Robertson, Alison (2005) Saxitoxin binding proteins: biological perspectives. PhD thesis, James Cook University.

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Abstract

Saxitoxin binding protein (STXBP) is a functional classification which describes all proteins capable of binding to the paralytic shellfish toxin (PST), saxitoxin (STX). Based on this functionality, this group includes the voltage gated sodium channels (VGSCs), pufferfish STX and tetrodotoxin (TTX) binding proteins (PSTBPs) and saxiphilin (SXPN) which was been isolated from the amphibian Rana catesbeiana. Various activities and relationships of bullfrog SXPN have been elucidated including the ability to inhibit papain, human cathepsin Band L and the substantial homology of the amino acid sequence to transferrins (TFs). However, the biological role of SXPN has not been thoroughly examined and remains a mystery. It is likely that a detoxification mechanism exists in animals exposed to PSTs, and may explain the defined STX binding activity of soluble STXBPs. Therefore, the main objective of this thesis was to examine various aspects of the biological relationship between STX and soluble STXBPs to determine whether these proteins provide a defensive arsenal against PST intoxication.

PreliIninary studies indicated that current methods for the detection of STXBPs were problematic and time intensive therefore several radio-receptor assays were developed and trialled to identify a suitable primary screening regimen for the detection and characterisation of these proteins. Assays utilising anion and cation exhange methods, protein binding and traditional charcoal radio-receptor methods were compared to published formats. A receptor binding filtration assay incorporating protein binding membranes of mixed cellulose esters (MCE) proved to be a robust method for the sensitive and accurate detection of STXBPs. This assay was easily converted for use as a PST screening tool and was validated in subsequent chapters using Bufo marinus plasma which is readily obtainable source of STXBPs.

With the aid of this optimised assay method, the diversity of soluble STX-specific receptors was investigated to extend previous phylogenetic surveys and identify any commonality between species that contain STXBPs. More than 1000 extracts, representing over 200 different species from the marine, freshwater and terrestrial environment were investigated, resulting in the discovery of eight novel STXBPs extending the known phylogenetic diversity of STXBPs to include species from Onychophora and Mollusca. Seven of these species were characterised as STX specific hydrophilic receptors based on their ability to exclusively bind STX. In addition, a STXBP likely to belong to the PSTBP group was identified in the toad fish, T. pleurogramma.

Further examination of species collected from a verified STX "Hot Spot" resulted in the identification of one additional STXBP in the extracts from the crab, Lophozozymus octodentatus. The occurrence of PSTs and diversity STX sources and vectors at this site were examined by means of radioreceptor assays including the centipede SXPN assay, rat brain synaptosome assay, liquid chromatography-fluorescence detection with post column oxidation, in addition to confirmatory mass spectrometric analysis. This study confirmed 3 new sources of PSTs in benthic food web of Port Hedland including the macroalgae, Sargassum sp. Jania sp.2 and Jania sp.3. However, the STX levels in these species did not explain the extreme levels of STX observed in some vectors. A number of new PST vectors were identified from the bivalves Tridacna squamosa, Pinctada albina sugilata, Saccostrea glomerata, Malleus regula and the first incidence of STX in the octopi, Octopus (Abdopus) sp. 5, was reported. The lack of widespread STXBPs in the intertidal STX "Hot Spot" did not conclusively support the toxin defence hypothesis as a likely biological role of STXBPs.

Finally, an animal model, B. marinus, was selected for an in depth analysis of STXBPs. STXBP levels in the toad were ubiquitous across all life stages and within all tissues with the exception of the venom glands, and reflected previous reports of saxiphilin distribution in the bullfrog Roo catesbeiana. Interestingly, a positive correlation was demonstrated between environmental temperature and levels of STX binding activity in toad plasma. The STXBP isolated from B. marinus plasma was successfully purified, revealing an estimated protein size of 93 kDa and 3 peptide sequences which facilitated degenerate PCR experiments. Cloned STXBP-specific fragments of cDNA from toad liver were then cloned and the corresponding translated amino acid sequences revealed homology to the C-lobe of both saxiphilin from R. catesbeiana (also known to contain the STX binding site) and a variety of TFs.

The biological role of soluble STXBPs remains a mystery but substantial advances have been made in terms of the diversity, function and relationships of these proteins. The potential application of STXBPs uncovered during this research, in both medical and research applications of PSP treatment and detection is substantial and the wealth of data collected will promote several new directions of research in this area.

Item ID:	34822
Item Type:	Thesis (PhD)
Keywords:	paralytic shellfish toxin; PST; radio-receptor assays; saxitoxin binding proteins; saxitoxin; STX; STXBP
Related URLs:	http://researchonline.jcu.edu.au/4645/ http://researchonline.jcu.edu.au/4205/ http://researchonline.jcu.edu.au/13950/
Additional Information:	Publications arising from this thesis are available from the Related URLs field. The publications are: Robertson, Alison, Negri, Andrew P., Burnell, James N., and Llewellyn, Lyndon E. (2006) Development and assessment of radioreceptor binding assays for the detection of saxitoxin binding proteins in biological extracts. Analytical Biochemistry, 356 (1). pp. 66-75. Llewellyn, Lyndon, Negri, Andrew, and Robertson, Alison (2006) Paralytic shellfish toxins in tropical oceans. Toxin Reviews, 25 (2). pp. 159-196. Robertson, Alison, Stirling, David, Robillot, Cedric, Llewellyn, Lyndon, and Negri, Andrew (2004) First report of saxitoxin in octopi. Toxicon, 44 (7). pp. 765-771.
Date Deposited:	21 Jul 2015 05:49
FoR Codes:	06 BIOLOGICAL SCIENCES > 0601 Biochemistry and Cell Biology > 060101 Analytical Biochemistry @ 100%
SEO Codes:	97 EXPANDING KNOWLEDGE > 970106 Expanding Knowledge in the Biological Sciences @ 50% 92 HEALTH > 9204 Public Health (excl. Specific Population Health) > 920406 Food Safety @ 50%
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