Chemical and Biological Studies of Cyclotides

(2006). Chemical and Biological Studies of Cyclotides PhD Thesis, Institute for Molecular Bioscience, University of Queensland.

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Thesis Title Chemical and Biological Studies of Cyclotides
School, Centre or Institute Institute for Molecular Bioscience
Institution University of Queensland
Publication date 2006
Thesis type PhD Thesis
Abstract/Summary The cyclotides are an interesting family of naturally occurring macrocyclic peptides that are highly stable and possess a diversity of biological activities, including uterotonic, haemolytic, anti-HIV, neurotensin antagonist, anti-microbial, trypsin inhibitory, anti-tumour/cytotoxic and insecticidal activity. Their chemical stability, as demonstrated by their resistance to proteolytic degradation and capacity to withstand extreme temperature and pH conditions, is attributed to their unique protein structural motif termed the cyclic cystine knot (CCK), which is composed of a cyclised peptide backbone and a knotted disulfide topology, where two disulfide bridges and their connecting backbone segments form an embedded ring that is threaded by a third disulfide bond. In this project, the cyclotides were investigated for their potential use as naturally occurring crop protection agents against a number of economically important agricultural pests. However, the study was broader than this and included the discovery of novel cyclotides, bioassays and NMR studies. Chapters 1 and 2 provide an introduction to the topics and describe the materials and methods, respectively. Chapter 3 describes the complete suite of cyclotides present in the African plant Oldenlandia affinis, characterised by LC-MS, MS-MS, total amino acid analyses and UV-visible scans. This study expanded the total number of known cyclotides in this plant to 17, of which nine new sequences were characterised. In addition, five cyclotide derivatives containing oxidation products of tryptophan were identified, including oxindolylalanine, n-formylkynurenine, and kynurenine derivatives. Furthermore, two linear derivatives of cyclotides were described. These acyclic derivatives have three intact disulfide bonds, and their N- and C- termini coincide with the hypothesised cleavage site from the precursor protein. This work has increased knowledge about sequence variation accommodated by the CCK scaffold, confirms its applicability as a template for drug design, and also shows the first natural degradation pathways for the cyclotides. These degradation pathways have important implications on the half-life of the cyclotides, and hence their persistence and environmental fate if used in agriculture. The The molluscicidal activity of members of the cyclotide family is reported in Chapter 4. Pure and crude cyclotide extracts from O. affinis and the ornamental groundcover Viola odorata were found to have molluscicidal activities against the most important pest in wetland rice production, the golden apple snail (GAS), Pomacea canliculata. Crude cyclotide mixtures from V. odorata and O. affinis both have molluscicidal activities, the former being more potent than the latter. The 24 h LC[subscript]50 for kalata B1, B2 and metaldehyde, a known molluscicide, were determined at 90, 54 and 105 µM, respectively. At 65 µM, pure cyclotides from V. odorata, namely cycloviolacin O1 and kalata S, both caused 100% GAS mortality while kalata B1, B2 and metaldehyde caused 78, 68 and 60% mortality, respectively. Kalata B7 was weakly active, while kalata B8 did not have molluscicidal activity. The possible downstream effects of the cyclotides on non-target freshwater (Nile tilapia) and saltwater (brine shrimp) marine organisms were investigated. The studies showed that kalata B1was only mildly toxic to brine shrimp compared to podophyllotoxin, a known cytotoxic agent. Similarly, kalata B2 was at least three times less toxic to Nile tilapia than rotenone, a known piscicidal agent. Autolytic degradation sites in present cyclotides have been identified in exposed inter-cysteine loops, particularly among the Trp and Gly residues. Knowledge of these autolytic degradation sites gives insights into how the half-life of these novel molluscicidal agents can be manipulated to ensure a balance between efficacy on pests and proper degradation in the environment. In Chapter 5, members of the cyclotide family were tested on three nematode species, Caenorhabditis elegans, Meloidogyne javanica and Radopholus similis, to investigate their potential use as nematicidal agents. The results show that the cyclotides have nematicidal activities comparable to, if not better than, the known nematicidal agent fenamiphos. Although active at hundreds of micromolar concentrations, the mortality rate increased with increasing time of treatment. It was also found that C. elegans, M. javanica and R. similis have different degrees of susceptibility to the peptides. Different cyclotides have varying haemolytic activities, but compared to melittin the cyclotides are only mildly haemolytic. The pesticidal mode of action of the cyclotides is yet to be fully elucidated but appears to involve more than just their capacity to disrupt cell membranes, their hydrophobicity, or their CCK topology. The varying pesticidal potencies of different cyclotides show that their activities are based on their sequence variations. Kalata B2 was not phytotoxic to tomatoes, suggesting that cyclotides can potentially be used as a soil drench or in seed treatment for nematode control without adverse effects on crops. Alternatively, transgenic plants can be created to specifically express cyclotides in their roots to protect them from nematode infestation. In Chapter 6, cyclotides with insecticidal activities were identified and their possible mode of action was elucidated. It was confirmed that fresh O. affinis leaves contain insecticidal agents. Cotton bollworm (Helicoverpa armigera) larvae fed with young fully-expanded leaves did not grow beyond first instar and died three days after treatment. In a search for putative insecticidal peptides in O. affinis, kalata B2 was identified as the major leaf-specific peptide that was expressed in both winter and summer. Kalata B5 was identified as a differentially-expressed peptide in the aerial parts of the plant during winter, when the plant is at its reproductive phase, possibly to protect its flowers and pods from insect attack. Therefore, the insecticidal properties of kalata B2 and B5 were investigated and compared against those of kalata B1. Assays on H. armigera larvae fed on an artificial soya bean diet showed that kalata B5 is a potent insecticidal peptide, killing 100% of the treated population after four days of treatment, while kalata B2 had a similar activity as kalata B1, with 27.5% and 20.0% mortality, respectively. Nevertheless, kalata B1 and B2 inhibited the growth and development of the surviving larvae by 75.4 and 70.8%, respectively. Results from a protein blot assay of the insect frass showed that the larvae did ingest the peptides from the artificial diet and that the peptides were not degraded even after passing through the insect gut. The possible modes of action of kalata B1, as demonstrated in the human body lice (Pedicularis humanus humanus) assays, was mainly systemic- and to some extent contact-, but not repellent-insecticidal. In Chapter 7, the three-dimensional structure of kalata B5 was determined by NMR spectroscopy and was compared to the structures of kalata B1 and B2 in an endeavour to elucidate the observed differences in insecticidal activity reported in Chapter 6. Kalata B5 was also compared to the prototypic bracelet cyclotide, cycloviolacin O1. Detailed structural studies were conducted on all four peptides by subjecting them to various pH and temperature conditions. Kalata B5 adopts a cyclic cystine knot motif. Its solution structure can be described as a well-defined bracelet structure consisting of 30 amino acids with a cyclised peptide backbone, a cystine knot core, a region of β-sheet, a 3[subscript]10 helix, and a number of β-turns. Although kalata B1 and B2 adopt a similar global fold, they lack the 3[subscript]10 helix in loop 3 that is present in kalata B5. Nevertheless, for all four peptides, it was found that residues in loop 3 interacted with the conserved Glu3 residue in loop 1 to stabilise the structure of the peptide during the deprotonation. While the amino acid composition of kalata B5 is significantly different to that of B1 and B2, many of the intra-molecular hydrogen-bonds identified were situated in similar places within the inter-cysteine loops. However, a number of residues in loops 2, 3 and 5 are not hydrogen bonded, and could potentially form hydrogen-bond interactions with a putative receptor. The surface representations of the three peptides highlighted these obvious differences and found that loops 2 and 3 in kalata B5 formed a region of hydrophobic patch, while in kalata B1 and B2 this region is predominantly hydrophilic. In addition, loop 5 in kalata B5 consists of charged residues, while this region is substituted by hydrophobic residues in kalata B1 and B2. Overall, this project has confirmed the potential use of the cyclotides as naturally occurring pesticidal agents against a number of economically important agricultural pests. The project elucidated the gross modes of action, the downstream effects on non-target aquatic organisms, the degradation pathways and the phytotoxic effects of the cyclotides. It also contributed in the discovery of novel cyclotides, including some modified and linear derivatives. Finally, this project resulted in the structure elucidation of kalata B5 and identified the important roles of particular residues in the activity and structural stability of the cyclotides.

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