Ramakrishnan, L.; (2010) Molecular characterization of novel ADP-ribosyl cyclases from the sea urchin. Doctoral thesis , UCL (University College London).

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Abstract

Calcium signalling is ubiquitous and regulates diverse cellular processes. Cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) are second messengers that are involved in calcium release from the intracellular organelles. These molecules are structurally and mechanistically distinct but synthesised by a common enzyme, ADP-ribosyl cyclase (ARC). The sea urchin has long been a model system for both calcium signalling and embryogenesis. In fact, cADPR and NAADP were both discovered in the sea urchin. However, molecular details of ARCs and their roles during development are limited. Recently three ARC isoforms: SpARC1, SpARC2 and SpARC3 were identified from the sea urchin. In this study, additional novel isoforms including SpARC4 were cloned from S.purpuratus, highlighting the further expansion of ARCs in basal deuterostomes. SpARC2, SpARC3 and SpARC4 were found to be glycoproteins tethered to the plasma membrane via a GPI-anchor. SpARC2 and SpARC4 were multi-functional and able to produce both cADPR and NAADP over a wide pH range. SpARC2 was a preferential base-exchanger and SpARC4 a preferential cyclase. A unique non-canonical active site tyrosine residue regulated the cyclisation: base-exchange activity ratio of SpARC4. Both SpARC2 and SpARC4 were poor hydrolases and unable to cyclise NGD, a NAD surrogate. A single non-conserved glycine residue in the “TLEDTL domain” of SpARC2 was responsible for its poor hydrolase activity. All SpARC isoforms were detectable in S.purpuratus egg and the majority of ARC activity was GPI-anchored. During the course of early development, SpARC isoforms were differentially expressed and the endogenous ARC activity also varied. Over-expression and knock-down of SpARC4 during embryo development interfered with gastrulation. These findings provide new insights into the molecular mechanisms of multifunctionality of this remarkable family of enzymes and suggest that the expression and activities of ARCs could be fine-tuned for production of specific calcium messengers during embryogenesis of S.purpuratus.

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