Development of Resources and Procedures for The Determination of The Saponin Secretome of The Crown-Of-Thorns Starfish

Stuart J H Smith

doi:10.25907/00969

Saponins are a diverse class of secondary metabolites that have been implicated in a variety of pharmacological applications, which has driven research in the field. While research into the elucidation and application of plant- and class Holothuroidea (i.e., sea cucumbers) -derived saponins is extensive, saponins in the class Asteroidea (i.e., seastars) have been largely overlooked and primarily limited to elucidation. The continual advancements in liquid chromatography techniques and mass spectrometry technologies have allowed for greater metabolite detection rates, including improved isolation and elucidation of saponins. These factors have significantly contributed to the expansion of known saponin structures isolated from marine invertebrates, however, prior to this Thesis research, no centralised reference had been available for saponins specific to marine invertebrates; rather information was scattered across multiple metabolic databases and hundreds of publications. Utilising available literature on marine animal derived saponins, in Chapter 2, a database was constructed containing 985 saponin related structures with the full categorisation of structure, elucidation source and chemical characteristics. The Marine Animal Saponin Database (MASD) was designed to allow for rapid identification of saponins from mass spectroscopy (MS) data. By leveraging the information gained through the curation of the MASD, a review was prepared (Chapter 3) that summarises currently recognised bioactivities for asteroid-derived saponins, supported by quantitative in vitro assays. While there have been some insights into the effects of asteroid-derived saponins as anti-microbial agents, the majority of research has been in relation to their haemolytic and anti-cancer properties. Yet expansion of research is required to determine their selectivity to non-tumourigenic cell types. Endemic to the tropical coral reefs of the Indo-pacific region, the coral-eating Crown-of-Thorns Starfish (CoTS; Acanthaster cf. solaris) inhabits an environment rich in benthic-hunting predators, necessitating the development of potent anti-predation defences to survive. Besides the array of enlarged sharp spines that provide physical deterrence, there exist an arsenal of toxic proteins and secondary metabolites secreted that can deter potential predators. The major secondary metabolites recognised as defensive molecules are saponins, due to their ability to embed into the lipid-bilayer of cells. In the past 60 years, CoTS population numbers have increased to pest status on various coral reefs worldwide, including the Great Barrier Reef (Australia). A deeper understanding of their saponin profile, both endogenous and exogenous (secreted) may assist with the development of a biocontrol. Equipped with the MASD, an isolation optimisation of asteroid-derived saponins was performed using CoTS body wall tissue (Chapter 4). An evaluation of liquid-liquid partitioning identified 43 saponin structures, however, there was low selectivity, resulting in saponins identified all partition extracts and large-scale sample losses. Alternatively, a comparable extraction utilising three different solid-phase extraction (SPE) cartridges, identifying 114 saponins with minimal evidence of sample losses. Analysis of the multiple elution stages indicated the SPE cartridges were most suitable for high volume, low concentration saponin extractions from CoTS. Finally, CoTS saponins released into the surrounding water (for defence and/or communication) was investigated (Chapter 5). CoTS-conditioned water was collected and processed using a serial SPE procedure and saponins identified using mass spectrometry and the MASD. Approximately 265 saponins were identified, with an 84% saponin compositional consistency between CoTS individuals (n=4, including 2 males and 2 females). Furthermore, the potential for sex-specific secretory saponins was investigated, however, no distinct changes in secretory saponin profile was observed. Lastly, while the serial SPE procedure was deemed effective at broad-spectrum identification of secreted saponins, semi-quantitative data for saponin composition was found to be highly inconsistent, indicating further optimisation is required for quantitative results. In summary, this PhD Thesis has set an important foundation for the determination of saponin profile from Asteroids. This has been achieved through the development of the MASD resource, which enabled rapid identification of saponins via MS analysis. This new database, allowed for bioactive saponin data to be quantitatively compared for the first time, highlighting structures of interest and exposing existing research gaps needing attention. The evaluation of existing procedures for their efficiency in the identification of saponins produced by CoTS individuals, identified a processing methodology which can provide consistent saponin identification using SPE cartridges. The resources and procedures developed in this thesis, allowed for the identification of endogenous saponins and those secreted into the surrounding water, demonstrating high compositional consistency, and providing the first evidence that CoTS secrete a defined saponin secretome.

Development of Resources and Procedures for The Determination of The Saponin Secretome of The Crown-Of-Thorns Starfish

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