Preparing 2-D protein extracts from Caenorhabditis elegans.
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Animals↗
Biomedical subjects
R Zwilling
Publications and source records attributed to R Zwilling.
Cloning and characterization of a cDNA coding for Astacus embryonic astacin, a member of the astacin family of metalloproteases from the crayfish Astacus astacus.
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.
Amino Acid Sequence↗
Phosphinic peptides, the first potent inhibitors of astacin, behave as extremely slow-binding inhibitors.
A series of phosphinic pseudo-peptides varying in length and composition have been designed as inhibitors of the crayfish zinc endopeptidase astacin, the prototype of the astacin family and of the metzincin superfamily of metalloproteinases. The most efficient phosphinic peptide, fluorenylmethyloxycarbonyl-Pro-Lys-PhePsi(PO2CH2)Ala-P ro-Leu-Val, binds to astacin with a Ki value of 42 nM, which is about three orders of magnitude below the corresponding values for previously used hydroxamic acid derivatives. However, the rate constants for association (kon = 96.8 M-1.s-1) and dissociation (koff = 4.1 x 10(-6) s-1) are evidence for the extremely slow binding behaviour of this compound. N-terminally or C-terminally truncated phosphinic analogues of this parent molecule are much less potent, indicating a critical role of the peptide size on the potency. In particular, omission of the N-terminal proline residue leads to a 40-fold increase in Ki which is mostly due to a 75-fold higher koff value. These findings are consistent with the previously solved crystal structure of astacin complexed with one of the phosphinic peptides, benzyloxycarbonyl-Pro-Lys-PhePsi(PO2CH2)Ala-Pro-O-methyl, Ki = 14 microM [Grams, Dive, Yiotakis, Yiallouros, Vassiliou, Zwilling, Bode and Stöcker (1996) Nature Struct. Biol. 3, 671-675]. This structure also reveals that the phosphinic group binds to the active site as a transition-state analogue. The extremely slow binding behaviour of the phosphinic peptides is discussed in the light of the conformational changes involving a unique 'tyrosine switch' in the structure of astacin upon inhibitor binding. The phosphinic peptides may provide a rational basis for the design of drugs directed towards other members of the astacin family which, like bone morphogenetic protein 1 (BMP1; i.e. the procollagen C-proteinase), have become targets of pharmacological research.
Binding Sites↗
Genomic organization of the zinc-endopeptidase astacin.
The crayfish digestive protease astacin is the first described member of the astacin family of zinc-endopeptidases, for which it is regarded as a prototype. We have isolated and characterized the genomic sequence of astacin which spans a region of 2616 bp. The coding sequence is distributed over five exons and is interrupted by four introns. It was observed that structurally and functionally essential units of the protein, like the three alpha-helices, the five beta-strands, the Zn-binding motif, and the Met turn are never disrupted by introns. The start site of transcription was determined by primer extension analysis, confirming the existence of a pre-pro-protein of 49 amino acids which so far had not been detectable at the protein level. In addition, when compared to the amino acid sequence of mature astacin, a carboxy-terminal extension of two additional amino acids was also found. The exon-intron pattern of the astacin gene was compared to those of three other astacin family members with known genomic sequences, i.e., tolloid of Drosophila, the fish hatching enzyme LCE, and the human BMP1 gene. In each of the four proteins one intron was found to be inserted in the codon for a similar Gly residue which is highly conserved in this position within the astacin family.
Amino Acid Sequence↗
X-ray absorption spectroscopy study of zinc coordination in tetanus neurotoxin, astacin, alkaline protease and thermolysin.
Tetanus and botulinum neurotoxins constitute a new group of Zn-endopeptidases which has been recently actively investigated with the purpose of correlating their biochemical properties to their neurobiocytosis inhibitory capacity. Crystallographic data show that Zn-endopeptidases are characterized by an active site with a Zn atom coordinated to two histidines and glutamate-bound water molecule. The two histidines and glutamate resides belong to the HEXXH motif which is characteristic of most Zn-endopeptidases. A forth metal ligand is a glutamate in thermolysin-like proteinases, but it is an histidine in the astacin family of proteinases and in alkaline protease. Astacin and alkaline protease possess a tyrosine as fifth Zn ligand, whose position in the case of alkaline protease could not be determined by X-ray crystallography. Not much is known about the atom arrangement around the active site in tetanus neurotoxin. In this work X-ray absorption spectroscopy has been used to obtain information on the Zn coordination mode in tetanus neurotoxin. The near-edge and extended fine-structure absorption spectra of this toxin are compared with those of astacin, alkaline protease and thermolysin. The present data and sequence information suggest a new pattern of Zn coordination in tetanus neurotoxin with one water molecule and three aromatic residues as metal ligands. These residues are the two histidines of the characteristic motif and a tyrosine which is tentatively identified with Tyr242, on the basis of sequence comparison and mutagenesis experiments. The mean distances of the Zn from the nearest coordinated atoms is reported. Our results indicate that alkaline protease, like astacin, also possesses a tyrosine as a fifth ligand.
Amino Acid Sequence↗
Structure of astacin with a transition-state analogue inhibitor.
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Binding Sites↗
Crystal structures, spectroscopic features, and catalytic properties of cobalt(II), copper(II), nickel(II), and mercury(II) derivatives of the zinc endopeptidase astacin. A correlation of structure and proteolytic activity.
The catalytic zinc ion of astacin, a prototypical metalloproteinase from crayfish, has been substituted by Co(II), Cu(II), Hg(II), and Ni(II) in order to probe the role of the metal for both catalysis and structure. Compared to Zn(II)-astacin, Co(II)- and Cu(II)-astacin display enzymatic activities of about 140 and 37%, respectively, while Ni(II)- and Hg(II)-astacin are almost inactive. The electron paramagnetic resonance spectrum of Cu(II)-astacin is typical of 5-fold coordinated copper(II), and its intense absorption maxima at 445 and 325 nm are probably due to ligand-metal charge-transfer transitions involving Tyr-149. This residue had been identified previously by x-ray crystallography of the zinc enzyme as a zinc ligand, in addition to three imidazoles and a glutamic acid-bound water molecule. We present now the refined high-resolution x-ray crystal structures of Cu(II)-, Co(II)-, and Ni(II)-astacin, which exhibit a virtually identical protein framework to the previously analyzed structures of Zn(II)-, apo-, and Hg(II)-astacin. In Co(II)- and Cu(II)-astacin, the metal is penta-coordinated similarly to the native zinc enzyme. In the Ni(II) derivative, however, an additional solvent molecule expands the metal coordination sphere to a distorted octahedral ligand geometry, while in Hg(II)-astacin, no ordered solvent molecule at all is observed in the inner coordination sphere of the metal. This indicates a close correlation between catalytic properties and ground-state metal coordination of astacin.
Amino Acid Sequence↗
Implications of the three-dimensional structure of astacin for the structure and function of the astacin family of zinc-endopeptidases.
Astacin, a zinc-endopeptidase from the crayfish Astacus astacus L., represents a structurally distinct group of metalloproteinases termed the 'astacin family'. This protein family includes oligomeric membrane-bound proteins with zinc proteinase domains found in rodent kidneys (meprins A and B) and human small intestine (N-benzoyl-L-tyrosyl-4-aminobenzoate hydrolase). Another branch of this family comprises morphogenetically active proteins, which induce bone formation (human bone morphogenetic protein 1), or which play specific roles during the embryonic development of amphibians, fishes, echinoderms, and insects. The X-ray crystal structure of astacin has recently been solved to a resolution of 0.18 nm [Bode et al. (1992) Nature 358, 164-167]. This structure is different from hitherto known metalloendopeptidase structures and has been used in the present study to analyze the structures of the other members of the astacin protein family. Computer-assisted modelling of the proteolytic domain of the alpha-subunit of meprin A based on the astacin structure is possible if five single and one double residue deletions and three single residue insertions are implied. The proteinase domains of the other astacins can be included in the model-based sequence alignment by introducing additionally three insertions and one deletion. All of these insertions and deletions are observed in loop segments connecting regular secondary structure elements and should leave the overall structure unaltered. The topology of residues forming the zinc-binding active site of astacin corresponds to almost identical arrangements in all other astacins, suggesting that these are likewise metalloproteinases. Based on this similarity, it is proposed that the active-site metal ion of the astacins is penta-coordinated by three histidine residues, a tyrosine residue and a water molecule in a trigonal bipyramidal geometry. Other remarkable common features are a hydrophobic cluster in the N-terminal domain and a conserved, solvent-filled cavity buried in the C-terminal domain. Most interestingly, the amino-termini of all astacins can be modelled to start in a corresponding internal water cavity as seen in the astacin template, where the terminal alanine residue forms a water-linked salt bridge to Glu103, directly adjacent to His102, the third zinc ligand. Therefore, an activation mechanism for the astacins reminiscent of that of the trypsin-like proteinases had been suggested, which now seems to be probable also for the other astacins. Besides these common traits, there are some minor differences which may have important consequences on the function of the astacins.(ABSTRACT TRUNCATED AT 400 WORDS)
Amino Acid Sequence↗
Refined 1.8 A X-ray crystal structure of astacin, a zinc-endopeptidase from the crayfish Astacus astacus L. Structure determination, refinement, molecular structure and comparison with thermolysin.
Astacin, a 200 residue digestive zinc-endopeptidase from the crayfish Astacus astacus L., is the prototype of the "astacin family", which comprises several membrane-bound mammalian endopeptidases and developmentally implicated regulatory proteins. Large trigonal crystals of astacin were grown, and X-ray reflection data to 1.8 A resolution were collected. The astacin structure has been solved by multiple isomorphous replacement using six heavy-atom derivatives, and refined to a crystallographic R-value of 0.158 applying stringent constraints. All 200 residues are clearly defined by electron density; 181 solvent molecules have been localized. Besides the native structure, the structures of Hg-astacin (with a mercury ion replacing the zinc) and of the apoenzyme were also refined. The astacin molecule exhibits a kidney-like shape. It consists of an amino-terminal and a carboxy-terminal domain, with a deep active-site cleft in between. The zinc ion, located at the bottom of this cleft, is co-ordinated in a novel trigonal-bipyramidal geometry by three histidine residues, a tyrosine and by a water molecule, which is also bound to the carboxylate side-chain of Glu93. The amino-terminal domain of astacin consists mainly of two long alpha-helices, one centrally located and one more peripheral, and of a five-stranded pleated beta-sheet. The amino terminus protrudes into an internal, water-filled cavity of the lower domain and forms a buried salt bridge with Glu103; amino-terminally extended pro-forms of astacin are thus not compatible with this structure. The carboxy-terminal domain of astacin is mainly organized in several turns and irregular structures. Because they share sequence identity of about 35%, the structures of the proteolytic domains of the other "astacin" members must be quite similar to astacin. Only a few very short deletions and insertions quite distant from the active-site distinguish their structures from astacin. The five-stranded beta-sheet and the two helices of the amino-terminal domain of astacin are topologically similar to the structure observed in the archetypal zinc-endopeptidase thermolysin; the rest of the structures are, in contrast, completely unrelated in astacin and thermolysin. The zinc ion, the central alpha-helix and the zinc-liganding residues His92, Glu93 and His96 of astacin are nearly superimposable with the respective groups of thermolysin, namely with the zinc ion, the "active-site helix", and His142TL, Glu143TL and His146TL of the zinc-binding consensus motif His-Glu-Xaa-Xaa-His (where Xaa is any amino acid residue).(ABSTRACT TRUNCATED AT 400 WORDS)
Amino Acid Sequence↗
Structure of astacin and implications for activation of astacins and zinc-ligation of collagenases.
Astacin, a digestive zinc-endopeptidase from the crayfish Astacus astacus L., is the prototype for the 'astacin family', which includes mammalian metallo-endopeptidases and developmentally regulated proteins of man, fruitfly, frog and sea urchin. Here we report the X-ray crystal structure of astacin, which reveals a deep active-site cleft, with the zinc at its bottom ligated by three histidines, a water molecule and a more remote tyrosine. The third histidine (His 102) forms part of a consensus sequence, shared not only by the members of the astacin family, but also by otherwise sequentially unrelated proteinases, such as vertebrate collagenases. It may therefore represent the elusive 'third' zinc ligand in these enzymes. The amino terminus of astacin is buried forming an internal salt-bridge with Glu 103, adjacent to His 102. Astacin pro-forms extended at the N terminus, as observed for some 'latent' mammalian astacin homologues, did not exhibit this 'active' conformation, indicating an activation mechanism reminiscent of trypsin-like serine proteinases.
Amino Acid Sequence↗
alpha2-Macroglobulin from hemolymph of the freshwater crayfish Astacus astacus.
1. A high mol. wt proteinase inhibitor has been purified from the haemolymph of the freshwater crayfish Astacus astacus. 2. The protein is a disulphide-bonded dimer (Mr 390,000) of two identical polypeptide chains (Mr 185,000). 3. The inhibitor displays a broad specificity and protects trypsin from inhibition by soybean trypsin inhibitor and thus is similar to vertebrate alpha 2-macroglobulin. 4. The alpha 2-macroglobulin-like inhibitor from Astacus interacts with bovine trypsin in an equimolar stoichiometry thereby decreasing tryptic hydrolysis of N-benzoyl-L-arginine-ethylester to 50% residual activity. In contrast, the activity of Astacus protease, a digestive zinc proteinase from crayfish toward succinyl-alanyl-alanyl-alanyl-4-nitroanilide is inhibited almost completely. 5. Sensitivity of the inhibitor to methylamine and autolytic cleavage suggests the presence of an internal thioester bond. 6. The N-terminal amino acid sequence of Astacus alpha 2-macroglobulin is strongly related to the alpha 2-macroglobulins from Pacifastacus leniusculus (91% identity) and from the lobster Homarus americanus (72% identity). In contrast, only 25% of the residues are identical with the alpha 2-macroglobulin from the horseshoe crab Limulus polyphemus. There is also a faint similarity to human complement protein C3 and human alpha 2-macroglobulin.
Amino Acid Sequence↗
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)
Amino Acid Sequence↗
Thiol containing compounds and amino acid hydroxamates as reversible synthetic inhibitors of Astacus protease.
Reversible synthetic inhibitors are characterized for Astacus protease, a 22,614-Da zinc containing neutral endopeptidase from the digestive tract of crayfish. Effective inhibition was demonstrated for several simple thiol containing compounds and a series of amino acid hydroxamates. Both classes of inhibitors had ID50 values ranging from 10(-2) to 10(-4) M for inhibition of hydrolysis of succinyl-Ala-Ala-Ala-p-nitroanilide. Tyrosine hydroxamate was found to be the most effective inhibitor with an ID50 of 175 microM and the mode of inhibition by this compound was determined to be of the simple noncompetitive type. In contrast to the other inhibitors tested, cysteine was seen to partially inactivate the enzyme in a time-dependent manner. The kinetics of this process was studied in detail using progress curve analysis. It was determined that cysteine was acting as a weak chelator and slowly establishing an equilibrium between metallo- and apoenzyme. In the presence of the strong zinc scavenger EDTA, cysteine can, in effect, function as a catalyst in transferring the metal from the protein to the secondary chelator at a rate 10,000 times faster than the rate of unassisted zinc dissociation. The series of amino acid hydroxamates served as probes into the microenvironment of the active site. Possible binding modes of the inhibitors are discussed on the basis of the relationship between the chemical nature of the inhibitor side chains and the strength of inhibition.
Amino Acids↗
Biosynthesis of Astacus protease, a digestive enzyme from crayfish.
For the first time, the site of biosynthesis of a well characterized invertebrate digestive enzyme is localized. The enzyme chosen, Astacus protease, is a zinc-metalloenzyme occuring in high concentration in the gastric fluid of the freshwater crayfish Astacus astacus. Enzyme production was stimulated in adult crayfish either by feeding or by removal of the gastric fluid. Immunohistochemistry, cytology and investigation with radioactive tracers demonstrate that in the hours following stimulation, new enzyme was produced in the F-cells of the midgut gland and subsequently discharged into the midgut gland lumen. The enzyme was then accumulated and stored extracellularly in the cardiac stomach in active form. The mechanism of enzyme production observed in Astacus differs considerably from vertebrates suggesting an alternative model for synthesis and storage of digestive enzymes.
Animals↗
Kinetic evidence for cooperative binding of two ortho-phenanthroline molecules to astacus protease during metal removal.
Kinetic evidence is presented that introduces a new possibility for a mechanism of metal removal from a protein by a chelator. Astacus protease is a 22,614 dalton zinc-metalloendopeptidase from the digestive tract of the freshwater crayfish. Recent studies have shown that it contains a single zinc atom and that removal of this metal yields inactive apo-enzyme, which can be reactivated upon readdition of zinc, cobalt, or copper. The enzyme is inactivated by metal chelators in a time and concentration dependent manner. The inactivation of Zn-Astacus protease by 1,10-phenanthroline (OP) can be monitored continuously in the presence of substrate. The concentration of substrate was found to have no effect on the inactivation rate, indicating that the chelator binding during inactivation is of the noncompetitive type. First-order rate constants for the inactivation process are seen to depend on the concentration of chelator in a sigmoidal manner. Based on mathematics analogous to that for cooperativity in enzyme-substrate kinetics, the deduction is made that there are two OP binding sites on the protein and that the rate of inactivation is related to the saturation of both sites with ligand. If one uses this model, the limiting rate constant of inactivation upon saturation of both sites with ligand is 6.76 x 10(-3) sec-1, and the half maximal rate occurs at an OP concentration of 6.52 mM. A mechanism is proposed wherein both protein bound chelators can cooperate during metal removal either by direct chelation of the metal or by allosteric means. The proposed model and the noncompetitive binding of chelator and substrate are discussed in relation to a recently proposed metal binding site.
Animals↗
Protease inhibitors from Ecballium elaterium seeds.
Several protease inhibitors were found in the seeds of a Cucurbitacea, Ecballium elaterium, and were separated from one another by affinity and molecular sieve chromatography. Three main trypsin isoinhibitors were purified by ion-exchange chromatography and the sequence of the major one, EETI II, was elucidated and compared with other inhibitors of the squash family. It is a peptide of M.W. 3020 of strong inhibitory activity (Ka = 8 x 10(11) M-1) against trypsin, showing high Gly content, six half-cystine residues, but devoid of histidine, threonine, tryptophan, and tyrosine residues.
Amino Acid Sequence↗
Amino acid sequence of a unique protease from the crayfish Astacus fluviatilis.
The amino acid sequence of a protease from the crayfish Astacus fluviatilis has been determined from overlapping sets of peptides derived largely by cleavage at Met, Lys, or Arg residues. The protein comprises 200 amino acid residues in a single polypeptide chain, corresponding to a molecular mass of 22,614 daltons. Two disulfide bonds link Cys-42 to Cys-198 and Cys-64 to Cys-84. The sequence of this invertebrate protease appears to be unique since it has no homologous relationship to any of the known protein sequences.
Amino Acid Sequence↗