Meprin β is a zinc-dependent metalloprotease of the astacin family highly expressed in kidney and intestine. Dysregulation of meprin β expression and activity was observed in several pathological conditions such as Alzheimer’s disease, cancer, fibrosis, and inflammatory bowel disease. Therefore, inhibition of meprin β could be a promising therapeutic option to treat pathological conditions influenced by this protease. However, available small-molecule compounds such as the broad-spectrum metalloproteinases inhibitors marimastat or actinonin lack specificity. Hence, more selective meprin β inhibitors needs to be developed. Here, we provide evidence that the synthetically generated cognate meprin β propeptide serves as highly specific inhibitor for the protease (IC50 = 1.35 µM), while other related metalloproteases such as meprin α and a disintegrin and metalloproteinases (ADAMs) are not affected. Employing a targeted approach revealed that removal of the last four C-terminal amino acids of the propeptide significantly improved its inhibitory capacity (IC50 = 0.68 µM). In cell-based experiments, treatment with the propeptide resulted in diminished meprin β activity and decreased cleavage of its substrates, including the interleukin-6 receptor (IL-6R) and the G protein-coupled receptor latrophilin-3 (LPHN3). Suitability was further confirmed in ex vivo cultivated organotypic brain slices, where the administration of the propeptide also resulted in decreased meprin β activity and diminished latrophilin-3 cleavage. Thus, not only the synthetic wildtype propeptide but especially a C-terminally truncated propeptide of meprin β represent highly specific inhibitors with potential for therapeutic application.

Meprin α and meprin β are zinc metalloproteases that are strongly expressed in intestinal and renal tissues and are expressed as homo- and heterodimers. In the kidney and intestine, they are involved in extracellular matrix assembly and modulation of inflammatory responses. However, meprin β has recently attracted attention because it generates Alzheimer's Disease (AD)-specific Aβ peptides and cleaves brevican, a major component of the perineuronal nets (PNNs) in the brain. PNNs stabilize synapses, thereby regulating plasticity and memory formation. Brevican cleavage correlated with impaired spatial memory formation and impaired CA1 long-term potentiation (LTP) in meprin β transgenic mice. Furthermore, numerous studies have shown the dysregulation of PNN components in AD. Still, the physiological and pathological functions of proteolytic PNN remodeling remain elusive. This study identified an essential role of meprin α in brevican cleavage. It enhanced meprin β's catalytic activity on brevican in co-expression. Moreover, an N-terminomics analysis identified novel meprin β substrates, neurocan, and receptor-type tyrosine-protein phosphatase zeta (RPTPζ) in the brain. Both are key components of PNNs. RPTPζ cleavage by meprin α and meprin β was confirmed in vitro. To assess the functional impact of meprin-mediated proteolysis on the brain extracellular matrix, PNNs and synaptic organization were investigated in vivo using immunofluorescence and electron microscopy. Meprin-mediated proteolysis disrupted PNN structure and decreased synapse density in the hippocampal CA1 region of meprin β transgenic mice. This identifies meprin-dependent PNN remodeling as a novel mechanism contributing to synaptic dysfunction.

Dysregulations within the epidermal proteolytic network can cause hyperproliferative and inflammatory disorders. Although the metalloprotease meprin α is localized in the stratum basale in healthy skin, increased levels are found in the upper epidermal layers in wound healing and psoriatic lesions. To investigate a link between meprin α expression and keratinocyte proliferation, we developed a mouse model for inducible expression of pathological meprin α levels (ie, K5Mα mice). K5Mα mice developed a skin phenotype characterized by hyperkeratosis, acanthosis, parakeratosis, and barrier defect. Keratinocyte hyperproliferation and local inflammation were induced upon induction of meprin α expression. By N-terminomics, we identified dermokine, a regulator of keratinocyte proliferation and epidermal immune response, as a putative substrate of meprin α. We validated the proteolysis and identified the cleavage site, which is highly conserved in mammals, suggesting that dermokine degradation by meprin α represents a central mechanism in wound healing and hyperproliferative skin diseases.

Systemic inflammatory response syndrome (SIRS) is a frequent critical condition in clinical patients marked by dysregulated immune activation and high mortality. Early initiation of appropriate interventions are important for patient outcome, but molecular markers for diagnosis are not SIRS-specific. We performed hematological analyses and health-status assessments on a transgenic disease mouse model that recapitulates elevated epidermal levels of the metalloprotease meprin α (K5Mα) reported in inflammatory skin diseases. In a cohort of intensive care patients that either developed SIRS (n = 19) or not (n = 29), we measured parameters associated with systemic inflammation and organ function as well as serum meprin α levels. K5Mα mice developed fatal SIRS characterized by hypothermia, severe weight loss, hypochromic microcytic anemia, neutrophilic leukocytosis and cytokine release syndrome. Serum concentrations of meprin α correlated with disease progression in K5Mα mice. We detected high meprin α levels in the serum of intensive care patients who developed SIRS but in none of the patients who did not develop SIRS. Serum meprin α levels significantly correlated with clinical parameters like C-reactive protein, procalcitonin and white blood cell count, but unlike all other measured inflammatory parameters allowed a clear identification of SIRS patients. We propose serum meprin α levels as a potential biomarker for SIRS. However, we would like to emphasize that due to our limited cohort size subsequent larger-scale, multicentered studies are warranted to validate our findings and potentially provide more detailed insight into whether there is an association between elevated meprin α serum levels and specific causes of SIRS or dysfunction of particular organ systems.

The emergence of Alzheimer's disease (AD) pathology has been the focus of multiple hypotheses, with amyloid β (Aβ) playing a central role due to its presence in both familial and sporadic AD. Therefore, a crucial aspect of AD research is understanding the generation of different Aβ species. Aβ peptides result from the proteolytic processing of Amyloid Precursor Protein (APP) by β- and γ-secretases, with BACE1 being the most prominent β-secretase. However, BACE1-overexpressing mouse models exhibit disadvantages, making them limited for AD research. Importantly, N-terminally truncated Aβ species, which constitute up to 70 % of Aβ in AD brains, are not generated by BACE1. In recent years, alternative proteases capable of cleaving APP have been identified, bridging the gap between N-terminally truncated Aβ species and BACE1-derived Aβ. Among these novel players, the metalloprotease meprin β has emerged as a risk factor in AD pathology, generating both N-terminally truncated and full-length Aβ species. Our primary objective was to develop a mouse model that more accurately resembles the pathology of AD beyond BACE1-overexpressing models, while simultaneously confirming APP cleavage of meprin β in the hippocampus and cerebral cortex. Overexpression of meprin β led to a marked increase in soluble Aβ levels, particularly in the hippocampus, indicating a higher vulnerability or elevated meprin β activity in this region compared to the cerebral cortex. Notably, this biochemical change occurred without any observable behavioral deficits, suggesting a region-specific role of meprin β in AD pathology that may extend beyond immediate functional impairment.

Keratinocyte proliferation and differentiation is regulated via proteolytic networks. Dysregulation of proteases within these networks can cause hyperproliferative and inflammatory skin disorders. In healthy skin the metalloprotease meprin α is localized in the stratum basale . In contrast, in wound healing tissue and psoriatic lesions increased meprin α levels are found in the upper epidermal layers. We developed a transgenic mouse model for inducible expression of pathological meprin α levels (K5Mα) to investigate its epidermal degradome and identify molecular links to keratinocyte proliferation and skin inflammation. K5Mα mice developed a severe skin phenotype characterized by hyperkeratosis, acanthosis and parakeratosis accompanied by increased transepidermal water loss and a strong inflammatory response within six days after induction of meprin α overexpression. Histological and molecular analyses showed that increasing meprin α expression correlates with meprin α activity and keratinocyte hyperproliferation. Proteomics analyses revealed massive changes in proteins associated with keratinocyte differentiation and epidermal barrier integrity already three days after induction. N-terminomics data indicated a dominant chymotryptic activity with elevated proteolytic turnover of proteins associated with the cytoskeleton, cellular stress responses and cell adhesion. By filtering for cleavage sites that match with the specificity of meprin α, we identified highly elevated dermokine-derived peptides. Subsequent mass spectrometric analyses validated dermokine as a novel substrate of meprin α and identified the cleavage site, which is highly conserved in mammals. Based on the striking similarities with the phenotype reported for dermokine αβγ-/- mice, we propose meprin α as a central regulator of keratinocyte proliferation and leukocyte recruitment by proteolytic inactivation of dermokine. Hence, pathological meprin α activity could be a driver of hyperproliferative, inflammatory skin disorders like psoriasis vulgaris. ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft

The metalloprotease meprin β is known for its multifunctional involvement in various physiological processes throughout the body including the brain. However, its broader functions within the brain besides amyloid β generation remain largely unexplored. To investigate this, we utilized a mouse model overexpressing meprin β in neurons within the cortex and hippocampus, regions crucial for learning and memory. Behavioral assessments, employing the Morris' Water Maze paradigm test, revealed impaired cognitive functions in animals overexpressing meprin β. Furthermore, electrophysiological recordings in hippocampal slices using multielectrode arrays showed an impaired long-term potentiation (LTP) in meprin β-overexpressing mice compared to wild-type counterparts. Intriguingly, concomitant with the LTP impairment, we observed an increased neuronal excitability. These findings underline the complicated interplay between meprin β abundance and behavioral manifestations, suggesting a broader impact on neural circuit dynamics. To elucidate the molecular mechanisms underlying these observed deficits, western blotting analyses were conducted to address the expression of glutamatergic receptors. Neither the expression of the N-methyl-D-aspartate (NMDA) nor the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor showed variation relative to each other. The application of N-terminomics identified brevican as a proteolytic substrate of meprin β and thus a potential key mediator linking meprin β overexpression to the observed effects. Previous studies have reported that brevican knockout in animal models influences learning and memory. Our data demonstrate that meprin β modulates brevican expression, likely contributing to the effects we have observed in our mouse model. These results shed light on the broader functional significance of meprin β in neurological processes.

OBJECTIVE:To fully understand why C-reactive protein (CRP) is usually only mildly elevated in active systemic lupus erythematosus (SLE), although interleukin-6 (IL-6) is increased, but is high in SLE patients with bacterial infections. METHODS:Sera and peripheral blood mononuclear cells (PBMCs) of SLE patients and healthy individuals were investigated. IL-6 and soluble IL-6 receptor (sIL-6R) were measured by ELISA. Membrane IL-6 receptor-α (CD126), gp130 (CD130) and signal transducer and activator of transcription 3 (STAT3) phosphorylation after IL-6 stimulation were analysed by flow cytometry. PBMCs and HepG2 liver cells were stimulated with various cytokines, and IL-6 receptor shedding was determined by immunoprecipitation and western blotting of supernatants. HEK-293 T cells were transfected with wild type CD126 or a shedding-resistant mutant, and soluble sIL-6R was measured following cytokine stimulation. RESULTS:While sIL-6R and IL-6 were increased with SLE activity, CD126 positive lymphocytes were decreased in SLE. IL-6 plus IFNα decreased CD126 on lymphocytes, but increased sIL-6R in their supernatant, detected by immunoprecipitation and western blotting. IL-6 plus IFNα on HepG2 liver cells resulted in a similar sIL-6R increase. Increased supernatant sIL-6R was seen in HEK-293 T cells transfected with wild type CD126, but not those transfected with the shedding-resistant mutant. STAT3 phosphorylation upon IL-6 stimulation was reduced. CONCLUSION:The combination of IL-6 and type I IFN induces shedding of CD126 to sIL-6R, shifting IL-6 signalling to trans-signalling. In situations of IL-6 excess over sIL-6R only, IL-6 or IL-6-sIL-6R complexes reach the liver and increase CRP. These findings not only explain the discrepancy in SLE, but also have implications for severe viral infection.

The metalloprotease meprin β is upregulated in neurons and astrocytes of Alzheimer’s disease patients’ brains. While the role of meprin β as the β-secretase of amyloid precursor protein (APP) has been characterized, its broader substrate profile within the brain remains largely unexplored. Hence, to identify additional substrates, we conducted N-terminomics of brain lysates from mice overexpressing meprin β in astrocytes employing the Hydrophobic Tagging-Assisted N-terminal Enrichment (HYTANE) strategy. We observed 3906 (82.2%) N-terminal peptides and identified seven new substrates that match meprin β in terms of localization and cleavage specificity. Of note, the meprin β overexpressing mice show mild cognitive impairments caused by amyloidogenic APP processing alongside hyperactivity and altered exploratory behavior seemingly independent of APP cleavage. Hence, latrophilin-3 was of particular interest, as latrophilin-3 defects are associated with hyperactivity in mice and human. In brain lysates from mice overexpressing meprin β in astrocytes as well as in cellulo, we validated the cleavage of latrophilin-3, resulting in the release of two N-terminal domains. These domains promote interactions with neuronal proteins such as fibronectin leucine-rich repeat transmembrane proteins, promoting adequate synapse formation. Thus, meprin β might affect synaptic integrity by cleaving interaction domains of latrophilin-3, potentially exacerbating the observed hyperactivity phenotype.

Several steps of cancer progression, from tumor onset to metastasis, critically involve proteolytic activity. To elucidate the role of proteases in cancer, it is particularly important to consider single-nucleotide variants (SNVs) that affect the active site of proteases, thereby influencing cleavage specificity, substrate processing, and thus cancer cell behavior. To facilitate systematic studies, we here present a targeted approach to determine the impact of cancer-associated protease variants (TACAP). Starting with the semiautomated identification of potential specificity-modulating SNVs, our workflow comprises mass spectrometry–based cleavage specificity profiling and substrate identification, localization, and inhibitor studies, followed by functional analyses investigating cancer cell properties. To demonstrate the feasibility of TACAP, we analyzed the meprin β R238Q variant. This amino acid exchange R238Q leads to a loss of meprin β’s characteristic cleavage preference for acidic amino acids at P1′ position, accompanied with changes in substrate pool and inhibitor affinity compared to meprin β wild type.

Proteolytic cell surface release ('shedding') of the prion protein (PrP), a broadly expressed GPI-anchored glycoprotein, by the metalloprotease ADAM10 impacts on neurodegenerative and other diseases in animal and in vitro models. Recent studies employing the latter also suggest shed PrP (sPrP) to be a ligand in intercellular communication and critically involved in PrP-associated physiological tasks. Although expectedly an evolutionary conserved event, and while soluble forms of PrP are present in human tissues and body fluids, for the human body neither proteolytic PrP shedding and its cleavage site nor involvement of ADAM10 or the biological relevance of this process have been demonstrated thus far. In this study, cleavage site prediction and generation (plus detailed characterization) of sPrP-specific antibodies enabled us to identify PrP cleaved at tyrosin 226 as the physiological and apparently strictly ADAM10-dependent shed form in humans. Using cell lines, neural stem cells and brain organoids, we show that shedding of human PrP can be stimulated by PrP-binding ligands without targeting the protease, which may open novel therapeutic perspectives. Site-specific antibodies directed against human sPrP also detect the shed form in brains of cattle, sheep and deer, hence in all most relevant species naturally affected by fatal and transmissible prion diseases. In human and animal prion diseases, but also in patients with Alzheimer`s disease, sPrP relocalizes from a physiological diffuse tissue pattern to intimately associate with extracellular aggregated deposits of misfolded proteins characteristic for the respective pathological condition. Findings and research tools presented here will accelerate novel insight into the roles of PrP shedding (as a process) and sPrP (as a released factor) in neurodegeneration and beyond.

Neurotoxic amyloid-β (Aβ) peptides cause neurodegeneration in Alzheimer’s disease (AD) patients’ brains. They are released upon proteolytic processing of the amyloid precursor protein (APP) extracellularly at the β-secretase site and intramembranously at the γ-secretase site. Several AD mouse models were developed to conduct respective research in vivo. Most of these classical models overexpress human APP with mutations driving AD-associated pathogenic APP processing. However, the resulting pattern of Aβ species in the mouse brains differs from those observed in AD patients’ brains. Particularly mutations proximal to the β-secretase cleavage site (e.g., the so-called Swedish APP (APPswe) fostering Aβ1-x formation) lead to artificial Aβ production, as N-terminally truncated Aβ peptides are hardly present in these mouse brains. Meprin β is an alternative β-secretase upregulated in brains of AD patients and capable of generating N-terminally truncated Aβ2-x peptides. Therefore, we aimed to generate a mouse model for the production of so far underestimated Aβ2-x peptides by conditionally overexpressing meprin β in astrocytes. We chose astrocytes as meprin β was detected in this cell type in close proximity to Aβ plaques in AD patients’ brains. The meprin β-overexpressing mice showed elevated amyloidogenic APP processing detected with a newly generated neo-epitope-specific antibody. Furthermore, we observed elevated Aβ production from endogenous APP as well as AD-related behavior changes (hyperlocomotion and deficits in spatial memory). The novel mouse model as well as the established tools and methods will be helpful to further characterize APP cleavage and the impact of different Aβ species in future studies.

The metalloproteases meprin α and meprin β are highly expressed in the healthy gut but significantly decreased in inflammatory bowel disease, implicating a protective role in mucosal homeostasis. In the colon, meprin α and meprin β form covalently linked heterodimers tethering meprin α to the plasma membrane, therefore presenting dual proteolytic activity in a unique enzyme complex. To unravel its function, we applied N-terminomics and identified galectin-3 as the major intestinal substrate for meprin α/β heterodimers. Galectin-3–deficient and meprin α/β double knockout mice show similar alterations in their microbiome in comparison to wild-type mice. We further demonstrate that meprin α/β heterodimers differentially process galectin-3 upon bacterial infection, in germ-free, conventionally housed (specific pathogen–free), or wildling mice, which in turn regulates the bacterial agglutination properties of galectin-3. Thus, the constitutive cleavage of galectin-3 by meprin α/β heterodimers may play a key role in colon host-microbiome homeostasis.

Since the discovery of meprins 40 years ago, significant progress has been made in understanding their roles in health and disease. Particularly the application of proteomic techniques for identification of substrates and cleavage site specificity, combined with the use of meprin knock-out mice have contributed to the understanding of the physiological and pathological functions of these fascinating proteases. Now, it is well known that meprin α and meprin β play important roles in inflammation, cancer, neurodegeneration, and fibrosis.Therefore, targeted inhibition of meprins might be a suitable therapeutic application to treat associated diseases.

The cell surface receptor cluster of differentiation 44 (CD44) is the main hyaluronan receptor of the human body. At the cell surface, it can be proteolytically processed by different proteases and was shown to interact with different matrix metalloproteinases. Upon proteolytic processing of CD44 and generation of a C-terminal fragment (CTF), an intracellular domain (ICD) is released after intramembranous cleavage by the γ-secretase complex. This intracellular domain then translocates to the nucleus and induces transcriptional activation of target genes. In the past CD44 was identified as a risk gene for different tumor entities and a switch in CD44 isoform expression towards isoform CD44s associates with epithelial to mesenchymal transition (EMT) and cancer cell invasion. Here, we introduce meprin β as a new sheddase of CD44 and use a CRISPR/Cas9 approach to deplete CD44 and its sheddases ADAM10 and MMP14 in HeLa cells. We here identify a regulatory loop at the transcriptional level between ADAM10, CD44, MMP14 and MMP2. We show that this interplay is not only present in our cell model, but also across different human tissues as deduced from GTEx (Gene Tissue Expression) data. Furthermore, we identify a close relation between CD44 and MMP14 that is also reflected in functional assays for cell proliferation, spheroid formation, migration and adhesion.

BMP-1/tolloid-like proteinases (BTPs) are major players in tissue morphogenesis, growth and repair. They act by promoting the deposition of structural extracellular matrix proteins and by controlling the activity of matricellular proteins and TGF-β superfamily growth factors. They have also been implicated in several pathological conditions such as fibrosis, cancer, metabolic disorders and bone diseases. Despite this broad range of pathophysiological functions, the putative existence of a specific endogenous inhibitor capable of controlling their activities could never be confirmed. Here, we show that procollagen C-proteinase enhancer-2 (PCPE-2), a protein previously reported to bind fibrillar collagens and to promote their BTP-dependent maturation, is primarily a potent and specific inhibitor of BTPs which can counteract their proteolytic activities through direct binding. PCPE-2 therefore differs from the cognate PCPE-1 protein and extends the possibilities to fine-tune BTP activities, both in physiological conditions and in therapeutic settings.

Cardiometabolic diseases, such as type 2 diabetes and cardiovascular disease, have a high public health burden. Understanding the genetically determined regulation of proteins that are dysregulated in disease can help to dissect the complex biology underpinning them. Here, we perform a protein quantitative trait locus (pQTL) analysis of 248 serum proteins relevant to cardiometabolic processes in 2893 individuals. Meta-analyzing whole-genome sequencing (WGS) data from two Greek cohorts, MANOLIS (n = 1356; 22.5× WGS) and Pomak (n = 1537; 18.4× WGS), we detect 301 independently associated pQTL variants for 170 proteins, including 12 rare variants (minor allele frequency < 1%). We additionally find 15 pQTL variants that are rare in non-Finnish European populations but have drifted up in the frequency in the discovery cohorts here. We identify proteins causally associated with cardiometabolic traits, including Mep1b for high-density lipoprotein (HDL) levels, and describe a knock-out (KO) Mep1b mouse model. Our findings furnish insights into the genetic architecture of the serum proteome, identify new protein-disease relationships and demonstrate the importance of isolated populations in pQTL analysis.

The gastric microbiome and inflammation play a key role in gastric cancer (GC) by regulating the immune response in a complex manner and by inflammatory events supporting carcinogenesis. Meprin β is a zinc endopeptidase and participates in tissue homeostasis, intestinal barrier function and immunological processes. It influences local inflammatory processes, dysbiosis and the microbiome. Here, we tested the hypothesis that meprin β is expressed in GC and of tumor biological significance. Four hundred forty whole mount tissue sections of patients with therapy-naive GC were stained with an anti-meprin β antibody. The histoscore and staining pattern were analyzed for each case. Following dichotomization at the median histoscore into a “low” and “high” group, the expression was correlated with numerous clinicopathological patient characteristics. Meprin β was found intracellularly and at the cell membrane of GC. Cytoplasmic expression correlated with the phenotype according to Lauren, microsatellite instability and PD-L1 status. Membranous expression correlated with intestinal phenotype, mucin-1-, E-cadherin-, β-catenin status, mucin typus, microsatellite instability, KRAS mutation and PD-L1-positivity. Patients with cytoplasmic expression of meprin β showed a better overall and tumor-specific survival. Meprin β is differentially expressed in GC and has potential tumor biological relevance. It might function as a tumor suppressor or promotor depending on histoanatomical site and context.

Alzheimer's disease (AD) is the most common form of dementia, however incurable so far. It is widely accepted that aggregated amyloid β (Aβ) peptides play a crucial role for the pathogenesis of AD, as they cause neurotoxicity and deposit as so-called Aβ plaques in AD patient brains. Aβ peptides derive from the amyloid precursor protein (APP) upon consecutive cleavage at the β- and γ-secretase site. Hence, mutations in the APP gene are often associated with autosomal dominant inherited AD. Almost thirty years ago, two mutations at the β-secretase site were observed in two Swedish families (termed Swedish APP (APPswe) mutations), which led to early-onset AD. Consequently, APPswe was established in almost every common AD mouse model, as it contributes to early Aβ plaque formation and cognitive impairments. Analyzing these APPswe-based mouse models, the aspartyl protease BACE1 has been evolving as the prominent β-secretase responsible for Aβ release in AD and as the most important therapeutic target for AD treatment. However, with respect to β-secretase processing, the very rare occurring APPswe variant substantially differs from wild-type APP. BACE1 dominates APPswe processing resulting in the release of Aβ1-x, whereas N-terminally truncated Aβ forms are scarcely generated. However, these N-terminally truncated Aβ species such as Aβ2-x, Aβ3-x and Aβ4-x are elevated in AD patient brains and exhibit an increased potential to aggregate compared to Aβ1-x peptides. Proteases such as meprin β, cathepsin B and ADAMTS4 were identified as alternative β-secretases being capable of generating these N-terminally truncated Aβ species from wild-type APP. However, neither meprin β nor cathepsin B are capable of generating N-terminally truncated Aβ peptides from APPswe. Hence, the role of BACE1 for the Aβ formation during AD might be overrepresented through the excessive use of APPswe mouse models. In this review we critically discuss the consideration of BACE1 as the most promising therapeutic target. Shifting the focus of AD research towards alternative β secretases might unveil promising alternatives to BACE1 inhibitors constantly failing in clinical trials due to ineffectiveness and harmful side effects.

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.