Astacins across all phyla minimally comprise a zinc-binding CD, which is preceded by an upstream propeptide or prodomain (PRO) for latency and a signal peptide (S) for targeting to the plasmalemma or the extracellular space in animals [(Gomis-Rüth et al., 2012a); Figure 1]. However, most astacins are multidomain proteins, which have acquired a diverse set of additional domains (Figure 1). We retrieved reported experimental crystal structures of a CD, a “MAM” domain [first identified in meprin, A5 protein and receptor protein tyrosine phosphatase μ; (Cismasiu et al., 2004)], a “TRAF” domain [reminiscent of tumour-necrosis-factor receptor-associated factor; (Park, 2018)] and PRO domains, and computed high-confidence computational models of the “CUB” domain [first identified at the sequence level in complement subcomponents C1r/C1s, Uegf and BMP1; (Bork and Beckmann, 1993)] and epidermal growth factor (EGF)-like domains (see the Methods section) for their molecular analysis.

The CD is ascribed to protein family Pfam-01400 (Figure 1) and spans ∼200 residues. It contains two or three disulfide bonds at variable positions (Gomis-Rüth et al., 2012a) and is divided into an upper N-terminal subdomain and a lower C-terminal subdomain by an extended active-site cleft, as first revealed by the crystal structure of archetypal crayfish astacin (Bode et al., 1992; Gomis-Rüth et al., 1993) (Figure 2A). The N-terminal subdomain is rich in regular secondary structure and contains a hallmark five-stranded β-sheet (β1–β5; Figure 2A), whose lowermost strand β4 frames the upper rim of the active-site cleft when viewed in the standard orientation of MPs (Gomis-Rüth et al., 2012b) (Figure 2A, left), and two helices: the “backing helix” and the “active-site helix”. The latter encompasses most of a characteristic zinc-binding motif (HEXXHXXGXXH; amino-acid one letter code; X stands for any residue), which is found across astacins and other metzincin families (Bode et al., 1993; Stöcker et al., 1993; Yiallouros et al., 2000; Gomis-Rüth et al., 2012a; Cerdà-Costa and Gomis-Rüth, 2014; Arolas et al., 2018). The helix includes the first two zinc-liganding histidines and the general base/acid glutamate required for catalysis (Arolas et al., 2018). After the glycine of the motif, the chain undergoes a sharp turn and enters the C-terminal subdomain, which is more irregular and just encompasses a short β-ribbon (β6–β7) and a “C-terminal helix” as regular secondary structure (Figure 2A). The C-terminal subdomain provides two more zinc ligands, viz., the third histidine of the motif and a downstream tyrosine, which is swung out in a “tyrosine-switch” motion upon substrate binding to stabilize the reaction intermediate during catalysis (Grams et al., 1996; Yiallouros et al., 2000). This tyrosine is found two positions after another conserved element within metzincins, the “Met-turn” methionine (Bode et al., 1993; Tallant et al., 2010), which creates a hydrophobic base for the metal-binding site (Tallant et al., 2010). The tyrosine and the methionine are embedded in a characteristic SIMHY-motif in astacins (Stöcker et al., 1993).

Finally, another structural characteristic of astacin CDs is an unaccessible N-terminus (Gomis-Rüth et al., 2012a; Guevara et al., 2022). Maturation cleavage occurs at a bond that is occluded in the zymogen, which entails that partial unfolding of the segment flanking the activation site and/or preliminary cleavages are required for activation (Guevara et al., 2010; Arolas et al., 2012; Guevara et al., 2022). Upon final cleavage, the first six or seven residues of the mature enzyme are amply repositioned and penetrate the mature enzyme moiety, so the first two or three residues are completely buried in the molecular structure (Gomis-Rüth et al., 1993; Arolas et al., 2012; Ran et al., 2020; Guevara et al., 2022). Moreover, the new N-terminus binds the “family-specific” glutamate immediately after the third zinc-binding histidine (Bode et al., 1993; Gomis-Rüth, 2003), either directly through its side chain or through the α-amino group via a solvent molecule (Figure 2A). This feature is unique among MPs and reminiscent of trypsin-like serine endopeptidases, which dedicate an aspartate next to the catalytic serine to bind the likewise buried mature N-terminus (Bode et al., 1986). The astacin glutamate is immediately followed by an XXRXDRD motif (Gomis-Rüth, 2003) whose charged residues establish interactions relevant for domain stability.

A MAM domain is found after the CD in meprins α and β, Limulus and Hydra astacins and other (potential) family members (Figure 1) (Arolas et al., 2012; Eckhard et al., 2021; Guevara et al., 2022). The crystal structure of human meprin β (Arolas et al., 2012; Eckhard et al., 2021) reveals that its MAM domain is a β-sandwich consisting of a four- and a five-stranded antiparallel β-sheet, which are twisted and rotated ∼25 degrees relative to each other (Figure 2B). The domain conforms to a jelly-roll architecture featuring two four-stranded Greek key motifs and is connected by two disulfide bonds. Furthermore, the domain has a sodium-binding site, at which the cation is octahedrally coordinated by six oxygens from side chains and the main chain of the protein (Figure 2B). The overall architecture of the domain conforms to the structural criteria defined for the MAM protein family (Pfam-00629), which was identified in silico in meprin α and β, A5 protein, and receptor protein tyrosine phosphatase μ (Beckmann and Bork, 1993). Comparison with other MAM domains reveals that the central β-sandwich is conserved but the loops responsible for functionality deviate, as well as the metal-binding capacity and arrangement (Aricescu et al., 2006; Yelland and Djordjevic, 2016). This domain appears to have adhesive functions and, in meprin β, it contributes to dimerization by bringing the CD and TRAF domains together (Arolas et al., 2012; Eckhard et al., 2021).

Uniquely for astacins, meprins α and β exhibit a TRAF domain downstream of the MAM domain (Figure 1) (Arolas et al., 2012; Eckhard et al., 2021). The crystal structure of human meprin β (Arolas et al., 2012; Eckhard et al., 2021) shows that this moiety features two twisted four-stranded antiparallel β-sheets, which are rotated ∼40 degrees relative to each other (Figure 2C, left) and give rise to a flatter sandwich than in MAM (compare Figure 2B, right and Figure 2C, right). The strands are connected by loops of variable length, which include two short helical segments plus a short β-ribbon and give rise to a double Greek key architecture. The second Greek key is inserted into the first one but does not form a jelly roll. The only cysteine of this domain (C⁴⁹²) is buried and unbound, the N- and the C-terminus are on contiguous β-strands of the front β-sheet (Figure 2C, left), the C-terminus protrudes from the top surface of the domain (Figure 2C, right). In general, the TRAF domain of meprin β resembles tumor-necrosis-factor receptor-associated factors, which are mediators of cell activation engaged in homo- and heterodimerization and originated the TRAF protein family (Pfam-00917) (Zapata et al., 2001).

Further relevant for astacins are CUB domains (Figure 1), which were first identified in complement subcomponents C1r/C1s, Uegf and BMP1 (Bork and Beckmann, 1993) and form protein family Pfam-00431. They occur in BTP-subfamily astacins including BMP1, as well as in echinoderm astacins, a paralogue within A. astacus and several other orthologues in up to five copies (Figure 1). According to a highly reliable AlphaFold computational model (see Figure 2D and the Methods section), the first CUB domain of human BMP1 would be a β-sandwich made of an antiparallel four-stranded β-sheet and a mixed parallel/antiparallel five-stranded β-sheet, which would be both partially twisted. Their strands would be nearly parallel (Figure 2D, left), in contrast to MAM (Figure 2B, left) and TRAF (Figure 2C, left), and connected by mostly short loops. Two disulfide bonds would crosslink the domain. CUB domains were apparently present in the last common ancestor of eumetazoans and are currently found in synaptic proteins (González-Calvo et al., 2022). Remarkably, combinations of CUB and MAM domains are found in neuropilins, which are receptors for axon guidance cues and play synaptic roles (González-Calvo et al., 2022). Moreover, a CUB domain is engaged in the “Venus-flytrap” mechanism of inhibition of endopeptidases by the human pan-peptidase tetrameric inhibitor α₂-macroglobulin. It participates in major structural rearrangement of the C-terminal half of the protomer, which further includes three more domains (Marrero et al., 2012; Goulas et al., 2017; Luque et al., 2022). A CUB domain also participates in the “snap-trap” mechanism of monomeric α₂-macroglobulin-related inhibitors from commensal and pathogenic bacteria such as Escherichia coli and Salmonella enterica (Wong and Dessen, 2014; Garcia-Ferrer et al., 2015; Goulas et al., 2017).

Next, EGF domains (Pfam-00008) are widely present in up to six copies in several astacins, including meprins, BTPs and proteins from nematodes and echinoderms (Figure 1). Generally, they are found in many animal proteins in the extracellular part of membrane-bound or secreted proteins (Bork et al., 1996). In meprin β, the EGF-like domain is considered a hinge domain, which moves the dimer from a membrane-proximal position for cleavage of transmembrane substrates, such as the amyloid precursor protein, to a membrane-distal position upon binding to its endogenous inhibitor fetuin B (Karmilin et al., 2019; Eckhard et al., 2021). We obtained a generally reliable AlphaFold computational model (see the Methods section) for the EGF-like domain of human BMP1 (see Figure 2E). It revealed a ∼40-residue structure cross-connected by three disulfide bonds for structural integrity and two β-hairpins, which overall conform to the standard architecture of these domains (Wouters et al., 2005).

Finally, large diversity is found across astacin PROs, which range between 34 and 486 residues and just share the motif FXGDI among animal orthologues (Guevara et al., 2010; Gomis-Rüth et al., 2012a). The PROs of crayfish astacin (Figure 2F), human meprin β (Figure 2G), bacterial myroilysin (Figure 2H) and horseshoe crab astacin (Figure 2I) have been structurally characterized. They revealed essentially unstructured peptides running along the cleft of the CD in the opposite direction of a true substrate, which precludes their intramolecular cleavage (Guevara et al., 2010; Arolas et al., 2012; Xu et al., 2017; Guevara et al., 2022). The catalytic solvent molecule bound to the catalytic zinc ion in the mature CD (Figure 2A) is replaced by either the aspartate of the motif in the three animal zymogens (Guevara et al., 2010; Arolas et al., 2012; Guevara et al., 2022) or a cysteine in the bacterial enzyme (Xu et al., 2017), which lacks the motif. These residues operate according to an “aspartate-switch” or “cysteine-switch” mechanism of latency, respectively. Such mechanisms have been also reported, among others, for the MPs fragilysin-3 from Bacteroides fragilis (Goulas et al., 2011) and matrix metalloproteinases (Springman et al., 1990; Rosenblum et al., 2007), respectively.

Multicellularity presumably originated several times in unicellular opisthokont holozoans, which have been suggested as precursors of metazoans [(Sebé-Pedrós et al., 2017; Berman 2019)]. To better understand the hierarchical clustering of the distinct phyla within Holozoa, which originate 1.3 billion years ago (Berman 2019), and to put our phylogenetic studies into context, we tentatively assembled a consensus dendrogram based on current literature (Figure 3) given the apparent disparity in the available models (see Section 3.2). This hypothesis entails that Holozoa would split into Teretosporea, themselves consisting of Corallochytrea/Pluriformea (alias Opisthokonta incertae sedis) and Ichthyosporea, and Filozoa. These, in turn, would divide into Filasterea and Choanozoa. The latter would consist of Choanoflagellata, which are unicellular flagellates, and Metazoa, which encompass the multicellular animals and date back to about 760 million years ago (Berman 2019). Up the tree, Bilateria would englobe animals with a plane of symmetry (including Xenacoelomorpha), except echinoderms, which evince post-larval (secondary) pentaradial symmetry. They sequentially would team up with Cnidaria, Placozoa, Porifera and Ctenophora to eventually form Metazoa (Figure 3).

We performed searches for astacins in several protein and gene databases (see Section 3.1), which revealed > 25,000 entries for potential peptidases of the M12A family. This is how astacins are defined in the MEROPS database of peptidases and their inhibitors [www.ebi.ac.uk/merops; (Rawlings and Bateman, 2021)]. In addition, > 12,000 sequences from > 1,000 species of identified and putative family members were found within family PF01400 within the PFAM database (Mistry et al., 2021). At this point, high-confidence manually curated sequence searches were performed with the sequence of the mature CD of crayfish astacin. The resulting hit sequences were verified to span the entire CD and contain the intact zinc-binding motif, as well as the family-specific glutamate followed by the XXRXDRD and SIMHY motifs, with just minimal conservative substitutions (Figure 4 reproduces selected aligned example sequences). They were further checked to contain a PRO with the zinc-blocking aspartate. A subgroup of sequences was chosen for alignments, phylogenetic tree construction and physiological considerations (Supplementary Table S1; Sections 2.4–2.7). In addition, Table 1 presents a selection of described and potential non-vertebrate metazoan astacins.

We consistently found astacin sequences from humans down the tree of life until the root of subphylum Vertebrata (a selection is provided by Supplementary Table S1), among which the jawless fishes (Agnatha) are most basal, with two extant genera: lampreys and hagfishes (Supplementary Table S1). Vertebrata associate with the subphyla Tunicata/Urochordata, which includes sea squirts and the base tunicate Ciona intestinalis, and Cephalochordata, which features the lancelet (amphioxus), to form the phylum Chordata (Figure 3). These taxa also evinced abundant astacins. In addition, we could find sequences for the phyla Echinodermata and Hemichordata within Ambulacraria, which together with Chordata form Deuterostomia. Within Ecdysozoa, we could retrieve sequences from Tardigrada and Arthropoda but not Onychophora within Panarthropoda; Priapulida but not Kinorhyncha or Loricifera within Scalidophora; and Nematoda but not Nematomorpha within Nematoida. Out of Gnatifera plus Chaetognatha, we could only get hits for Rotifera. As to Lophotrochozoa, we found astacins in Annelida, Bryozoa, Brachiopoda and Mollusca. Next, we identified potential orthologues within Platyhelminthes but not Mesozoa and Gastrotricha. All these phyla constitute Protostomia, which together with Deuterostomia form Nephrozoa (Figure 3). The latter give rise to Bilateria together with Xenacoelomorpha, for which we could find sequences in two organisms (Table 1). Finally, we could identify astacins in all the remaining more primitive metazoan phyla: Cnidaria, Placozoa, Porifera and Ctenomorpha. All these results (Figure 3; Table 1) suggest that astacins are systematically present at least down to the root of Metazoa.

Choanoflagellata have been discussed as animal precursors for a long time, since they are similar to choanocytes of sponges (Hickman et al., 2020). However, a search for astacins within Monosiga brevicolis (King et al., 2008), Salpingoeca rosetta [formerly Proterospongia sp.; (Fairclough et al., 2013)] and Monosiga ovata of this phylum did not reveal significantly similar proteins. In addition, organisms from the next related taxa, Filasterea (Capsaspora owczarzaki), Ichthyosporea (Sphaeroforma arctica) and Corallochytrea/Pluriformea (Corallochytrium limacisporum and Syssomonas multiformis), which together with Metazoa and Choanoflagellata constitute the Holozoa (Figure 3), did not contain potential orthologs either in the generally available databases. At this point, we got access to unpublished genome sequences from a series of organisms at the root of Holozoa, viz., the Choanoflagellata Acanthoeca spectabilis, Choanoeca perplexa, Diaphanoeca grandis, Salpingoeca dolichothecata, Salpingoeca infusorum, Salpingoeca kvevrii, Salpingoeca macrocollata, Salpingoeca punica, Salpingoeca urceolata and Stephanoeca diplocostata; the Filastereum Ministeria vibrans; and the Ichthyosporea Amoebidium parasiticum, Abeoforma whisleri and Pirum gemmata [I. Ruiz-Trillo & M. Leger, personal communication, and (Torruella et al., 2015; Carr et al., 2017; Grau-Bové et al., 2017)]. We identified several potential astacin orthologues in Diaphanoeca grandis (e.g., comp29462_c0_seq1:1552770, comp29716_c0_seq2:1462332 and comp33115_c0_seq4:851467), Salpingoeca dolichothecata (e.g., comp24492_c1_seq1:7011852, comp14956_c0_seq2:1981400 and comp26781_c0_seq6:13261), Amoebidium parasiticum (Amoebidium_parasiticum_Apar_comp21710_c1_seq1m30654/1239) and Ministeria vibrans (Ministeria_vibrans_Mvib_g2222t1/16202), whose sequences are provided in Supplementary Figure S1. Altogether, these findings would suggest that astacins probably originate in the root of Holozoa according to Darwinian descent.

A selection of invertebrate and vertebrate sequences (Supplementary Table S1) enabled us to construct a phylogenetic tree for metazoan astacins (Figure 5), which was solely based on a sequence alignment of the respective CDs. Three sequences encompassed multiple CDs, which originate in the most basal sponge Amphimedon qeeenslandica (UniProt code [UP] A0A1X7U9V1), the nematode hookworm Ancylostoma caninum (UP A0A368GTC8) and the blow fly Lucilia cuprina (UP A0A0L0BYD0). This tree does not mirror the phylogenesis of organisms presented in Figure 3 since it contains both orthologous and paralogous astacins. Indeed, members of the distinct subfamilies evinced separate clustering (Figure 5). The tree is not comprehensive, since only a selection of astacins comprising the minimal setup of typical sequential and structural motifs (as outlined above) were included. Nevertheless, certain clusters of astacins can be recognized. These are, starting clockwise in the upper left quadrant of the circular tree (Figure 5), the 1) hatching enzymes, which originate from the same root as ovastacin; 2) ShKT-carrying astacins from chordates, nematodes, cnidarians, priapulids and arthropods (chelicerates and crustaceans), which include the prototypal crayfish astacin; 3) a clade containing the meprins; 4) a second cluster of ShKT-astacins, mostly from cnidarians (right centre); 5) proteins rich in MAM, EGF and CUB domains (right bottom); and 6) the BTPs (left bottom).

Searches in non-holozoan Eukaryota including plants, fungi and the fungus-like Oomycota revealed merely < 400 sequences scattered across Alveolata, Stramenopila, Rhizaria, Archaeplastida (Haptista), Excavata (Discoba) and some Amoebozoa clades. By contrast, no astacins were detected in any of the other eukaryotic taxa except for the silver mallet wood Rhodamnia argentea [GenBank code (GB) XP_030553468], the only plant orthologue retrieved. However, this entry was debunked as a contamination with a tolloid-like chelicerate astacin from the wheat curl mite Aceria tosichella (UP A0A8B8R4B3).

The observation of astacin-like proteins within Stramenopila is remarkable since this taxon alone already accounts for ∼200 of the hits. These are heterokonts that were formerly grouped into fungi (Holomycota) but currently are considered to be closer to brown algae than to fungi (Sebé-Pedrós et al., 2017). They further include the Oomycetes (“egg fungi”), a clade containing many parasitic/saprophytic organisms, which in turn encompass most of the hits within Stramenopila. Among them is Aphanomyces astaci, a well-known parasite of the North American crayfish Cambarus clarkii, which developed resistance against this pest. However, when American crayfish were brought to Europe in the late 19th century, A. astaci infection caused the “crayfish plague” in the endogenous crayfish population (A. astacus), which almost caused its extinction (Diéguez-Uribeondo and Söderhäll, 1993). Oomycetes are known champions of horizontal gene transfer (HGT) (Koonin et al., 2001; Keeling and Palmer, 2008), thereby gathering enzymes useful to target their prey (Judelson, 2017). This genetic transfer route could thus explain the generally scattered but locally focused presence of astacin CDs, which is inconsistent with Darwinian descent, in eukaryotes outside Holozoa.

HGT could also account for the sporadic occurrence of astacin-like peptidases in archaea, bacteria and viruses, which would thus also be xenologues (Koonin et al., 2001). Examples of archaeal sequences were found in Candidatus korarcheota (UP A0A662SFB1), Nitrosopumilus sp. (GB MCA9827382), Methanotrichaceae archaeon (GB MBN1323470) and Halobacteriales archaeon QH_6_64_20 (GB PSP40402). Viral sequences were restricted to Lutzomyia reovirus 2 (UP A0A0H4M9A8). The more populous bacterial examples were from Bacillus cereus (GB WP_235610182), Acinetobacter baumanii (GB WP_207273295), Klebsiella pneumoniae (GB NAU77905), Bacillus thuringiensis (GB WP_228528809), Legionella pneumophila (GB WP_061484376), Bacillus mycoides (GB WP_186320991), among others. Overall, the vast majority of bacterial astacin hosts live in intimate contact with animals, which would facilitate HGT of genes from eukaryotes to prokaryotes. Among them are those of the biochemically studied proteins flavastacin from Flavobacterium meningosepticum [also known as Elizabethkingia meningoseptica; UP Q47899; (Tarentino et al., 1995)] and myroilysins from Myroides profundi (UP B5B0E6) and Myroides sp. CSLB8 (UP A0A0P0DZ84) (Xu et al., 2017; Ran et al., 2020). In the latter case, zymogenic latency was shown to follow a different mechanism from the animal forms [see Section 2.1; (Guevara et al., 2022)], which would further support an HGT event as the origin of its presence in the bacterium. This is reminiscent of the aforementioned fragilysin-3, which originates in a member of the human colon microbiota. Its CD was proposed to be an adamalysin/ADAM xenologue acquired by HGT from the host that separately evolved to derive a distinct mechanism of latency (Goulas et al., 2011; Goulas et al., 2013).

In the phylum Chordata, subphylum Vertebrata, order Mammalia, six genes encode astacin peptidases [bmp1, tll1, tll2, mepa, mepb and astl; http://degradome.uniovi.es/met.html; (Quesada et al., 2009)]. The first three genes code for the BMP1, TLL1 and TLL2 proteins, which belong to the BTPs (Wozney et al., 1988; Nguyen et al., 1994). They are characterized by an arrangement of five CUB domains and two EGF-like domains C-terminal of the CD and are present in virtually all metazoan phyla (Figures 1, 5). BTPs are important for dorsoventral patterning in metazoan embryogenesis (Sato and Sargent, 1990; Holley et al., 1996; de Robertis, 2008; de Robertis and Tejeda-Muñoz, 2022). In deuterostomes, they are also crucial for extracellular matrix assembly through the processing of precursors of matrix components, growth factors and their receptors (Kessler et al., 1996; Ge and Greenspan, 2006). The CUB and EGF-like domains of these astacins bear important exosites for substrate recognition and targeting toward the extracellular matrix (Sieron et al., 2000; Garrigue-Antar et al., 2004; Ge and Greenspan, 2006; Hintze et al., 2006; Hopkins et al., 2007; Wermter et al., 2007). Although BTPs are closely linked with bilaterian morphogenesis, which supposedly originated in “Urbilateria” (de Robertis, 2008; de Robertis and Tejeda-Muñoz, 2022), they are not restricted to Bilateria and are also present in radially symmetric metaozans, Cnidaria (Saina et al., 2009) and Ctenophora (Pang et al., 2011). Related proteins with slightly deviating CUB/EGF arrangements were also found in the basal placozoan species Trichoplax adhaerens (CUBEGF_TRIAD) and in several other astacins (Figure 5). Taken together, BTPs are pan-metazoan astacins with essential functions in embryogenesis, which exert further additional functions in several metazoan phyla.

Two other human astacin genes, mepa and mepb, encode meprin α and meprin β for which orthologs have only been detected among vertebrates. Both meprins are membrane bound but meprin α is released already in the trans-Golgi network by furin cleavage and stays membrane bound only in association with meprin β. The latter is a “sheddase”, which releases cell-surface proteins such as growth factors, cytokines, receptors, as well as amyloid precursor protein through cleavage at its β-secretase site. Deregulation of meprins leads to neurodegenerative diseases, changes in barrier function (such as in the blood brain barrier), inflammatory bowel disease, fibrosis, nephritis and cancer (Sterchi et al., 2008; Arolas et al., 2012; Becker-Pauly and Pietrzik, 2016; Arnold et al., 2017; Eckhard et al., 2021; Gindorf et al., 2021; Bayly-Jones et al., 2022; Werny et al., 2022). The unique domain composition of meprins includes MAM, TRAF and EGF-like domains (Figures 1, 5), and although MAM- and EGF-containing astacins have been identified in other metazoan phyla (see Figures 1, 5; Supplementary Table S1), none of these are apparently membrane bound or exhibit comparable physiological potential to vertebrate meprins. Finally, our database searches unravelled meprin-like astacin-CDs also in basal vertebrates such as lamprey (MEPβ_PETMA) and hagfish (MEPα_EPTBU), which just encompass the S, PRO, CD and MAM moieties (see Figures 1, 5; Supplementary Table S1), similarly to a reported horseshoe crab enzyme [LASTMAM_LIMPO; (Becker-Pauly et al., 2009; Guevara et al., 2022)].

The sixth human astacin is ovastacin, which is encoded by the astl gene and is expressed only in oocytes among mammals (Burkart et al., 2012). Absence of ovastacin results in subfertility, since it is released during the cortical reaction after intrusion of a sperm cell and causes hardening of the zona pellucida of the extracellular matrix surrounding the egg. This provides rigidity and robustness to the resulting embryo until its implantation in the uterus (Stöcker et al., 2014; Körschgen et al., 2017). Ovastacin has the basic domain composition S-PRO-CD, which is followed by a disordered domain of unknown function. This domain stays connected with the oolemma after the release of the enzyme into the perivitelline space during the cortical reaction, which suggests a function in membrane anchoring and shedding of ovastacin (Körschgen et al., 2017). A similar function to ovastacin was reported for alveolin from the medaka fish Oryzias latipes, which likewise hardens the envelope of the fertilized egg (zygote) in bony fishes (Shibata et al., 2000).

In egg-laying vertebrates like fishes, amphibians, reptiles and birds, a specialized group of astacin MPs termed hatching enzymes has evolved (Nagasawa et al., 2022). They are absent from mammals and involved in the cleavage of the eggshell. They optionally contain an additional pair of cysteine residues in the N-terminal subdomain of their mature CDs compared to crayfish astacin, as well as extra C-terminal CUB domains (Figures 1, 5). Hatching enzymes are also present in egg-laying invertebrates, such as the crayfish A. astacus, which in addition to the prototypic digestive astacin archetype possesses the “Astacus embryonic astacin” (AEA_ASTAS in Figure 5; Supplementary Table S1; Geier and Zwilling, 1998). Finally, a reproductive astacin was also described from Drosophila seminal plasma (SEMP1_DROME; Figure 5; Supplementary Table S1). This MP is involved in a proteolytic cascade that triggers sperm capacitation and thus regulates fertility in the fruitfly (LaFlamme et al., 2012; LaFlamme and Wolfner, 2013; LaFlamme et al., 2014).

The genome analysis of the roundworm Caenorhabditis elegans uncovered 40 astacins termed “nematode astacins” (Möhrlen et al., 2003; Park et al., 2010). Moreover, in a comprehensive analysis of 154 helminth species of the phyla Nematoda and Platyhelminthes, many of them parasitic, an enormous radiation of astacins was also observed (Martín-Galiano and Sotillo, 2022). Most remarkable are the > 100 different additional domains that occur downstream of the CD in variable combinations, thus yielding an enormous functional versatility for these proteins. These domains do not only include protein-protein and protein-carbohydrate interacting domains, but also additional enzymatic functions, such as trypsin-like serine peptidases and hydroxylases.

Particularly striking are astacins containing “ShKT” domains, which mimic a toxin from the sea anemone Stychodactyla helianthus that blocks potassium channels (Castañeda et al., 1995). We found such ShKT-astacins in Nematoda (CAEEL, ONCVO, ANCCA and TRISP; see Figure 5; Table 1; Supplementary Table S1), Plathyhelminthes (SCHMD, ECHMU and SCHJA), Arthropoda (PENVA and DAPMA), Priapulida (PRICA), Bryozoa (BUGNE and LINUN), Mollusca (MYTCO) and Cnidaria (HYDVU, HYDEC, PODCA, NEMVE), as well as in the lower chordates sea squirt (HALRO and CIOIN) and lancelet (BRABE). In Figure 5, ShKT-astacins are labelled with pink branch tips (see Supplementary Table S1). These astacins are mostly expressed in epithelia forming barriers to the environment or in the digestive tract, which suggests functions in protection and defence, as well as preservation of the epithelial integrity (Park et al., 2010; Isolani et al., 2018). However, considering the parasitic lifestyle of many of the organisms harbouring ShKT-astacins, the combination of proteolytic and toxin domains may also challenge the respective host (Moran et al., 2013; Martín-Galiano and Sotillo, 2022). Similarly, toxicity has also been reported for astacins lacking ShKT domains from spider venoms (Trevisan-Silva et al., 2010), in which other proteins may take over the role of the latter domains in linking proteolytic activity with specific toxicity. This is reminiscent of the snake venom MPs from the adamalysin/ADAM family, for which forms spanning only the CD are not haemorrhagic while those encompassing further C-terminal disintegrin-like and cysteine-rich domains may be haemorrhagic (Fox and Serrano, 2009; Gomis-Rüth et al., 2013; Herrera et al., 2018). Finally, many ShKT-astacins carry additional CUB, EGF, MAM, etc. domains that may serve to modulate activity. Examples are the morphogenetically active Hydra vulgaris ShKT-proteins HMP1, HMP2 and HAS7 (Figure 5; Supplementary Table S1). While HMP1 is involved in head formation and head regeneration (Yan et al., 1995), HMP2 has a function in foot morphogenesis (Yan et al., 2000) and HAS7 specifically cleaves Hydra WNT3 during head morphogenesis and thereby restricts head organizer formation (Ziegler et al., 2021; Holstein, 2022).

A surprising function for an astacin peptidase was very recently reported for the sea star Asterias rubens, which underpins the enormous versatility of nature’s toolbox. In zoology textbooks [e.g., (Hickman et al., 2020)], the locomotion of Asteroidea on a surface is usually explained as the concerted action of a multitude of tiny sucking pods in the podia of the tube feet lining the oral face of the animal’s arms. However, pod attachment is apparently not based on suction but on a secreted glue consisting of adhesive matrix proteins that are left on the surface after detachment. The latter is mediated by an astacin MP spanning a CD and a CUB domain (CUB_ASTRU; see Figure 5; Supplementary Table S1), which is specifically secreted by de-adhesive gland cells and releases the adhesive material from the surface of the tube feet (Algrain et al., 2022).

Sequence searches were performed with the Psi-Blast program (Altschul and Koonin, 1998) within UniProt (https://www.uniprot.org) and GenBank (https://www.ncbi.nlm.nih.gov/genbank) using standard parameters, as well as with the Ensembl genome browser (http://www.ensembl.org), within the Choanobase database (King, 2005) and the Merops database [https://www.ebi.ac.uk/merops; (Rawlings and Bateman, 2021)]. Literature searches for described astacins were performed with the keyword “astacin*” within PubMed (https://pubmed.ncbi.nlm.nih.gov), which retrieved 342 papers. Hits were manually curated.

The currently available trees for holozoans in zoology textbooks such as (Hickman et al., 2020) present several discrepancies with models proposed by recent research publications (Cannon et al., 2016; Giribet et al., 2019; Laumer et al., 2019). These are based on massive sequence data generated in the last decade by increasingly affordable sequencing methods, first through the Illumina MiSeq platform and more recently through MinION sequencing (Santos et al., 2020), which are also partially applicable to natural history collections (Folk et al., 2021). As an example, phylum Chaetognatha was considered a separate clade at the same level as Spiralia and Ecdysozoa, while it is currently envisaged as a sister clade of Gnathifera within Spiralia (Kocot et al., 2017). Also setting phylum Porifera at the root of Metazoa contradicts recent models, which choose Ctenophora for this place (Giribet et al., 2019; Laumer et al., 2019). Finally, recent work proposed new models at the base of Holozoa, with Ichtyosporea and Corallochytrea/Pluriformea forming Teretosporea, which together with Filozoa give rise to Holozoa (Torruella et al., 2015; Arroyo et al., 2020). Accordingly, we assembled a dendrogram for holozoans based on consensus information extracted from these and other recent publications (Ryan et al., 2010; Ruggiero et al., 2015; Torruella et al., 2015; Cannon et al., 2016; Kocot et al., 2017; Lu et al., 2017; Sebé-Pedrós et al., 2017; Whelan et al., 2017; Adl et al., 2019; Giribet et al., 2019; Laumer et al., 2019; Marlétaz et al., 2019; Sogabe et al., 2019; Schoch et al., 2020; Schulze and Kawauchi, 2021).

Amino-acid sequence alignments and phylogenetic trees were computed using the Seaview program (http://doua.prabi.fr/software/seaview) (Galtier et al., 1996; Gouy et al., 2010). Alignments were performed with Clustal Omega within Seaview (Sievers et al., 2011) using default parameters. Manual adjustment of the S₁’ regions in Figure 4 was performed based on overlays of the X-ray crystal structures of crayfish astacin [Protein Data Bank (PDB) access codes 1AST, 1QJI] and zebrafish hatching enzyme 1 (PDB 3LQB). Phylogenetic trees were calculated with PhyML using the maximum likelihood approach as implemented in Seaview (Guindon et al., 2010). The BLOSUM62 scoring matrix was used and 100 bootstrap replications were computed in each case. Trees were represented with Figtree (http://tree.bio.ed.ac.uk).

Precise and accurate computational models of specific astacin domains were obtained with the AlphaFold program (Jumper et al., 2021; Tunyasuvunakool et al., 2021). To this aim, the program was locally installed in a high-performance computing cluster operated with linux and the amino acid sequences were fed into the program, which was run employing standard parameters. Quality of the predicted models was monitored through the average predicted local distance difference test (pLDD1) value. Values exceeding 90% are considered to originate in high-accuracy models, while those above 80% correspond to generally correct models for the backbone (Tunyasuvunakool et al., 2021). The unique EGF-like domain of human BMP1 (residues 547–590, see UP P13497) was predicted as a generally correct model for the backbone given an average pLDD1 value of 82.5%. Moreover, the first CUB domain of human BMP1 (residues 321–434, see UP P13497) was predicted highly accurately (average pLDD1 = 91.5%). Three-dimensional structure figures were prepared using Chimera (Goddard et al., 2018).