Functional characterization and expression of molluscan detoxification enzymes and transporters involved in dietary allelochemical resistance

Abstract

Understanding how organisms deal with potentially toxic or fitness-reducing allelochemicals is important for understanding patterns of predation and herbivory in the marine environment. The ability of marine consumers to tolerate dietary toxins may involve biochemical resistance mechanisms, which increase the hydrophilicity of compounds and facilitate their active efflux out of sensitive cells and tissues. While several allelochemical-responsive detoxification enzymes have been sequenced and functionally characterized in terrestrial invertebrates feeding on chemically defended host plants, there is virtually no information concerning the role of these biotransformation enzymes that may mediate feeding tolerance in marine invertebrates. The objective of this research was to assess the diversity and dietary regulation of cytochrome P450s (CYP), glutathione S-transferases (GST) and ABC transporters in the generalist marine gastropod Cyphoma gibbosum feeding on a variety of chemically defended gorgonian corals, and to identify those dietary natural products that act as substrates for these proteins.Molecular and proteomic techniques identified both allelochemically-responsive CYPs, and constitutively expressed GSTs and transporters in Cyphoma digestive glands. Inhibition of Cyphoma GST activity by gorgonian extracts and selected allelochemicals (i.e., prostaglandins) indicated that gorgonian diets are likely to contain substrates for molluscan detoxification enzymes. In vitro metabolism studies with recombinant CYPs suggested those Cyphoma enzymes most closely related to vertebrate fatty acid hydroxylating enzymes may contribute to the detoxification ofichthyodeterrent cyclopentenone prostaglandins found in abundance in selected gorgonian species. Finally, the presence and activity of multixenobiotic resistance transporters in Cyphoma and the co-occuring specialist nudibranch, Tritonia hamnerorum, suggests these efflux transporters could function as a first line of defense against dietary intoxication. Together, these results suggest marine consumers that regularly exploit allelochemicalrich prey have evolved both general (GST and ABC transporters) and allelochemicalspecific (CYP) detoxification mechanisms to tolerate prey chemical defenses.Thesis Supervisor: Mark E. Hahn Title: Senior Scientist, Biology Department, WHOI COMPLETE ABSTRACT Intense consumer pressure in tropical marine ecosystems not only structures the organization and functioning of marine communities, but has a profound effect on the phenotype of resident organisms. In order to persist in the midst of extreme predation and herbivory, sessile benthic marine organisms, afforded with no means of escape, often produce chemical defenses, termed allelochemicals, which render their tissues unpalatable or toxic to most potential consumers. Despite the fitness-reducing consequences of consuming noxious/toxic prey, less mobile consumers, frequently drawn to their hosts by the chemical refugia they provide, are able to tolerate the toxicological challe...

Proteomic identification, cDNA cloning and enzymatic activity of glutathione S-transferases from the generalist marine gastropod, Cyphoma gibbosum

“…Thus, high expression of GST mu isoforms may allow C. gibbosum to tolerate the chemical defenses of its host and subsequently feed longer than would otherwise be possible. Preliminary studies have demonstrated that prostaglandins found in gorgonians significantly inhibit Cyphoma GST activity in vitro [49], consistent with the idea that gorgonian prostaglandins may be substrates for Cyphoma GSTs.…”

“…Rather, the high GST activity may reflect an adaptation that facilitates consumption of allelochemical-rich prey. Additional findings indicate that C. gibbosum GSTs are expressed constitutively at high levels regardless of the gorgonian diet, providing further support for this hypothesis [49]. Whether high GST activity is common among marine species that feed exclusively on chemically defended food is unknown, and may depend upon the specific suite of allelochemicals present in the diet.…”

Biochemical Warfare on the Reef: The Role of Glutathione Transferases in Consumer Tolerance of Dietary Prostaglandins

“…HPLC separation of affinity-purified GSTs identified fourteen unique peaks (see Figure S1 ). HPLC peak 1 was previously identified as a theta-class GST, while HPLC peaks 2 thru 14 were identified as mu-class GST subunits [44] . HPLC peaks 4 and 8 represented the majority of expressed GST subunits at 25% and 68%, respectively.…”

“…Cytosolic and affinity-purified GSTs were isolated from Cyphoma digestive gland samples as described in [44] . Briefly, cytosolic GSTs were isolated by homogenizing digestive glands (n = 39) separately in buffer (0.1 M potassium phosphate, 1 mM EDTA, 1 mM DTT, 1.15% potassium chloride, protease inhibitor cocktail (1×); pH 7.5), and differentially centrifuging the homogenates to obtain the cytosolic fraction containing the soluble GST pool.…”

“…Enzyme activity was measured using CDNB as a substrate by the method of [69] optimized for C. gibbosum [14] , [44] in a microplate format. The reaction mixture (in a final volume of 200 µL) contained 0.1 M potassium phosphate buffer, 1.0 mM EDTA, pH 7.5, 1 mM CDNB, 1 mM reduced GSH and 2 µg of cytosolic protein or 3.3–6.4 ng of affinity-purified GST sample.…”