The mode of action of cyclotides: Functional studies of the prototypic cyclotide kalata B1

Yen-Hua Huang (2010). The mode of action of cyclotides: Functional studies of the prototypic cyclotide kalata B1 PhD Thesis, Institute for Molecular Bioscience, The University of Queensland.

       
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Author Yen-Hua Huang
Thesis Title The mode of action of cyclotides: Functional studies of the prototypic cyclotide kalata B1
School, Centre or Institute Institute for Molecular Bioscience
Institution The University of Queensland
Publication date 2010-05
Thesis type PhD Thesis
Supervisor Professor David Craik
Dr Michelle Colgrave
Dr Andrew Kotze
Total pages 178
Total colour pages 130
Total black and white pages 48
Subjects 06 Biological Sciences
Abstract/Summary Cyclotides are topologically unique plant-derived cyclic peptides present in plants from Rubiaceae (coffee), Violaceae (violet) and Cucurbitaceae families. Their primary function in plants is proposed to be defense-related, based on their potent insecticidal activity. Members of this family of peptides incorporate the exceptional features of a head-to-tail cyclic backbone and a cystine knot motif, the combination of which imparts extraordinary stability to these molecules, and thus makes the cyclotide framework valuable for drug design applications. Kalata B1, the prototypic cyclotide which has a range of biologically important activities, including insecticidal and nematocidal activities, might potentially be a solution to the prevalence of resistance to current pesticides. To develop cyclotides as novel agricultural agents, it is necessary to understand the structural and biological basis for their reported anthelmintic activities. The aim of this thesis is to investigate the mode of action of kalata B1, which is believed to involve membrane interactions, through biophysical, chemical and biological approaches, to delineate the fundamental mechanism responsible for its nematocidal activity. The membrane-lytic properties of kalata B1 were examined using a range of biophysical techniques and reported in Chapters 3 of this thesis. Kalata B1 was shown to cause leakage of vesicle contents from large unilamellar vesicles with a preference for model membranes containing phosphoethanolamine, especially those incorporating sphingomyelin and cholesterol. Using the same dye leakage experiments, the lytic activity of a suite of alanine mutants of kalata B1 showed that substitution of residues in the well-defined hydrophobic patch of kalata B1 decreased its lytic activity dramatically. In addition, several residues responsible for the lytic activity of kalata B1 were found to cluster separately to form a bioactive patch, which was believed to be involved in a self-association step before pore-formation. Electrophysiological experiments provided unequivocal evidence of kalata B1-induced pore formation in liposome patches, and the conductive pores formed were observed to expand over time. The pore-forming propensity of kalata B1 was further confirmed using giant unilamellar vesicles (GUVs) encapsulating different molecular sizes of fluorescently-tagged dextran and is reported in Chapter 4. A representative group of three other cyclotides, kalata B2, B7 and B8, was also examined and compared with kalata B1. Kalata B2 and B7 appeared to cause dye leakage from GUVs by inducing membrane permeation similar to kalata B1, but an alternative lytic mechanism was observed from kalata B8 since total membrane disruption was observed in the presence of this peptide. The results suggested that the mode of action of cyclotides differs depending on sequence, having significant effects with surface hydrophobicity and distribution of surface charge. In Chapter 5, the effect of incorporating positively charged residues in kalata B1 on nematocidal and haemolytic activity is reported. A lysine scanning mutagenesis study of kalata B1 was conducted, where each of the non-cysteine residues were successively replaced with lysine. The suite of peptides was assayed against the larvae of two sheep nematodes, Haemonchus contortus and Trichostrongylus colubriformis. The data clearly highlighted residues that were crucial for anthelmintic activity in kalata B1. Disruption of either the hydrophobic or bioactive patches by incorporation of lysine in these regions abolished nematocidal and haemolytic activities of kalata B1, confirming that the continuity of these two patches are essential for its biological activity. Furthermore, it was demonstrated that the nematocidal activity of kalata B1 could be increased by incorporation of basic amino acids in strategic locations. The data indicated the presence of a third important face on the opposite side to the bioactive patch, since lysine substitution in this face resulted in dramatic improvement in anthelmintic activity. This face was termed the “amendable face” in this thesis. The incorporation of a lysine residue into kalata B1 enabled labelling of this peptide with biotin to help localise the molecule in the adult H. contortus nematodes following ingestion. Chapter 6 reports that a biotinylated lysine mutant of kalata B1 interacted with the lipid-rich epicuticle layer of adult H. contortus and the interaction may contribute to the death of the nematodes. The combined findings in this chapter suggest that cyclotides do not need to be ingested by the worms to exert their toxic effects, but an interaction with the external surface alone is toxic. In summary, the overall findings from this thesis show that kalata B1 is a pore-forming cyclotide, and its nematocidal and haemolytic activities can be attributed to its membrane-active properties. The observed lytic mechanisms of kalata B1 and its lysine mutants reveal that the distribution of basic residues and continuity of the hydrophobic patch on the surface of cyclotides are important modulators of their reported activities. The results from this thesis provide a mechanistic explanation for the diversity of biological functions ascribed to this fascinating family of peptides. The findings should facilitate future investigations of other cyclotide members and the development of the cyclotide framework in agricultural and pharmaceutical applications.
Keyword Kalata B1
Cyclotides
Circular Proteins
Peptides
sheep nematodes
cyclotide-membrane interactions
Additional Notes Page numbers that should be printed in colour: 23-49, 51-76, 79-83, 85-93, 95-125, 127-131, 133-141, 143-146, 148-150, 155-164, and 167

 
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