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We previously reported that five astacin-like metalloproteases with thrombospondin type-1 repeats (Tasts) located on the sperm surface are a promising candidate as the protease involved in sperm penetration of the vitelline coat (VC) during fertilization of the ascidian Ciona intestinalis type A (Phlebobranchia). However, whether such a protease is involved in the fertilization of other ascidians is unknown. Here, we investigated the effects of four metalloprotease inhibitors on the fertilization of the ascidian Halocynthia roretzi (Stolidobranchia). Three metalloprotease inhibitors, GM6001, TAPI-0, and TAPI-1, strongly inhibited fertilization at 33 and 11 μM, whereas TAPI-2 weakly inhibited fertilization at 33 μM. In contrast, GM6001NC (negative control) had no effect on fertilization at 100 μM. Furthermore, GM6001 had no inhibitory effect on the fertilization of VC-deprived eggs. The metalloprotease appears to function at the middle or late stage of fertilization. Ten Tast genes were identified in the H. roretzi genome database, among which four genes (HrTast1, HrTast2b, HrTast2c, and HrTast3c) possessed a single transmembrane domain in the N-terminal region. These four genes are transcribed in the testis and ovary, as revealed by RT-PCR. Anti-HrTast2c IgG raised against a peptide corresponding to the Zn-binding consensus sequence weakly inhibited fertilization at 0.5 mg/mL. These results led us to propose that sperm astacin-like metalloproteases may be involved in sperm penetration of the VC during H. roretzi fertilization. [ABSTRACT FROM AUTHOR]

BACKGROUND: Plutella xylostella (Linnaeus) is a destructive pest of cruciferous crops due to its strong reproductive capacity and extensive resistance to pesticides. Seminal fluid proteins (SFPs) are the main effective factors that determine the reproductive physiology and behaviour of both sexes. Although an increasing number of SFPs have been identified, the effects of astacins in SFPs on agricultural pests have not yet been reported. Here, we elucidated the mechanisms by which Sast1 (seminal astacin 1) regulates the fertility of Plutella xylostella (L.). RESULTS: PxSast1 was specifically expressed in the testis and accesssory gland. CRISPR/Cas9‐induced PxSast1 knockout successfully constructed two homozygous mutant strains. Sast1 impaired the fertility of P. xylostella by separately regulating the reproductive capacity of males and females. Loss of PxSast1, on the one hand, significantly decreased the ability of males to mate and fertilize, mainly manifested as shortened mating duration, reduced mating competitiveness and decreased eupyrene sperm production; on the other hand, it significantly inhibited the expression of chorion genes in females, resulting in oogenesis deficits. Simultaneously, for mated females, the differentially expressed genes in signalling pathways related to oogenesis and chorion formation were significantly enriched after PxSast1 knockout. CONCLUSION: These analyses of the functions of PxSast1 as the regulator of spermatogenesis and oogenesis establish its importance in the fertility process of P. xylostella, as well as its potential as a promising target for genetic regulation‐based pest control. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

During the evolution of astacin metalloprotease family genes, gene duplication occurred, especially in the lineage of teleosts, in which several types of astacins containing six conserved cysteines (c6ast) emerged. One of them is patristacin, originally found in syngnathid fishes, such as pipefishes and seahorses. Patristacin is expressed in the brood pouch and is present on the same chromosome as other c6ast (pactacin and nephrosin) genes. We first surveyed all the genes from 33 teleost species using a genome database, and characterized the genes by phylogenetic analysis. Pactacin and nephrosin gene homologs were found from all the examined species with only few exceptions, while patristacin gene homologs were found from only several lineages. The patristacin gene homologs were found as multicopy genes in most species of Percomorpha, one of the diverged groups in teleosts. Further diversification of the gene occurred during the evolution of Atherinomorphae, one of the groups in Percomorpha. Fishes of Atherinomorphae possess two types of patristacin, belonging to subclades 1 and 2. Among the Atherinomorpha, we chose the southern platyfish to examine the patristacin gene expression. Platyfish possess eight patristacin gene homologs, called XmPastn1, 2, 3, 4, 5, 7, 10, and 11. Of these genes, only XmPastn2 belongs to subclade 1, while the other seven belong to subclade 2. Only XmPastn2 showed strong expression in several organs of adult platyfish, as observed in reverse‐transcription polymerase chain reaction of RNA extracts. Cells expressing XmPastn2 were predominantly mucus‐secreting cells found in epidermis around the jaw, as revealed by in‐situ hybridization. This result suggests that XmPastn2 is secreted and may contribute to mucus formation or secretion. Research highlights: Neprhosin and pactacin genes were already established in Euteleostei. However, the patristacin gene appeared during the evolution of Euteleostei, and further diversification into two groups (subclades 1 and 2) occurred during evolution. [ABSTRACT FROM AUTHOR]

Bone morphogenetic protein 1 (BMP1), a member of the astacin family of zinc‐metalloproteases, proteolytically cleaves the low‐density lipoprotein receptor (LDLR) within its ligand‐binding domain, reducing the binding and cellular uptake of LDL‐cholesterol. Here, we aimed to determine whether astacin proteases other than BMP1 may also cleave LDLR. Although human hepatocytes express all six astacin proteases, including the meprins and mammalian tolloid, we found through pharmacological inhibition and genetic knockdown that only BMP1 contributed to the cleavage of LDLR in its ligand‐binding domain. We also found that the minimum amino acid change required to render mouse LDLR susceptible to cleavage by BMP1 is mutation at the P1′ and P2 positions of the cleavage site. When expressed in cells, the resulting humanised‐mouse LDLR internalised LDL‐cholesterol. This work provides insight into the biological mechanisms regulating LDLR function. [ABSTRACT FROM AUTHOR]

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.

[Display omitted] • The astacin domain is involved in 145 unique domain architectures in helminths. • The astacin domain is linked to numerous roles related to the parasitic lifestyle. • Module recombination indicates convergent evolution in independent nematode taxa. • Specific key residues suggest substrate adaptation in astacin subfamilies. • Parasite astacin studies should consider functional astacin specialisation. Helminths secrete a plethora of proteins involved in parasitism-related processes such as tissue penetration, migration, feeding and immunoregulation. Astacins, a family of zinc metalloproteases belonging to the peptidase family M12, are one of the most abundantly represented protein families in the secretomes of helminths. Despite their involvement in virulence, very few studies have addressed the role of this loosely defined protein group in parasitic helminths. Herein, we have analysed the predicted proteomes from 154 helminth species and confirmed the expansion of the astacin family in several nematode taxa. The astacin domain associated with up to 110 other domains into 145 unique domain architectures, where CUB and ShK constitute the principal and nearly independent bi-domain frameworks. The presence of co-existing domains suggests promiscuous adaptable functions to several roles. These activities could be related either to substrate specificity or to higher-order functions, such as anti-angiogenesis and immunomodulation, where the astacin domain would play an accessory role. Furthermore, some phylogenetically restricted mutations in the astacin domain affected residues located at the active cleft and binding sub-pockets, suggesting adaptation to different substrate specificities. Altogether, these findings suggest the astacin domain is a highly adaptable module that fulfils multiple proteolytic needs of the parasitic lifestyle. This study contributes to the understanding of helminth-secreted astacins and, ultimately, provides the foundation to guide future investigations about the role of this diverse family of proteins in host–parasite interactions. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

BMP‐1/tolloid‐like proteinases belong to the astacin family of human metalloproteinases, together with meprins and ovastacin. They represent promising targets to treat or prevent a wide range of diseases such as fibrotic disorders or cancer. However, the study of their pathophysiological roles is still impaired by the lack of well‐characterized inhibitors and the questions that remain regarding their selectivity and in vivo efficiency. As a first step towards the identification of suitable tools to be used in functional studies, we have undertaken a systematic comparison of seven molecules known to affect the proteolytic activity of human astacins including three hydroxamates (FG‐2575, UK383,367, S33A), the protein sizzled, a new phosphinic inhibitor (RXP‐1001) and broad‐spectrum protease inhibitors (GM6001, actinonin). Their efficacy in vitro, their cellular toxicity and efficacy in cell cultures were thoroughly characterized. We found that these molecules display very different potency and selectivity profiles, with hydroxamate FG‐2575 and the protein sizzled being very powerful and selective inhibitors of BMP‐1, whereas phosphinic peptide RXP‐1001 behaves as a broad‐spectrum inhibitor of astacins. Their use should therefore be carefully considered in agreement with the aim of the study to avoid result misinterpretation.

A crucial step for tumor cell extravasation and metastasis is the migration through the extracellular matrix, which requires proteolytic activity. Hence, proteases, particularly matrix metalloproteases (MMPs), have been discussed as therapeutic targets and their inhibition should diminish tumor growth and metastasis. The metalloproteases meprin α and meprin β are highly abundant on intestinal enterocytes and their expression was associated with different stages of colorectal cancer. Due to their ability to cleave extracellular matrix (ECM) components, they were suggested as pro-tumorigenic enzymes. Additionally, both meprins were shown to have pro-inflammatory activity by cleaving cytokines and their receptors, which correlates with chronic intestinal inflammation and associated conditions. On the other hand, meprin β was identified as an essential enzyme for the detachment and renewal of the intestinal mucus, important to prevent bacterial overgrowth and infection. Considering this, it is hard to estimate whether high activity of meprins is generally detrimental or if these enzymes have also protective functions in certain cancer types. For instance, for colorectal cancer, patients with high meprin β expression in tumor tissue exhibit a better survival prognosis, which is completely different to prostate cancer. This demonstrates that the very same enzyme may have contrary effects on tumor initiation and growth, depending on its tissue and subcellular localization. Hence, precise knowledge about proteolytic enzymes is required to design the most efficient therapeutic options for cancer treatment. In this review, we summarize the current findings on meprins' functions, expression, and cancer-associated variants with possible implications for tumor progression and metastasis. [ABSTRACT FROM AUTHOR]

BMP‐1/tolloid‐like proteinases belong to the astacin family of human metalloproteinases, together with meprins and ovastacin. They represent promising targets to treat or prevent a wide range of diseases such as fibrotic disorders or cancer. However, the study of their pathophysiological roles is still impaired by the lack of well‐characterized inhibitors and the questions that remain regarding their selectivity and in vivo efficiency. As a first step towards the identification of suitable tools to be used in functional studies, we have undertaken a systematic comparison of seven molecules known to affect the proteolytic activity of human astacins including three hydroxamates (FG‐2575, UK383,367, S33A), the protein sizzled, a new phosphinic inhibitor (RXP‐1001) and broad‐spectrum protease inhibitors (GM6001, actinonin). Their efficacy in vitro, their cellular toxicity and efficacy in cell cultures were thoroughly characterized. We found that these molecules display very different potency and selectivity profiles, with hydroxamate FG‐2575 and the protein sizzled being very powerful and selective inhibitors of BMP‐1, whereas phosphinic peptide RXP‐1001 behaves as a broad‐spectrum inhibitor of astacins. Their use should therefore be carefully considered in agreement with the aim of the study to avoid result misinterpretation. BMP‐1/tolloid‐like proteinases (BTPs) are prominent members of the astacin subgroup of metalloproteinases which are currently evaluated as therapeutic targets in several diseases. We report a detailed analysis of seven potential BTP inhibitors, both synthetic and natural molecules, in terms of potency, toxicity and selectivity towards meprins. This study emphasises the importance of carefully choosing the best tool for each application. [ABSTRACT FROM AUTHOR]

Planarian flatworms possess extraordinary regenerative capability and body plasticity, which rely on a composite population of stem cells, the neoblasts. Despite impressive advances have been recently achieved in the knowledge of neoblast biology, few is still known about factors that are released by differentiated tissues and influence the neoblast fate. Extracellular matrix (ECM) is a fundamental component of the stem cell niche and its remodeling affects stem cell fate. Here we provide the characterization of the astacin gene family of metalloproteinases in planarians, good candidate enzymes for generating dynamicity in the ECM. Ten and eighteen astacin isoforms were identified in the planarian species Schmidtea mediterranea and Dugesia japonica, respectively. Besides the already characterized Smedolloid, in Schmidtea mediterranea are present eight astacins with a minimal structure (a signal peptide, an activation domain and a Zn-binding catalytic domain), that are colocalized in large cells organized in a peculiar, not yet morphologically characterized, two-ring-shaped structure located in the middle of the body. A single astacin, characterized by a ShK toxin domain in its C-terminal region, has been found to be produced in gastrodermal cells. [ABSTRACT FROM AUTHOR]

Proteins of the ShK superfamily are characterized by a small conserved domain (ShKT), first discovered in small venom peptides produced by sea anemones, and acting as specific inhibitors of voltage-dependent and calcium-activated K+ channels. The ShK superfamily includes both small toxic peptides and larger multifunctional proteins with various functions. ShK toxins are often important components of animal venoms, where they perform different biological functions including neurotoxic and immunosuppressive effects. Given their high specificity and effectiveness, they are currently regarded as promising pharmacological lead compounds for the treatment of autoimmune diseases. Here, we report on the molecular analysis of ShKT domain containing proteins produced by the Mediterranean vampire snail Cumia reticulata, an ectoparasitic gastropod that feeds on benthic fishes. The high specificity of expression of most ShK transcripts in salivary glands identifies them as relevant components of C. reticulata venom. These ShK proteins display various structural architectures, being produced either as single-domain secretory peptides, or as larger proteins combining the ShKT with M12 or CAP domains. Both ShKT-containing genes and their internal ShKT domains undergo frequent duplication events in C. reticulata, ensuring a high level of variability that is likely to play a role in increasing the range of their potential molecular targets.

Background: Spiders are known for their predatory efficiency and for their high capacity of digesting relatively large prey. They do this by combining both extracorporeal and intracellular digestion. Whereas many high throughput ("-omics") techniques focus on biomolecules in spider venom, so far this approach has not yet been applied to investigate the protein composition of spider midgut diverticula (MD) and digestive fluid (DF). Results: We here report on our investigations of both MD and DF of the spider Nephilingis (Nephilengys) cruentata through the use of next generation sequencing and shotgun proteomics. This shows that the DF is composed of a variety of hydrolases including peptidases, carbohydrases, lipases and nuclease, as well as of toxins and regulatory proteins. We detect 25 astacins in the DF. Phylogenetic analysis of the corresponding transcript(s) in Arachnida suggests that astacins have acquired an unprecedented role for extracorporeal digestion in Araneae, with different orthologs used by each family. The results of a comparative study of spiders in distinct physiological conditions allow us to propose some digestion mechanisms in this interesting animal taxon. Conclusion: All the high throughput data allowed the demonstration that DF is a secretion originating from the MD. We identified enzymes involved in the extracellular and intracellular phases of digestion. Besides that, data analyses show a large gene duplication event in Araneae digestive process evolution, mainly of astacin genes. We were also able to identify proteins expressed and translated in the digestive system, which until now had been exclusively associated to venom glands. [ABSTRACT FROM AUTHOR]

Loxosceles spiders' venom comprises a complex mixture of biologically active toxins, mostly consisting of low molecular mass components (2–40 kDa). Amongst, isoforms of astacin-like metalloproteases were identified through transcriptome and proteome analyses. Only LALP1 ( Loxosceles Astacin-Like protease 1) has been characterized. Herein, we characterized LALP3 as a novel recombinant astacin-like metalloprotease isoform from Loxosceles intermedia venom. LALP3 cDNA was cloned in pET-SUMO vector, and its soluble heterologous expression was performed using a SUMO tag added to LALP3 to achieve solubility in Escherichia coli SHuffle T7 Express LysY cells, which express the disulfide bond isomerase DsbC. Protein purification was conducted by Ni-NTA Agarose resin and assayed for purity by SDS-PAGE under reducing conditions. Immunoblotting analyses were performed with specific antibodies recognizing LALP1 and whole venom. Western blotting showed linear epitopes from recombinant LALP3 that cross-reacted with LALP1, and dot blotting revealed conformational epitopes with native venom astacins. Mass spectrometry analysis revealed that the recombinant expressed protein is an astacin-like metalloprotease from L. intermedia venom. Furthermore, molecular modeling of LALP3 revealed that this isoform contains the zinc binding and Met-turn motifs, forming the active site, as has been observed in astacins. These data confirmed that LALP3, which was successfully obtained by heterologous expression using a prokaryote system, is a new astacin-like metalloprotease isoform present in L. intermedia venom. [ABSTRACT FROM AUTHOR]

Body color is an important quality and determines the value of koi. Fish are unable to fully synthesize carotenoid pigments. Therefore, carotenoid needs to be added to their diets. This study determined the effect of astacin on the growth and body color enhancement in healthy red-white ornamental carp (Cyprinus carpio var. koi). Fish were fed diets containing 0 (control), 50, 100, 150, 200, or 250 mg astacin/kg diet for 8 weeks. At the end of the experiment, samples of red-white, red, and white skin, head, scales, and fin ray were collected for carotenoid content analysis and visual observation. Fish weight and specific growth rate were significantly higher in fish fed the 150, 200, and 250 mg diets than in fish fed the control while the feed conversion rates in the 200 and 250 groups were significantly lower than in the control. Carotenoid deposition in the skin, head, scale, and fin ray of fish fed diets containing astacin was significantly higher than that those fed the control. In addition, a higher astacin content resulted in a brighter body color. The highest carotenoid concentration was achieved when 250 mg astacin/kg feed was added. Astacin was mainly deposited in the skin, scales, and head. [ABSTRACT FROM AUTHOR]

Abstract: Astacin, acting as the prototype of astacin family and of the metzincin superfamily, is a mononuclear zinc enzyme that catalyzes the hydrolysis of polypeptides and proteins. In the present article, the reaction mechanism of astacin has been investigated using density functional theory (DFT) method. Using a model of the active site constructed on the basis of the X-ray crystal structure of astacin, the potential energy surface for catalytic reaction has been mapped and the transition states and intermediates along the reaction pathway are characterized. The calculations give general support to the previously proposed mechanism by experiments, which mainly involve nucleophilic attack on carbonyl group of substrate and C―N bond cleavage, and reveal more detailed mechanistic features. The Glu93 functions as a crucial general base to activate the zinc-bound water and the resulting zinc-bound hydroxide acts as the real nucleophile. It is demonstrated that there exists a “reactant region”, where the interconversion between zinc-bound water and zinc-bound hydroxide occurs rapidly. The rate-limiting step is predicted to be the nucleophilic attack, which leads to an anionic gem-diolate tetrahedral intermediate. During the catalysis, zinc ion provides main catalytic power by stabilizing the developing charge of tetrahedral intermediate, while the Tyr149 residue also partially contributes to the catalysis by stabilizing the anionic intermediate. In addition, it is shown that the Cys64 plays roles in assisting in binding and orientating the substrate via electrostatic interaction. [Copyright &y& Elsevier]

The article discusses a study which found that astacin proteases cleave dentin sialophosphoprotein (Dspp) to generate dentin phosphoprotein (Dpp). The researchers incubated fluorescence resonance energy transfer (FRET) peptides containing the amino acid context of the Dpp cleavage site or a mutant version of that context. They isolated Dspp proteoglycan from dentin power and incubated it with the three enzymes that cleaved Dspp-FRET at the G-D bond. Findings revealed that astacins are expressed by odontoblasts as shown by the developing porcine molars.

The metalloproteases meprin α and β are expressed in several tissues, leukocytes, and cancer cells. In skin, meprins are located in separate layers of human epidermis indicating distinct physiological functions, supported by effects on cultured keratinocytes. Meprin β induces a dramatic change in cell morphology and a significant reduction in cell number, whereas in vitro evidence suggests a role for meprin α in basal keratinocyte proliferation. Meprins are secreted as zymogens that are activated by tryptic proteolytical processing. Here, we identify human kallikrein-related peptidases (KLKs) 4, 5, and 8 to be specific activators of meprins. KLK5 is capable of activating both metalloproteases. Interestingly, KLK4 and 8 cleave off the propeptide of meprin β only, whereas in contrast plasmin exclusively transforms meprin α to its mature form. Moreover, we show that proKLK7 is processed by meprins. N-terminal sequencing revealed cleavage by meprin β two amino acids N-terminal to mature KLK7. Interestingly, this triggering led to an accelerated activation of the serine protease in the presence of trypsin, but not of other tryptic KLKs, such as KLK2, 4, 5, 8, or 11. In summary, we demonstrate a specific interaction between meprin metalloproteases and kallikrein-related peptidases, revealing possible interactions within the proteolytic web. [ABSTRACT FROM AUTHOR]

The review deals with the properties of astacin family of zinc-dependent metalloproteinases. These enzymes exhibit arylamidase activity, which is not typical for metalloproteinases. Special attention is paid to physiological functions of the astacin proteinases and to the influence of domain composition and posttranslational modifications on the activity and stability of these enzymes. [ABSTRACT FROM AUTHOR]

Abstract: In this work, we report the cloning, heterologous expression, and characterization of two novel astacin proteases from the chelicerate Limulus polyphemus (horseshoe crab), designated as LAST (Limulus astacin) and LAST_MAM (Limulus astacin containing a MAM domain), respectively. The expression pattern showed ubiquitous occurrence of LAST_MAM, while LAST was predominantly restricted to the eyes and brain, indicating a function in the nervous system. Both enzymes contain the characteristic metzincin-type zinc-binding region and Met turn. While LAST is made up only of the typical prodomain and astacin-like protease domain, LAST_MAM contains an additional MAM (meprin A5 protein tyrosine phosphatase μ) domain, which so far only has been found in few astacins such as the vertebrate meprin Hydra and squid enzymes, and in a number of other extracellular proteins such as A5 protein and tyrosine phosphatase μ. These gave rise to the designation MAM for this protein module. MAM domains have been shown to be responsible for protein oligomerization in meprin proteases and tyrosine phosphatase μ. Since the horseshoe crab has kept its body plan for almost half a billion years, it is therefore a privileged organism for the study of protease evolution. In this context, we could show by phylogenetic analysis that this protease is not related to the other MAM-domain-containing astacins indicating different evolutionary origins of these proteins. Moreover, we clearly demonstrated the divergent evolvement of the MAM module itself, and not only with regard to proteases. However, there are some unique functional features that are not shared by other members of this protein family. For example, LAST_MAM is the only astacin protease known so far that is active in its zymogen form, indicating that the presence of the N-terminal propeptide does not prevent proteolytic activity. [Copyright &y& Elsevier]

Abstract: Procollagen C-peptidase, also known as bone morphogenetic protein 1 (BMP-1), is a multidomain, zinc endopeptidase of the astacin M12A family. BMP-1 is the prototype of a small group of proteases that have key roles in extracellular matrix formation and morphogenesis. BMP-1, its splice form mTLD, and the related proteases TLL-1 and TLL-2 are considered as promising drug targets for the treatment of excessive fibrosis and muscle wasting. We report here the crystal structures of the protease domains of human BMP-1 and the closely related Tolloid-like protease 1 (TLL-1). The crystal structures reveal an unexpected conformation of a cysteine-rich loop within the active site, and suggest that a flap movement is required in order to allow substrate binding. On the basis of these substantial differences between the BMP-1 and astacin active sites, a structural basis for their differing substrate specificities is proposed. [Copyright &y& Elsevier]

Abstract: The astacins are a subfamily of the metzincin superfamily of metalloproteinases. The first to be characterized was the crayfish enzyme astacin. To date more than 200 members of this family have been identified in species ranging from bacteria to humans. Astacins are involved in developmental morphogenesis, matrix assembly, tissue differentiation and digestion. Family members include the procollagen C-proteinase (BMP1, bone morphogenetic protein 1), tolloid and mammalian tolloid-like, HMP (Hydra vulgaris metalloproteinase), sea urchin BP10 (blastula protein) and SPAN (Strongylocentrotus purpuratus astacin), the ‘hatching’ subfamily comprising alveolin, ovastacin, LCE, HCE (‘low’ and ‘high’ choriolytic enzymes), nephrosin (from carp head kidney), UVS.2 from frog, and the meprins. In the human and mouse genomes, there are six astacin family genes (two meprins, three BMP1/tolloid-like, one ovastacin), but in Caenorhabditis elegans there are 40. Meprins are the only astacin proteinases that function on the membrane and extracellularly by virtue of the fact that they can be membrane-bound or secreted. They are unique in their domain structure and covalent subunit dimerization, oligomerization propensities, and expression patterns. They are normally highly regulated at the transcriptional and post-translational levels, localize to specific membranes or extracellular spaces, and can hydrolyse biologically active peptides, cytokines, extracellular matrix (ECM) proteins and cell-surface proteins. The in vivo substrates of meprins are unknown, but the abundant expression of these proteinases in the epithelial cells of the intestine, kidney and skin provide clues to their functions. [Copyright &y& Elsevier]

Human growth and development are conditioned by insulin-like growth factors (IGFs), which have also implications in pathology. Most IGF molecules are sequestered by IGF-binding proteins (IGFBPs) so that exertion of IGF activity requires disturbance of these complexes. This is achieved by proteolysis mediated by IGFBP proteases, among which the best characterised is human PAPP-A, the first member of the pappalysin family of metzincins. We have previously identified and studied the only archaeal homologue found to date, Methanosarcina acetivorans ulilysin. This is a proteolytically functional enzyme encompassing a pappalysin catalytic domain and a pro-domain involved in maintenance of latency of the zymogen, proulilysin. Once activated, the protein hydrolyses IGFBP-2 to -6 and insulin chain β in vitro. We report here that ulilysin is also active against several other substrates, viz (azo)casein, azoalbumin, and extracellular matrix components. Ulilysin has gelatinolytic but not collagenolytic activity. Moreover, the proteolysis-resistant skeletal proteins actin and elastin are also cleaved, as is fibrinogen, but not plasmin and α1-antitrypsin from the blood coagulation cascade. Ulilysin develops optimal activity at pH 7.5 and strictly requires peptide bonds preceding an arginine residue, as determined by means of a novel fluorescence resonance energy transfer assay, thus pointing to biotechnological applications as an enzyme complementary to trypsin. [ABSTRACT FROM AUTHOR]

Meprins are members of the astacin family of metalloproteases expressed in epithelial tissues, intestinal leukocytes and certain cancer cells. In mammals, there are two homologous subunits, which form complex glycosylated disulfide-bonded homo- and heterooligomers. Both human meprin α and meprin β cleave several basement membrane components, suggesting a role in epithelial differentiation and cell migration. There is also evidence that meprin β is involved in immune defence owing to its capability of activating interleukin-1β and the diminished mobility of intestinal leukocytes in meprin β-knockout mice. Here we show for the first time by reverse transcription PCR, immunoblotting and immunofluorescence analyses that meprins are expressed not only in mammals, but also in the zebrafish Danio rerio. In contrast to the human, mouse and rat enzymes, zebrafish meprins are encoded by three genes, corresponding to two homologous α subunits and one β subunit. Observations at both the mRNA and protein level indicate a broad distribution of meprins in zebrafish. However, there are strikingly different expression patterns of the three subunits, which is consistent with meprin expression in mammals. Hence, D. rerio appears to be a suitable model to gain insight into the basic physiological functions of meprin metalloproteases. [ABSTRACT FROM AUTHOR]

Procollagen C-proteinase (PCP) removes the C-terminal pro-peptides of procollagens and also processes other matrix proteins. The major splice form of the PCP is termed BMP1 (bone morphogenetic protein 1). Active BMP1 is composed of an astacin-like protease domain, three CUB (complement, sea urchin Uegf, BMP1) domains and one EGF-like domain. Here we compare the recombinant human full-length BMP1 with its isolated proteolytic domain to further unravel the functional influence of the CUB and EGF domains. We show that the protease domain alone cleaves truncated procollagen VII within the short telopeptide region into fragments of similar size as the full-length enzyme does. However, unlike full-length BMP1, the protease domain does not stop at this point, but degrades its substrate completely. Moreover, the protease domain cleaves other matrix proteins such as fibronectin, collagen I and collagen IV, which are left intact by the full-length enzyme. In addition, we show for the first time that thrombospondin-1 is differently cleaved by both BMP1 and its catalytic domain. In summary, our data support the concept that the C-terminal domains of BMP1 are important for substrate recognition and for controlling and restricting its proteolytic activity via exosite binding. [ABSTRACT FROM AUTHOR]

Abstract: The tissue-invasive nematode Onchocerca volvulus causes skin and eye pathology in human onchocerciasis. While the adult females reside sessile in subcutaneous nodules, the microfilariae are abundantly released from the nodules, males and juvenile worms migrate through the host tissue. Matrix-degrading metallo- and serine proteinases have been detected in excretory-secretory worm products that may be essential for migration of the mobile stages. In this study, a 1713bp long cDNA encoding for a putative proteinase of O. volvulus has been isolated. The predicted protein sequence includes a signal peptide indicating secretion to the extracellular space, a propeptide, an astacin-like protease domain, an EGF-like and a CUB-domain, thereby identifying the protein as a member of the astacin family of zinc endopeptidases. Onchoastacin, Ov-AST-1, is most closely related to a subfamily comprising nematode astacins including Caenorhabditis and Ancylostoma. Ov-AST-1 was expressed as a recombinant protein in baculovirus-infected insect cells and exhibited enzymatic activity. The exposure of onchoastacin to the host immune system is indicated by demonstration of IgG reacting with the recombinant Ov-AST-1 and with two peptides of the protein. Since a homologous metalloproteinase is part of a promising hookworm vaccine, Ov-AST-1 may be a candidate for intervention strategies in filarial infections. [Copyright &y& Elsevier]

Abstract: Human pappalysin-1 is a multi-domain metalloprotease engaged in the homeostasis of insulin-like growth factors and the founding member of the pappalysin family within the metzincin clan of metalloproteases. We have recently identified an archaeal relative, ulilysin, encompassing only the protease domain. It is a 262-residue active protease with a novel 3D structure with two subdomains separated by an active-site cleft. Despite negligible overall sequence similarity, noticeable similarity is found with other metzincin prototypes, adamalysins/ADAMs and matrix metalloproteinases. Ulilysin has been crystallised in a product complex with an arginine–valine dipeptide occupying the active-site and positions and in a complex with the broad-spectrum hydroxamic acid-based metalloprotease inhibitor, batimastat. This molecule inhibits mature ulilysin with an IC50 value of 61μM under the conditions assayed. The binding of batimastat to ulilysin evokes binding to vertebrate matrix metalloproteases but is much weaker. These data give insight into substrate specificity and mechanism of action and inhibition of the novel pappalysin family. [Copyright &y& Elsevier]

Abstract: The astacin family metalloproteinase is a family of zinc-dependent endopeptidases which play crucial roles in embryonic development, bone growth and morphogenesis. A cDNA clone encoding a putative astacin-like metalloproteinase (pf-ALMP) was isolated from hemocytes of pearl oyster, Pinctada fucata. The novel metalloproteinase presents a molecular organization close to the astacins, but has a novel C-terminal domain with cysteine arrays. RT-PCR analysis revealed that pf-ALMP was expressed dramatically high in hemocytes, which was affected by lipopolysaccharides (LPS) challenge. High expression of pf-ALMP was also found in gill, gonad and digestion gland, and in situ hybridization demonstrated that pf-ALMP was expressed in the epithelia cells of these tissues. Substrate analysis studies indicated that the recombinant pf-ALMP catalytic domain could digest gelatin. Interestingly, the pf-ALMP also could be involved in cell proliferation processes and the cysteine arrays were necessary for the proliferative activity. Taken together, these studies also help to further understand the functions of astacins which may be related to the processes of molluscan inflammatory response, embryo development, proliferation and shell formation. [Copyright &y& Elsevier]

Abstract: Digestive fluid of the araneid spider Argiope aurantia is known to contain zinc metallopeptidases. Using anion-exchange chromatography, size-exclusion chromatography, sucrose density gradient centrifugation, and gel electrophoresis, we isolated two lower-molecular-mass peptidases, designated p16 and p18. The N-terminal amino acid sequences of p16 (37 residues) and p18 (20 residues) are 85% identical over the first 20 residues and are most similar to the N-terminal sequences of the fully active form of meprin (β subunits) from several vertebrates (47–52% and 50–60% identical, respectively). Meprin is a peptidase in the astacin (M12A) subfamily of the astacin (M12) family. Additionally, a 66-residue internal sequence obtained from p16 aligns with the conserved astacin subfamily domain. Thus, at least some spider digestive peptidases appear related to astacin of decapod crustaceans. However, important differences between spider and crustacean metallopeptidases with regard to isoelectric point and their susceptibility to hemolymph-borne inhibitors are demonstrated. Anomalous behavior of the lower-molecular-mass Argiope peptidases during certain fractionation procedures indicates that these peptidases may take part in reversible associations with each other or with other proteins. A. aurantia digestive fluid also contains inhibitory activity effective against insect digestive peptidases. Here we present evidence for at least thirteen, heat-stable serine peptidase inhibitors ranging in molecular mass from about 15 to 32 kDa. [Copyright &y& Elsevier]

Abstract: Astacin metalloprotease family members function in a wide variety of biologic events, including cell differentiation and morphogenesis during embryonic development and adult tissue differentiation. We previously isolated and characterized an astacin-like squid metalloprotease (ALSM). To elucidate the embryonic expression of ALSM, we performed immunohistochemical analysis with specific antibodies and examined the expression profiles of ALSM isoforms by in situ hybridization analysis. Tissue distribution and expression were also examined in adult spear squid. mRNA expression of ALSM isoforms I and III was first detected in newly hatched squid and was restricted to the liver. No mRNA signals were detected in other tissues even in adult squids. At the protein level, both isoforms were prominent in the liver of embryos and later in digestive organs of adult squid. Both isoforms were also detected in muscle tissues, including mantle and tentacle muscle. Staining for ALSM III was also identified in the iris and in tissues near the eye in squid embryos. However, no reactive bands were detected by immunoblotting of adult squid eyes. Thus, ALSM is initially expressed at the late stage of embryogenesis in spear squid, and expression is restricted to the liver. Thereafter, ALSM isoforms function in various tissues in an isoform-dependent manner. [Copyright &y& Elsevier]

Abstract: Strongyloides stercoralis, an important nematode pathogen of humans, is transmitted by contact with soil contaminated with the microscopic larvae of the parasite. We determined the cDNA sequence and deduced amino acid structure of a metallo-proteinase that is abundantly transcribed expressed by infective stage larvae of S. stercoralis. This deduced structure of the enzyme revealed a multi-domain protein that included an NH2-terminal peptidase. This peptidase consisted of a signal peptide, a pro-enzyme region, and a mature peptidase domain that included the metal ion co-ordinating motifs, HETSHALGVIH and SIMHY (“Met-turn”), characteristic of the catalytic active site of members of the metzincin superfamily of zinc metallo-endopeptidases. It was phylogenetically and structurally similar to astacin from the digestive gland of the crayfish Astacus astacus, to the HCH-1 peptidase of Caenorhabditis elegans required for hatching and migration of a post-embryonic neuroblast, and to the morphogenetically important peptidases, bone morphogenetic protein-1 (BMP-1) and Drosophila tolloid. In addition, the Strongyloides enzyme, designated strongylastacin, includes a central epidermal growth factor (EGF) domain followed by a carboxyl CUB (complement sub component C1r/C1s/embryonic sea urchin protein Uegf/bone morphogenetic protein) domain. Inspection of the dbEST database revealed the presence of at least 9 transcript clusters that are related to greater or lesser extent to strongylastacin; based on these expressed sequence tags, strongylastacin was expressed only in the infective third stage larvae, whereas other transcript clusters were expressed both in filariform and rhabditiform stages or only in the rhabditiform stage. Based on the deduced sequence, structure, and expression profile, strongylastacin is the probable candidate for the zinc-dependent metalloprotease, Ss40, known to be deployed by larvae of S. stercoralis to penetrate human skin to initiate infection. [Copyright &y& Elsevier]

Molting is required for progression between larval stages in the life cycle of nematodes. We have identified four mutant alleles of a Caenorhabditis elegans metalloprotease gene, nas-37, that cause incomplete ecdysis. At each molt the cuticle fails to open sufficiently at the anterior end and the partially shed cuticle is dragged behind the animal. The gene is expressed in hypodermal cells 4 hours before ecdysis during all larval stages. The NAS-37 protein accumulates in the anterior cuticle and is shed in the cuticle after ecdysis. This pattern of protein accumulation places NAS37 in the right place and at the right time to degrade the cuticle to facilitate ecdysis. The nas-37 gene has orthologs in other nematode species, including parasitic nematodes, and they undergo a similar shedding process. For example, Haemonchus contortus molts by digesting a ring of cuticle at the tip of the nose. Incubating Haemonchus larvae in extracted exsheathing fluids causes a refractile ring of digested cuticle to form at the tip of the nose. When Haemonchus cuticles are incubated with purified NAS-37, a similar refractile ring forms. NAS-37 degradation of the Haemonchus cuticle suggests that the metalloproteases and the cuticle substrates involved in exsheathment of parasitic nematodes are conserved in free-living nematodes. [ABSTRACT FROM AUTHOR]

Functional analysis using RNAi was performed on eleven genes for metalloproteases of the M12A family in Cae-norhabditis elegans and the interference of the C17G1.6 gene (nas-37) was found to cause incomplete molting. The RNAi of the C26C6.3 gene (nas-36) also caused a similar molting defect but not so severely as that of the nas-37 gene. Both the genes encode an astacin-like metalloprotease with an epidermal growth factor (EGF)-like domain, a CUB domain, and a thrombospondin-1 domain, in this order. The promoter-driven green fluorescent protein (GFP) expression analysis suggested that they are expressed in hypodermal cells throughout the larval stages and in the vulva of adult animals. In the genetic background of rde-1(ne219), where RNAi does not work, the molting defect caused by the nas-37 interference was observed when the transgenic wild-type rde-1 gene was expressed under the control of the dpy-7 promoter, known to be active in the hypodermal cells, but not under the control of the myo-3 promoter, active in the muscular cells. Therefore these proteases are thought to be secreted by the hypodermal cells and to participate in shedding of old cuticles. [ABSTRACT FROM AUTHOR]

Metalloendopeptidases are present across all kingdoms of living organisms; they are ubiquitous and widely involved in metabolism regulation through their ability either to extensively degrade proteins or to selectively hydrolyze specific peptide bonds. They must be subjected to exquisite spatial and temporal control to prevent this vast potential from becoming destructive. These enzymes are mostly zinc-dependent and the majority of them, named zincins, possess a short consensus sequence, HEXXH, with the two histidines acting as ligands of the catalytic zinc and the glutamate as the general base. A subclass of the zincins is characterized by a C-terminally elongated motif, HEXXHXXGXXH/D, with an additional strictly conserved glycine and a third zinc-binding histidine or aspartate. Currently, representative three-dimensional structures of six different proteinase families bearing this motif show, despite low sequence similarity, comparable overall topology. This includes a substrate-binding crevice, which subdivides the enzyme moiety into an upper and a lower subdomain. A common five-stranded β-sheet and two α-helices are always found in the upper subdomain. The second of these helices encompasses the first half of the elongated consensus sequence and is therefore termed the active-site helix. Other shared characteristics are an invariant methionine-containing Met-turn beneath the catalytic metal and a further C-terminal helix in the lower subdomain. All these structural features identify the metzincin clan of metalloendopeptidases. This clan is reviewed from a structural point of view, based on the reported structures of representative members of the astacins, adamalysins, serralysins, matrixins, snapalysins, and leishmanolysins, and of inhibited forms, either by specific endogenous protein inhibitors or by zymogenic pro-domains. Moreover, newly available genomic sequences have unveiled novel putative metzincin families and new hypothetical members of existing ones. [ABSTRACT FROM AUTHOR]

Meprins are zinc-endopeptidases of the astacin family, which are expressed as membrane-bound or secreted forms in renal and intestinal brush-border membranes of mouse, rat and man. There are two types of meprin subunits, α and β, which form disulfide-bonded homo- and heterodimers; further oligomerization is mediated by non-covalent interactions. Both subunits are translated as proenzymes that have to be activated by removal of an N-terminal propeptide. In the gut, the most probable activator is trypsin. In addition, plasmin has been shown to activate the human α subunit in colorectal cancer tissue. In the present study we have overexpressed the human meprin α subunit and a His-tagged soluble tail-switch-mutant of meprin β in Baculovirus-infected insect cells. The recombinant homo-oligomeric proteins were purified by gel filtration and affinity chromatography with yields of up to 10 mg/l cell culture medium and analyzed with regard to their activation mechanism. While both α and Β homo-oligomers are activated by trypsin, only meprin α homo-oligomers are processed to their mature form by plasmin. These results indicate a different accessibility of the propeptide in meprin homo-oligomers and suggest an explanation for the appearance of meprin hetero-oligomers consisting of active α, but latent β subunits. [ABSTRACT FROM AUTHOR]

We have identified a novel Tolloid-like metalloprotease, called Xolloid-related (Xlr), that is expressed during early Xenopus development. Transcripts for xlr are localized to the marginal zone of mid-gastrulae and are most abundant in ventral and lateral sectors. At neurula stages xlr is strongly expressed around the blastopore and in the pharyngeal endoderm, and more weakly expressed throughout the ventral half of the embryo. Transcripts are detected in the nervous system, particularly the hindbrain and spinal cord, and tailbud of tailbud stage embryos, with weaker expression in the anterior nervous system, otic vesicle, heart, and pronephric duct. Transcription of xlr is increased by BMP4 and decreased by Noggin and tBR, indicating that xlr is regulated by BMP signalling. Injection of xlr mRNA inhibits dorsoanterior development and the dorsal axis inducing ability of coinjected chordin, but not noggin or tBR, mRNA. Xlr conditioned media cleaves Chordin in vitro, indicating that this protease may regulate the availability of Chordin in vivo. [Copyright &y& Elsevier]

We have cloned four cDNAs encoding astacin-like squid metalloproteases (ALSMs)-I and -II from the Japanese common squid and ALSMs-I and -III from the spear squid. Analysis of the deduced amino acid sequences revealed that ALSMs possess a signal peptide and a pro-sequence followed by an astacin-like catalytic domain and an MAM (m¯eprin, A¯5 protein, receptor protein-tyrosine phosphatase μ¯) domain. Phylogenetic analysis revealed that ALSM corresponds to a new cluster of astacins. To analyze the function of the MAM domain, wild-type ALSM and an MAM-truncated mutant were expressed in a baculovirus expression system. The expressed protein encoding full-length ALSM hydrolyzed myosin heavy chain as effectively as native ALSM, whereas the MAM-truncated mutant possessed no protease activity, suggesting that the MAM domain contributes to substrate recognition. ALSM has been isolated from squid liver and mantle muscle. However, analysis with a specific antibody generated against ALSM indicated the presence of ALSM in a wide variety of tissues. ALSM was located in the extracellular matrix of mantle muscle cells. Thus, ALSM is a secreted protease, as are other members of the astacin family. The extracellular localization raises the possibility of substrates other than myosin. The physiological role of ALSM remains unknown, at this time. [ABSTRACT FROM AUTHOR]

The astacin family of zinc endopeptidases was named after the digestive enzyme astacin isolated from the crayfish Astacus astacus. Employing a reverse transcription/PCR strategy with degenerate oligonucleotide primers specific for two signature seqences of the astacin family, we have isolated a 1602-bp cDNA from embryos of developing A. astacus eggs, which was designated Astacus embryonic astacin (AEA). This cDNA was found to code for an astacin-like protease domain which accounts for the N-terminal half of the predicted protein. The C-terminal half mainly consists of two complement subcomponent C1r/C1s/embryonic sea urchin protein Uegf/bone morphogenetic protein 1 (CUB) domains. The metalloprotease domain displays an amino acid sequence identity of 42 % with astacin. A higher sequence similarity was found to astacin family members that act as hatching enzymes in different species, e.g. chorioallantoic membrane protein 1 (CAM-1; from quail) and Xenopus hatching enzyme (formerly UVS.2), both of which show 54 % identity, and high and low choriolytic enzymes (HCE and LCE) from the teleost Oryzias latipes (52 % and 48 % identity, respectively). A relationship to astacin-like hatching enzymes is further supported by a phylogenetic analysis of the protease domains. Expression of AEA mRNA in developing embryos was found to be restricted to unhatched juveniles (larvae) during the last 8 days before hatching. AEA transcripts could not be detected in various tissues of adult animals or in eggs and embryos from an earlier developmental stage. AEA expression starts about 8 days prior to hatching, followed by a strong (18-fold) induction with a maximum at day 4 before hatching. Newly hatched juveniles were found not to express the AEA mRNA. [ABSTRACT FROM AUTHOR]

A novel isoform of myosinase was purified to homogeneity from liver of spear squid by sequential chromatographies using SP Sephadex, hydroxylapatite, Zn/Co chelating affini-ty, and TSK-gel G2000SW columns. Myosinase activity was detected as a single peak of 45-kDa protein by gel filtration. The novel isoform of myosinase specifically hydrolyzed a rabbit skeletal muscle myosin heavy chain into products of 120 and 100 kDa in the presence of Co2+ ions, and the cleavage site in the myosin heavy chain was quite different from those of two known myosinase isoforms, I and II. Therefore, we named the novel isoform myosinase III. Myosinase III was also distinguishable from myosinase I by its amino-ter-minal sequence. The sequence showed similarity to an internal sequence of the astacin family. [ABSTRACT FROM AUTHOR]

The astacin protease Meprin β represents an emerging target for drug development due to its potential involvement in disorders such as acute and chronic kidney injury and fibrosis. Here, we elaborate on the structural basis of inhibition by a specific Meprin β inhibitor. Our analysis of the crystal structure suggests different binding modes of the inhibitor to the active site. This flexibility is caused, at least in part, by movement of the C-terminal region of the protease domain (CTD). The CTD movement narrows the active site cleft upon inhibitor binding. Compared with other astacin proteases, among these the highly homologous isoenzyme Meprin α, differences in the subsites account for the unique selectivity of the inhibitor. Although the inhibitor shows substantial flexibility in orientation within the active site, the structural data as well as binding analyses, including molecular dynamics simulations, support a contribution of electrostatic interactions, presumably by arginine residues, to binding and specificity. Collectively, the results presented here and previously support an induced fit and substantial movement of the CTD upon ligand binding and, possibly, during catalysis. To the best of our knowledge, we here present the first structure of a Meprin β holoenzyme containing a zinc ion and a specific inhibitor bound to the active site. The structural data will guide rational drug design and the discovery of highly potent Meprin inhibitors. [ABSTRACT FROM AUTHOR]