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W Stöcker

Publications and source records attributed to W Stöcker.

At least 19 recordsLinked to original sources
PubMed1998

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↗
PubMed1998

Serum antibodies against membranous labyrinth in patients with "idiopathic" bilateral vestibulopathy.

To investigate the possibility of an autoimmune mechanism in idiopathic bilateral vestibulopathy (IBV), we screened patients' sera for antibodies against inner ear structures. IgG antibodies against membranous labyrinth (ampulla, semicircular canals, saccule and utricle) were detected in 8 of 12 patients by immunofluorescence on rat inner ear cryosections. All but one serum of 22 healthy controls and the sera of 6 patients with known autoimmune disorders showed only background staining. Low-titre anti-nuclear IgM antibodies were present in three control sera and one IBV serum. High-titre anti-nuclear IgM was found in a patient with lupus erythematosus and in one with scleroderma. Anti-nuclear IgM was not organ-specific. No human serum used contained detectable anti-vascular preformed antibodies. Cross-reactivity to sections of liver, kidney, cornea, brain and skeletal muscle was absent. Double-staining for IgG and F-actin, the primary constituent of hair cell cilia, did not show predominant Ig-coating of sensory hair cells. Immunosuppressive therapy in 3 IBV patients did not improve the disorder, probably owing to irreversible loss of sensory and neural structures. These data suggest that the bulk of anti-labyrinthine autoantibodies may be an epiphenomenon, yet a small subgroup of organ-specific autoantibodies may synergize with a cellular response in the development of vestibular lesions.

Autoantibodies↗
PubMed1998

Dysregulation of insulin-like growth factors in a case of generalized acquired lipoatrophic diabetes mellitus (Lawrence Syndrome) connected with autoantibodies against adipocyte membranes.

We report on a 33-year-old male patient with generalized acquired lipodystrophy, insulin resistant diabetes mellitus and acanthosis nigricans (Lawrence Syndrome). First probable symptoms of lipodystrophy (weight loss, shrinkage of subcutaneous fatty tissue, and loss of muscular strength) became evident three years ago, with the onset of diabetes mellitus occurring about six months later. The patient suffered from the following clinical symptoms: IDDM with increasing insulin-requirement, extreme reduction of fatty tissue, fatty liver hepatitis with elevated liver enzymes, glomerulopathy, muscular and neuropathic pains, as well as hypertriglyceridaemia. A basal C-peptide concentration is rather high. Definitely, the endogenous insulin secretion is increased. In other words, insulin resistance is documented. In an effort to identify the pathogenetic mechanisms of lipoatrophic diabetes mellitus in this patient and to develop a therapeutic strategy, antibodies against different tissues and endocrinologic regulation were investigated. It was possible to demonstrate the presence of serum autoantibodies against lipocytes of the subcutis and other tissues, against hepatic stellate cells, together with autoantibodies against different endocrine organs. By studying the basis of diabetic abnormalities relating to the growth hormone (GH), the insulin-like growth factor (IGF) dynamics in this patient, i.e. reductions of GH, IGF-I, IGF-II, IGF-Binding protein (IGF-BP) 2 and IGF-BP 3, were detected. An immunosuppressive treatment strategy was not beneficial.

Adipocytes↗
PubMed1997

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↗
PubMed1996

Combined detection of CD44 isoforms by exon-specific RT-PCR and immunohistochemistry in primary human brain tumors and brain metastases.

Expression of CD44 has been implicated in tumor growth and metastasis. Here we demonstrate CD44 expression in primary human brain tumors (n = 44) and brain metastases (n = 7) by RT-PCR and immunohistochemistry. Standard CD44 was found to be expressed by the majority of primary brain tumors and brain metastases. For the first time to our knowledge, CD44 expression is demonstrated for acoustic neurinomas and pituitary adenomas. Exon-specific analysis by RT-PCR and indirect immunofluorescence revealed expression of alternatively spliced CD44 isoforms in the group of brain metastases only. However, in one glioblastoma multiforme, expression of CD44v5 and CD44v6 was found immunohistochemically. This tumor took an unusual clinical course giving rise to multiple intrahepatic and lymph node metastases. Quantitatively different expression of standard CD44 in gliomas versus meningiomas is reported (p < 0.01).

Alternative Splicing↗
PubMed1996

[Cytokine therapy and the endocrine system: is it necessary to monitor more than the thyroid gland?].

UNLABELLED: Cytokines and Endocrine System: Is it Meaningful to Monitor More than the Thyroid Gland? AIM: It was the aim of this study to evaluate the occurrence, the degree, and clinical relevance of immunological phenomena in endocrine and non-endocrine organs as seen under therapy with interferon-alpha (IFN-alpha) and/or interleukin-2 (Il-2). PATIENTS AND METHODS: In 61 patients (age: 50.2 +/- 12.8 years) receiving cytokines as treatment for hepatological or hemato-oncological diseases, parameters of the thyroid gland, the gonadal system, the adrenal gland the pituitary gland, parameters of water and electrolyte-balance, and of bone metabolism were measured. All patients were treated with interferon in a mean dosage of 15.3 +/- 10.5 mio U subcutaneously per week. Additionally, 15 patients were treated with interleukin-2 (36.5 +/- 22.3 mio U subcutaneously per week). Among other assays, patients "sera were screened for the existance of autoantibodies against 30 different antigenic substrates using an indirect immunofluorescence technique (IIF). RESULTS: Pts, treated with IFN-alpha and IL-2 (n = 13) showed a significant induction of thyroid antibodies against thyroglobulin (anti-TG): 61 +/- 82 U/ml vs. 1000 +/- 2352 U/ml (p < 0.05) and against thyroid-peroxidase (anti-TPO): 162 +/- 538 vs. 468 +/- 1071 U/ml (p < 0.05). In pts, only treated with IFN-alpha (n = 26), the increase of anti-TG from 69 +/- 97 to 134 +/- 178 U/ml and of anti-TPO from 20 +/- 21 to 21 +/- 19 U/ml was insignificant. Patients with a combined treatment of IFN-alpha and Il-2 were mainly affected: 2 patients developed hypo- and 5 hyperthyroidism. Thus, regular controls of thyroid function during the entire duration of therapy is justified. We were unable to show any influence of the duration of treatment or dosage of cytokines on the endocrine and non-endocrine parameters investigated. In addition, 8 different AAB (against smooth/striated muscles, parietal cells, nuclear parts, myelin, keratin, endothel, and neuroendothel) were detected by IIF which did not change under therapy. Two patients newly developed AAB during therapy, however none of them suffered from clinical symptoms. AAB against endocrine organs other than the thyroid gland were not detected. No other endocrine system showed alterations of its function. CONCLUSION: Based on the data of this study we conclude that the thyroid gland should be regularly monitored for immunological and functional changes during cytokine therapy. The results do not support a general recommendation for a routine screening of other, non-thyroidal endocrine systems for autoimmune phenomena and alterations of function in each patient.

Adult↗
PubMed1995

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.

Amino Acid Sequence↗
PubMed1995

Astacin.

Explore the source record for details and available documents.

Amino Acid Sequence↗
PubMed1995

Structural features of a superfamily of zinc-endopeptidases: the metzincins.

A large number of zinc endopeptidases contain an HEXXHXXGXXH consensus motif in their catalytic site (single letter code; X is any amino acid residue). These enzymes can be grouped into four distinct families, the astacins, the adamalysins, the serralysins and the matrix metalloproteinases (matrixins). Despite a low degree of sequence similarity, their catalytic modules are topologically similar. A topology derived sequence alignment suggests that the four families form a superfamily, called the metzincins because of a perfectly superimposable methionine residue close to the zinc-binding active site. Topological similarity to the thermolysin-like enzymes indicates that these enzymes may have had a common ancestor.

Amino Acid Sequence↗
PubMed1994

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↗
PubMed1994

[Stiff-man syndrome. Report of 4 cases].

Four patients are presented who exhibited progressive muscular rigidity in both legs, the thoracolumbar, paraspinal and the abdominal muscles. In only one patient, there was an initial involvement of the shoulder girdle muscles. Electromyography in all four patients at rest recorded continuous electric activity resembling an interference pattern. Following sudden exteroceptive stimuli the activity increased in amplitude and density this corresponded clinically to painful spasms. All patients had oligoclonal banding in CSF. In the serum and CSF of three patients IgG subtype autoantibodies were detected. These have been found to be directed against GABAergic nerve terminals in the rat and human cerebellum and, more specifically, to glutamic acid decarboxylase. All patients improved on clonazepam. Trials of intermittent high-dose methylprednisolone administration gave relief from rigidity in one patient and permitted reduction of clonazepam in another. Intravenous immunoglobulins however had no effect in one patient.

Adult↗
PubMed1993

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↗
PubMed1993

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↗
PubMed1992

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↗
PubMed1992

The 70 kD heat shock protein (hsp 70) in soil invertebrates: a possible tool for monitoring environmental toxicants.

The expression of hsp 70 after heat shock or exposure to heavy metals/molluscicides was investigated by fluorography or immunoblot in three diplopods (Glomeris marginata, Cylindroiulus punctatus, Tachypodoiulus niger), two slugs (Deroceras reticulatum, Arion ater), and one isopod (Oniscus asellus). In O. asellus, hsp 70 expression occurred after heat shock and also after lead treatment, whereby a solution of 100 mg/kg Pb2+ was sufficient. Animals of the same species taken from a heavy metal polluted site in the vicinity of a lead/zinc smelter also showed the presence of hsp 70. The comparison of laboratory and field experiments demonstrated the suitability of O. asellus for monitoring tests. In contrast, the blot pattern after contamination with 1,000 mg/kg Pb2+ (in the mentioned diplopods) or different concentrations of the molluscicide Cloethocarb (BASF, FRG) (slugs) showed no differences compared to the respective control group.

Animals↗