BMP-1 and the astacin family of metalloproteinases: A potential link between the extracellular matrix,growth factors and pattern formation

Members of the astacin family of metalloproteinases such as human bone morphogenetic protein 1 (BMP-1) have previously been linked to cell differentiation and pattern formation during development through a proposed role in the activation of latent growth factors of the TGF-β superfamily. Recent finding⁽¹⁾ indicate that BMP-1 is identical to pro-collagen C-proteinase, which is a metalloproteinase involved in extracellular matrix (ECM) formation. This observation suggests that a functional link may exist between astacin metalloproteinases, growth factors and cell differentiation and pattern formation during development. Taken together, current studies indicate that BMP-1 and possibly other astacin metalloproteinases are multifunctional enzymes that act directly on growth factors and the ECM. In combination, these dual actions would have profound effects on developmental processes.     

The metzincins--topological and sequential relations between the astacins, adamalysins, serralysins, and matrixins (collagenases) define a superfamily of zinc-peptidases.

The three-dimensional structures of the zinc endopeptidases human neutrophil collagenase, adamalysin II from rattle snake venom, alkaline proteinase from Pseudomonas aeruginosa, and astacin from crayfish are topologically similar, with respect to a five-stranded beta-sheet and three alpha-helices arranged in typical sequential order. The four proteins exhibit the characteristic consensus motif HEXXHXXGXXH, whose three histidine residues are involved in binding of the catalytically essential zinc ion. Moreover, they all share a conserved methionine residue beneath the active site metal as part of a superimposable "Met-turn." This structural relationship is supported by a sequence alignment performed on the basis of topological equivalence showing faint but distinct sequential similarity. The alkaline proteinase is about equally distant (26% sequence identity) to both human neutrophil collagenase and astacin and a little further away from adamalysin II (17% identity). The pairs astacin/adamalysin II, astacin/human neutrophil collagenase, and adamalysin II/human neutrophil collagenase exhibit sequence identities of 16%, 14%, and 13%, respectively. Therefore, the corresponding four distinct families of zinc peptidases, the astacins, the matrix metalloproteinases (matrixins, collagenases), the adamalysins/reprolysins (snake venom proteinases/reproductive tract proteins), and the serralysins (large bacterial proteases from Serratia, Erwinia, and Pseudomonas) appear to have originated by divergent evolution from a common ancestor and form a superfamily of proteolytic enzymes for which the designation "metzincins" has been proposed. There is also a faint but significant structural relationship of the metzincins to the thermolysin-like enzymes, which share the truncated zinc-binding motif HEXXH and, moreover, similar topologies in their N-terminal domains.     

Families of metalloendopeptidases and their relationships.

Crystal structures available for four metalloendopeptidases have revealed zinc ligands for these enzymes. New sequence information has made it possible to compare the primary structures of the zinc-binding site in metalloendopeptidases. A scheme based on the zinc-binding site is proposed to classify metalloendopeptidases into five distinct families: thermolysin, astacin, serratia, matrixin, and snake venom metalloproteinases. Two histidines and one glutamate are zinc-ligands in the thermolysin family. Three histidines and one tyrosine are zinc ligands in the other four families, which are further distinguished by the identity of the residue following the third histidine and by the environment surrounding the tyrosine.     

Structure, expression, and developmental function of early divergent forms of metalloproteinases in Hydra

Metalloproteinases have a critical role in a broad spectrum of cellular processes ranging from the breakdown of extracellular matrix to the processing of signal transduction-related proteins. These hydrolytic functions underlie a variety of mechanisms related to developmental processes as well as disease states. Structural analysis of metalloproteinases from both invertebrate and vertebrate species indicates that these enzymes are highly conserved and arose early during metazoan evolution. In this regard, studies from various laboratories have reported that a number of classes of metalloproteinases are found in hydra, a member of Cnidaria, the second oldest of existing animal phyla. These studies demonstrate that the hydra genome contains at least three classes of metalloproteinases to include members of the 1) astacin class, 2) matrix met-alloproteinase class, and 3) neprilysin class. Functional studies indicate that these metalloproteinases play diverse and important roles in hydra morphogenesis and ce     

Structure,expression, and developmental function of early divergent forms of metalloproteinases in hydra

Metalloproteinases have a critical role in a broad spectrum of cellular processes ranging from the breakdown of extracellular matrix to the processing of signal transduction-related proteins. These hydrolytic functions underlie a variety of mechanisms related to developmental processes as well as disease states. Structural analysis of metalloproteinases from both invertebrate and vertebrate species indicates that these enzymes are highly conserved and arose early during metazoan evolution. In this regard, studies from various laboratories have reported that a number of classes of metalloproteinases are found in hydra, a member of Cnidaria, the second oldest of existing animal phyla. These studies demonstrate that the hydra genome contains at least three classes of metalloproteinases to include members of the 1) astacin class, 2) matrix metalloproteinase class, and 3) neprilysin class. Functional studies indicate that these metalloproteinases play diverse and important roles in hydra morphogenesis and cell differentiation as well as specialized functions in adult polyps. This article will review the structure, expression, and function of these metalloproteinases in hydra.     

Matrilysin: Expression, purification, and characterization

The expression vector pGEX-2T under the control of the IPTG-inducibletac promotor is effective for the production of a fusion protein of glutathione transferase (GST, 26 kDa) and promatrilysin (28 kDa) separated from the C-terminus of GST by a thrombin cleavage site. Zwittergen (palmityl sulfobetaine), 2%, solubilizes the fusion protein that is found associated with inclusion bodies. The solubilized fusion protein is purified by affinity chromatography on GSH agarose. Promatrilysin is obtained by thrombin cleavage either on the column or after GSH elution of the fusion protein. Mono S chromatography of the recovered protein yields homogeneous promatrilysin. The zinc content of promatrilysin and its activated enzyme product is slightly greater than 2 mol of zinc per mole of protein. The results indicate that the matrix metalloproteinases (MMPs) contain two metal-binding sites at which zinc is firmly bound and possibly a third site at which it is weakly bound. Primary sequence alignments for all the MMPs have a sequence homologous to the zinc-binding site of astacin,HExxHxxGxxH, suggesting one of the zinc sites is a catalytic one, in agreement with the known inhibition of these enzymes by chelators. However, the other zinc-binding site(s) likely reflect the different ways that astacin and the MMP subfamilies are stabilized, i.e., disulfides in astacin and metal ions in the MMPs.     

Kinetics of nitroanilide cleavage by astacin

The investigation of the catalytic properties of astacin, a zinc-endopeptidase from the crayfish Astacus astacus L., has gained importance, because the enzyme represents a novel, structurally distinct family of metalloproteinases which also includes a human bone morphogenetic protein (BMP1). Astacin releases nitroaniline from succinyl-alanyl-alanyl-alanyl-4-nitroanilide (Suc-Ala-Ala-Ala-pNA), a substrate originally designed for pancreatic elastase. This activity was unexpected since only few metalloproteinases cleave small nitroanilide substrates, and, moreover, the primary specificity of astacin toward protein substrates is determined by short, uncharged amino-acid sidechains in the P'1-position, i.e. the new N-terminus after cleavage. The specificity constants, kcat/Km, for the release of nitroaniline from substrates of the general structure Suc-Alan-pNA (n = 2, 3, 5) and Alan-pNA (n = 1, 2, 3) increase with the number of alanine residues. The longest peptide, Suc-Ala(-)-Ala-Ala-Ala-Ala-pNA, is the only one out of eleven substrates used in this study, which is cleaved at two positions by astacin. The first cleavage yields Suc-Ala(-)-Ala and Ala-Ala-Ala-pNA. From the resulting C-terminal fragment, Ala-Ala-Ala-pNA, a second cut releases nitroaniline. The 1200-fold higher specificity constant observed for the first as compared to the second cleavage in Suc-Ala-Ala-Ala-Ala-Ala-pNA reflects the preference of astacin for true peptide bonds and also the importance of a minimum length of the substrate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of pro-astacin. Immunological and model peptide studies on the processing of immature astacin, a zinc-endopeptidase from the crayfish Astacus astacus.

To contribute knowledge of the processing and activation of invertebrate proteolytic enzymes, we studied the metalloprotease astacin, a digestive enzyme from the freshwater crayfish Astacus astacus (decapod crustacean). It is the prototype of the protein family of astacins, members of which occur in organisms from bacteria to man and are involved in a variety of physiological reactions. According to its genomic structure, astacin is produced as a zymogen [Geier, G., Jacob, E., St?cker, W. & Zwilling, R. (1997) Arch. Biochem. Biophys. 337, 300-307]. To localize and follow the processing of pro-astacin in different parts of the digestive tract, we synthesized two peptides covering the pro part of pro-astacin and raised antibodies against them. In addition, antiserum against the whole active astacin was produced. Using immunohistochemical investigation, we detected pro-astacin in the F cells of the hepatopancreas and all the way into the tubular lumen and the collecting ducts of this gland. Immunoblot assays revealed only active astacin, and never pro-astacin, present in the cardiac stomach. We conclude from these studies that astacin is secreted into the lumen of the hepatopancreatic tubules in its pro form and is activated on its way to the stomach. To investigate which of the two endopeptidases found in the digestive tract of crayfish, astacin or trypsin, is responsible for cleaving the propeptide from pro-astacin, we synthesized different peptides that mimick the activation site. MS analysis of the cleavage products of astacin and trypsin showed that astacin is capable of catalyzing its own activation. Any contribution of trypsin would require the successive action of an aminopeptidase. Substituting glycine for arginine at position -1 of the activation site does not prevent astacin activity. As most members of the astacin protein family have basic amino-acid residues in this position, in these cases also astacin-specific cleavage would be possible.     

First structure of a snake venom metalloproteinase: a prototype for matrix metalloproteinases/collagenases.

Adamalysin II, a 24 kDa zinc endopeptidase from the snake venom of Crotalus adamanteus, is a member of a large family of metalloproteinases isolated as small proteinases or proteolytic domains of mosaic haemorrhagic proteins from various snake venoms. Homologous domains have recently been detected in multimodular mammalian reproductive tract proteins. The 2.0 A crystal structure of adamalysin II reveals an ellipsoidal molecule with a shallow active-site cleft separating a relatively irregularly folded subdomain from the calcium-binding main molecular body composed of a five-stranded beta-sheet and four alpha-helices. The folding of the peptide fragment containing the zinc-binding motif HExxHxxGxxH bears only a distant resemblance to thermolysin, but is identical to that found in astacin, with the three histidines and a water molecule (linked to the glutamic acid) likewise constituting the zinc ligand; adamalysin II lacks a fifth (tyrosine) zinc ligand, however, leaving its zinc ion tetrahedrally co-ordinated. Furthermore, adamalysin II and astacin share an identical active-site basement formed by a common Metturn. Due to their virtually identical active-site environment and similar folding topology, the snake venom metalloproteinases (hitherto called adamalysins) and the astacins (and presumably also the matrix metalloproteinases/mammalian collagenases and the Serratia proteinase-like large bacterial proteinases) might be grouped into a common superfamily with distinct differences from the thermolysin family.     

Retrospective analysis of a secondary structure prediction: the catalytic domain of matrix metalloproteinases.

Secondary structure prediction of the catalytic domain of matrix metalloproteinases is evaluated in the light of recently published experimentally determined structures. The prediction was made by combining conformational propensity, surface probability, and residue conservation calculated for an alignment of 19 sequences. The position of each observed secondary structure element was correctly predicted with a high degree of accuracy, with a single beta-strand falsely predicted. The domain fold was also anticipated from the prediction by analogy with the structural elements found in the distantly related metalloproteinases thermolysin, astacin, and adamalysin.     

Developmental roles of the BMP1/TLD metalloproteinases

The astacin family (M12A) of the metzincin subclan MA(M) of metalloproteinases has been detected in developing and mature individuals of species that range from hydra to humans. Functions of this family of metalloproteinase vary from digestive degradation of polypeptides, to biosynthetic processing of extracellular proteins, to activation of growth factors. This review will focus on a small subgroup of the astacin family; the bone morphogenetic protein 1 (BMP1)/Tolloid (TLD)-like metalloproteinases. In vertebrates, the BMP1/TLD-like metalloproteinases play key roles in regulating formation of the extracellular matrix (ECM) via biosynthetic processing of various precursor proteins into mature functional enzymes, structural proteins, and proteins involved in initiating mineralization of the ECM of hard tissues. Roles in ECM formation include: processing of the C-propeptides of procollagens types I-III, to yield the major fibrous components of vertebrate ECM; proteolytic activation of the enzyme lysyl oxidase, necessary to formation of covalent cross-links in collagen and elastic fibers; processing of NH2-terminal globular domains and C-propeptides of types V and XI procollagen chains to yield monomers that are incorporated into and control the diameters of collagen type I and II fibrils, respectively; processing of precursors for laminin 5 and collagen type VII, both of which are involved in securing epidermis to underlying dermis; and maturation of small leucine-rich proteoglycans. The BMP1/TLD-related metalloproteinases are also capable of activating the vertebrate transforming growth factor-beta (TGF-beta)-like "chalones" growth differentiation factor 8 (GDF8, also known as myostatin), and GDF11 (also known as BMP11), involved in negative feedback inhibition of muscle and neural tissue growth, respectively; by freeing them from noncovalent latent complexes with their cleaved prodomains. BMP1/TLD-like proteinases also liberate the vertebrate TGF-beta-like morphogens BMP2 and 4 and their invertebrate ortholog decapentaplegic, from latent complexes with the vertebrate extracellular antagonist chordin and its invertebrate ortholog short gastrulation (SOG), respectively. The result is formation of the BMP signaling gradients that form the dorsal-ventral axis in embryogenesis. Thus, BMP1/TLD-like proteinases appear to be key to regulating and orchestrating formation of the ECM and signaling by various TGF-beta-like proteins in morphogenetic and homeostatic events.     

Purification and cloning of an endogenous protein inhibitor of carp nephrosin, an astacin metalloproteinase

Nephrosin is a newly discovered member of the astacin family. It is a secreted proteinase and is present in carp head kidney, kidney, and spleen, all of which are responsible for immune and hematopoietic functions in fish. A complex formed by nephrosin and its inhibitor was purified from carp kidney extract by heparin affinity column chromatography. The presence of the nephrosin-inhibitor complex in different tissues was examined by immunoblotting with polyclonal antisera against the purified nephrosin inhibitor and nephrosin. Both nephrosin and the nephrosin inhibitor were present mainly in gill, head kidney, kidney, and spleen. In addition, we have cloned the cDNA encoding the nephrosin inhibitor. There are two different cDNA clones possibly resulting from two different genes, and the long form contains unique tandem repeat sequences in the 3'-end. The deduced primary structure of nephrosin inhibitor is similar to that of fetuin-A, a mammalian protein present in blood, liver, cerebrospinal fluid, and cerebral cortex during fetal development. Treatment with both N-glycosidase F and O-glycosidase removed the carbohydrate moiety of the nephrosin inhibitor and decreased the apparent molecular mass from 40 to 30 kDa. The nephrosin inhibitor seems to be synthesized in liver and then secreted to the blood as a precursor. When it was distributed into hematopoietic tissues, it was processed from 67 to 40 kDa and acquired inhibitory activity. This processing phenomenon of fetuin has not been reported elsewhere. Importantly, the presence of an endogenous inhibitor of nephrosin is the first report of this kind for astacin enzymes. It is very likely that endogenous tissue inhibitors may also be present for the regulation of other astacin enzymes.     

Activation mechanism of pro-astacin: role of the pro-peptide,tryptic and autoproteolytic cleavage and importance of precise amino-terminal processing

Astacin (EC 3.4.24.21) is a prototype for the astacin family and for the metzincin superfamily of zinc peptidases, which comprise membrane-bound and secreted enzymes involved in extracellular proteolysis during tissue development and remodelling. Generally, metzincins are translated as pro-enzymes (zymogens), which are activated by removal of an N-terminal pro-peptide. In astacin, however, the mode of zymogen activation has been obscured, since the pro-form does not accumulate in vivo. Here we report the detection of pro-astacin in midgut glands of brefeldin A-treated crayfish (Astacus astacus) by immunoprecipitation and mass spectrometry. We demonstrate that the pro-peptide is able to shield the active site of mature astacin as a transient inhibitor, which is degraded slowly. In vitro studies with recombinant pro-astacin in the absence of another protease reveal a potential of auto-proteolytic activation. The initial cleavage in this autoactivation appears to be an intramolecular event. This is supported by the fact that the mutant E93A-pro-astacin is incapable of autoactivation, and completely resistant to cleavage by mature astacin. However, this mutant is cleaved by Astacus trypsin within the pro-peptide. This probably reflects the in vivo situation, where Astacus trypsin and astacin work together during pro-astacin activation. In a first step, trypsin produces amino-terminally truncated pro-astacin derivatives. These are trimmed subsequently by each other and by astacin to yield the mature amino terminus, which forms a salt-bridge with Glu103 in the active site. The disruption of this salt-bridge in the mutants E103A and E103Q results in extremely heat labile proteins, whose catalytic activities are not altered drastically, however. This supports a concept according to which the linkage of Glu103 to the precisely trimmed amino terminus is a crucial structural prerequisite throughout the astacin family.     

Critical amino acids in the active site of meprin metalloproteinases for substrate and peptide bond specificity

The protease domains of the evolutionarily related alpha and beta subunits of meprin metalloproteases are approximately 55% identical at the amino acid level; however, their substrate and peptide bond specificities differ markedly. The meprin beta subunit favors acidic residues proximal to the scissile bond, while the alpha subunit prefers small or aromatic amino acids flanking the scissile bond. Thus gastrin, a peptide that contains a string of five Glu residues, is an excellent substrate for meprin beta, while it is not hydrolyzed by meprin alpha. Work herein aimed to identify critical amino acids in the meprin active sites that determine the substrate specificity differences. Sequence alignments and homology models, based on the crystal structure of the crayfish astacin, showed electrostatic differences within the meprin active sites. Site-directed mutagenesis of active site residues demonstrated that replacement of a hydrophobic residue by a basic amino acid enabled the meprin alpha protease to cleave gastrin. The meprin alphaY199K mutant was most effective; the corresponding mutation of meprin betaK185Y resulted in decreased activity toward gastrin. Peptide cleavage site determinations and kinetic analyses using a variety of peptides extended evidence that meprin alphaTyr-199/betaLys-185 are substrate specificity determinants in meprin active sites. These studies shed light on the molecular basis for the substrate specificity differences of astacin metalloproteinases.     

Comparative analysis of binding sites of human meprins with hydroxamic acid derivative by molecular dynamics simulation study

Meprins are complex and highly glycosylated multi-domain enzymes that require post-translational modifications to reach full activity. Meprins are metalloproteases of the astacin family characterized by a conserved zinc-binding motif (HExxHxxGFxHExxRxDR). Human meprin-α and -β protease subunits are 55% identical at the amino acid level, however the substrate and peptide bond specificities vary markedly. Current work focuses on the critical amino acid residues in the non-primed subsites of human meprins-α and -β involved in inhibitor/ligand binding. To compare the molecular events underlying ligand affinity, homology modeling of the protease domain of humep-α and -β based on the astacin crystal structure followed by energy minimization and molecular dynamics simulation of fully solvated proteases with inhibitor Pro-Leu-Gly-hydroxamate in S subsites were performed. The solvent accessible surface area curve shows a decrease in solvent accessibility values at specific residues upon inhibitor binding. The potential energy, total energy, H-bond interactions, root mean square deviation and root mean square fluctuation plot reflect the subtle differences in the S subsite of the enzymes which interact with different residues at P site of the inhibitor.     

Structure, expression, and developmental function of early divergent forms of metalloproteinases in Hydra

Metalloproteinases have a critical role in a broad spectrum of cellular processes ranging from the break-down of extracellulax matrix to the processing of signal transduction-related proteins. These hydrolyticfunctions underlie a variety of mechanisms related to developmental processes as well as disease states.Structural analysis of metalloproteinases from both invertebrate and vertebrate species indicates that theseenzymes are highly conserved and arose early during metazoan evolution. In this regard, studies from vari-ous laboratories have reported that a number of classes of metalloproteinases are found in hydra, a memberof Cnidaria, the second oldest of existing animal phyla. These studies demonstrate that the hydra genomecontains at least three classes of metalloproteinases to include members of the 1) astacin class, 2) matrix met-alloproteinase class, and 3) neprilysin class. Functional studies indicate that these metalloproteinases playdiverse and important roles in hydra morphogenesis and cell differentiation as well as specialized functionsin adult polyps. This article will review the structure, expression, and function of these metalloproteinasesin hydra.     

Identification and characterization of hydra metalloproteinase 2 (HMP2): a meprin-like astacin metalloproteinase that functions in foot morphogenesis

Several members of the newly emerging astacin metalloproteinase family have been shown to function in a variety of biological events, including cell differentiation and morphogenesis during both embryonic development and adult tissue differentiation. We have characterized a new astacin proteinase, hydra metalloproteinase 2 (HMP2) from the Cnidarian, Hydra vulgaris. HMP2 is translated from a single mRNA of 1.7 kb that contains a 1488 bp open reading frame encoding a putative protein product of 496 amino acids. The overall structure of HMP2 most closely resembles that of meprins, a subgroup of astacin metalloproteinases. The presence of a transient signal peptide and a putative prosequence indicates that HMP2 is a secreted protein that requires post-translational processing. The mature HMP2 starts with an astacin proteinase domain that contains a zinc binding motif characteristic of the astacin family. Its COOH terminus is composed of two potential protein-protein interaction domains: an "MAM" domain (named after meprins, A-5 protein and receptor protein tyrosine phosphatase mu) that is only present in meprin-like astacin proteinases; and a unique C-terminal domain (TH domain) that is also present in another hydra metalloproteinase, HMP1, in Podocoryne metalloproteinase 1 (PMP1) of jellyfish and in toxins of sea anemone. The spatial expression pattern of HMP2 was determined by both mRNA whole-mount in situ hybridization and immunofluorescence studies. Both morphological techniques indicated that HMP2 is expressed only by the cells in the endodermal layer of the body column of hydra. While the highest level of HMP2 mRNA expression was observed at the junction between the body column and the foot process, immunofluorescence studies indicated that HMP2 protein was present as far apically as the base of the tentacles. In situ analysis also indicated expression of HMP2 during regeneration of the foot process. To test whether the higher levels of HMP2 mRNA expression at the basal pole related to processes underlying foot morphogenesis, antisense studies were conducted. Using a specialized technique named localized electroporation (LEP), antisense constructs to HMP2 were locally introduced into the endodermal layer of cells at the basal pole of polyps and foot regeneration was initiated and monitored. Treatment with antisense to HMP2 inhibited foot regeneration as compared to mismatch and sense controls. These functional studies in combination with the fact that HMP2 protein was expressed not only at the junction between the body column and the foot process, but also as far apically as the base of the tentacles, suggest that this meprin-class metalloproteinase may be multifunctional in hydra.     

The astacin family of metalloendopeptidases

Molecular cloning of a human intestinal brush border metalloendopeptidase (N-benzoyl-L-tyrosyl-p-aminobenzoic acid hydrolase, PPH) and a mouse kidney brush border metalloendopeptidase (meprin A) has revealed 82% identity in the NH2-terminal amino acid sequences (198 residues) of the mature enzymes. Furthermore, searching of protein sequence data bases with the inferred peptide sequences as probes revealed strong similarities to astacin, a crayfish digestive protease, and an NH2-terminal domain of a human bone morphogenetic protein (BMP-1). Meprin A and PPH both have approximately 30% identity with astacin and BMP-1. Multiple alignment analysis indicated that 37 residues, including 3 cysteine residues, are strictly conserved for the four proteins in a sequence frame equivalent to the complete 200-amino acid astacin sequence. The four proteins contain a zinc-binding motif (HEXXH), found at the active site of most metalloendopeptidases, within an extended sequence of HEXXHXXGFXHE which is unique to this subgroup of metalloendopeptidases. In addition, the four proteins have 54% identity in a 24-amino acid sequence that includes the putative active site. A fifth protein, Xenopus laevis developmentally regulated protein UVS.2, also shares sequence identity with the metalloendopeptidases. These data provide strong evidence for an evolutionary relationship of these proteins. It is suggested that this new family of metalloendopeptidases be called the "astacin family."