Distribution and diversity of ROS-generating enzymes across the animal kingdom, with a focus on sponges (Porifera)

The ROS generators, SOD and NOX (including Duox), both represent ancient and large enzyme families that are widespread across the animal kingdom. Genes encoding both are present in all of the 19 metazoan species that we assessed (Tables 1 and 2; Fig. 2), but it is notable that the gene numbers vary considerably between species (from 1 to 25; Tables 1 and 2). Below we discuss the dynamic content of these gene families across the animal kingdom. For each of the two enzyme families, we draw upon our new findings from the phylum Porifera — considered sister to all other animal phyletic lineages — to provide new insights into the evolution of these gene families since the dawn of the Metazoa more than 700 million years ago.

Superoxide dismutase (SOD)

The SOD family has been evolving for some ~ 2.5 (Ga) billion years of the Earth’s history, under vastly divergent selective pressures and with prolific cross-domain horizontal gene transfer events [15, 38, 41, 42]. In the animal kingdom alone, we observe various metalloforms and their subfamilies are not distributed equally across the metazoan tree, and neither are their localisations to various subcellular compartments. Consistent with previous analyses based on smaller numbers of animal taxa, we find that CuZn SOD1 and Mn SOD2 are the most conserved widespread SOD subfamily members [40, 43, 44]. However, the broader taxonomy that we present here extends the known SOD diversity by revealing the widespread metazoan prevalence of an atypical CuZnSOD, named Rsod, and an additional MnSOD subfamily member, SOD2X.

Evolution of the SOD family

Amongst the CuZnSODs, it is widely accepted that cytoplasmic SOD1 arose first and later gave rise to the extracellular SOD3 with the addition of signal peptides [12, 40, 44, 45].Our analysis reveals that SOD1 forms a strongly supported monophyletic group comprising metazoan, fungi, plant, algae and amoebozoan species (93%), whilst SOD3 is paraphyletic. We found metazoan species commonly have at least one additional CuZnSOD outside of SOD1 or Rsod, but these sequences are diverse and do not necessarily classify as SOD3; not all sequences encode a signal peptide, and many possess additional domains not typically associated to CuZnSOD. Indeed, together, we found these unclassified CuZnSOD sequences with those previously annotated as SOD3 formed six independent monophyletic groups, and three further independent branches (Fig. 3).

Thus, our results strongly support the hypothesis that SOD3 has arisen via multiple, independent evolutionary events in different metazoan lineages [40, 44, 45]. Moreover, some of these “SOD3” branches are separated by considerable distances, and many are not localised extracellularly as is typically described for SOD3 in vertebrates, nematodes and arthropods [40, 44] (Fig. 4). Considering that localisation of SOD dictates its signalling pathway involvement, we suggest that these unclassified CuZnSOD sequences likely exhibit diverse functionality across the metazoan phyla. Given this, we further suggest that the term SOD3 is not sufficient to capture the complete diversity of paraphyletic CuZnSODs that have independently arisen, on multiple occasions across the metazoans.

Interestingly, most of the sponges we survey lack any additional CuZnSOD SOD3-like gene, which we suggest may be functionally substituted by the extracellular Rsod that is encoded by all sponge species except for O. carmela that does encode an extracellular SOD3. As with SOD1, the Rsod monophyletic clade comprises representatives from multiple kingdoms and thus supports previous suggestions that Rsod belongs to an ancient group of proteins [44]. Based on these observations, we propose that Rsod predates SOD3 and that this explains the predominant absence of SOD3 in phylum Porifera where Rsod is common.

Of the various SOD metalloforms, it has most commonly been hypothesised that MnSOD (homologous to non-animal FeSOD) is ancestral, with CuZnSOD arising later, after the great oxidation event (reviewed by [12, 15, 42]). Alternatively, [38] recently proposed instead that CuZnSOD first arose in the Archean, predating Fe/MnSOD that subsequently appeared much later in the mid-Proterozoic. In support of this, here, we find SOD1 is the closest monophyletic clade to CuZnSODs from Archean cyanobacterial lineages Pseudanabaena sp. and Gloeobacter violaceus [38] (Fig. 3). Moreover, unlike MnSOD2 where the metazoans form an independent monophyletic group (87%), the SOD1 clade comprises genes from multiple kingdoms, reflecting an ancient evolutionary origin that predates separation of the eukaryotic kingdoms (Fig. 3). SOD1 sequences are also more variably localised; they are found within the nucleus, ER, mitochondria, cell membrane and extracellularly across five metazoan species (Fig. 4; Additional file 3). Conversely, Mn SOD2 are consistently localised within the mitochondria, indicating a comparatively greater degree of conservation (Fig. 3).

We must also consider the prevalence of cross-domain horizontal gene transfer. Evolutionary analyses of bacterial SOD indicate CuZnSOD was lost as cyanobacteria diversified within new ecological niches [46] and then later spread via horizontal transmission between non-cyanobacterial phyla likely multiple times, resulting in the distribution found today [38, 42]. Thus, it seems possible the distribution of metazoan SODs may similarly have arisen via horizontal acquisition across the domains.

An atypical CuZnSOD — Rsod — is widespread in the animal kingdom

We reveal a widespread metazoan presence of an atypical CuZnSOD, phylogenetically distant from both SOD1 and SOD3, that is largely localised either extracellularly or membrane bound (green shape, Fig. 3). These atypical CuZnSOD sequences share sequence and structural similarity to a D. melanogaster sequence named Rsod, “Related to SOD” (Dmel\CG31028, FBgn0051028), clustering together within a single, well-supported monophyletic clade [40] (Fig. 3). The structure of Rsod is unique from other known CuZnSODs, comprising multiple CuZnSOD domains (between 2 and 6) often with signal peptide and/or transmembrane regions (Fig. 1B). Rsod is considered to belong to an ancient group of proteins containing CuZnSOD homology domains [40], but has been little explored since its initial description, and its function is not yet known.

To date, Rsod sequences have been identified across the animal kingdom, including in insects, fish and the urochordate Ciona intestinalis and also in fungi [40, 45], BmSOD6, Accession Nos. LC229593 from 45]. Here, we additionally identify Rsod homologues within six sponges, echinoderms, cephalochordate (Branchiostoma floridae) and two vertebrates, indicating the widespread prevalence of this atypical CuZnSOD (Fig. 3). Notably, we show here for the first time that Rsod is particularly abundant and diversified within phylum Porifera, and especially within T. wilhelma (class Demospongiae) and S. ciliatum (class Calcarea). Subcellular localisation predictions indicate that most Rsod sequences we identified are either extracellular (21 sequences) or cell membrane bound (23 sequences), except five sequences localised within the cytoplasm in sponges (1 in A. queenslandica, X. bergquistia, E. muelleri and 2 in T. wilhelma), four in the vacuole/lysome (1 in B. floridae, and T. wilhelma and 2 in S. ciliatum) and one within the nucleus (Fig. 4; Additional file 3). Intriguingly, five species of Porifera, the tunicate C. intestinalis and Ascomycota fungi Phaeosphaeria nodorum, that do not encode any other extracellular CuZnSOD (e.g. SOD3), all encode both extracellular and membrane forms of Rsod; the exception is E. muelleri that encodes a cytoplasmic Rsod instead of membrane bound. In contrast, O. carmela, Acanthaster planci, Danio rerio, Xenopus tropicalis, D. melanogaster and Bombyx mori (BmSOD6; Kobayashi et al. 2019), all of which do encode extracellular SOD3, have only the cell membrane localised form (Fig. 4; Additional file 3). Thus, we suggest the extracellular Rsod in these species may functionally replace an extracellular SOD3. That said, B. floridae and Strongylocentrotus purpuratus comprise only extracellular localised Rsod, despite also encoding extracellular SOD3 (Fig. 4).

To date, the possible function of Rsod remains unknown. Kobayashi et al. assessed the responsiveness of different SOD genes within silk moth (B. mori) under various oxidative stressors and found Rsod gene (BmSOD6) is mostly expressed within the testes on day 3 of fifth instar larvae, indicating a role in removing ROS generated during spermatogenesis [47]. Additionally, honeybees (Apis mellifera) exposed to caging stress for up to 4 weeks showed a constant upregulation of only two genes, namely Rsod and another antioxidant thioredoxin-1 (Trx-1) [48]. In crown of thorns starfish, A. planci, maintained in captivity, one Rsod homologue (gbr.190.13.t1) is upregulated in three tissues (skin, tube feet and spines), and a second homologue (gbr.190.14.t1) in skin only, compared to wild populations [49].

The NADPH oxidase family

Much of the current understanding of NOX function is based on mammals, but variation in NOX subfamily membership across the metazoan and beyond indicates NOX functions are also likely to be variable. In accordance with 30 and 31, we find the calcium-regulated subfamilies have the earliest origin and are the widest distributed, followed by subunit-regulated NOXs that first appeared in fungi and amoeba, and for which we find the sponges (Porifera) have the earliest metazoan NOX2 co-ortholog (Fig. 5). However, neither the calcium- nor subunit-regulated NOXs are universally found in all metazoans, nor is any single NOX subfamily member. This is consistent with previous analyses based on smaller numbers of taxa [31, 32] showing large variability in gene number across the metazoan tree, indicative of lineage-specific gene duplication and apparent gene loss involving all NOX gene family members.

There are no core NOX subfamilies present across the Metazoa

NOX gene duplication and losses have been commonly documented but are not yet well understood [32]. Whilst some species encode multiple (up to 7) sequences for a single NOX, others lack the subfamily entirely (Table 2). Different NOXs are documented to function with precise, spatiotemporal specificity (Nathan and Cunningham-Bussel 2013; Sies and Jones 2020). Thus, it is somewhat surprising to find extensive gene losses in M. leidy that comprises just a single NOX5, and Caenorhabditis elegans and D. melanogaster that each comprise only two calcium-regulated NOXs (Table 2; Fig. 2). Whilst plants similarly encode a single subfamily, RBOHs that are NOX5-like homologues, species possess up to 10 different members of these in the subfamily [31, 50]. This raises the question of how species may compensate the absence of key subfamily members, NOX2, NOX5 and Duox, that each have disparate described signalling roles [27, 29, 51].

One possibility is that where a single NOX subfamily is represented by multiple genes, each gene exhibits distinct spatio-temporal specificity. For instance, S. ciliatum and C. intestinalis encode multiple Duox enzymes but lack NOX5. Considering that both Duox and NOX5 may be activated by calcium-sensitive EF-hand domains, the multiple Duox genes may compensate for the missing NOX5 function, as has been predicted in rodents [52]. That said, N. vectensis (Cnidaria) lacks both these calcium-regulated NOXs but does encode a subunit-regulated NOX4 that is absent in other basal metazoans. An alternative possibility is that missing subfamily members may be compensated for by the existing NOX genes having a broader range of functions that depend on their spatiotemporal localisation. As an example, NOX2 was documented first in phagocytic cells of sea urchin and mouse oocytes, where it was determined to function in the “oxidative burst” during phagocytosis [53, 54] but since has been identified in diverse cell types, and with diverse functions [reviewed in 23].

The ecological niche of an organism also will impact its redox states [2, 55] and thus likely the redox machinery it requires. The ctenophore, M. leidyi that encodes a single NOX5, exhibits daily vertical migrations [56, 57], perhaps in response to high irradiance levels, that are known to influence localised redox states [58]. We propose that vertical migratory species such as M. leidyi may also be able to mediate O2•− production via timing their daily migrations. Indeed, [59] show that antioxidant protection in cetaceans differs between shallow-diving and deep-diving habitats, as reflected by their O2•− production and antioxidant levels [60]. The metabolic activity of vertical migrations may sufficiently influence ROS generation in the mitochondrial ETC, endoplasmic reticulum or peroxisome to compensate fewer NOX enzymes. For example, M. leidyi encodes a comparatively wide range of CuZnSODs that are localised to those ROS-generating subcellular compartments, which may reflect greater ROS generation during cellular and metabolic processes (Table 1; Figs. 2 and 3).

Class Demospongiae encode a novel, calcium-regulated NOX

Within class Demospongiae (phylum Porifera), we identify here for the first time a structurally unique and phylogenetically distant NOX, which we call DspNOX (Fig. 6). The C-terminal region of DspNOX shares sequence and structural similarity with animal NOX5, comprising the core NOX domain features, and except for in the freshwater demosponge E. muelleri, is EF-hand calcium sensitive. DspNOX is ~ 700 residues longer than NOX5 and red algae NOXC/D and has a unique domain structure towards the N-terminal region that likely confers additional functionality and modes of activation.

Specifically, the N-terminal region of DspNOX comprises three novel elements (Fig. 6D, E). First, there is a PAS domain (PF00989) that functions as a sensory unit for diverse signals, including chemoreception, redox, photons and voltage, and thus acts as a highly versatile signal transducer [61]. It is well known as a light sensor, helping entrain the canonical circadian clock, but is also important in abiotic stress responses and innate immunity [62, 63]. The presence of both EF-hand and PAS suggests that, in addition to calcium, DspNOX could be regulated directly by abiotic factors. Second, DspNOX contains a long intrinsically disordered protein (IDP) region, defined as 30 or more consecutive disordered residues [64, 65]. The nature of IDP interactions (high specificity, but low affinity) increases a protein’s possible interactions and functional plasticity, making them well suited for signalling and regulatory functions [66]. Third, DspNOX contains five (or three within E. muelleri) additional transmembrane regions encoded within either a domain of unknown function DUF2427 (PF10348) or in the eukaryotic cytochrome b561 (Cyt-b561; PF03188).

Cyt-b561-containing proteins are an enzyme family of transmembrane, ascorbate-dependant oxidoreductases, most well known for their role in recycling ascorbate (i.e. vitamin C) via electron transfer from two heme b groups across the membrane [67]. Cyt-b561 enzymes may also be involved with iron metabolism, first described in the mammalian duodenal Cyt b561 (Dcytb), essential in the uptake of dietary nonheme iron (Fe+3) [68]. Dcytb uses ascorbate in the cytoplasm as an electron donor to reduce either Fe+3 into soluble, ferrous iron (Fe+2) or monodehydroascorbate (MDHA), depending on substrate availability [69]. Considering that the fenton reaction of Fe+2 with O2•− generates the highly reactive hydroxyl radical (•OH), it is particularly interesting that DspNOX has the potential to produce both these molecules. Indeed, because of the significant damage that •OH causes if not contained, organisms have evolved under strong selection to keep ferrous iron and ROS apart [1]. Thus, we suggest that DspNOX may provide an adaptive mechanism to keep Fe+2 away from O2•− if the ASC binding sites are on the same side as O2•− generation.

NOX or pre-NOX genes are distributed right across all the eukaryotic supergroups, except for Rhizaria [17, 25]. It is hypothesised that an enzyme similar to the red algal NOXD gave rise to animal NOX5, via acquisition of calcium-binding motifs [31, 70, 71]. Interestingly, similar to DspNOX, red algal NOXD and amoebozoan NOXC also comprise long IDP regions, which thus may indicate ancestral calcium-regulated NOXs (Fig. 6B, C). Red algae similarly encode four additional transmembrane domains, although these are located between two NADPH-binding site sub-regions and thus predicted to function only as an anchor to the membrane [70]. Results from our phylogenetic analysis reveal that red algae NOXD is sister to DspNOX (Fig. 5). Although the possibility of long-branch attraction cannot be discounted, the described structural similarities of these two proteins and the consistent placement of NOXC/D with previous assessments [31] together suggest this relationship is unlikely to be an artefact. Thus, we propose that DspNOX also represents an ancient NOX lineage that is likely a lineage-specific innovation within the class Demospongiae (phylum Porifera).

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