Abstract
BackgroundDespite the profound variation among marine consumers in tolerance for allelochemically-rich foods, few studies have examined the biochemical adaptations underlying diet choice. Here we examine the role of glutathione S-transferases (GSTs) in the detoxification of dietary allelochemicals in the digestive gland of the predatory gastropod Cyphoma gibbosum, a generalist consumer of gorgonian corals. Controlled laboratory feeding experiments were used to investigate the influence of gorgonian diet on Cyphoma GST activity and isoform expression. Gorgonian extracts and semi-purified fractions were also screened to identify inhibitors and possible substrates of Cyphoma GSTs. In addition, we investigated the inhibitory properties of prostaglandins (PGs) structurally similar to antipredatory PGs found in high concentrations in the Caribbean gorgonian Plexaura homomalla.Principal Findings Cyphoma GST subunit composition was invariant and activity was constitutively high regardless of gorgonian diet. Bioassay-guided fractionation of gorgonian extracts revealed that moderately hydrophobic fractions from all eight gorgonian species examined contained putative GST substrates/inhibitors. LC-MS and NMR spectral analysis of the most inhibitory fraction from P. homomalla subsequently identified prostaglandin A2 (PGA2) as the dominant component. A similar screening of commercially available prostaglandins in series A, E, and F revealed that those prostaglandins most abundant in gorgonian tissues (e.g., PGA2) were also the most potent inhibitors. In vivo estimates of PGA2 concentration in digestive gland tissues calculated from snail grazing rates revealed that Cyphoma GSTs would be saturated with respect to PGA2 and operating at or near physiological capacity.SignificanceThe high, constitutive activity of Cyphoma GSTs is likely necessitated by the ubiquitous presence of GST substrates and/or inhibitors in this consumer's gorgonian diet. This generalist's GSTs may operate as ‘all-purpose’ detoxification enzymes, capable of conjugating or sequestering a broad range of lipophilic gorgonian compounds, thereby allowing this predator to exploit a range of chemically-defended prey, resulting in a competitive dietary advantage for this species.
Highlights
Glutathione S-transferases (GSTs, EC 2.5.1.18) comprise a large superfamily of enzymes whose soluble members primarily function as detoxification enzymes, facilitating the conjugation of a diverse array of hydrophobic electrophilic xenobiotics by the nucleophilic attack of glutathione [1]
The relative proportion of each glutathione Stransferases (GSTs) subunit, represented by separate HPLC peaks and calculated based on HPLC peak area, did not differ significantly as a function of gorgonian diet when expressed either as percent of all subunits present or when normalized to the amount of affinity-purified GST sample injected on to the HPLC column. These results indicate that while GST activity is constitutively expressed at high levels in Cyphoma digestive gland, both GST activity and subunit abundance are unaffected by gorgonian diet
The mean percent inhibition from five affinity-purified GST concentrates was used to calculate IC50 values. bKi values were calculated using the Cheng-Prusoff equation with an Km value of 0.41 mM for CDNB obtained from the average of two independent experiments; a CDNB substrate concentration of 1 mM; and IC50 values listed in the table above. cIn an attempt to constrain the likely range of calculated Ki values, test calculations using Km values for CDNB ranging from 0.1 mM to 1.0 mM resulted in Ki values for 15(S)-prostaglandin A2 (PGA2) of 6.0 to 37.7 mM. doi:10.1371/journal.pone.0008537.t001
Summary
Glutathione S-transferases (GSTs, EC 2.5.1.18) comprise a large superfamily of enzymes whose soluble members primarily function as detoxification enzymes, facilitating the conjugation of a diverse array of hydrophobic electrophilic xenobiotics by the nucleophilic attack of glutathione [1]. GST isoforms purified from polyphagous herbivores that regularly consume isothiocyanate-containing cruciferous plants were able to metabolize a broader range of isothiocyanate allelochemicals in comparison to GSTs from specialists that did not consume crucifers and lacked the ability to conjugate isothiocyanates [4]. These findings imply that the evolution of generalist GST forms favors promiscuous catalytic activity presumably needed to cope with the breadth of dietary toxins encountered; this mirrors results for other consumer counter-defense proteins (i.e., cytochrome P450s) whereby more catalytically flexible detoxification enzymes may promote a greater degree of polyphagy [8]. We investigated the inhibitory properties of prostaglandins (PGs) structurally similar to antipredatory PGs found in high concentrations in the Caribbean gorgonian Plexaura homomalla
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