The astacins are a family of metallopeptidases (MPs) that has been extensively described from animals. They are multidomain extracellular proteins, which have a conserved core architecture encompassing a signal peptide for secretion, a prodomain or prosegment and a zinc-dependent catalytic domain (CD). This constellation is found in the archetypal name-giving digestive enzyme astacin from the European crayfish Astacus astacus. Astacin catalytic domains span ∼200 residues and consist of two subdomains that flank an extended active-site cleft. They share several structural elements including a long zinc-binding consensus sequence (HEXXHXXGXXH) immediately followed by an EXXRXDRD motif, which features a family-specific glutamate. In addition, a downstream SIMHY-motif encompasses a "Met-turn" methionine and a zinc-binding tyrosine. The overall architecture and some structural features of astacin catalytic domains match those of other more distantly related MPs, which together constitute the metzincin clan of metallopeptidases. We further analysed the structures of PRO-, MAM, TRAF, CUB and EGF-like domains, and described their essential molecular determinants. In addition, we investigated the distribution of astacins across kingdoms and their phylogenetic origin. Through extensive sequence searches we found astacin CDs in > 25,000 sequences down the tree of life from humans beyond Metazoa, including Choanoflagellata, Filasterea and Ichtyosporea. We also found < 400 sequences scattered across non-holozoan eukaryotes including some fungi and one virus, as well as in selected taxa of archaea and bacteria that are pathogens or colonizers of animal hosts, but not in plants. Overall, we propose that astacins originate in the root of Holozoa consistent with Darwinian descent and that the latter genes might be the result of horizontal gene transfer from holozoan donors.

The metalloproteinase ovastacin is released by the mammalian egg upon fertilization and cleaves a distinct peptide bond in zona pellucida protein 2 (ZP2), a component of the enveloping extracellular matrix. This limited proteolysis causes zona pellucida hardening, abolishes sperm binding, and thereby regulates fertility. Accordingly, this process is tightly controlled by the plasma protein fetuin‐B, an endogenous competitive inhibitor. At present, little is known about how the cleavage characteristics of ovastacin differ from closely related proteases. Physiological implications of ovastacin beyond ZP2 cleavage are still obscure. In this study, we employed N‐terminal amine isotopic labeling of substrates (N‐TAILS) contained in the secretome of mouse embryonic fibroblasts to elucidate the substrate specificity and the precise cleavage site specificity. Furthermore, we were able to unravel the physicochemical properties governing ovastacin–substrate interactions as well as the individual characteristics that distinguish ovastacin from similar proteases, such as meprins and tolloid. Eventually, we identified several substrates whose cleavage could affect mammalian fertilization. Consequently, these substrates indicate newly identified functions of ovastacin in mammalian fertilization beyond zona pellucida hardening.

The horseshoe crab Limulus polyphemus is one of few extant Limulus species, which date back to ∼250 million years ago under the conservation of a common Bauplan documented by fossil records. It possesses the only proteolytic blood-coagulation and innate immunity system outside vertebrates and is a model organism for the study of the evolution and function of peptidases. The astacins are a family of metallopeptidases that share a central ∼200-residue catalytic domain (CD), which is found in >1000 species across holozoans and, sporadically, bacteria. Here, the zymogen of an astacin from L. polyphemus was crystallized and its structure was solved. A 34-residue, mostly unstructured pro-peptide (PP) traverses, and thus blocks, the active-site cleft of the CD in the opposite direction to a substrate. A central `PP motif' (F35-E-G-D-I39) adopts a loop structure which positions Asp38 to bind the catalytic metal, replacing the solvent molecule required for catalysis in the mature enzyme according to an `aspartate-switch' mechanism. Maturation cleavage of the PP liberates the cleft and causes the rearrangement of an `activation segment'. Moreover, the mature N-terminus is repositioned to penetrate the CD moiety and is anchored to a buried `family-specific' glutamate. Overall, this mechanism of latency is reminiscent of that of the other three astacins with known zymogenic and mature structures, namely crayfish astacin, human meprin β and bacterial myroilysin, but each shows specific structural characteristics. Remarkably, myroilysin lacks the PP motif and employs a cysteine instead of the aspartate to block the catalytic metal.

The metalloproteinase ovastacin is released by the mammalian egg upon fertilization and cleaves a distinct peptide bond in zona pellucida protein 2, a component of the enveloping extracellular matrix. This limited proteolysis causes zona pellucida hardening, abolishes sperm binding and thereby regulates fertility. Accordingly, this process is tightly controlled by the plasma protein fetuin-B, an endogenous competitive inhibitor. At present, little is known about how the cleavage characteristics of ovastacin differ from closely related proteases. Physiological implications of ovastacin beyond ZP2 cleavage are still obscure. In this study, we employed N-terminal amine isotopic labeling of substrates (N-TAILS) contained in the secretome of mouse embryonic fibroblasts to elucidate the substrate specificity and the precise cleavage site specificity. Furthermore, we were able to unravel the physicochemical properties governing enzyme-substrate interactions. Eventually, we identified several potential physiological substrates with significance for mammalian fertilization. These data suggest that ovastacin might regulate sperm-oocyte interaction and fertility beyond zona pellucida hardening. Graphical Abstract

BACKGROUND:The Hydra head organizer acts as a signaling center that initiates and maintains the primary body axis in steady state polyps and during budding or regeneration. Wnt/beta-Catenin signaling functions as a primary cue controlling this process, but how Wnt ligand activity is locally restricted at the protein level is poorly understood. Here we report a proteomic analysis of Hydra head tissue leading to the identification of an astacin family proteinase as a Wnt processing factor.RESULTS:Hydra astacin-7 (HAS-7) is expressed from gland cells as an apical-distal gradient in the body column, peaking close beneath the tentacle zone. HAS-7 siRNA knockdown abrogates HyWnt3 proteolysis in the head tissue and induces a robust double axis phenotype, which is rescued by simultaneous HyWnt3 knockdown. Accordingly, double axes are also observed in conditions of increased Wnt activity as in transgenic actin::HyWnt3 and HyDkk1/2/4 siRNA treated animals. HyWnt3-induced double axes in Xenopus embryos could be rescued by coinjection of HAS-7 mRNA. Mathematical modelling combined with experimental promotor analysis indicate an indirect regulation of HAS-7 by beta-Catenin, expanding the classical Turing-type activator-inhibitor model.CONCLUSIONS:We show the astacin family protease HAS-7 maintains a single head organizer through proteolysis of HyWnt3. Our data suggest a negative regulatory function of Wnt processing astacin proteinases in the global patterning of the oral-aboral axis in Hydra.

The encounter of oocyte and sperm is the key event initiating embryonic development in mammals. Crucial functions of this existential interaction are determined by proteolytic enzymes, such as acrosin, carried in the sperm head acrosome, and ovastacin, stored in the oocyte cortical granules. Ovastacin is released upon fertilisation to cleave the zona pellucida, a glycoprotein matrix surrounding the oocyte. This limited proteolysis hardens the oocyte envelope, and thereby provides a definitive block against polyspermy and protects the developing embryo. On the other hand, acrosin, the renowned and most abundant acrosomal protease, has been thought to enable sperm to penetrate the oocyte envelope. Depending on the species, proteolytic cleavage of the zona pellucida by acrosin is either essential or conducive for fertilisation. However, the specific target cleavage sites and the resulting physiological consequences of this proteolysis remained obscure. Here, we treated native mouse zonae pellucidae with active acrosin and identified two cleavage sites in zona pellucida protein 1 (ZP1), five in ZP2 and one in ZP3 by mass spectrometry. Several of these sites are highly conserved in mammals. Remarkably, limited proteolysis by acrosin leads to zona pellucida remodelling rather than degradation. Thus, acrosin affects both sperm binding and mechanical resilience of the zona pellucida, as assessed by microscopy and nanoindentation measurements, respectively. Furthermore, we ascertained potential regulatory effects of acrosin, via activation of latent pro-ovastacin and inactivation of fetuin-B, a tight binding inhibitor of ovastacin. These results offer novel insights into the complex proteolytic network modifying the extracellular matrix of the mouse oocyte, which might apply also to other species.

Significance Membrane-bound metallopeptidases cut substrates as “sheddases” on the cell surface. Due to the irreversibility of peptide bond cleavage, they are regulated by specific, colocalizing protein inhibitors. We solved the crystal structure of the complex between the ectomoiety of the human integral membrane sheddase, meprin β (Mβ), and its only reported endogenous protein inhibitor, fetuin-B (FB), which reveals a large particle of ∼250-kDa and ∼160-Å maximal dimension. Our results unveiled a sophisticated “raised elephant trunk” mechanism as the structural basis for the very strong inhibition of Mβ by FB. Moreover, we derived a functional model for Mβ on the cell membrane, which foresees a transition between a membrane-proximal and a membrane-distal position for catalytic and inhibitory functions, respectively.

Astacin metalloproteinases, in particular meprins α and β, as well as ovastacin, are emerging drug targets. Drug-discovery efforts have led to the development of the first potent and selective inhibitors in the last few years. However, the most recent compounds are based on a highly flexible tertiary amine scaffold that could cause metabolic liabilities or decreased potency due to the entropic penalty upon binding to the target. Thus, the aim of this study was to discover novel conformationally constrained scaffolds as starting points for further inhibitor optimization. Shifting from flexible tertiary amines to rigid heteroaromatic cores resulted in a boost in inhibitory activity. Moreover, some compounds already exhibited higher activity against individual astacin proteinases compared to recently reported inhibitors and also a favorable off-target selectivity profile, thus qualifying them as very suitable chemical probes for target validation.

After fertilization, the oocyte-specific metalloproteinase ovastacin is released and cleaves the zona pellucida protein 2 (ZP2), making the zona pellucida impermeable to sperm. Before fertilization, the zona remains permeable because previously released ovastacin is inhibited by fetuin-B. Consequently, in the absence of fetuin-B, ZP2 cleavage occurs prematurely and leads to infertility of female fetuin-B deficient mice. In contrast, fetuin-B/ovastacin double-deficient oocytes show a permanently permeable zona with intact ZP2. In this study, we asked if the elastic modulus of the zona pellucida informs about ZP2 cleavage and thus could serve as a new reference of oocyte fertility. Therefore, we determined the elastic modulus of mouse oocytes by nanoindentation as a direct measure of mechanical zona hardening. The elastic modulus reflects ZP2 cleavage, but with more than double sensitivity compared to immunoblot analysis. The elastic modulus measurement allowed to define the range of zona hardening, confined by the extreme states of the zona pellucida in fetuin-B and ovastacin-deficient oocytes with cleaved and uncleaved ZP2, respectively. We present here nanoindentation as a method to quantify the effect of potential contributing factors on the zona hardening of individual oocytes. To demonstrate this, we showed that mechanical hardening of the zona pellucida is forced by recombinant ovastacin, inhibited by additional administration of fetuin-B, and unaffected by zinc. Since the change in elastic modulus is induced by ZP2 cleavage, an automated elastic modulus measurement of oocytes may serve as a novel sensitive, non-destructive, marker-free, and observer-unbiased method for assessing individual oocyte quality.

The Hydra head organizer acts as a signaling center that initiates and maintains the primary body axis in steady state polyps and during budding or regeneration. Wnt/beta-Catenin signaling functions as a primary cue controlling this process, but how Wnt ligand activity is locally restricted at the protein level is poorly understood. Here we report the identification of an astacin family proteinase as a Wnt processing factor. Hydra astacin-7 (HAS-7) is expressed from gland cells as an apical-distal gradient in the body column, peaking close beneath the tentacle zone. HAS-7 siRNA knockdown abrogates HyWnt3 proteolysis in the head tissue and induces a robust double axis phenotype, which is rescued by simultaneous HyWnt3 knockdown. Accordingly, double axes are also observed in conditions of increased Wnt levels as in transgenic actin::HyWnt3 and HyDkk1/2/4 siRNA treated animals. HyWnt3 -induced double axes in Xenopus embryos could be rescued by co-injection of HAS-7 mRNA. Mathematical modelling combined with experimental promotor analysis indicate an indirect regulation of HAS-7 by beta-Catenin, expanding the classical Turing-type activator-inhibitor model. Our data suggest a negative regulatory function of Wnt processing astacin proteinases in the global patterning of the oral-aboral axis in Hydra .

Despite huge progress in hormonal therapy and improved in vitro fertilization methods, the success rates in infertility treatment are still limited. A recently discovered mechanism revealed the interplay between the plasma protein fetuin-B and the cortical granule-based proteinase ovastacin as novel key-mechanism in the regulation of fertilization. Upon sperm-egg fusion, cleavage of a distinct zona pellucida component by ovastacin destroys the sperm receptor, enhances zona robustness and eventually provides a definitive block against polyspermy. An untimely onset of this zona hardening prior to fertilization would consequently result in infertility. Physiologically, this process is controlled by fetuin-B, an endogenous ovastacin inhibitor. Here we aimed at the discovery of small molecular inhibitors of ovastacin that could mimic the effect of fetuin-B. Hence, these compounds could be useful lead structures for the development of specific ovastacin inhibitors that can be utilized in infertility treatment or in vitro fertilization.

Vertebrate fetuins are multi-domain plasma-proteins of the cystatin-superfamily. Human fetuin-A is also known as AHSG, α2-Heremans-Schmid-glycoprotein. Gene-knockout in mice identified fetuin-A as essential for calcified-matrix-metabolism and bone-mineralization. Fetuin-B deficient mice, on the other hand, are female infertile due to zona pellucida 'hardening' caused by the metalloproteinase ovastacin in unfertilized oocytes. In wildtype mice fetuin-B inhibits the activity of ovastacin thus maintaining oocytes fertilizable. Here we asked, if fetuins affect further proteases as might be expected from their evolutionary relation to single-domain-cystatins, known as proteinase-inhibitors. We show that fetuin-A is not an inhibitor of any tested protease. In stark contrast, the closely related fetuin-B selectively inhibits astacin-metalloproteinases such as meprins and ovastacin, but not astacins of the tolloid-subfamily, nor any other proteinase. The analysis of fetuin-B expressed in various mammalian cell types, insect cells, and truncated fish-fetuin expressed in bacteria, showed that the cystatin-like domains alone are necessary and sufficient for inhibition. This report highlights fetuin-B as a specific antagonist of ovastacin and meprin-metalloproteinases. Control of ovastacin was shown to be indispensable for female fertility. Meprin inhibition, on the other hand, renders fetuin-B a potential key-player in proteolytic networks controlling angiogenesis, immune-defense, extracellular-matrix-assembly and general cell-signaling, with implications for inflammation, fibrosis, neurodegenerative disorders and cancer.

Human fetuin-B plays a key physiological role in human fertility through its inhibitory action on ovastacin, a member of the astacin family of metallopeptidases. The inhibitor consists of tandem cystatin-like domains (CY1 and CY2), which are connected by a linker containing a “CPDCP-trunk” and followed by a C-terminal region (CTR) void of regular secondary structure. Here, we solved the crystal structure of the complex of the inhibitor with archetypal astacin from crayfish, which is a useful model of human ovastacin. Two hairpins from CY2, the linker, and the tip of the “legumain-binding loop” of CY1 inhibit crayfish astacin following the “raised-elephant-trunk mechanism” recently reported for mouse fetuin-B. This inhibition is exerted by blocking active-site cleft sub-sites upstream and downstream of the catalytic zinc ion, but not those flanking the scissile bond. However, contrary to the mouse complex, which was obtained with fetuin-B nicked at a single site but otherwise intact, most of the CTR was proteolytically removed during crystallization of the human complex. Moreover, the two complexes present in the crystallographic asymmetric unit diverged in the relative arrangement of CY1 and CY2, while the two complexes found for the mouse complex crystal structure were equivalent. Biochemical studies in vitro confirmed the differential cleavage susceptibility of human and mouse fetuin-B in front of crayfish astacin and revealed that the cleaved human inhibitor blocks crayfish astacin and human meprin α and β only slightly less potently than the intact variant. Therefore, the CTR of animal fetuin-B orthologs may have a function in maintaining a particular relative orientation of CY1 and CY2 that nonetheless is dispensable for peptidase inhibition.

Mammalian fetuin-A and fetuin-B are abundant serum proteins with pleiotropic functions. Fetuin-B is a highly selective and potent inhibitor of metallopeptidases (MPs) of the astacin family, which includes ovastacin in mammals. By inhibiting ovastacin, fetuin-B is essential for female fertility. The crystal structure of fetuin-B was determined unbound and in complex with archetypal astacin, and it was found that the inhibitor has tandem cystatin-type modules (CY1 and CY2). They are connected by an exposed linker with a rigid, disulfide-linked `CPDCP-trunk', and are followed by a C-terminal region (CTR) with little regular secondary structure. The CPDCP-trunk and a hairpin of CY2 form a bipartite wedge, which slots into the active-site cleft of the MP. These elements occupy the nonprimed and primed sides of the cleft, respectively, but spare the specificity pocket so that the inhibitor is not cleaved. The aspartate in the trunk blocks the catalytic zinc of astacin, while the CY2 hairpin binds through a QWV X GP motif. The CY1 module assists in structural integrity and the CTR is not involved in inhibition, as verified by in vitro studies using a cohort of mutants and variants. Overall, the inhibition conforms to a novel `raised-elephant-trunk' mechanism for MPs, which is reminiscent of single-domain cystatins that target cysteine peptidases. Over 200 sequences from vertebrates have been annotated as fetuin-B, underpinning its ubiquity and physiological relevance; accordingly, sequences with conserved CPDCP- and QWV X GP-derived motifs have been found from mammals to cartilaginous fishes. Thus, the raised-elephant-trunk mechanism is likely to be generally valid for the inhibition of astacins by orthologs of fetuin-B.

Matrix metalloproteinases (MMPs) are secreted as proenzymes, containing propeptides that interact with the catalytic zinc, thereby controlling MMP activation. The MMP-9 propeptide is unique in the MMP family because of its post-translational modification with an N-linked oligosaccharide. ProMMP-9 activation by MMP-3 occurs stepwise by cleavage of the propeptide in an aminoterminal (pro-AT) and carboxyterminal (pro-CT) peptide. We chemically synthesized aglycosyl pro-AT and pro-CT and purified recombinant glycosylated pro-ATSf-9 . First, we report new cleavage sites in the MMP-9 propeptide by MMP-3 and neutrophil elastase. Additionally, we demonstrated with the use of western blot analysis a higher resistance of glycosylated versus aglycosyl pro-AT against proteolysis by MMP-3, MMP-9, meprin α, neutrophil elastase and by protease-rich synovial fluids from rheumatoid arthritis patients. Moreover, we investigated the effect of glycosylation on proteolytic activation of human proMMP-9 with the use of zymography and dye-quenched gelatin cleavage analysis. Compared to recombinant Sf-9 proMMP-9 glycoforms, larger oligosaccharides of human neutrophil proMMP-9 increased resistance against proteolytic activation. Additionally, proMMP-9 from Congenital Disorder of Glycosylation patients, compared to healthy controls, showed a higher activation rate by MMP-3. Finally, we demonstrated that glycan-galectin-3 interactions reduced proMMP-9 activation. In conclusion, modification of MMP-9 propeptide glycosylation is a fine-tuning mechanism and co-determines the specific activity of MMP-9 in physiology and pathology. ENZYMES: MMP-9 EC 3.4.24.35, MMP-3 EC 3.4.24.17, meprin α EC 3.4.24.18, neutrophil elastase EC 3.4.21.37, trypsin EC 3.4.21.4 and PNGase F EC 3.5.1.52.

STUDY QUESTIONHow and where is pro-ovastacin activated and how does active ovastacin regulate zona pellucida hardening (ZPH) and successful fertilization?STUDY FINDINGOvastacin is partially active before exocytosis and pre-hardens the zona pellucida (ZP) before fertilization.WHAT IS KNOWN ALREADYThe metalloproteinase ovastacin is stored in cortical granules, it cleaves zona pellucida protein 2 (ZP2) upon fertilization and thereby destroys the ZP sperm ligand and triggers ZPH. Female mice deficient in the extracellular circulating ovastacin-inhibitor fetuin-B are infertile due to pre-mature ZPH.STUDY DESIGN, SAMPLES/MATERIALS, METHODSWe isolated oocytes from wild-type and ovastacin-deficient (Astlnull) FVB mice before and after fertilization (in vitro and in vivo) and quantified ovastacin activity and cleavage of ZP2 by immunoblot. We assessed ZPH by measuring ZP digestion time using α-chymotrypsin and by determining ZP2 cleavage. We determined cellular distribution of ovastacin by immunofluorescence using domain-specific ovastacin antibodies. Experiments were performed at least in triplicate with a minimum of 20 oocytes. Data were pre-analyzed using Shapiro-Wilk test. In case of normal distribution, significance was determined via two-sided Student's t-test, whereas in case of non-normal distribution via Mann-Whitney U-test.MAIN RESULTS AND THE ROLE OF CHANCEMetaphase II (MII) oocytes contained both inactive pro-ovastacin and activated ovastacin. Immunoblot and ZP digestion assays revealed a partial cleavage of ZP2 even before fertilization in wild-type mice. Partial cleavage coincided with germinal-vesicle breakdown and MII, despite the presence of fetuin-B protein, an endogenous ovastacin inhibitor, in the follicular and oviductal fluid. Upon exocytosis, part of the C-terminal domain of ovastacin remained attached to the plasmalemma, while the N-terminal active ovastacin domain was secreted. This finding may resolve previously conflicting data showing that ovastacin acts both as an oolemmal receptor termed SAS1B (sperm acrosomal SLLP1 binding protein; SLLP, sperm lysozyme like protein) and a secreted protease mediating ZP2 cleavage.LIMITATIONS, REASONS FOR CAUTIONFor this study, only oocytes isolated from wild-type and ovastacin-deficient FVB mice were investigated. Some experiments involved oocyte activation by the Ca2+ ionophore A23187 to trigger ZPH.WIDER IMPLICATIONS OF THE FINDINGSThis study provides a detailed spatial and temporal view of pre-mature cleavage of ZP2 by ovastacin, which is known to adversely affect IVF rate in mice and humans.LARGE SCALE DATANone.STUDY FUNDING AND COMPETING INTEREST(S)This work was supported by the Center of Natural Sciences and Medicine and by a start-up grant of the Johannes Gutenberg University Mainz to W.S., and by a grant from Deutsche Forschungsgemeinschaft and by the START program of the Medical Faculty of RWTH Aachen University to J.F. and W.J.D. There are no competing interests to declare.