The Discovery, Isolation and Characterisation of Cyclic Trypsin Inhibitors from Momordica cochinchinensis and other Cyclotides

Philip Nguyencong (2010). The Discovery, Isolation and Characterisation of Cyclic Trypsin Inhibitors from Momordica cochinchinensis and other Cyclotides PhD Thesis, The Institute for Molecular Bioscience, The University of Queensland.

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Author Philip Nguyencong
Thesis Title The Discovery, Isolation and Characterisation of Cyclic Trypsin Inhibitors from Momordica cochinchinensis and other Cyclotides
School, Centre or Institute The Institute for Molecular Bioscience
Institution The University of Queensland
Publication date 2010-11
Thesis type PhD Thesis
Supervisor Professor David J. Craik
Total pages 204
Total colour pages 27
Total black and white pages 177
Subjects 06 Biological Sciences
Abstract/Summary This thesis describes studies on peptides, known as cyclotides, from plants. These fascinating proteins are the only known molecules that have a head-to-tail cyclic backbone and a cystine knot structural motif. These features make them very stable, and because cyclotides have a range of interesting biological activities they have attracted attention as leads in peptide-based drug design. Chapter 1 provides an overview of the background to this thesis and defines the scope of the work. Chapter 2 describes the methods used and the remaining chapters describe the experimental studies (Chapter 3–6), and summarise the findings (Chapter 7). Cyclic trypsin inhibitors (CTIs), also known as cyclic knottins, are the newest sub-class of cyclotides, and are unique in that these cyclotides are the only class with well-established linear homologues known as the squash trypsin inhibitors. This situation allows the opportunity to compare the differences between cyclic and linear homologues on the DNA level to reveal further insights into the processing mechanism of cyclotides. The construction of a genomic DNA library for Momordica cochinchinensis was attempted but due to difficulties, described in Chapter 3 of this thesis, construction was unsuccessful. However, a partial DNA sequence to the MCoTI-II peptide was determined via a combination of molecular biology procedures. Although the full precursor sequence remains elusive, this study revealed that the CTI precursor resembles the precursor sequences reported for cyclotides in the Möbius and bracelet sub-classes. This finding is supported by the fact that linker regions in the CTI precursor contain highly conserved elements that exist in cyclotide precursors, including a Leu/Ile residue two positions upstream and downstream of the mature peptide domain. In addition to this, the MCoTI-II peptide sequence is also arranged within the mature peptide domain with greater similarity to cyclotide clones rather than to squash inhibitor clones. This finding also suggests that the processing and cyclisation of CTIs occurs in a similar fashion to the other cyclotide sub-classes. The CTI sub-class of cyclotides currently only consists of two peptides, MCoTI-I and MCoTI-II, with the second peptide being more abundantly expressed. These two peptides are only be found in the seeds of M. cochinchinensis much like their linear counterparts, the squash trypsin inhibitors, which are expressed in the seeds of various Cucurbitaceae plants. M. cochinchinensis also contains linear squash trypsin inhibitors and it is a wonder why new CTIs have not yet been discovered in other Cucurbitaceae plants that express squash trypsin inhibitors as well. A study described in Chapter 4 of this thesis describes the field trip I undertook to collect various Cucurbitaceae samples and the chemical extraction procedure I used to isolate and identify possible CTIs. Results from this study, however, suggest that MCoTI-I and MCoTI-II are still the only known CTIs. New CTIs can also be discovered on the nucleic acid level, although the full precursor sequences is required so that a unique probe or primer site can be determined to optimise CTI targeting. Cyclotides from the bracelet and Möbius sub-classes have had several precursor sequences elucidated and a unique primer binding site specific for cyclotide precursors has also been previously determined by a colleague. Chapter 5 describes the use of this cyclotide specific primer and several others to isolate cyclotide clones from Viola hederacea, and in particular the clone to the vhl-2 cyclotide. This clone was found to contain high sequence identity to a previously discovered cyclotide clone, Vok1, which was isolated from the plant Viola odorata, with up to 96.6% identity in the Pro-domain of the precursors. These two plants, V. hederacea and V. odorata are geographically distant, which rules out the homology of these two genes being attributed to a recent gene transfer event. The results, however, can be described by a gene duplication event that occurred in an ancient ancestor of the two species. The haemolytic ability of the peptide vhl-2 was also analysed in this study and it was found to induce the haemolysis of red blood cells at a level similar to the cyclotide kalata B2. Sequence identity between the two peptides is 73% and so it is suggested that a structural comparison between the peptides will reveal more information about the haemolytic ability of cyclotides. Cyclotide clones have revealed a multitude of information regarding the ancestry of the peptides and the processing and cyclisation events required to form mature cyclic cyclotides. So when a colleague isolated the first linear cyclotide, Violacin A, I performed a study, as described in Chapter 6 of this thesis, to determine the precursor to this peptide, the Vov-1 clone. The results of this study revealed that Vov-1 contains a premature stop codon, a result of a single nucleotide polymorphism, which prevented the expression of important processing residues that would have enabled the cyclisation of the peptide into a mature cyclotide. Overall, these studies aimed to shed more light into the origins and the diversity of cyclotides, with special focus on the CTI sub-class of cyclotides. The studies determined that the CTIs are processed in a similar fashion to other cyclotides. New CTIs remain elusive and improvements to the discovery process may eventually find more peptides in this sub-class. The discovery of cyclotide clones is an alternative way to determine new cyclotide sequences and analysis of these clones has shed more light into cyclotide ancestry. Unique cyclotide-like clones such as the Vov-1 clone for Violacin A, has also given insight into the processing and cyclisation mechanism for mature cyclotides. Overall, the studies that were performed in my research have added to the current knowledge of cyclotides and they have also opened up questions and avenues of research to further enrich this knowledgebase.
Keyword Cyclotides
Circular Proteins
Molecular Biology
Cyclic Trypsin Inhibitors
Momordica cochinchinensis
Additional Notes 21, 25, 27, 31, 37, 40, 47, 68, 73, 77, 80, 107, 110, 125, 126, 129, 131, 132, 137, 143, 145, 150, 155, 184, 196, 197, 199

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Created: Mon, 27 Jun 2011, 16:42:52 EST by Mr Philip Nguyencong on behalf of Library - Information Access Service