Insights into phosphatase-activated chemical defense in a marine sponge holobiont

Abstract

Marine sponges often contain potent cytotoxic compounds, which in turn evokes the principle question of how marine sponges avoid self-toxicity. In a marine sponge Discodermia calyx, the highly toxic calyculin A is detoxified by the phosphorylation, which is catalyzed by the phosphotransferase CalQ of a producer symbiont, "Candidatus Entotheonella" sp. Here we show the activating mechanism to dephosphorylate the stored phosphocalyculin A protoxin. The phosphatase specific to phosphocalyculin A is CalL, which is also encoded in the calyculin biosynthetic gene cluster. CalL represents a new clade and unprecedently coordinates the heteronuclear metals Cu and Zn. CalL is localized in the periplasmic space of the sponge symbiont, where it is ready for the on-demand production of calyculin A in response to sponge tissue disruption.

Fig. 1. Calyculin A biosynthetic gene cluster and bioconversion process between calyculin A and phosphocalyculin A. (A) Calyculin biosynthetic gene cluster. The ORFs encoding the NRPS and PKS responsible for the assembly line of calyculin biosynthesis are highlighted in orange. The other ORFs in blue are putative tailoring enzymes except for CalQ, which has been identified as the calyculin A-specific phosphotransferase. The putative functions of calJ–Y genes are listed in Table S1, ESI. (B) The activation/inactivation mechanism of phosphocalyculin A/calyculin A through dephosphorylation/phosphorylation in the marine sponge D. calyx.

Fig. 2. Identification of the native phosphocalyculin A phosphatase. (A) Chromatogram of LW-803 gel filtration. The elution time of the native phosphatase was 10.7 min, suggesting that the molecular weight is 45 ± 3 kDa (orange spots indicate the profile of each calibration marker: 670–1 kDa). (B) 10% SDS-PAGE of the purified native protein (CBB stained). Lane M is molecular weight markers. The three lanes represent fractions eluted between 10.5–12.0 min. The protein bands were subjected to the PMF analysis (see Fig. S2, ESI†). (C) Amino acid sequences of native phosphatase and recombinant CalL detected by PMF. The peptide fragments detected from both the recombinant and native CalL proteins, highlighted in green. The fragments only detected with recombinant CalL are highlighted in yellow. Plain regions were not detected from both proteins. The fragments detected by LC–MS/MS are shown as bold letters. The MS/MS fragmentation patterns of the digested amino acids of both native and recombinant CalL are compared in Fig. S2 (ESI†). The signal peptidase I cleavage site and the start methionine in the previously predicted ORF are pointed out with red and black triangles, respectively.

Fig. 3. Alignment of the five metal-coordinating motifs. Amino acid sequences of CalL, its homologs, and representative enzymes (purple acid phosphatases, pyrophosphatases; and nucleases) are aligned and five metal-coordinating motifs (I–V) were showed. The MPE domain has two catalytic metals at the active site, and each is coordinated by four residues (residues for metal I, orange box; metal II, yellow box; I and II, black box). The NCBI codes were described in parentheses after species names.

Fig. 4. Localization of CalL and its reaction place in the producer cells. To visualize the accumulation of free phosphoric acid in filamentous bacterial cells, cells were stained with malachite green reagent (MG). The left panels are the images of filamentous bacterial cells before staining. The right panels show high-magnification images of stained cells. Scale bars, 10 μm.