

Reza Salavati, Ms. Bibiana Gonzalez-Santana, Ms. Rency Mathew, Dr. Desiré Nsangou, Dr.

Linhua Zhang, Mr. Fabio DaSilva, Dr. Karine Thivièrge, Dr. Corine Demanga, Mrs. Jenny Eng,

Mr. Anand Chakroborty, Ms. Caroline Mahlig, Ms. Vanessa Dufour, Mr. Anthony Sassi and Ms.

Rona Strasser. I would like to add that I had the greatest opportunity to be guided by Dr. Sophie

Cotton, my committee member and mentor.

This project would not have been possible without the collaboration of Dr. Paul McVeigh and his laboratory for providing us with the required Macrostomum lignano cDNA and worm protein extracts in addition to his correspondences which allowed us to better understand the flatworm and diatoms, and also Dr. Mostafa Zamanian for providing us with Schmidtea mediterranea gene sequences that would not be otherwise available for phylogeny purposes.

Finally I would like to thank my family who has encouraged me to achieve higher education, Dr. Bill Nye “The Science Guy” who inspired me at a young age to pursue science,

Dr. Malcolm Baines who pushed me forward, Dr. Greg Marczynski who taught me the basics and Dr. Richard Phillips Feynman, who taught me the meaning of integrity in science.

Abstract

The objective of this study was to explore Macrostomum lignano, a free-living organism, as a model organism for parasitic trematodes, such as Fasciola and Schistosoma, in order to better understand the role of their cysteine proteases (cathepsins). Using a bioinformatics approach, two novel cysteine proteases genes (mlcl1 and mlcb2) were identified and phylogenetically characterized. These genes were synthesized, cloned into the yeast secretory system Pichia pastoris (Invitrogen), and functionally-active recombinant proteins were expressed and purified. These recombinant peptidases were then characterized biochemically in terms of activity and stability in various conditions including temperature, salinity and pH.

Antibodies specific for the recombinant proteins were generated through a peptide or whole- protein immunization, and tested against both the recombinant proteins and the worm extract proving that the proteins exist in the worm. These studies lay the foundation for further investigations on the biological function of the cysteine peptidases using RNAi and confocal microscopy. In conclusion, M. lignano is a tractable model organism for its parasitic counterparts.

L’objectif de cette recherche était d’explorer l’organisme, vivant en liberté dans la nature,

Macrostomum lignano en tant qu’organisme modèle pour ses cousins parasites, Fasciola et

Schistosoma, et de mieux comprendre le rôle de leurs protéases cystéines (cathepsins). En utilisant une approche bioinformatique, deux nouveaux gènes de protéases cystéines (mlcl1 et mlcb2) ont été découverts et caractérisés phylogénétiquement. Ces gènes ont été synthétisés, clonés dans un système de sécrétion employant la levure Pichia pastoris (Invitrogen) et exprimés en tant que protéases recombinantes et actives. Ces protéases recombinantes ont alors été

5 caractérisées biochimiquement en termes d’activité et de stabilité dans diverses conditions telles que la température, la salinité et le pH. Des anticorps spécifiques aux protéines recombinantes ont été générés en immunisant des mammifères avec des séquences de peptides ou la protéine recombinante entière, et ont été testés avec l’extrait du vers prouvant ainsi que les protéines sont bel et bien exprimées. Ces études jettent la base pour l’investigation sur la fonction biologique des protéases cystéines par l’emploi du RNAi et de la microscopie confocale. En conclusion, M. lignano est un organisme modèle tractable pour ses cousins parasites.

Abbreviations 3D Three Dimensions AMC 7-Amino-4-methylcoumarin Asn Asparagine BMGY Buffered Glycerol-Complex Media BMMY Buffered Methanol-Complex Media BSA Bovine Serum Albumin CA-074 (L-3-trans-(Propylcarbamyl)oxirane-2-carbonyl)-L-isoleucyl-L-proline CAA-0225 ((2S,3S)-oxirane-2,3-dicarboxylic acid Cys Cysteine DNA Deoxyribonucleic acid DTT Dithiothreitol E-64 N-[N-(L-3-trans-carboxyirane-2-carbonyl)-L-leucyl]-agmatine EST Expressed Sequence Tag ERFNIN Glutamate Arginine Phenylalanine Asparagine Isoleucine Asparagine GNFD Glycine Asparagine Phenylalanine Aspartate Gy Gray Unit (equivalent to the absorption of one joule, in ionizing radiation, per kg) His Histidine IFNγ Interferon-gamma IgG Immunoglobulin G KM Michaelis-Menten constant Kcat Catalytic constant Ki Inhibitory constant NCBI National Center for Biotechnology Information OD Optical Density PAD Pichia Adenine Dropout PBS Phosphate Buffered Saline PPT Parts per thousand RNAi Ribonucleic Acid Interference SDS Sodium Dodecyl Sulfate SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis SSC Saline Sodium Citrate tBLASTn Protein-Nucleotide 6-Frame Translation Basic Local Alignment Search Tool WHO World Health Organization YNB Yeast Nitrogen Base YPD Yeast extract Peptone Dextrose Z-F-R-AMC Styrene-Phenylalanine-Arginine-7-Amino-4-Methylcoumarin Z-L-R-AMC Styrene-Leucine-Arginine-7-Amino-4-Methylcoumarin Z-P-R-AMC Styrene-Proline-Arginine-7-Amino-4-Methylcoumarin Z-R-R-AMC Styrene- Arginine-Arginine-7-Amino-4-Methylcoumarin

List of Tables

Chapter 2

Table 1. Kinetic parameters of MlCL1. KM is the Michaelis-Menten constant, Kcat represents the time required for 1 unit of enzyme to process 1 unit of substrate and Kcat/KM is the catalytic efficacy.

Chapter 3

Table 1. Kinetic parameters of MlCB2. KM is the Michaelis-Menten constant, Kcat represents the time required for 1 unit of enzyme to process 1 unit of substrate and Kcat/KM is the catalytic efficacy of the enzyme.

List of Figures

Chapter 2

Figure 1. Annotated protein sequence of MlCL1. This signal peptide sequence and the inhibitory peptide sequence are highlighted in grey and yellow respectively. The S4, S3, S2, S1 and S1’ binding sites are shown. The catalytic triad typical of cathepsins (C25, H159 and N175) is shown. The custom mutation made is also show (N130-Q130).

Figure 2. Cloning manipulations of MlCL1. MlCL1 (green) is synthesized in the pUC57 vector; using the restriction enzymes KpnI and MlyI, the gene is cut out and the corresponding fragments are separated in the agarose gel. The pPinkα-HC vector is cut at the restriction site directly adjacent to the signal peptide sequence using StuI and MlyI. Using a T4 ligase, the gene and vector are ligated together; to test which E. coli colony contained the correctly ligated vector, the purified DNA is restricted once more using NdeI and KpnI digestion which either shows a 6kbp and a 1.2kbp band (no MlCL1) or a 6kbp and a 2.5kbp band (MlCL1 present). Using SpeI, the vector is linearized and transformed into P. pastoris via electroporation.

Figure 3. Phylogenetic tree of cathepsins L and B. MlCL1 is a plesion of the crown CL1 group in the cathepsin L1 clade. Its ancestor is common to the ancestor of the cathepsins L1 of the Schistosoma species. Tree is a distance-based tree using pro-enzyme sequences. A maximum likelihood tree agreed with the tree presented above although the supporting values were not similar.

Figure 4. Colony-Blot of P. pastoris. Only a few colonies actively express the recombinant MlCL1; all positive colonies were picked but only colonies 11, 23 and 24 showed the most promising results with yields greater than colonies 3, 7 and 18.

Figure 5. Western Blot and SDS-PAGE of MlCL1 using the cathepsin inhibitor E-64. Lane L represents the molecular weight marker, A contains neither DTT nor E-64 in the loading dye, B

8 contains DTT in the loading dye, C contains E-64 without DTT and D has both E-64 and DTT. Lane W contains MlCL1 incubated at 37 °C for 1 hour with DTT and no inhibitors. All enzymes are activated and mature.

Figure 6. pH profile of MlCL1 using Z-F-R-AMC. The enzyme has a broad range for activity between pH 4.5 and 6.5. Activity steadily drops below pH 4.5 and drastically drops above pH 7.0.

Figure 7. MlCL1 activity at 4 °C and 37 °C. The enzyme works at colder temperatures as expected but at much higher levels than anticipated (60% of maximum activity).

Figure 8. MlCL1 activity at different salinities. Activity is unaffected by the salt concentration of the enzyme; no significant changes even past 50 ppt have been noted.

Figure 9. MlCL1 stability over time at 37 °C in pH 5.5 and in presence of DTT. A massive activity variation is observed between time 0 and 1 hour; it is proposed that the inactive enzymes are activating under these conditions. The p-value for the first 4 hours indicates that the means are not significantly different (p = 0.7215). The enzyme’s half-life is 24 hours and even after 2 days, the enzyme is still active.

Figure 10. Stability of MlCL1 after being exposed at different temperatures for 1 hour. MlCL1 appears to be very stable between 4 and 42 °C. Stability is significantly hindered at 50 °C and all enzymatic activity is lost at 60 °C.

Figure 11. Stability of MlCL1 in the presence of NaCl at different concentrations. Brackish salt water is typically between 0.5 and 30 ppt. MlCL1 only loses about 20% of activity past 1 ppt and maintains the trend even past 30 ppt.

Figure 12. Dixon plot of the inhibitor E-64. The plot is that of a tight binding mechanism that competes for the active site with the substrate Z-F-R-AMC.

Figure 13. Dixon plot of the inhibitor CAA0225. The plot is that of a tight binding mechanism that competes for the active site with the substrate Z-F-R-AMC. The Ki is very small in comparison to E-64 (375 times lower).

Chapter 3

Figure 1. Annotated protein sequence of MlCB2. This signal peptide sequence and the inhibitory peptide sequence are highlighted in grey and yellow respectively. The S4, S3, S2, S1 and S1’ binding sites are shown. The catalytic triad typical of cathepsins (C25, H159 and N175) is shown. The catalytic diad, characteristic of cathepsins B in the occluding loop is also highlighted (H110-H111). The custom mutation made is also shown (H171-Q171).

Figure 2. Edited MlCB2 DNA and translated amino acid sequence. The MlyI restriction site (orange) is added at the beginning of the pro-enzyme sequence. The catalytic triad (C25, H179,

N185) are highlighted in red as individual residues; the catalytic diad of the occluding loop (H110-H111) is highlighted (bright green). The Aspartate residue (D171) is mutated and highlighted under CAA and Q171 (yellow). A glycine and a proline residues (dark blue) are added at the end of the sequence followed by a hexahistidine sequence (bright blue) using different codons. The stop codon is moved past the hexahistidine tag and followed by a Kpn1 restriction site (green).

Figure 3. Phylogenetic tree of cathepsins L and B. MlCB2 is a plesion of the crown CB2 group in the cathepsin B2 clade. Its ancestor is common to the ancestor of the cathepsins B2 of Trichobilharzia and Schistosoma species. Tree is a distance-based tree using pro-enzyme sequences. A maximum likelihood tree agreed with the tree presented above although the supporting values were not similar.

Figure 4. Cloning manipulations of MlCB2. MlCB2 (green) is synthesized in the pUC57 vector; using restriction enzymes KpnI and MlyI, the gene is cut out and corresponding fragments are separated in the agarose gel. The pPinkα-HC vector is cut at the restriction site directly adjacent to the signal peptide sequence using StuI and MlyI. Using a T4 ligase, the gene and vector are ligated together; to test which E. coli colony contained the correctly ligated vector, the purified DNA is restricted once more using NdeI and KpnI digestion which either shows a 9kbp and a 1kbp band (no MlCB2) or a 9kbp and a 2kbp band (MlCB2 present). Using SpeI, the vector is linearized and transformed into P. pastoris via electroporation.

Figure 5. Colony immunoblot results. Colony epiplate with resulting colonies (A), the immunoblot results (B) indicating expression of the enzyme by the colony. Each immunoblot colony retains its original shape from the epiplate which helps in identifying which produce enzymes and which do not. The shadows are artifacts caused by the sliding of the film during the development of the immunoblot.

Figure 6. Western Blot of MlCB2 (purified and culture media). Columns A and B represent the purified protein production of MlCB2 by colonies 11 and 19 respectively. Following are media from each respective culture and day post-induction (A1 is colony 11 on day 1, B2 is colony 19 on day 2 and so forth). MlCB2 clones require a single day to generate enough pro-enzyme. After 2 days the enzymes activate within the culture (and slightly on day 1). On day 3 the enzyme starts to degrade substantially and also starts to complex with itself.

Figure 7. Coomassie Blue Stain (A), respective Western Blot using anti-hexahistidine tag antibodies (B) and Western Blot using anti-MlCB2 rat antibodies (C). The Western blot reveals that the protein degrades down to small fragments containing the His-tag but not in high enough concentrations to be seen on the Coomassie stain; the protein also seems to complex with copies of itself when highly concentrated. The anti-MLCB2 antibodies bind to the mature enzyme but very weakly to the zymogen.

Figure 8. Activity of MlCB2 at various pH using Z-L-R-AMC as a substrate. The enzyme exhibits a broad range of pH optima ranging from 5.5 in sodium acetate [0.1M] to 7.5 in sodium phosphate [0.1M].

Figure 9. Activity of MlCB2 at various temperatures. MlCB2 retains up to 80% of its maximum activity at 4 °C. The enzyme is most active at room temperature and also at 37 °C.

Figure 10. Activity of MlCB2 at various salinities. The enzymatic activity marginally increases at higher salt concentrations. Overall the enzyme functions well from 80 to 100% regardless of the salinity.

Figure 11. Stability of MlCB2 at various temperatures. The stability is assessed through the enzymatic activity; if the enzyme is no longer stable, its activity should decrease drastically. MlCB2 cannot withstand temperatures above 42 °C; it is almost completely ablated at 50 °C and inactive at 60 °C.

Figure 12. Stability of MlCB2 at various salinities. The enzyme was incubated at a much lower volume (10 times lower) than the total reaction volume in order to minimize the effect of salinity on the activity. MlCB2’s stability is unaffected by salinity including high salt concentrations such as 50 ppt.

Figure 13. Stability of MlCB2 at various times. The enzyme was incubated with 4 mM DTT at 22 °C for 0, 0.5, 1, 2, 4, 8 and 24 hours. The enzyme is stable and has optimal activity at time 0. 50% of maximum activity is lost after 2 hours and complete loss of activity occurs after 24 hours.

Figure 14. SDS-PAGE of the digestion of BSA by MlCB2. L is the ladder, A, B, C, D, E, F and G are the aliquots isolated from the master mix at times 0, 0.5, 1, 2, 4, 6 and 8 hours respectively. MlCB2 is present at the expected size of 27 kDa while BSA is present much higher between 50 and 75 kDa. After 2 hours (D), 2 bands of BSA get completely digested and only after 8 hours (G) is the total BSA digested. The lanes MlCB2 and BSA represent the MlCB2 and BSA control lanes respectively.

Figure 15. Inhibition of MlCB2 and MlCL1 by CA-074. MlCB2 is selectively inhibited by the compound at its lowest concentration (0.250 µM) while MlCL1 is only slightly inhibited at much higher concentrations (which would be expected since the substrate concentration for MlCL1 is 3 µM).

Figure 16. Dixon plot of MlCB2 in the presence of the competitive inhibitor CA-074. Experiment was replicated 3 times with similar results pointing for a Ki of 0.007445 µM.

Figure 17. Dixon plot of MlCB2 in the presence of the competitive inhibitor E-64. Experiment was replicated 3 times with similar results pointing for a Ki of 0.04224 µM.

Chapter 1 - Introduction

Introduction

Helminth, or worm, parasites are a major problem, especially in developing countries.

Annual losses to the agricultural communities amount to US$2-3.2 billion (Rehman and Jasmer,

1999; McManus and Dalton, 2006), most of which are losses in livestock such as sheep and cattle. Some helminths (such as Fasciola or Schistosoma) are also zoonotic and are responsible for over 200 million human infections (WHO, 2001; Molyneux, 2006). Much effort has been made to understand the life cycle of these parasites, their general anatomy, host-parasite interactions and the chemotherapeutic drugs that are used to treat their pathologies. As such, there has been a focus primarily on the parasitic helminths rather than any other member of the platyhelminths family.

The proteins flatworms secrete to invade and interact with their host are mainly what make the flatworms such formidable parasites (Cesari et al., 2000; Dvorak et al., 2005). The functions of these secreted proteins range from being digestive to immunosuppressive

(McKerrow et al., 1991; Darani et al., 1997). Due to their complex life-cycle and multiple life- cycle stages, protein expression also varies with the stage. Parasitic flatworms are very arduous to work with because they require an intermediate snail host, need varying conditions depending on the life cycle stage in vitro, have strict nutritional requirements and are very difficult to manipulate genetically (Wilson, 2011). While RNA interference (RNAi) techniques have been developed and work relatively well with larval stages (e.g. the cercariae of schistosomes) their success in modifying adult phenotype is highly dependent on the targeted gene and knockdowns sometimes do not exhibit any phenotypic difference (Wilson, 2011).

Although parasitic trematodes are related to other flatworms not much attention has been given to their free-living counterparts. Schistosoma mansoni is the best studied parasitic

13 trematode to date due to its medical and economical relevance. Parasites of the genus Fasciola also receive much attention due to their global economic impact (Wilson, 2011). On the other hand, only a few free-living planarians including Schmidtea mediterranea, Dugesia and the newly discovered Macrostomum lignano have been studied. Wilson (2011) noted that, although all the parasitic and free-living organisms are related, the fields of research do not generally overlap and consequently not much communication between groups and their experiments have been made. A simple example of this would be whole mount in situ hybridization (WISH) which was first applied to the free-living S. mediterranea in 1999 (Sanchez and Newmark, 1999;

Sanchez et al., 2002) while in S. mansoni, it was applied in 2007 (Dillon et al., 2007). The converse is also true; mass-spectrometric proteomic analyses of S. mansoni started in 2004

(Curwen et al., 2004) while S. mediterranea’s analyses started in 2011 (Adamidi et al., 2011;

Fernandez-Taboada et al., 2011). Therefore, more studies need to be performed on free-living planarians in order to integrate and compare this with the known information on parasites, and more discussions between research groups on the results of their experiments need to ensue in order to better understand the evolutionary changes that occurred and led to parasitism.

Parasitic Trematodes

In general, free-living planarians live and breed in the environment as a simple egg-to- juvenile-to-adult cycle. By contrast, parasites have multiple alternate stages in other hosts. For example, Schistosoma parasites start off as eggs which hatch into juvenile miracidia that infect an intermediate gastropod host, a snail. Once it penetrates the snail, the miracidium undergoes a round of differentiation into the cercarial form. Upon contact with its host skin the schistosomal cercaria will actively penetrate it, losing its tail in the process. Fasciola shares a similar life cycle

14 but larvae of these parasites enter their hosts by penetrating the intestinal wall following ingestion of dormant cyst. Once inside their hosts these parasites will undergo their final metamorphosis into an adult and become sexually mature. Parasites of the genus Clonorchis and

Dracunculus possess almost the same life cycle but in these cases the cercaria are ingested orally within an intermediate host. These parasites infect copepods and are then ingested by humans either accidentally when drinking unclean waters or when eating fish that have eaten copepods previously.

Parasitic trematodes, therefore, have very complex life cycles and vary gene expression accordingly at different stages. It follows that these trematodes require different culture conditions for each stages and are difficult to maintain vitro for lengthy periods. By contrast, free-living planarians are much easier to work with than their parasitic counterparts.

Free-Living Planarians

Free-living planarians are bilaterally symmetric organisms from the platyhelminths phylum (Pearse, 1994). They are usually flat and have an anterior head and a posterior tail

(Pearse, 1994). On their dorsal surface they bear two eye spots that track light and exhibit negative phototaxis, i.e. they travel away from light. On the ventral surface, they are equipped with a mouth and cilia. These organisms have not evolved an anus hence they can only ingest and regurgitate their meal (Ruppert et al., 2004). Free-living planarians do not freely swim but instead, they glide on surfaces which is facilitated by a mucus layer secreted from two lines of cells on their ventral surface (Pearse, 1994). Although they are equipped with cilia and it was thought that the cilia played an important role in locomotion, when the cilia are paralyzed, locomotion still occurs, however. When the muscles are paralyzed locomotion is absent

15 indicating that the flatworms' muscles are the primary source of locomotion (Pearse, 1994).

Free-living planarians have two methods of reproducing, asexually and sexually

(Kobayashi et al., 2009). Sexual reproductive organs are fully developed only during mating seasons, and, being hermaphrodites, they bear both ovaries and testes (Pearse, 1994; Lázaro et al., 2009). Following mating, their reproductive organs degenerate only to be regenerated when the mating season returns (Pearse, 1994). Although hermaphrodites, they cannot self-fertilize and thus require the presence of a mate. When mating, the two worms come together and lay their ventral surfaces against one another. On the ventral surface, the penis is inserted inside the genital pore and sperm is deposited in the copulatory sac. The sperm subsequently travels to the ovaries and fertilizes the eggs (Pearse, 1994). Once fertilized, the eggs pass down the oviducts where they are equipped with yolk cells originating from yolk glands. The yolk cells function to provide a nutrient reserve to the eggs. Once eggs reach the genital chamber, they are enveloped and form a capsule that is then released into the water. Several weeks later, they hatch as sexually immature juveniles (Pearse, 1994; Kobayashi et al., 2009).

The asexual reproduction process is quite peculiar in some planarians. It can occur as a result of wounding (such as a bilateral cut) or spontaneously (Pearse, 1994; Kobayashi et al.,

2009). When spontaneously reproducing, the tail begins to pull apart and latch onto a substrate as the anterior portion continues its movement. Once they are separated, each part regenerates the corresponding organs that are missing (Pearse, 1994; Lázaro et al., 2009).

Planarians such as Dugesia have an advanced system of regeneration (Agata, 1999).

Throughout their bodies are stocks of neoblasts that, when wounded, activate and start the regeneration process (Bronsted 1942; Pearse, 1994). Some free-living planarians can be cut bilaterally and two organisms will result (Egger 2008). If the head is cut bilaterally, the planarian

16 will regenerate two heads (Pearse, 1994). This property has become the main focus of many research laboratories that exploit planarians as model organisms for regeneration (Mouton,

2009). Planarians, such as M. lignano, have neoblasts that can withstand up to 200 Gy of radiation and still maintain their regenerative properties (De Mulder 2010).

Feeding in Planarians

Planarians have adapted to many environments and have different feeding styles but are still related to each other phylogenetically. Planarians are typically scavengers and usually feed on dead carcasses that they come across. Some planarians such as Polycelis felina hunt their own kind in a classical predator-prey interaction (Pearse, 1994; Alonso and Camargo, 2011). Usually through ambushes, they will attack and latch onto their prey while dragging their own bodies on the ground to slow them down. The predator will then latch onto the ground itself and pull on its prey. Finally it will bite and wound the prey until it stops resisting before finally feasting on its carcass. Other planarians, for example M. lignano, feed on smaller organisms such as algae or protozoans while planarians such as Bdelloura live a commensal lifestyle with their host and feed off its dead parts (in this case the horseshoe crab) (Pearse, 1994).

The remaining of the planarians have adopted a symbiotic lifestyle. From those symbionts, commensals diverged into either mutualism or parasitism (Pearse, 1994). Because of their tight association with their hosts and co-evolutionary pressures, the parasitic flatworms have become more specialized and diverged in anatomy and biochemistry from their ancestors.

For example, the class Monogenea, composed mainly of simple fish ecto-parasites, have adapted to grip on their hosts using a haptor organ and feed on the mucus, blood or epithelium (Pearse,

1994). Some endoparasites have developed immunosuppressants, such as F. hepatica (Dalton et

17 al., 2003), in order to live for long periods in their host, while others created new methods to penetrate their host tissues. Parasitism has also impacted free-living planarian features that are no longer present in parasites. S. mansoni is no longer a hermaphrodite and Fasciolae and

Schistosomae cannot regenerate.

Macrostomum lignano

M. lignano is a free-living planarian that was first discovered inhabiting the brackish waters of Lignano, Italy (Ladurner, 2005). This flatworm is in fact more circular shape than flat and lives mainly in moist sandy areas (Ladurner et al., 2006). It has a large mouth (hence macro-

(large) stomum (mouth)) but no anus; it has been reported to ingest and regurgitate its food

(Ladurner, 2005). It is a member of the class Turbellaria, which has been regarded as the point of origin of all parasitic platyhelminths (also known as a paraphyletic class) (Pearse, 1994 ).

M. lignano lacks any circulatory or respiratory systems but manages to maintain oxygen and waste transfers through simple passive diffusion and its relatively small size (Pearse, 1994).

For this reason, it cannot survive in locations that are susceptible to dehydration as they will succumb to massive fluid losses. They lack a cuticle, a layer of dead cells, but are equipped with a single layer of cells that bear cilia and have a locomotive funtion (Ladurner, 2005)..

M. lignano has a strictly sexual reproduction. It will lay one egg every day throughout the year. It doesn't have a mating season like other free-living planarians. The eggs hatch after 5 days and a juvenile is released that will mature after 18 days. M. lignano cannot reproduce asexually

(Ladurner, 2005)..

M. lignano regenerative properties are quite extensive: if cut transversally, the head will be able to regenerate the entire posterior, including gonads; the posterior end, however, will die.

This regenerative property is due to M. lignano's totipotent stem cell system. These cells, called neoblasts, remain idle in S-phase before progressing into mitosis (Egger, 2006; Ladurner, 2008).

Being totipotent cells, when regenerating, these cells can differentiate into any of the available cell types in M. lignano. The neoblasts also confer to the planarian its extensive plasticity. After

3 months of starvation, the flatworm will shrink to the size of a juvenile hatchling through the process of autophagy (Gonzalez-Estevez et al., 2007; Ladurner et al., 2008; Gonzalez-Estevez and Sato, 2010). When fed once more, it will regain its normal size due to the neoblasts regenerating the lost structures. These stem cells have also shown extreme resistance to radiations: 5 Gray units (Gy) is enough to kill an average human; the worm can tolerate up to

200 Gy (De Mulder et al., 2010).

Due to these properties, M. lignano has been heralded as a model organism for regeneration research (Ladurner et al., 2008). As such, laboratories such as Peter Ladurner’s

(University of Innsbruck, Austria) and Eugene Berezikov's (Hubrecht Institute, Utrecht, The

Netherlands) are doing extensive regenerative studies on this organism. Various techniques have been perfected for this, some of which are still difficult to apply to parasites; for example, RNA interference is easily achievable through a soaking method (Pfister et al., 2008; Sekii et al.,

2009).

In the phylogenetic context M. lignano is a plesion of the crown group of parasitic trematodes. It shares a common ancestry with the ancestor that later gave rise to all parasitic trematodes. It is more closely related to an ancestor common to Schistosoma mansoni and

Schistosoma japonicum. Schmidtea mediterranea was another candidate free-living worm we had considered for our studies but due to its phylogenetic ancestory being more distant to the parasitic ancestor and having growth issues in the laboratory setting (Sanchez, personal

19 correspondance), M. lignano appears to be more suited for parasite-comparative studies.

Phytoplankton: Nitzschia curvilineata

Diatoms are important to understand in the context of free-living planarians since they are the flatworms’ main food source. Due to their ubiquitous nature and highly nutritional contents, diatoms (and phytoplanktons in general) tend to be at the base of marine food chains and depend on bacteria to provide the appropriate substrates for their photosynthesis (Boney,

1975).

The phytoplankton Nitzschia curvilineata is a ubiquitous diatom of the class

Bacilariophyceae. It is corymbiform (boat-shaped) and smaller than a millimeter yet still visible.

Diatoms have a unique cell wall as it is made from silicate (SiO2) known as the “frustule”

(Kroger and Wetherbee, 2005; Boney, 1975). The frustule is a two part shell that is composed by two valves: the epitheca and the hypotheca. The epitheca overlaps the hypotheca at an indented girdle region similar to how a larger shell covers a smaller shell with an ovoid space between them. The final construct is held together by a pectinaceous band and sometimes the presence of small cusps. Although it might seem like the diatom is essentially trapped in its own theca, the shell has raphes (which are essentially pores) on the valve surfaces which allows the medium to reach the plasma membrane before transporting it to the cytoplasm and allow feeding of substrate.

During division, the epitheca remains the epitheca while the hypotheca becomes the new epitheca in the daughter cells (Kroger and Wetherbee, 2005); as such only a new hypotheca is ever synthesized. Since there has been no observed cell size diminution it is assumed the frustule grows with the daughter cells (Kroger and Wetherbee, 2005).

N. curvilineata appears brown-green due to its chloroplasts and xanthophyll pigments. In addition, it produces and stores highly concentrated starch and lipids in vacuoles. This organism is also autotrophic as well as lithotrophic, hence it derives energy from inorganic compounds. It has been show that diatoms in this family can metabolize various other minerals such as cobalt or copper and use them as electron transporters in absence of iron (Manimaran, 2012).

Peptidases

In order to catabolize their food, most organisms use peptidases to break proteins into simpler amino acids. In trematodes, the function of peptidases range quite a lot. Peptidases are necessary for the trematodes' survival and pathogenicity making them an attractive target to inhibit or prevent infection by parasitic trematodes such as Fasciola or Schistosoma. In addition, peptidases are also very immunogenic and could be used as components for vaccines in order to prevent future infections in livestock, and possibly even in humans (Dalton et al., 2003).

Understanding the structural and biochemistry of peptidases in relation to their function, and how they are expressed and regulated is fundamental in order to explain the role in parasitism. In addition, comparing peptidases from one species to another can generate interesting knowledge about what makes them pathogenic. Identifying similarities between peptidases could lead to a chemical compound that can inhibit all parasitic peptidases.

Peptidases have been classified using three main properties by Rawlings and Barrett

(1993) namely structure, homology and function. Some peptidases are exopeptidases and can only cleave residues on the N- or C-terminal end of a polypeptide. Peptidases exhibit very particular specificities; for example, a carboxypeptidase (exopeptidase) will cleave a single amino acid residue from the C-terminus; a tripeptidylpeptidase (exopeptidase) will cleave a

21 tripeptide from the N-terminus (Kasny et al, 2009). On the other hand, endopeptidases cleave polypeptides at specific sites inside the protein sequence.

Almost a third of all trematode's peptidases are serine peptidases (Kasny et al, 2009).

Their catalytic triad consists of a histidine, aspartate and serine residues. Of these serine peptidases, the Schistosoma cercarial elastase (CE) is a notable example; this peptidase degrades skin macromolecules and allows the cercaria to penetrate the skin of its host (Newport et al,

1988; McKerrow and Salter, 2002; Salter et al., 2000; Curwen and Wilson, 2003). Other proteases include the papain-like cysteine peptidases, also known as cathepsins, exploit cysteine in their activite site rather than serine (see below). These perform several functions, for example the Schistosoma mansoni’s cathepsin L2 (SmCL2) is suggested to be involved in the reproduction of the platyhelminth (Dillon et al., 2007; Bogitish et al., 2001) while SmCL3 is suggested to be involved in the digestion of food (Dvorak et al., 2009)..

Metalloproteases are another family of proteases that use specific metal cations to activate water molecules which catalyze peptide bond cleavage. These metal cations can be zinc, cobalt, manganese, nickel or copper. Cleavage of the peptide bond is performed by water molecules that have been activated by the metal cation (water molecule has been reduced to a hydroxyl ready to perform a nucleophilic attack on the peptide bond). These peptidases tend to be exopeptidases when they have a single cation but can be both exo- and endopeptidases when they possess two or more cations.

Aspartic peptidases differ from the other proteases because they can interact with other zymogens. These peptidases can not only activate themselves but they can also trans-activate other peptidases. The parasitic aspartic peptidases do not break down tissues such as collagen but instead, function to break down haemoglobulin (Choi et al., 2006; Delcroix et al., 2006).

Cathepsins

Cathepsins (or cysteine peptidases) are a class of endopeptidases that have been divided into many subgroups depending on their substrate specificities and structures: this family includes subgroups B, C, F, H, K, L, O, S, V, W, and X for instance which are all present in humans (Rawlings and Barrett, 2004). Flatworms are more primitive than humans and only have three subgroups, B, C and L (Robinson et al., 2008). Our laboratory is mainly interested in the cathepsins B and L from the parasitic organisms Fasciola and Schistosoma. The largest family of cathepsins is the papain-like peptidases which are sulphydryl-dependant. They also have a specific conserved catalytic triad of three amino acids, cysteine, histidine and asparagine, of which the cysteine residue is the main catalytic amino acid (hence "cysteine peptidases").

These peptidases can function both intracellularly and extracellularly which has made them a good serological tool in confirming whether or not livestock is being infected by parasites such as Paragonimus or Fasciola (Sajid and McKerrow, 2002; Dalton et al., 2003). Due to this reason, they have become the main focus for vaccine research against trematodes. Additionally, these cathepsins have also been the reason behind the ineffectiveness of the host's own immune response because they cleave antibodies and promote immune evasion (McKerrow et al., 2006).

Amongst the cathepsin families, the most important one is the papain-like peptidase.

These cathepsins require the presence of a thiol group to function properly. Unlike other peptidases, these are expressed as pre-pro-enzymes and require two cleavage steps to become activated (Collins et al., 2004). The pre- domain consists of a signal peptide while the pro- domain is the typical pro-sequence inhibiting the protein (Sajid and McKerrow, 2002; Collins et al., 2004). When exposed to the proper conditions (for example, the slightly acidic gut lumen), the pro-enzyme will autocatalytically cleave its own native pro- inhibitor. Two of the most

23 conserved features of this cathepsin family are its CGSCWAFS motif and its fully folded bi-lobed structure (Lecaille et al., 2002). The most common function attributed to these peptidases in the world of parasitic trematodes is the degradation and digestion of haemoglobulin, a major source of amino acids which are required for the survival of the parasite.

The cathepsin family has a specific triad of residues that are conserved across all species and that catalyze the peptidase activity. These residues are Cys25, His159 and Asn175 in papain

(Lecaille et al., 2002). The Cys25 and His159 residues are absolutely required to catalyze the reaction while the Asn175 may or may not be present to act as a stabilizer. It is the imidazolium ring of His159 and the sulphydryl group of Cys25 that together form the reactive thiolate- imidazolium charged reactive site that catalyzes the peptidase activity (Lecaille et al., 2002; Sajid and McKerrow, 2002). Cathepsins have seven binding subsites within the active site (S4, S3, S2,

S1, S1’, S2’ and S3’ respectively). The catalytic cleavage site is located between the S1 and S1' subsites. Cathepsins' specificities are tied to their pocket sites, more specifically their S2 and S3 sites (Choe et al., 2006). Cathepsins have a native inhibitor peptide (their pro-domain) that is subsequently cleaved when activated. Inhibitors have been developed to mimic the peptide such as E-64 (Z-F-A-CH2N) and Z-F-F-CH2N (Mason et al., 1985).

An important family is the cathepsin L family. The Schistosoma mansoni cathepsin L 1

(SmCL1) and the Schistosoma japonicum cathepsin L 1 (SjCL1) had been erroneously named in the past but is, in fact, are part of a separate protease groups classified as cathepsin F (Rawlings et al., 2008). Cathepsin Ls are very immunogenic and also immunomodulatory; it has been shown that the Fasciola cathepsin L1 and L2 may suppress the Th1 immune response and IFNγ production in infected hosts (Dalton et al., 2003; Donnelly et al., 2010). Due to this important immunogenic property, cathepsin L has been used as a prime candidate in vaccine research

24 against Fasciola (Dowd et al., 1995; Spithill and Dalton, 1998; Anderson et al., 1999). Using a recombinant protein, vaccinated livestock had a much better immune response and much less fascioliasis-associated morbidity (Dalton et al., 2003).

Cathepsins L also have two conserved motifs, an ERFNIN motif and a GNFD motif in the pro-peptide (Li et al., 2012). They are endopeptidases and generally tend to be active at an optimal acidic pH; SmCL2 for instance is optimally active between a pH range of 3.0 to 6.5 and peaking at 5.35 (Brady et al., 2000; Dalton and Brindley, 1997) and loses activity at a pH of 7.0 and above (Brady et al., 2000). These cathepsins are closely related to human cathepsins S and K

(Tort et al., 1999). The cathepsin L’s specificity is mainly due to its S1 and S2 sites where the S2 site prefers to accommodate aliphatic amino acids and the S1 site conversely prefers aromatic amino acids (Choe et al., 2006). A cathepsin L specific inhibitor was developed and called

CAA0225. CAA0225 is a derivative of E-64 but designed to have very specific and high affinity for L-type cathepsins (Takahashi et al., 2009).

Cathepsins B are another type of cathepsins that are also predominant in parasitic trematodes. Around 15 cathepsins B1 and seven cathepsins B2 genes from nine different trematodes have been identified (Kasny et al., 2009). Cathepsins B have both endo- and exopeptidase capabilities due to a specific 30 amino acid string known as an “occluding loop”

(Beckman et al., 2006; Caffrey et al., 2002; Law et al., 2003; Meemon et al., 2004). The occluding loop is a conserved motif that has a conserved double histidine motif (H110-H111) also known as the “catalytic diad”. The exopeptidase activity specifically cleaves dipeptides from the C-terminus of substrates (Illy et al., 1997; Krupa et al., 2002). An inhibitor specific for cathepsins B, known as CA-074 (N-[L-3-trans-propylcarbamoyloxirane-2-carbonyl]-Ile-Pro-

OH), acts as a competitive inhibitor by competing for S2 site binding (Murata et al., 1991). A

25 derivative of CA-074 had an isopropyl group replaced with a phenol and named MB-074 and is also specific for cathepsins B (Greenbaum et al., 2000).

The specific subgroups of cysteine peptidases L and B are the main focus of the research described in this thesis as they are the most important peptidases in the parasitic organisms of interest in our laboratory, Fasciola and Schistosoma. Once cathepsin homologs from the free- living flatworm M. lignano are found and characterized, they can be used as models for their parasitic counterparts to better understand their biochemistry and biological function to help us learn more about the evolutionary pressures that parasitism has imposed on these cathepsins.

CHAPTER 2 - Characterization of the Free-Living Macrostomum lignano Cathepsin L1

1. Introduction

Parasitic trematodes such as Schistosoma mansoni infect humans and are responsible for an estimated 200 million infections each year (WHO, 2001; Molyneux, 2006). Other trematodes such as Fasciola are important veterinary pathogens causing annual losses which amount to US$

2-3.2 billion, the majority being livestock such as sheep and cattle (Anderson et al., 1999;

McManus and Dalton, 2006).

These trematodes are very efficient at infecting, burrowing and digesting host tissues while also subverting their immune response to prolong their stay in the host (McKerrow et al.,

1991; Darani et al., 1997). Most of these functions are performed by the parasites’ secreted proteins (Cesari et al. 2000, Dvorak et al., 2005). The secreted proteins of interest, in this case, are the papain-like cysteine proteases (also known as cathepsins).

Cathepsins are ubiquitous in nature; all organisms have these proteins including viruses.

There are many different families of cathepsins and clans: the one of interest here are the Clan

C1A Cathepsin L. The cathepsins L use the typical cathepsin catalytic triad (Cys25, His159 and

Asn175) (Lecaille et al., 2002) and are expressed as a pre-pro-enzyme which carries a signal peptide, a pro-domain which contains an inhibitory peptide and a mature enzyme which contains the catalytic region (Sajid and McKerrow, 2002; Collins et al., 2004). The catalytic active site of cathepsins L have seven binding subsites (S4 to S3') where the catalytic cleavage site is located between the S1 and S1'. Cathepsins L and F tend to be similar but cathepsins L have an ERFNIN motif that cathepsins F do not (Li et al., 2012).

One of the main challenges in studying these enzymes in vivo is the parasites’ susceptibility to starve to death. Already these parasites are often fragile outside their host and therefore they cannot be studied properly in this regard. A solution to this problem is turning to

28 model related free-living organisms. Since no model organisms have been determined, M. lignano, a Macrostomidae of the Turbellaria class and free-living platyhelminth, relative of the parasitic trematodes, is currently being studied as a model organism for Schistosoma mansoni and other parasitic trematodes. In order to study it, cathepsins must first be identified and have their homologues phylogenetically traced back into the parasites.

M. lignano is a platyhelminth that resides in the brackish waters of Lignano, Italy

(Ladurner, 2005). This flatworm is monoecious and feeds mainly on diatoms (Ladurner, 2005). It has a typical free-living flatworm life-cycle: adults lay eggs, eggs hatch into juveniles, juveniles mature into adults (Ladurner, 2005). Unlike other flatworms, it has a continuous reproductive cycle and can lay an egg every 10-14 days (Ladurner, 2005); other flatworms tend to have a specific mating cycle (Pearse, 1994; Lázaro et al., 2009). It possesses an extensive ability to regenerate tissue damage as long as its head is intact due to the presence of totipotent cells. What makes this flatworm a good candidate to become a model organism for investigation of proteases in feeding and regeneration is its resilience to starvation and rough conditions: if starved, it will simply digest itself until the adult worm reaches the size of the juvenile; when presented food, it will simply regenerate the lost structures (Gonzalez-Estevez et al., 2007; Ladurner et al., 2008;

Gonzalez-Estevez and Sato, 2010).

In this study, we have identified, cloned and produced a recombinant cathepsin L from the free-living flatworm M. lignano. Its enzyme kinetic properties were characterized in addition to its general biochemical properties and antibodies were produced against the recombinant enzyme.

2. Materials and Methods

2.1 Gene Identification and Phylogeny

The mlcl1 gene was discovered by tBLASTn in the Macrostomum EST database

(http://flatworm.uibk.ac.at/macest/blast.php ) by using Schistosoma mansoni’s cathepsin L1

(SmCL1), L2 (SmCL2) and Fasciola hepatica’s cathepsin L1 (FhCL1) are sequence probes.

MlCL1 was the only complete gene found in the database. The gene was not found in the genome database when it was interrogated and neither did fragments list any cathepsins L.

All phylogeny was done using full-length protein sequences (including both signal and inhibitory peptides) in order to maximize scoring potential around conserved areas. Schmidtea mediterranea sequences were obtained from Dr. Brett Pearson (University of Toronto, Canada) and Dr. Mostafa Zamanian (Institute of Parasitology, McGill, Canada) while all other sequences were obtained from NCBI’s protein database. Multiple sequence alignment and distance trees were generated using the computer software Geneious 5.4.6 (Biomatters).

2.2 Gene Editing

The cDNA encoding the M. lignano cathepsin L1 gene (mlcl1) was synthesized by

Genscript (New Jersey, U.S.) and inserted into the pUC57 vector. The synthesized gene was codon-optimized for expression in the yeast Pichia pastoris, and did not include the signal peptide-encoding region (identified using SignalP), and had the addition of a segment encoding for a glycine and proline followed by a hexahistidine tag before the stop codon. A potential N- glycosylation site was altered by replacing the codon for asparagine (N130 - ACG) with one encoding a glutamine (Q130 - CAA) to avoid glycosylation by Pichia pastoris (see Figure 1).

The gene was also flanked by a MlyI restriction site at the 5’ end and a KpnI restriction site after the stop codon at the 3’ end.

2.3 Cloning

All cloning was performed according to the PichiaPink Manual (Invitrogen). The mlcl1 gene was excised from pUC57 using the restriction enzymes MlyI and KpnI-HF. The yeast vector pPinkα-HC was linearized using StuI and KpnI-HF. The mlcl1 gene was then inserted into the linearized vector using a T4 ligase. Final construct was then linearized using SpeI and transformed into Pichia pastoris by electroporation using GenePulser II from Bio-Rad (1500V charging voltage, 200Ω resistance and 25 µF capacitance). The transformed yeast was then plated onto PAD agar and subsequently screened for successful transformants. To make sure that the correctly ligated product was selected, another digestion using KpnI and NdeI was performed

(see Figure 3) which would cut the vector in pieces of 7 kb and 2 kb (gene inserted) or 7 kb and 1 kb (no gene).

2.4 Recombinant Protein Production, Purification and Quantification

Protein production was performed according to the PichiaPink Manual (Invitrogen) but at a larger scale volume (12 L) and the OD reached in BMGY was 14. The supernatant was collected during the third day of the induction phase and filtered through a 0.45 µm pore filter and then another 0.22 µm pore filter to eliminate unwanted cellular waste.

MlCL1 was purified using affinity column chromatography. Nickel beads (1 mL) were washed and equilibrated in a column buffer at pH 8.0. The yeast culture supernatant was mixed

(1:4) with the column buffer and the pH was readjusted due to P. pastoris’ ability to acidify its environment. The supernatant was then passed through the column using gravity flow. Once all supernatant passed, the beads were washed once with wash buffer and eluted with elution buffer that had a lower pH (6.00) and higher concentration of imidazole (300 mM). The protein was then dialyzed using a dialysis cassette (Thermo Scientific) with a molecular weight cutoff of 20

000 Daltons in 2 L of PBS. MlCL1 was then aliquoted (250 µL) and stored at -20º C. MlCL1 was quantified using Bradford assay as indicated per the manufacturer’s instructions (BioRad).

P. pastoris can lose their plasmids and after repetitive inductions the colony stocks lack the ability to produce MlCL1; to obtain potent secretors, P. pastoris had to be re-plated on PAD agar and selected anew.

2.5 Colony Blot

White colonies that appeared on the PAD agar were picked and placed on YPD agar and left at 29º C to grow overnight. The colonies were lifted onto a nitrocellulose membrane and transposed (colonies being on top) onto a YNB/2% methanol agar plate. The plate was inverted and then incubated at 29º C overnight. The membrane was then transferred colony side up into a

32 series of petri dishes containing 3 mm filter papers soaked in the following solutions: 10% SDS,

Denaturing solution, Neutralizing solution, Neutralizing solution (second) and 2X SSC at room temperature. Transfer was performed for 10, 5, 5, 5 and 15 min, respectively, ensuring no bubbles were formed. The membrane was then washed twice with PBS/0.5% Tween 20 for 10 minutes followed by blocking using 5% milk in PBS/0.5% Tween 20. Anti-His-Tag primary antibodies were added 1:2500 and the membrane was incubated overnight at 4º C. The membrane was washed three times with PBS/0.5% Tween 20 at room temperature and incubated for an hour after adding 5% milk in PBS/0.5% Tween 20 and anti-mouse IgG-Peroxidase conjugate (1:1000). Once the membrane was dried, SuperSignal West Femto was added for 5 minutes before drying the membrane again and exposing it to a Kodak film.

2.6 Antibody Production and Purification

MlCL1 antibodies were developed against a specific peptide sequence. Using a 3D threading method, a peptide sequence that was on the outside of the folded mature enzyme was identified and sent off to Genscript for antibody production in a rabbit. The 3D threading method was a basic sequence alignment between FhCL1 (which has a known crystal structure) and

MlCL1; once immunogenic sites were identified by Genscript, they were selected on their location in the 3D model of FhCL1. The sequence selected was QAAESRCQYQRSRV and had an added cysteine group at the C-terminus to be conjugated to ovalbumin.

Antibodies and their peptide epitope are sent together for testing. Using a SulfoLink

Immobilization Kit for Peptides (Thermo SCIENTIFIC), the peptides were conjugated onto beads into a column and antibodies were passed through and washed before being eluted to ensure that only polyclonal antibodies specific to the peptide remained. The elution buffer used

33 was 0.2 M glycine•HCl at pH 2.5 and the neutralization buffer used was 1M Tris•HCl at pH 8.5.

All other reagents and procedures were done according to the instruction manual.

2.7 Kinetics and Substrate Specificities of MlCL1

To determine the KM (Michaelis-Menten constant) and Kcat, 0.5 µg (100.75 nM) (100.75 nM) of MlCL1 was used per wells. Substrate concentrations were 200, 100, 50, 25, 12.5, 6.25,

3.125 and 1 µM. DTT (2 mM) was added to the enzyme and pre-incubated for 15 minutes at 37º

C to activate it (this pre-incubation step was done for every other following assay). The buffer used was sodium phosphate [0.1 M] at a pH of 6.5. Substrates used were Z-F-R-AMC, Z-L-R-

AMC, Z-R-R-AMC and Z-P-R-AMC. All results were obtained in triplicates. The Kcat was obtained after leaving the reaction at 37º C overnight and obtaining the endpoint the following morning. The values were computed using SigmaPlot (Systat Software, Inc.).

2.8 pH Profile of MlCL1

The enzymatic activity of MlCL1 was tested over a range of pH in different buffers, all having a concentration of 0.1 M: sodium acetate (pH 4.0, 5.0, 5.0, 5.5), sodium citrate (pH 5.5,

6.0) and sodium phosphate (pH 6.0, 6.5, 7.0, 7.5, 8.0). Substrates used were Z-L-R-AMC, Z-F-

R-AMC, Z-R-R-AMC and Z-P-R-AMC, all at a concentration of 3 mM in a 200 µL reaction over an hour at 37º C. All substrate pH profiles were done in triplicates. To eliminate outliers, Z- scores were calculated for every reactions and all scores |z| = 3.0 or more were deemed outliers and were removed.

2.9 Activity of MlCL1 at Different Temperatures

To determine activity of MlCL1, the substrate Z-F-R-AMC was used based on the kinetic data which showed it was the most efficiently cleaved substrate. Temperatures chosen were 4º C, room temperature (22º C) and 37º C. Reaction mix contained 0.1 µg (20.15 nM) of MlCL1, 2 mM DTT and Z-L-R-AMC (3 µM) in sodium acetate pH 5.0. Enzyme and buffer were cooled down to 4º C before DTT was added to make sure temperature did not play a role in enzyme degradation or activation prior to starting the assay. Once substrate was added, the reactions were moved to their respective temperatures and left for incubation for an hour. All reactions were done in pentaplicates. Activity was assessed using endpoint fluorescence. Z-scores were calculated for every reactions and all scores |z| = 3.0 or more were deemed outliers and were removed.

2.10 Activity of MlCL1 at Different Salinities

Salinity was measured in parts per thousand (ppt or grams of NaCl per kg of water).

Salinities used for the assay were 0.5, 1, 5, 10, 15, 30 and 50 ppt (brackish waters range from 0.5 to 30 ppt). Controls for each salinity were made to ensure that the increasing sodium chloride concentrations did not affect the fluorochrome’s stability. All reactions were done in triplicates.

Reaction mix contained 0.1 µg (20.15 nM) of MlCL1, 2 mM DTT and Z-F-R-AMC (3 µM) in sodium acetate pH 5.0 and sodium chloride. All reactions were done at 37º C. All results were obtained in triplicates. Z-scores were calculated for every reactions and all scores |z| = 3.0 or more were deemed outliers and were removed.

2.11 Stability of MlCL1 at Different Times

To determine stability at different time points, all reactions were identical and initiated at the same time before being frozen at -20º C. Enzyme mix consisted of 4 mM DTT, 0.1 µg (20.15 nM) of MlCL1 and sodium acetate buffer pH 5.0 with a total volume of 100 µL. All samples were frozen at their designated time points and thawed simultaneously at room temperature before being added to a substrate mix consisting of 3 mM Z-F-R-AMC and sodium acetate buffer in 100 µL. The designated time points were 0, 0.5, 1, 2, 4, 8, 24 and 48 hours where at time 0, the DTT was added and the mixture was immediately frozen at -20º C. Once all enzyme mixtures were thawed and mixed with the substrate solution, their reaction rate was measured over an hour at 37º C. All results were obtained in triplicates.

2.12 Stability of MlCL1 at Different Temperatures

The stability of the enzyme at different temperatures was determined by monitoring enzyme activity. The enzyme mix contained 4 mM DTT, 0.1 µg (20.15 nM) of MlCL1 and sodium acetate pH 5.0. The enzyme mix was pre-incubated at 4º C for 10 minutes before they were allocated to their respective temperatures for an hour: 4º C, room temperature (22º C), 37º

C, 42º C, 50º C and 60º C. The enzyme mix was then added to a substrate mix of 3 mM Z-L-R-

AMC and sodium acetate pH 5.0 and immediately measured for its activity rate at 37º C for an hour. All results were obtained in triplicates.

2.13 Stability of MlCL1 at Different Salinities

The stability of the enzyme at different salinities was determined through its activity. In order to ensure that only the stability of the enzyme and not its activity was affected by the salinity, a small 20 µL enzyme mix was prepared with 4 mM DTT, 0.1 µg (20.15 nM) of

MlCL1, sodium acetate pH 5.0 and various salt concentrations (0, 0.5, 1, 5, 10, 15, 30 and 50 ppt). The enzyme mix was incubated at 37º C for an hour. The enzyme mix was then added to a substrate mix of 3.0 µM Z-F-R-AMC and sodium acetate pH 5.0 in a total volume of 200 µL and immediately measured for its activity at 37º C for an hour. All results were obtained in triplicates.

2.14 Inhibition of MlCL1 by E-64 and CAA0225

E-64 (general C1A clan inhibitor) and CAA0225 (cathepsin L-specific inhibitor) were tested to see their efficacy against the protease. Using a 96-well plate, the inhibitor was added and serially diluted two-fold in 100 µL with a final first concentration of 0.5 µM. Substrate was added afterwards at three concentrations: 6, 12 and 24 µM (half KM, KM and double KM).

Finally, 0.1 µg (20.15 nM) of enzyme was added to each well and immediately measured for its activity at 37º C for an hour. All results were obtained in triplicates. The software SigmaPlot was used to obtain the Dixon plot and a tight-binding inhibitor algorithm was used to obtain the Ki.

3. Results

3.1 Phylogeny

According to the phylogenetic tree (both distance and maximum likelihood), M. lignano’s cathepsin L1 shared a common ancestry with that of Schmidtea Mediterranea;

37 furthermore, it is a plesion group of the schistosome’s cathepsin L1(japonicum) and L3

(mansoni). It does not share any ancestry with the cathepsins L from Fasciola (see Figure 2).

Additionally, an ERFNIN and a GFND motif were found, further supporting the hypothesis of it being a cathepsin L (see Figure 1).

Figure 2. Phylogenetic tree of cathepsins L and B. MlCL1 is a plesion of the crown CL1 group in the cathepsin L1 clade. Its ancestor is common to the ancestor of the cathepsins L1 of the Schistosoma species. Tree is a distrance-based tree using pro-enzyme sequences. A maximum likelihood tree agreed with the tree presented above although the supporting values were not similar.

3.2 Cloning and Colony Blot

The cloning of the recombinant MlCL1 gene (rMlCL1) was successful. An Escherichia coli colony contained the insert in large quantities and was grown afterwards for plasmid extraction. The transformation of P. pastoris was also successful and the positive colonies on the

PAD agar were then tested for expression using the colony blot.

The colony blot revealed that only a subset of colonies produced the desired protein

(MlCL1); colonies 11, 23 and 24 were chosen for large scale protein production due to their opacities when compared to the other colonies and based on preliminary cultures’ results (see

Figure 4). Not all colonies expressed the protein.

pUC57 pPinkα-HC

KpnI StuI KpnI MlyI

rMlCL1 T4 Ligase

KpnI

NdeI

SpeI

Figure 3. Cloning manipulations of MlCL1. MlCL1 (green) is received in the pUC57 vector; using the restriction enzymes KpnI and MlyI, the gene is cut out and the corresponding fragments are isolated in the agarose gel. The pPinkα-HC vector is cut at the restriction site directly adjacent to the signal peptide sequence using StuI and MlyI. Using a T4 ligase, the gene and vector are ligated together; to test which E. coli colony contained the correctly ligated vector, the purified DNA is restricted once more using NdeI and KpnI digestion which either shows a 6kbp and a 1.2kbp band (no MlCL1) or a 6kbp and a 2.5kbp band (MlCL1 present). Using SpeI, the vector is linearized and transformed into P. pastoris via electroporation.

3.3 Protein Purification and Quantification

Once dialysis was complete, proteins were run on an SDS-PAGE gel to check for purity and a Western Blot to ensure that those bands are truly MlCL1. SDS-PAGE revealed a band at

25 kDa which is the predicted size of the mature and active enzyme. All bands turned out to be

MlCL1 according to the Western Blot (see Figure 5). The enzyme can autocatalytically activate itself through a cis mechanism in the culture environment. Subsequent protein production and purification yielded fully active and mature enzymes but at much lower concentrations; the enzyme may be toxic to the yeast culture (see Figure 5). Protein concentrations measured were of

168 µg/µL.

W L L A B C D 150 150 100 100

75 75

50 50

3 3

25 2

Figure 5. Western Blot and SDS-PAGE of MlCL1 using the cathepsin inhibitor E-64. Lane L represents the molecular weight marker, A contains neither DTT nor E-64 in the loading dye, B contains DTT in the loading dye, C contains E-64 without DTT and D has both E-64 and DTT. Lane W contains MlCL1 incubated at 37 °C for 1 hour with DTT and no inhibitors. All enzymes are activated and mature.

3.4 Kinetics and Substrate Specificities

MlCL1 is able to cleave substrates Z-L-R-AMC and Z-F-R-AMC (see Table 1). It poorly

cleaves Z-R-R-AMC and has little to no affinity and activity towards Z-P-R-AMC. Because the

substrate amount could not exceed 200 µM, we assumed that the km was higher than that and

that it basically cannot bind Z-P-R-AMC at all.

Table 1. Kinetic parameters of MlCL1. K is the Michaelis-Menten constant, K represents the time M cat required for 1 unit of enzyme to process 1 unit of substrate and Kcat/KM is the catalytic efficacy.

3.5 pH Profile of MlCL1

The optimal pH of reaction for substrates Z-F-R-AMC is a broad range from 4.5 in sodium acetate buffer [0.1M] to 6.5 in sodium phosphate buffer [0.1M]. Z-L-R-AMC has a broad range also from 5.5 in sodium acetate buffer [0.1M] to 6.5 in sodium phosphate buffer [0.1M]

(see Figure 6). Due to the low and insignificant rates (when compared to those of Z-L-R-AMC and Z-F-R-AMC), Z-R-R-AMC optimal pH could not be determined as all rates in absolute values ranged from 0.05 to 0.650 RFU/min (as opposed to 8-20 RFU/min in the case of Z-L-R-

AMC and Z-F-R-AMC).

MlCL1 pH profile with Z-F-R-AMC

100

60 % Activity % 40

0 4 4.5 5 5.5 6 6.5 7 7.5 8 pH

Figure 6. pH profile of MlCL1 using Z-F-R-AMC. The enzyme has a broad range for activity between pH 4.5 and 6.5. Activity steadily drops below pH 4.5 and drastically drops above pH 7.0. Buffer seems to play a role in activity at pH 5.5 where sodium citrate is more adequate.

MlCL1 Activity at different temperatures

120

100

60 % Activity % 40

0 4 37

Temperature (°C)

Figure 7. MlCL1 activity at 4 °C and 37 °C. The enzyme works at colder temperatures as expected but at much higher levels than anticipated (60% of maximum activity).

3.6 Activity of MlCL1 at Different Temperatures and Salinities

MlCL1 has an optimal activity temperature of 37º C. The enzyme is still active at about

60% maximum activity when at 4º C (see Figure 7). MlCL1 is completely unaffected by salinity which is what would be expected for the flatworm to be able to live freely in the brackish waters of Lignano (see Figure 8).

Figure 8. MlCL1 activity at different salinities. Activity is unaffected by the salt concentration of the enzyme; no significant changes even past 50 ppt have been noted.

MlCL1 Stability and Time

100

60 % Activity % 40

0 0 10 20 30 40 50 Time (hours)

3.7 Stability at Different Times, Temperatures and Salinities

MlCL1 is very stable at 37º C and only loses 50% of its activity after 24 hours (see

Figure 9). There is an observed increase in the activity from time 0 to 1 hour which could be explained by the fact that the enzyme population is mixed between active and inactive forms. In that period, the active forms remain active while the inactive pro- forms start to activate.

MlCL1 is very stable at 4º C, 22º C, 37º C, 42º C and loses most of its activity at 50º C before being completely inactivated at 60º C (see Figure 10). MlCL1 is stable in high salt conditions; past 1 ppt, the activity drops significantly to 75-80% but remains unaffected by higher salt concentrations (see Figure 11).

Anti-peptide antibodies successfully bound to the recombinant MlCL1 but not to the protein MlCL1 from worm extract during Western Blots. Once the antibodies were purified, antibodies clearly and successfully detected MlCL1 in the worm extract when added in a 1:500 ratio. In addition, purified antibodies had no specific binding to the ladder and a markedly drop in background.

MlCL1 Stability and Temperature

100

Activity% 40

4 22 37 42 50 60 Temperature ( °C) Figure 10. Stability of MlCL1 after being exposed at different temperatures for 1 hour. MlCL1 appears to be very stable between 4 and 42 °C. Stability is significantly hindered at 50 °C and all enzymatic activity is lost at 60 °C.

* * * * * *

Figure 11. Stability of MlCL1 in the presence of NaCl at different concentrations. Brackish salt water is typically between 0.5 and 30 ppt. MlCL1 only loses about 20% of activity at 1 ppt and maintains the trend even above 30 ppt.

3.8 Inhibition of MlCL1 by E-64 and CAA022

MlCL1 was successfully inhibited by both E-64 (general cysteine protease inhibitor) and

CAA0225 (derivative of E-64 specific to L-type cathepsins). The Ki for E-64 was 0.0124 µM

(see Figure 12) and the Ki for CAA0225 was 0.0000330 µM (see Figure 13). Therefore the protease is 375 times more sensitive to CAA022 than E-64.

MlCL1 was also tested with CA-074 to assess whether or not CA-074 was truly specific to cathepsins B. CA-074 did not have any significant effect on MlCL1 except at very high concentrations (30 µM or greater) and plateaued while the cathepsin B control was completely inhibited at all concentrations tested (see Figure 15, section 2).

Dixon

0.25

0.20

0.15

0.10

(RFU/min) 1/Rate

0.05

-0.03 -0.02 -0.01 0.00 0.01 0.02 0.03 0.04 [Inhibitor] (µM)

Vmax = 80.5 S = 6 Km = 36.4 S = 12 Ki = 0.01247 S = 24 E = 0.01084

Figure 12. Dixon plot of the inhibitor E-64. The plot is that of a tight binding mechanism that competes for the active site with the substrate Z- F-R-AMC. 47

Dixon

0.35

0.30

0.25

0.20

0.15

1/Rate (RFU/min) 1/Rate 0.10

0.05

-0.002 0.000 0.002 0.004 0.006 0.008 0.010 0.012 [Inhibitor] (µM)

Vmax = 9. S = 6 Km = 0.6914 S = 12 Ki = 3.301e-5 S = 24 E = 0.01595 Figure 13. Dixon plot of the inhibitor CAA0225. The plot is that of a tight binding mechanism that competes for the active site with the substrate Z- F-R-AMC. The Ki is very small in comparison to E-64 (375 times lower).

CHAPTER 3 - Characterization of the Free-Living Macrostomum lignano Cathepsin B2

1. Introduction

A second full-length sequence encoding a cysteine protease was identified when interrogating the M. lignano EST database. Upon multiple sequence alignment, it was a clear match in both length and conserved motifs to that of the cathepsin B family’s. When sought out in the genomic database, a 100% identity hit was found with two intron sequences. Since cathepsins B are also extensively used by the trematode parasites Fasciola and Schistosoma, this became another focus in the project. Once synthesized and characterized, information on the homologues could be inferred much like the cathepsin L1 in M. lignano.

Once the proper phylogeny was constructed, it became apparent that this peptidase was a plesion of the cathepsin B2 clade of Schistosoma mansoni and Trichobilharzia regenti/szidati.

SmCB2 was extensively studied by Dr. Caffrey’s group in the adult stages of the parasite but no function was ever discovered (Caffrey et al., 2002). It was suggested, because it was localized in the parenchyma and tegument’s tubercules, that it was most likely serving a biogenesis or turnover function. In T. regenti, TrCB2 was mainly studied in the cercarial stage and a putative skin penetrative function was implied due to the localization of the enzyme in the post-acetabular glands of the parasite (Dolečková et al., 2009). The enzyme could potentially digest skin, collagen and elastin but not haemoglobulin.

Considering the fact that both SmCB2 and TrCB2 are homologous, that they have putative functions in different life-cycle stages and that they share a common ancestry with the free-living flatworm, much more investigation is needed to understand the phylogeny of this gene and what its function in the free-living organisms is since these do not possess a cercaria stage.

In this study, we describe the identification, cloning and production of a functional recombinant cathepsin B from the free-living flatworm M. lignano. Its enzyme kinetic properties were characterized in addition to its biochemical properties. Antibodies were produced against the recombinant enzyme for future immunocytochemical studies.

2. Materials and Methods

2.1 Gene Identification and Phylogeny

The mlcb2 gene (see Figure 1) was discovered by tBLASTn in the M. lignano EST database (http://flatworm.uibk.ac.at/macest/blast.php) using S. mansoni’s cathepsin B1

(SmCB1), S. mansoni B2 (SmCB2) and F. hepatica’s cathepsin B1 (FhCB1) as queries. MlCB2 was the only complete cathepsin B gene found in the database. The genome database was similarly interrogated using the same queries and the DNA sequence returned was identical to that found in the EST database.

All phylogenetic analyses were performed using full-length cathepsin B sequences

(including both signal peptide and pro-domain). Schmidtea mediterranea sequences were obtained from Dr. Bret Pearson (University of Toronto, Canada) and Dr. Mostafa Zamanian

(Parasitology Institute, McGill, Canada) while all other sequences were obtained from NCBI’s protein database. Multiple sequence alignment and distance trees were generated using the computer software Geneious 5.4.6 (Biomatters).

2.2 Gene Editing

A cDNA encoding the M. lignano cathepsin B2 gene (mlcb2) was synthesized by

Genscript (New Jersey, U.S.) and inserted into the pUC57 vector. The synthesized gene was codon-optimized for expression in the yeast Pichia pastoris, and did not include the signal peptide-encoding region (identified using SignalP). A potential N-glycosylation site was altered by replacing the codon for asparagine (AAC, N225) for a glutamine (CAA, Q225) (this was done to avoid glycosylation by Pichia pastoris). It also had the addition of a segment encoding for glycine and proline followed by a hexahistidine tag before the stop codon (this was to facilitate downstream purification of expressed protein). The gene was also flanked by a MlyI restriction site at the 5’ end and a KpnI restriction site after the stop codon at the 3’ end. See Figure 2 for a graphical representation of the previously described steps.

Figure 2. Edited MlCB2 DNA and translated amino acid sequences. The MlyI restriction site [orange] is added at the beginning of the pro-enzyme sequence. The catalytic triad (C25, H179, N185) are highlighted in red as individual residues; the catalytic diad of the occluding loop (H110-H111) is highlighted (bright green). The Aspartate residue (D171) is mutated and highlighted under CAA and Q171 (yellow). A glycine and a proline residues (dark blue) are added at the end of the sequence followed by a hexahistidine sequence (bright blue) using different codons. The stop codon is moved past the hexahistidine tag and followed by a Kpn1 restriction site (green).

2.3 Cloning

All cloning was performed according to the PichiaPink Manual (Invitrogen). The mlcb2 gene was excised from pUC57 using the restriction enzymes MlyI and KpnI-HF. The yeast vector pPinkα-HC was linearized using StuI and KpnI-HF. The mlcb2 gene was then inserted into the linearized vector using a T4 ligase. The final construct was then linearized using SpeI and transformed into Pichia pastoris by electroporation. The transformed yeast was then plated

53 onto PAD agar and subsequently screened for successful transformants. To make sure that the correctly ligated product was selected, another digestion using KpnI and NdeI was performed

(see Figure 4) which would cut the vector in pieces of 7 kb and 2 kb (gene inserted) or 7 kb and 1 kb (no gene).

2.4 Colony Blot

Denaturing solution, Neutralizing solution, Neutralizing solution (second) and 2X SSC at room temperature. Transfer was performed for 10, 5, 5, 5 and 15 mins. respectively, ensuring no bubbles were formed. The membrane was then washed twice with PBS/0.5% Tween 20 for 10 minutes followed by blocking using 5% milk in PBS/0.5% Tween 20. Anti-His-Tag primary antibodies were added 1:2500 and the membrane was incubated overnight at 4º C. The membrane was washed three times with PBS/0.5% Tween 20 at room temperature and incubated for an hour after adding 5% milk in PBS/0.5% Tween 20 and anti-mouse IgG-Peroxidase conjugate (1:1000). Once the membrane was dried, SuperSignal West Femto was added for 5 minutes before drying the membrane again and exposing it to a Kodak film.

2.5 Recombinant Protein Production, Purification and Quantification

Protein production was performed according to the PichiaPink Manual (Invitrogen) but at a larger scale volume (4L) and the OD reached in BMGY was 8. In addition, one drop (≈35 µL) of antifoam 204 (Sigma) was added to prevent loss of moisture and loss of supplemental methanol added during induction phase. The supernatant was collected during the third day of the induction phase and filtered through a 0.45 µm pore filter and then another 0.22 µm pore filter to eliminate unwanted cellular debris.

MlCB2 was purified using affinity column chromatography. Nickel beads (1 mL) were washed and equilibrated in a column buffer at pH 8.0. The yeast supernatant was mixed (1:4) with the column buffer and the pH was readjusted due to P. pastoris’ ability to acidify its environment. The supernatant was then passed through the column using gravity flow. Once all supernatant passed, the beads were washed once with wash buffer and eluted with elution buffer that had a lower pH (6.00) and higher concentration of imidazole (300 mM).

The eluted protein was then dialyzed using a dialysis cassette (Thermo Scientific) with pores of 20 000 Daltons in 2 L of PBS. MlCB2 was then aliquoted (250 µL) and stored at -20º C.

MlCB2 was quantified using Bradford assay as indicated per the manufacturer’s instructions

(BioRad).

2.6 Antibody Production and Purification

MlCB2 antibodies were prepared by immunizing three rats (Wistar strain) with the recombinant protein by the antibody company Medimabs (Royalmount, Montreal, Canada). Each rat was immunized with 200 µg of proteins on days 1, 15, 26, 56, 76 and boosted on the 90th day.

The final bleed sera were harvested on the 105th day. The antibodies were purified using affinity

55 chromatography using MlCB2 enzymes as antigens (final concentration was 1.83 mg/mL).

Purity and specificity was tested on recombinant MlCB2 through Western Blots with purified antibodies being diluted 1:10 000 000.

2.7 Kinetics and Substrate Specificities

To determine the KM (Michaelis-Menten constant) and Kcat, 0.25 µg of MlCB2 was used per wells. Substrate concentrations were 320, 160, 80, 40, 20, 10, 5 and 2.5 µM. DTT (2 mM) was added to the enzyme and pre-incubated for 30 minutes at 37º C to activate it. The buffer used was 0.1 M sodium acetate pH 4.5. Substrates used were Z-F-R-AMC, Z-L-R-AMC, Z-R-R-

AMC and Z-P-R-AMC. All results were obtained in triplicates.

Kcat was obtained after leaving the reaction at 37º C overnight and obtaining the endpoint the following morning. All calculated values were computed using SigmaPlot (Systat Software,

Inc.).

2.8 pH Profile of MlCB2

The enzymatic activity of MlCB2 was tested over a spectrum of pHs in different buffers, all having a concentration of 0.1 M: sodium acetate (pH 4.0, 4.5, 5.0, 5.5), sodium citrate (pH

5.5, 6.0) and sodium phosphate (pH 6.0, 6.5, 7.0, 7.5, 8.0). Substrates used were Z-L-R-AMC, Z-

F-R-AMC, Z-R-R-AMC and Z-P-R-AMC, at a concentration of 20 µM in a 200 µL reaction over an hour at 37º C. The enzyme (0.1 µg) was pre-incubated at 37º C for 45 minutes with 4 mM DTT to make sure all enzymes were activated. All substrate pH profiles were performed in triplicate and repeated 3 times.

2.9 Activity of MlCB2 at Different Temperatures

To determine the optimal temperature for MlCB2, the substrate Z-L-R-AMC was used since our kinetic data showed it was the most efficiently cleaved substrate. Temperatures chosen were 4º C, room temperature (22º C) and 37º C. Reaction mix contained 0.25 µg of MlCB2, 2 mM DTT and Z-L-R-AMC (20 µM) in sodium citrate pH 6.0. Enzyme and buffer were cooled down to 4º C before DTT was added to make sure temperature did not play a role in enzyme degradation or activation prior to starting the assay. Once substrate was added, the reactions were moved to their respective temperatures and left for incubation for an hour. All reactions were performed in pentaplicate. Activity was assessed using endpoint fluorescence.

2.10 Activity of MlCB2 at Different Salinities

Salinity was measured in parts per thousand (ppt). Salinities used for the assay were 0.5,

1, 5, 10, 15, 30 and 50 ppt (brackish waters range from 0.5 to 30 ppt). Control experiments were initially performed at each salinity to ensure that the sodium chloride concentrations did not affect the fluorochrome’s stability. All reactions were done in triplicates. Reaction mix contained

0.25 µg of MlCB2, 2 mM DTT and Z-L-R-AMC (20 µM) in sodium citrate pH 6.0 and sodium chloride. All reactions were performed at room temperature (22º C) due to the instability of

MlCB2 at 37º C. All results were obtained in triplicates.

2.11 Stability of MlCB2 at Different Times

We determined the longevity of MlCB2 activity at 37° C which provides an indicator of enzyme stability. All incubations were initiated at the same time and the samples taken at each timepoint (0, 0.5, 1, 2, 4, 8, 24 and 48 hours) and frozen at -20º C. One day after the final

57 timepoint the samples were then thawed at the same time and assayed for enzymatic activity.

Enzyme mix consisted of 4 mM DTT, 0.25 µg of MlCB2 in sodium citrate buffer pH 5.5 with a total volume of 100 µL. Once thawed, a substrate solution added to enzyme mix consisting of Z-

L-R-AMC (20 µM) and sodium citrate buffer pH 6.0 in 100 µL. The reaction rate was measured over an hour at 37º C. All results were obtained in triplicates.

2.12 Stability of MlCB2 at Different Temperatures

The stability of the enzyme at different temperatures was also assessed. The enzyme mix contained 4 mM DTT, 0.25 µg of MlCB2 and sodium citrate pH 5.5. The enzyme mix was pre- incubated at 4º C for 5 minutes before they were allocated to their respective temperatures for an hour: 4º C, room temperature (22º C) and 37º C. The enzyme mix was then added to a substrate mix of Z-L-R-AMC (20 µM) and sodium citrate pH 6.0 at 37º C for an hour. All results were obtained in triplicates.

2.13 Stability of MlCB2 at Different Salinities

In order to ensure that only the stability of the enzyme and not its activity was affected by the salinity, a small 20 µL enzyme mix was prepared with 4 mM DTT, 0.25 µg of MlCB2, sodium citrate pH 5.5 and various salt concentrations (0, 0.5, 1, 5, 10, 15, 30 and 50 ppt). The enzyme mix was incubated at 37º C for an hour. The enzyme mix was then added to a substrate mix of Z-F-R-AMC (20 µM) and sodium citrate pH 6.0 in a total volume of 200 µL and immediately measured for its activity at 37º C for an hour. All results were obtained in triplicates.

2.14 Digestion of Natural Substrates by MlCB2

To determine whether or not MlCB2 can digest naturally occurring substrates such as

BSA, 8 µg of enzyme was incubated with 24 µg of substrate in a 160 µL reaction in sodium acetate pH 4.5 and 2 mM DTT. Aliquots were taken at different time points (0, 0.5, 1, 2, 4, 6 and

8 hours), frozen at -20 °C overnight and thawed on ice before being run on an SDS-PAGE gel.

All results were done in triplicates.

2.15 Inhibition of MlCB2 by CA-074 and E-64

CA-074 (Sigma-Aldrich) is a known inhibitor specific to cathepsins B via competitive tight-binding of the mature protein. To calculate the Ki, 0.1 µg of enzyme was pre-incubated in sodium acetate buffer pH 5.5 for 1 hour at 37° C. During the incubation, substrate and inhibitor were loaded into a 96 well plate in a manner to have the substrate concentration constant per group while diluting the inhibitor two-fold by serial dilution. The chosen substrate concentrations were 40, 80 and 160 µM (or .5(KM), KM and 2(KM)); the adequate starting concentration of CA-074 was determined to be 0.125 µM and serially diluted 6 times; for E-64, it was determined to be 1 µM. The last well was used as a negative control (no inhibitor). Results were measured at 37°C over a 60 minute time period. Extrapolation of results was performed using the software SigmaPlot (Systat Software, Inc.) and using the Dixon plot. The Ki for both

CA-074 and E-64 was calculated using the tight competitive binding formulae. All assays were done under reducing conditions (2 mM DTT).

3. Results

3.1 Phylogeny

According to the phylogenetic tree (see Figure 3), M. lignano’s cathepsin B2 shared a common ancestry with that of Schmidtea mediterranea. Furthermore it is a plesion group of the schistosome’s cathepsin B2. MlCB2 is paraphyletic, meaning it shares many primitive traits to that of its ancestor and thus branches out earlier in the phylogenetic tree than the crown group.

The phylogenetic tree also indicates that the cathepsins B from Fasciola were all derived from one ancestral inherited cathepsin B that later, in the Fasciolas, was duplicated repeatedly and mutated afterwards (which is not the case for Schistosoma or Trichobilharzia). MlCB2 is more similar to SmCB2 in terms of overall sequence than to the Fasciola cathepsins B.

Figure 3. Phylogenetic tree of cathepsins L and B. MlCB2 is a plesion of the crown CB2 group in the cathepsin B2 clade. Its ancestor is common to the ancestor of the cathepsins B2 of Trichobilharzia and Schistosoma species. Tree is a distance-based tree using pro- enzyme sequences. A maximum likelihood tree agreed with the tree presented above although the supporting values were not similar.

3.2 Cloning and Colony Blot

Cloning of MlCB2 was successful and a KpnI and NdeI digest of the final construct revealed that three clones contained rmlcb2 (see Figure 4). Those were chosen, linearized and inserted into Pichia pastoris and a subsequent colony blot was performed to detect protein production.

The colony blot (see Figure 5) revealed that all colonies selected produced the desired protein (MlCB2); colonies 5, 11 and 19 were chosen for large scale protein production due to their opacities on film (thus indicating a stronger production of the recombinant enzyme) when compared to the other colonies.

pUC57 pPinkα-HC

KpnI StuI MlyI MlyI

rMlCB2 T4 Ligase

KpnI

NdeI

SpeI

Figure 4. Cloning manipulations of MlCB2. MlCB2 (green) is received in the pUC57 vector; using the restriction enzymes KpnI and MlyI, the gene is cut out and the corresponding bands were isolated the agarose gel. The pPinkα-HC vector is cut at the restriction site directly adjacent to the signal peptide sequence using StuI and MlyI. Using a T4 ligase, the gene and vector are ligated together; to test which E. coli colony contained the correctly ligated vector, the purified DNA is restricted once more using NdeI and KpnI digestion which either shows a 9kbp and a 1kbp band (no MlCB2) or a 9kbp and a 2kbp band (MlCB2 present). Using SpeI, the vector is linearized and 61 transformed into P. pastoris via electroporation.

A B

Figure 5. Colony immunoblot results. In panel A, the colony epiplate with resulting colonies; in panel B, the immunoblot results where the darker the colony is the more enzymes are being produced. Each immunoblot colony retains its original shape from the epiplate which helps in identifying which produce enzymes and which do not. The shadows are artifacts caused by the sliding of the film during the development of the immunoblot.

3.3 Protein Purification and Quantification

Samples were also taken over time (3 days) in order to assess what the protein production pattern was (see Figure 6); it turned out that after 1 day of induction, there was about 1 mg of proteins already produced. We recommend overnight induction considering the stability of the enzyme over time at room temperature and 37 °C.

Recombinant MlCB2 was isolated from yeast medium by nickel chelate affinity chromatography. Once dialysis was complete, proteins were run on an SDS-PAGE gel to check for purity and developed on a Western Blot to ensure that those bands are truly MlCB2 (see

Figure 7). SDS-PAGE revealed bands around 37 kDa which is the predicted size of the pro- enzyme and a band at 27 kDa which is the predicted size of the mature and active enzyme. All bands were MlCB2 according to the Western Blot which indicates that the enzyme is purified.

Protein concentrations measured were 1 µg/µL. Higher and lower bands were observed on the

Western blot indicating degradation and complexing of the protein with itself; these were too low in amounts to be seen on the Coomassie stain.

L A B A1 A2 A3 B1 B2 B3

Figure 6. Western Blot of MlCB2 (purified and culture media). Columns A and B represent the purified protein production of MlCB2 by colonies 11 and 19 respectively. Following are media from each respective culture and day post- induction (A1 is colony 11 on day 1, B2 is colony 19 on day 2 and so forth). MlCB2 clones require a single day to generate enough pro-enzyme. After 2 days the enzymes activate within the culture (and slightly on day 1). On day 3 the enzyme starts to degrade substantially and also starts to complex with itself.

250 150 100 75

A B C

Figure 7. Coomassie Blue Stain (A), respective Western Blot using anti-hexahistidine tag antibodies (B) and Western Blot using anti-MlCB2 rat antibodies (C). The western blot reveals that the protein degrades down to small fragments containing the His-tag but not in high enough concentrations to be seen on the coomassie 63 stain; the protein also seems to complex with copies of itself when highly concentrated. The anti-MLCB2 antibodies bind to the mature enzyme but very weakly to the zymogen. 3.4 Antibodies Production and Purification

The anti-MlCB2 rat antibodies specifically bound to the mature MlCB2 but not to the zymogen (see Figure 7. C). The coarse sera from the Wistar rats could not bind MlCB2 clearly and had a lot of background signal associated with them. On the other hand, the purified antibodies were very specific to the enzyme. The optimal dilution found was 1:10 000 000 when using 100 ng of MlCB2.

3.5 Kinetics and Substrate Specificities of MlCB2

MlCB2 is to be able to cleave all the presented substrates (Z-L-R-AMC, Z-F-R-AMC, Z-

R-R-AMC and Z-P-R-AMC). MlCB2’s Kcat/KM indicates that it prefers leucine at the P2 position over phenylalanine, followed by arginine and proline (see Table 1). A typical cathepsin B behavior: a high affinity and low processing of arginine at the P2 position was observed

Table 1. Kinetic parameters of MlCB2. KM is the Michaelis-Menten constant, Kcat represents the time

required for 1 unit of enzyme to process 1 unit of substrate and Kcat/KM is the catalytic efficacy of the enzyme.

3.6 pH Profile of MlCB2

The optimal pH of reaction for substrates Z-L-R-AMC is 6.0 and 7.5 in sodium phosphate buffer (see Figure 8). The enzyme activity drops but is still above 80% maximum activity between pH 5.5 and 7.0, reaches 60% activity at pH 4.5 and 5.0 before dropping off down to 40% at pH 4.0 and 8.0. The enzyme can only activate fully at pH 4.5, 5.5 and 7.0.

Figure 8. Activity of MlCB2 at various pH using Z-L-R-AMC as a substrate. The enzyme exhibits a broad range of pH optima ranging from 5.5 in sodium acetate [0.1M] to 7.5 in sodium phosphate [0.1M].

3.7 Activity at Different Temperatures and Salinities of MlCB2

MlCB2 has an optimal activity temperature of 22º C (see Figure 9). The enzyme is still active at about 85% maximum activity when at 4º C and 90% maximum activity when at 37º C.

The lower catalytic activity at 37º C could be explained by how unstable the enzyme appears to be at that temperature over long periods of time.

MlCB2 activity is unaffected by changes in salinity (see Figure 10). MlCB2 can resist up to 50 ppt and the average % activity does seem decrease significantly but meaninglessly in terms of absolute rates.

3.8 Stability at Different Times, Temperatures and Salinities of MlCB2

MlCB2 is not very stable at 37º C and loses 50% of its activity after 4 hours before completely breaking down after 8 hours (see Figure 13). MlCB2 is very stable between 4º C and

42º C but beyond that, it loses its activity and cannot even function past 42º C (see Figure 11).

MlCB2 is impervious to changes in salinity (see Figure 12). Its activity levels remain constant in all ranges of salinities tested: from 0 ppt (control) to 50 ppt (salt water). The assay was also constructed in a way that the main stability solution (enzyme, salt, buffer and DTT) was in inferior volumes (20 µL) than the final volume (200 µL) of recorded activity. In this way, the activity was not affected by the salinity and only the stability was assessed.

3.9 Digestion of Natural Substrates by MlCB2

MlCB2 is able to digest BSA at pH 4.5. BSA was present at time 0 hours as 4 bands between 50 and 75 kDa (which was predicted) and MlCB2 was also present as bands at 37 and

27 kDa (see Figure 14). After 2 hours of digestion, the 2 lowest bands of BSA were absent indicating their digestion and finally, all BSA bands were gone after 8 hours of digestion.

Figure 9. Activity of MlCB2 at various temperatures. MlCB2 retains up to 80% of its maximum activity at 4 °C. The enzyme is most active at room temperature and also at 37 °C.

MlCB2 Activity and Salinity

100

60 % Activity % 40

0 50 30 15 10 5 1 0.5 0 ppt (g of NaCl per kg of H2O) Figure 10. Activity of MlCB2 at various salinities. The enzymatic activity marginally increases at higher salt concentrations. Overall the enzyme functions well from 80 to 100% regardless of the salinity. 67

MlCB2 Stability and Temperature

100

60 % Activity % 40

0 4 22 37 42 50 60 Temperature ( °C)

MlCB2 Stability and Salinity

100

Activity % 40

0 50 30 15 10 5 1 0.5 0

ppt (g of NaCl per kg of H2O)

MlCB2 Stability and Time at 22 °C

100

Activity % 40

0 0 4 8 12 16 20 24 Time (hours)

L 100 MlCB2 BSA A B C D E F G 75

3.10 Inhibition of MlCB2 by CA-074 and E-64

Preliminary testing indicated that CA-074 does selectively inhibit MlCB2 as opposed to non-cathepsins B, such as MlCL1 (see Figure 15). Primary concentrations were too high and reached 100% inhibition even at their lowest (0.250 µM). To measure the Ki, much more diluted concentrations of CA-074 were used. CA-074 is a potent inhibitor of MlCB2.

Activity assays have shown that the enzyme is completely inhibited by E-64 (assays not shown). According to the Dixon plot, the compound E-64 has a Ki of 0.04224 µM at 37° C and pH 5.5 using Z-L-R-AMC as a competitive substrate (Figure not shown). CA-074 is a much more potent inhibitor than E-64 for MlCB2 by a factor of 5.

According to the Dixon plot, the compound CA-074 has a Ki of 0.007445 µM at 37° C and pH 5.5 using Z-L-R-AMC as a competitive substrate (see Figure 16).

MlCL1 and MlCB2 Activity in the Presence of CA-074

100

60 MlCB2

% Activity % MlCL1 40

0 0 5 10 15 20 25 30 Concentration of CA-074 (µM)

Dixon

0.04 0.006

0.005 0.03

0.004

0.02 0.003

1/Rate (RFU/min) 1/Rate 1/Rate (RFU/min) 1/Rate 0.002

0.01 0.001

-0.04-0.08 -0.02 -0.060.00 0.02-0.04 0.04-0.020.06 0.000.08 0.100.02 0.12 0.040.14 [Inhibitor][Inhibitor] (µM)

Vmax Vmax = = 894.8 1380.4 SS = = 40 40 Km Km = = 82.6 89. SS = = 80 80 Ki Ki = = 0.007445 0.0218 SS = = 160 160 E = 0.05434 Figure 16. Dixon plot of MlCB2 in the presence of the competitive inhibitor CA-

074. Experiment was replicated 3 times with similar results pointing for a Ki of 0.007445 µM.

Dixon

0.06

0.05

0.04

0.03

0.02 (RFU/min/mg) 1/Rate

0.01

-0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 [Inhibitor] (µM)

Vmax = 1267.9 S = 40 Km = 108.9 S = 80 Ki = 0.04224 S = 160 E = 0.1388 Figure 17. Dixon plot of MlCB2 in the presence of the competitive inhibitor E-64. Experiment was replicated 3 times with similar results pointing for a Ki of 0.04224 µM.

CHAPTER 4 – Discussion and Conclusion

Discussion

The platyhelminth M. lignano is a plesion of the ancestral crown group that later split into other free-living platyhelminths and parasitic trematodes. Two genes encoding cysteine peptidases of the free-living organism M. lignano were identified, expressed and biochemically characterized in vitro. Consistent with the phylogenetic placing of M. lignano, both peptidase genes are plesion to the parasitic trematode crown groups. However, both genes are much more closely related to the ancestral gene that was later passed onto the parasites.

Based on its high sequence similarities to the cathepsin L from other species, the first cathepsin was termed MlCL1, where “1” represents the fact that this was the first to be discovered in this organism. MlCL1 is a plesion of SmCL3 and SjCL. Although Neobenedia has a more primitive and closer related cathepsin L (termed CL1 gene) to SmCL3 and SjCL no studies beyond the gene structure has been performed. Interestingly, we found that Fasciola has no homologous cathepsin peptidase gene to that found in M. lignano. The phylogenetic pattern regarding Fasciola cathepsin L seems to be a single gene inheritance, gene multiplication and diversification. In contrast, Schistosoma appears to have inherited three separate cathepsin L genes. Because of its phylogenetic position, MlCL1 is a good model gene for its parasitic counterparts. Phenotypically, the same function should remain in both parasitic and free-living organism and should they be knocked down or knocked out, an identical if not similar phenotype should be observed.

The MlCL1 protein structure has only one different feature that sets it apart from the other cathepsins L; that is, it has an unusually long pro-peptide sequence (at least 30 amino acids longer). Because of this particular feature, it resides in the same clade as SmCL3 and SjCL.

SmCL3 (discovered by Dvorak et al. in 2009) also has a pro-peptide region that is unusually

73 long with approximately 30 additional amino acids sequence which are predicted to form an alpha helix rather than a coil (Maestro , Schrödinger, LLC, New York, NY). The Schistosoma japonicum Cathepsin L (an ortholog of SmCL3) also has a pro-peptide region with an unusual extension of about 30 amino acids. MlCL1 contains a conserved ERFNIN and GNFD motif like

SjCL and SmCL3. We recommend that MlCL1 would instead be renamed MlCL3 to avoid confusion between genetic identities. By extension, we also suggest that SjCL be re-termed

SjCL3 (SmCL3-like) to be consistent with the phylogenetic analysis.

MlCL1’s ORF suggests that it is a member of the C1 family of Clan A cysteine peptidases; the presence of an ERFNIN and GFND motif and the typical catalytic triad support such conclusion. The recombinant MlCL1 was produced as a precursor and successfully activated in the presence of DTT indicating that the enzyme can activate itself though a cis mechanism. The predicted protein size for the mature enzyme on SDS-PAGE was 25 kDa. Bands were observed at those positions on both SDS-PAGE and Western Blot.

MlCL1 was inhibited by the general Clan CA peptidase inhibitor E-64 much like SmCL3

-1 -1 was decribed to be (Ki observed when using E-64 was 26.5 nM s ) (Dvorak et al, 2009). E-64 exhibits a Ki of 12.47 nM in the case of MlCL1 which means it is twice as sensitive as SmCL3 to the inhibitor. CAA0225, a derivative of E-64 also inhibited MlCL1 but was 375 times more potent than E-64 and specific for the cathepsin L. CA-074, a cathepsin B-specific inhibitor, did not inhibit MlCL1.

Kinetics studies revealed that MlCL1 is able to bind leucine and phenylalanine at the P2 position and to successfully cleave substrates such as Z-F/L-R-AMC. On the other hand,

MlCL1 cannot bind arginine very well or cleave it adequately. It also cannot bind proline and even less so cleave it. The order of preference of MlCL1 to the substrates was F>L. These

74 observations are consistent with the parasitic SmCL3 which also preferred hydrophobic residues at the P2 site and in order, tryptophan, tyrosine, leucine, phenylalanine and valine. The kinetics

- of SmCL3 when using Z-Phe-Arg-AMC consisted of a KM = 20.2 µM and a Kcat/KM = 410 mM

1 -1 s . The KM for MlCL1 in the presence of Z-Phe-Arg-AMC = 12.6 ± 3.4 µM and the Kcat/KM =

8.15964 mM-1s-1. By comparison MlCL1 has a two-fold greater affinity for the substrate but is

50 times less efficient per mM. In this regard, MlCL1 is much more active and potent than

SmCL3 when it comes to binding, processing and releasing its substrate. SmCL3 resembles human cathepsin V (an L-like cathepsin) in its S2 specificity while also having the capability of accepting aspartate at the P2 and P3 positions, much like human cathepsin F. Although not tested in this study, it would be an interesting experiment to carry out in the future to better understand the evolutionary relationship between cathepsins L, F and V and how their specificities changed over time.

The pH optimum of MlCL1 is very broad; a bell-shaped curve was observed between pH

5.0 and 6.5 in sodium acetate, citrate and phosphate [0.1 M] for Z-F-R-AMC. For Z-L-R-AMC, the optimal pH was 5.0 also in sodium acetate [0.1 M] (data not shown). SmCL3 however is active over a broader pH range, between pH 5.0 and 8.5 with a pH optimum of 6.5; the enzyme was also stable between pH 4.0 and 6.0. Surprisingly the pH optima of the parasite’s cathepsins were broader than that of the free-living flatworm.

Activity of MlCL1 is not affected by the salinity of its environment and could perform even in 50 ppt which is not the typical environment encountered by the flatworm. The enzyme could also function at about 60% capacity at 4° C while also being able to work at much higher temperatures such as 50° C. The broad range of temperature, activity and pH of the enzyme shows how much of a potent versatility it has and how it could be used biotechnologically at

75 various conditions most other enzymes could not work at. Unfortunately, no studies on activity/stability and salinity have been performed in parasites.

MlCL1 is very stable. It can resist high temperatures such as 42° C, high salinities such as 50 ppt and has a half-life of 24 hours. In vitro studies show that MlCL1 cannot process bovine serum albumin (BSA) (data not shown).

Antibodies produced against the cathepsin are specific to it alone and no other cathepsin

(FhCL1 and SmCB1). They showed the presence of a band at the expected size of a mature enzyme, 25 kDa, where native MlCL1 should be and this was repeatable but only the purified antibodies were specific enough to detect it. This is strong evidence that the gene is expressed and processed to an active enzyme.

Based on the phylogenetic, biochemical and kinetic results, MlCL1 can be expected to be localized in the same compartment as SmCL3. Quantitative PCR indicates that SmCL3 is predominantly expressed in the gut of the parasite during the mammalian infective stages although it is much less abundant relative to other gut-associated proteases in S. mansoni adults by 50 to 1000 fold and is 100 fold less than SmCB2, a tegument-associated protein

Immunolocalization confirmed qPCR data and showed that the enzyme is localized in the gastrodermis of both adult sexes and also within the vitellaria of the female adults. The studies still remain to be done to confirm similar localization.

The second cathepsin discovered was termed MlCB2, based on its high sequence similarity and phylogenetic position relative to other cathepsin B proteases of flatworms, particularly SmCB2, TsCB2 and TrCB2. The MlCB2 gene structure, mlcb2, is different from the

SmCB2 gene (the TrCB2 genomic sequence has not yet been revealed): mlcb2 has two introns

76 and three exons while smcb2 has three introns and four exons with introns being located in different locations (Caffrey et al. 2001).

The protein’s ORF suggests that this cathepsin is a member of the C1 family of Clan A cysteine peptidases; this included the typical catalytic triad (Cys25, His175 and Asn185) and the presence of an occluding loop (from D258 to N288) that is unique to this family. The presence of the occluding loop motif indicates the possibility of the enzyme to possess a peptidyl-dipeptidase

(or exopeptidase) activity (Musil et al., 1991). This is supported by the presence of the conserved

H110 and H111 catalytic diad which are involved in the exopeptidase activity of cathepsins B in general.

The M. lignano cathepsin B2 (MlCB2) is a plesion of SmCB2, TrCB2 and TsCB2.

SmCB2 is found in the parenchyma of the adult worm (Caffrey et al. 2001) while TrCB2 was localized in the post-acetubular glands of the cercaria larval stage (Dolečková et al. 2009). It is quite unusual that a homologous gene would be found in two different tissues and life cycle stages of two very closely related organisms but further and more detailed studies on temporal expression and tissue localisation is needed on these peptidases to help clarify their complete functions.

The recombinant MlCB2 was produced by P. pastoris as a zymogen precursor containing the inhibitory pro-domain, and was successfully activated to the mature form in the presence of

DTT. The enzyme was also shown to be able to activate itself in the absence of DTT and presence of E-64 suggesting that the enzyme can activate itself through a cis mechanism

(autocatalytic activation). For future reference, an overnight induction is sufficient to produce about 1 mg of enzyme.

The predicted sizes of the pro-enzyme and mature enzyme were 37 kDa and 28 kDa, respectively. Bands at those positions were observed on both SDS-PAGE and Western Blot.

Western Blot revealed bands at lower positions such as 20, 15 and 10 and were assumed to be products of degradation from the N-terminal end as they retained the hexahistidine tag.

Kinetics studies revealed that the enzyme is able to bind leucine, phenylalanine, arginine and proline at the P2 position and to successfully cleave substrates such as Z-L/F/R/P-R-AMC.

The order of the P2 preference of MlCB2 using these substrates were L>F>R>P. A typical cathepsin B high affinity and low turnover rate for arginine was noted. The enzyme activity using various substrates with variations at the P2 position indicated that SmCB2 preferred Z-

Phe-Arg-AMC three times more than Z-Val-Arg-AMC or Z-Leu-Arg-AMC while preferring Z-

Arg-Arg-AMC the least.

The pH optima of MlCB2 activation (transition between pro-enzyme and mature enzyme form) are 4.5, 5.5 and 7.0. The pH optima of MlCB2 activity (cleavage of substrate) is a broad range between pH 5.5 and 7.0. SmCB2 can hydrolyze Z-Phe-Arg-AMC and Z-Arg-Arg-AMC at pH optima 5.0 and 5.5, respectively; sharp activity peaks with 80% maximum activity boundaries at pHs 4.5 and 5.5 for Z-Phe-Arg-AMC and 5.0 to 5.5 for Z-Arg-Arg-AMC were observed and no substrate hydrolysis was detected at pHs above 6.5. The enzyme kinetics

-1 -1 revealed that native SmCB2 had a Kcat/KM of 625 mM s while the recombinant enzyme had a

-1 -1 Kcatt/KM of 1111 mM s when testing the Z-Phe-Arg-AMC substrate. For Z-Arg-Arg-AMC,

-1 -1 the Kcat/KM measured were 9 and 14 mM s for the native and recombinant enzyme respectively. No pH optima or kinetics were reported using other substrates. MlCB2 is thus more active than SmCB2 while maintaining similar substrate preferences and pH ranges. TrCB2 prefers lysine at the P1 position followed by arginine, whereas the enzyme prefers methionine,

78 valine, serine and glutamine (>50% maximum activity) at the P2 position and does readily accept arginine, phenylalanine or proline (less than 10% maximum activity).TrCB2, despite its phylogenetic relationship to MlCB2, does not share similar kinetic or biochemical properties.

Activity of MlCB2 correlates with temperature and salinity. MlCB2 is able to operate at

80% maximum activity at low temperatures such as 4° C which would be expected since the organism lives in brackish waters and the average temperature of sea water is ranged from -4° C to 4° C. What was most surprising is that the enzyme also functions well at higher temperatures such as 22° C, 37° C and 42° C. However, the enzyme is sensitive to much higher temperatures and it was observed that, given the higher activity at 37° C, the degradation rate was consequently much higher. At 37° C, the enzymatic activity is completely lost after 8 hours. On the other hand, when the enzyme was produced and purified at 4° C over a period of 4 days, it remained 100% stable its higher stability at low temperatures and higher volatility at higher temperatures. MlCB2 is only slightly affected by increasing salinity. In fact it shows resilience to salinity increase past 0.5 ppt and up to 50 ppt. Once stability of the enzyme to various salt concentrations was tested, it clearly showed that the enzyme itself is impervious to increases in salinity and the activity levels measured were constant.

MlCB2 does not resist time or temperature very well compared to other enzymes. When compared to another cathepsin from M. lignano (MlCL1; see other section), this enzyme degrades at lower temperatures and much faster. It can be hypothesized that unlike MlCL1,

MlCB2 has a higher turnover rate or does not need to be active for long periods of time.

In vitro studies showed that MlCB2 can cleave bovine serum albumin (BSA) after 8 hours of incubation. The smaller BSA fragments (50 kDa) were digested with relative ease and quickly (2 hours) but the larger (75 kDa) fragments were only digested after 8 hours.

MlCB2 is inhibited by both CA-074 (Cathepsin B specific) and E-64 (general cysteine protease inhibitor). When the zymogen was exposed to E-64 it could not activate to its mature form; this was visible on both SDS-PAGE and during enzyme activity assays. CA-074 proved to be twice as potent as E-64 when competitively inhibiting MlCB2 at 37 °C and pH 5.5 under reducing conditions (2 mM DTT). Ca-074 and E-64 are both potent inhibitors of TrCB2

(Dolečková et al. 2009).

SmCB2 was found in the parenchyma of both adult sexes, but in higher quantities in the male than the female parasites specifically in the parenchyma and the dorsal and lateral tubercules of the tegument (Caffrey et al. 2001). TrCB2 was localized in the post-acetubular glands of the parasite cercariae while no signal was observed in any other part of the cercaria’s body (Dolečková et al. 2009). Confocal microscopy was not performed on any other stage of the parasite’s life cycle and there is no knowledge as to whether or not it is present in the adult stage

(Dolečková et al. 2009). Using quantitative PCR, Dolečková et al. (2010) also revealed that

TrCB2 was expressed in all life-cycle stages of the parasite with the lowest expression being at the cercarial stage. Dr. Martin Kašný and his collaborators are currently investigating the cathespin B of the related parasite Trichobilharzia szidati (TsCB2) although results are pending

(cited in Kašný et al., 2011). The postulated functions of SmCB2 are biogenesis and tegumental turnover and/or some unknown role at host-parasite interface. It was suggested by Dolečková et al. (2010) that the cercariae of S. mansoni and S. japonicum produce cathepsins B2 but no experimental immunolocalization showing this was reported. Further investigation using confocal microscopy in M. lignano is necessary to confirm the localization of MlCB2 and to better infer on its putative function which can then be used to model either that of TrCB2/TsCB2 or SmCB2.

When comparing MlCB2 to the TrCB2 and the SmCB2, it is clear that SmCB2 retained more properties from the older MlCB2 than TrCB2 did. The MlCB2 S2 binding site accepts phenylalanine, leucine and arginine similarly to SmCB2. The active site of MlCB2, however, has not yet been investigated using methionine, valine, serine or glutamine as substrates. The pH optima of MlCB2 begins to peak at pH 5.0 but is much broader than SmCB2 and can hydrolyze substrates at a pH greater than 6.5. Unlike TrCB2, MlCB2 can hydrolyze BSA.

While two cysteine peptidase genes were discovered, produced and characterized in this study, four other cathepsin L fragments and two other cathepsin B fragments were found in the

EST database. Through PCR, those fragments could be sequenced, extracted and studied. Once the EST and genomic databases are completely assembled more cathepsins could be discovered, their phylogenetic positions could be determined and their biochemistry and functions explored

Although it is interesting that Fasciola has no published cathepsins homologous to the ones we discovered, we have evidence that the Fasciola hepatica and Fasciola gigantica cathepsin B9 (personal communication, Anand Chakroborty, Queens University Belfast, Ireland) is a homologue that has not yet been characterized, localized and with no known function that can be inferred upon using M. lignano as a model organism.

Conclusion

Future research should involve knockdown of the MlCB2 using RNAi to better understand the function of the cathepsin B in M. lignano. RNAi studies can also be carried side

81 by side with CA-074 or CAA-0225 which can selectively inhibit all cathepsins B and L, respectively, at low doses. This will allow us to understand further what the role of these enzymes are and through phylogenetic and bioinformatics approaches, give us a better understanding of SmCB2, TrCB2 and TsCB2, whose functions remain unknown and assumed.

RNAi and confocal microscopy together can also be used to better understand under what conditions MlCB2 and MlCL1 are expressed or repressed and the resulting information can provide inference on how their actual functions and regulation in their parasitic counterparts.

In support of our original hypothesis; Macrostomum lignano is a very attractive candidate to be a model organism for parasitic trematodes. It is very easily worked with in the laboratory setting, has homologous cysteine proteases found in parasitic trematodes and also has a lot of untapped potential for future research.

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