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Biomedical subjects

H Tschesche

Publications and source records attributed to H Tschesche.

At least 19 recordsLinked to original sources

A 25 kDa alpha 2-microglobulin-related protein is a component of the 125 kDa form of human gelatinase.

Besides the monomeric mammalian 95 kDa progelatinase, two additional forms, a disulfide-bridged 220 kDa dimer and a 125 kDa form were isolated from human PMN leukocytes. The 125 kDa progelatinase was identified as a covalently linked, disulfide-bridged heterodimer formed of the monomer with a 25 kDa protein. This 25 kDa protein was isolated from gelatinase bound to the affinity support of gelatin-Sepharose and eluted by DTE-containing buffer. The amino acid sequence of tryptic peptides of this protein revealed homology with an alpha 2-microglobulin-related protein from rats, a protein so far unknown in humans.

Alpha-Globulins

Synthesis of tissue inhibitor of metalloproteinase-1 (TIMP-1) in human hepatoma cells (HepG2). Up-regulation by interleukin-6 and transforming growth factor beta 1.

Metalloproteinases and their specific inhibitors, believed to play a role in extracellular matrix metabolism, are regulated by inflammatory cytokines. Here we have addressed the question of whether liver, the major site of synthesis of plasma proteinase inhibitors, is also capable of synthesizing the tissue inhibitor of metalloproteinase-1 (TIMP-1). We show at mRNA and protein levels that TIMP-1 is expressed in differentiated human hepatoma cells (HepG2) and that its synthesis is up-regulated by interleukin-6 (IL-6), transforming growth factor beta 1 and phorbol 12-myristate 13-acetate. The physiological role of this phenomenon is underlined by the fact that lipopolysaccharide administration into rats in vivo, as well as IL-6-stimulation of rat hepatocytes in primary culture, also leads to an increase of TIMP-1 mRNA in liver cells.

Carcinoma, Hepatocellular

Formation of a covalent Hg-Cys-bond during mercurial activation of PMNL procollagenase gives evidence of a cysteine-switch mechanism.

A common method for the activation of mammalian metalloproteinases is the use of mercurial compounds. Activation of PMNL procollagenase by soluble mercurials takes place as a three-step mechanism with a final intermolecular loss of the PRCGVPD autoinhibitor region. In this study covalently bound mercury in the form of mercurial agarose was chosen to probe activation of PMNL procollagenase. Activation was not achieved, since the final intermolecular cleavage with removal of the PRCGVPD motif could not take place. An intermediate form of the enzyme was bound to the column. Its N-terminal sequence determination proved cleavage of the Asp64-Met65 peptide bond leaving the cysteine of the propeptide domain for covalent attachment to the mercurial agarose. This gives further evidence of a cysteine-switch mechanism involving Cys71.

Amino Acid Sequence

Mercurial activation of human PMN leucocyte type IV procollagenase (gelatinase).

Autoproteolytic activation and processing of human polymorphonuclear leucocyte (PMNL) type IV procollagenase (gelatinase) was initiated by HgCl2 and was investigated by kinetic analysis and N-terminal sequence determination of the reaction products. In the first instance the propeptide domain was lost by subsequent cleavage of the Asp15-Leu16, Glu40-Met41, Leu52-Leu53 and Ala74-Met75 peptide bonds. The PRCGVPD sequence motif (residues Pro78-Asp84), which is conserved in all metalloproteinases and expected to be relevant for latency, remained uncleaved at the activated enzyme. The generated intermediate was further processed by three C-terminal cleavages. The Glu666-Leu667, Ala506-Glu507 and Ala398-Leu399 bonds were hydrolysed successively. From the fragmentation products we were able to conclude that three released fragment peptides contained unpaired free cysteine with the residues Cys497, Cys653, Cys683. Cleavage of the first C-terminal peptide bond resulted in the loss of one of the conserved Cys residues of the hemopexin-like domain, whereas the Cys residue of the PRCGVPD motif was retained at the fully active enzyme. The possibility of an entirely different activation mechanism for PMNL type IV procollagenase is discussed.

Amino Acid Sequence

Isolation and characterization of tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) from human rheumatoid synovial fluid.

The tissue inhibitors of metalloproteinases TIMP-1 and TIMP-2 were purified to apparent homogeneity from human rheumatoid synovial fluid (HRSF). The inhibitors were isolated by dissociation of non-covalent gelatinase/TIMP complexes. TIMP-1 migrated as a single polypeptide with Mr 28,500 on SDS-PAGE, while the Mr of TIMP-2 was 21,000. The inhibitory activity was stable under heat and acid pH. N-terminal sequence data were obtained for the first 15 residues of both inhibitors and showed identity to the human fibroblast inhibitors TIMP-1 and TIMP-2. This is the first demonstration that TIMP-1 and TIMP-2 can be directly purified from human rheumatoid synovial fluid. The complex formation between the metalloproteinase inhibitors and leucocyte metalloproteinases was shown by immunoblotting.

Amino Acid Sequence

Origin of cervical collagenase during parturition.

Cervical biopsy specimens were obtained under standard conditions from the posterior lip of the uterine cervix in 105 patients. A significant increase of collagenase activity was observed during parturition as determined with an assay with iodine 125-labeled native triple-helical collagen type I as the substrate. The collagenase was not likely to originate from cervical fibroblasts because in situ hybridization failed to detect synthesis of the specific procollagenase messenger ribonucleic acid. However, migration of polymorphonuclear leukocytes into the cervical stroma occurred on onset of labor, and an antibody specific for human leukocyte collagenase that did not cross react with fibroblast collagenase revealed the presence of the enzyme in the granules of polymorphonuclear leukocytes and subsequently in the extracellular matrix of the cervix. Therefore it is likely that the cells critically involved in collagen degradation during cervical dilatation are not resident fibroblasts but rather polymorphonuclear leukocytes emigrating from blood vessels.

Blotting, Northern

Latent collagenase and gelatinase from human neutrophils and their activation.

Procollagenase M(r) 85,000 (SDS-PAGE) was purified from buffy coat to homogeneity and represents a stable single polypeptide chain forming the entire proenzyme. The procollagenase can be activated by various proteinases, e.g. trypsin, chymotrypsin, cathepsin G, kallikrein and stromelysin and by different mercurial compounds. Proteolytic conversion of the latent enzyme to the active form by chymotrypsin is accompanied by a molecular weight reduction to an apparent M(r) 64,000. This active enzyme lacks the first 79 N-terminal residues. Activation by trypsin leads to a latent intermediate of apparent M(r) 70,000, lacking 48 N-terminal residues. The active enzyme is therefore generated upon prolonged incubation with trypsin by further cleavage of 22 N-terminal residues. Another latent intermediate form with apparent M(r) 69,000 is generated from the proenzyme upon incubation with leukocyte elastase by N-terminal cleavage of 53 or 64 residues, respectively. However, latent collagenase cannot be activated by plasmin. Activation by different mercurial compounds finally results in the formation of active collagenase with apparent M(r) 64,000. In contrast to the proenzyme, active collagenase can autolyse to give active M(r) 57,000 and 45,000 intermediates and two M(r) 28,000 fragments. Purification of latent leukocyte gelatinase yields three final products with apparent M(r) 98,000, 125,000 and 220,000 (SDS-PAGE; non reduced). Upon reduction, only the M(r) 98,000 form can be detected. The latent gelatinase can be activated in a similar manner as collagenase. Proteolytic activation by trypsin leads after N-terminal cleavage to an active gelatinase with sequence homology to leukocyte collagenase.

Amino Acid Sequence

Migration of polymorphonuclear leukocytes through human amnion membrane--a scanning electron microscopic study.

Scanning electron microscopy has been used to examine the changes in the basement membrane structure induced by polymorphonuclear leukocytes during leukodiapedesis. A diapedesis model based on a Boyden chamber fitted with a human amnion membrane simulated inflammatory processes during which leukocytes are stimulated to leave blood vessels and to penetrate basement membranes. In the Boyden chamber, a chemotactic gradient was developed from a solution of 10(-7) M formyl-methionyl-leucyl-phenylalanine, which induced the cells to migrate through the membrane. Our observations suggest that leukocytes, like tumor cells, emigrate in a three-step process. In the first instance, they adhere to the basement membrane. A local partial proteolysis follows, which is caused by secreted metalloproteinases, especially by gelatinase. The partial dissolution of the matrix facilitates cell penetration. Active locomotion and squeezing through the residual membrane matrix allows the cells to penetrate without complete local membrane destruction. The last step involves migration of the cells through the connective tissue barrier to the site of infection. The type I collagen fibres which form this loose stroma tissue are no obstacle and can be pushed aside without proteolytic degradation.

Amnion

Mercurial activation of human polymorphonuclear leucocyte procollagenase.

The mechanism of human polymorphonuclear leucocyte (PMNL) procollagenase activation by HgCl2 was investigated by kinetic and sequence analysis of the reaction products. HgCl2 activated PMNL procollagenase by intramolecular autoproteolytic cleavage of the Asn53-Val54 peptide bond to generate a collagenase species of Mr 65000, which was immediately converted into a second intermediate collagenase form by further autoproteolytic cleavage of the Asp64-Met65 peptide bond within the propeptide domain. This intermediate form (Met65 N-terminus) reached maximum concentrations after 45 min and displayed only about 40% of the maximum available enzymatic activity. Final activation was obtained after autoproteolytic cleavage of either Phe79-Met80 or Met80-Leu81 peptide bonds. Furthermore, activation in the presence of TIMP-1 did not suppress the intramolecular autoproteolytic cleavage of the Asn53-Val54 peptide bond. Complete inhibition of further autoproteolytic decay of the enzyme or generated peptides was observed, which was obviously due to complex formation between the intermediate collagenase form (Val54 N-terminus) and inhibitor, which was visualized using the Western blot technique. Thus PMNL procollagenase activation by HgCl2 followed a three-step activation mechanism which is entirely different from the known activation mechanisms of the fibroblast matrix metalloproteinases.

Amino Acid Sequence

Recombinant aprotinin homologue with new inhibitory specificity for cathepsin G.

The substitution of amino acids in the reactive site of aprotinin, a bovine serine proteinase inhibitor with potent activity against trypsin, plasmin and tissue kallikrein, led to a change in specificity of the inhibitor. Twelve new aprotinin variants prepared by recombinant DNA technology and expressed in Escherichia coli clearly demonstrated that the neighbouring groups of the P1 residue, in particular P'2, contribute to the specificity of the inhibitor, while earlier investigations on semisynthetically prepared variants revealed the importance of the P1 residue in dominating the inhibitory specificity. Recombinant aprotinin variants which act specifically against chymotrypsin-like proteinases, were obtained by substitution of the amino acids in position P1 and P'2 by hydrophobic amino acids like phenylalanine, tyrosine and leucine. Some of these variants, particularly those with phenylalanine or leucine substitutions, were also found to exhibit inhibitory activity against cathepsin G with an equilibrium constant of dissociation Ki of 10(-8) M. Inhibitory specificity against cathepsin G was not found in any semisynthetic variant prepared earlier.

Amino Acid Sequence

Inactivation of human plasma C1-inhibitor by human PMN leucocyte matrix metalloproteinases.

Highly purified human polymorphonuclear (PMN) leucocyte matrix metalloproteinases, collagenase and gelatinase, cleaved human plasma C1-inhibitor at the carboxyl site of Ala439 (P6). This led to a concomitant loss of C1-inhibitor activity. An additional cleavage site, at the carboxyl site of Ser441 (P4), was observed during PMN leucocyte gelatinase-induced inactivation, and a minor fragment of the plasma C1-inhibitor was generated.

Amino Acid Sequence

Release of proteinases from stimulated polymorphonuclear leukocytes. Evidence for subclasses of the main granule types and their association with cytoskeletal components.

The secretion of matrix-degrading proteinases and protein components involved in the production of cytotoxic metabolites is an important step in the sequence of defense reactions executed by polymorphonuclear leukocytes (PMNL) in response to stimulation. In the present report, we have analyzed degranulation of PMNL stimulated either with soluble synthetic peptides fLeu-Phe (fMet, formylmethionyl), or fAhx-Leu-Phe-Ahx-Tyr-Phe (Ahx, aminohexyl) which trigger chemotaxis and degranulation, or with opsonized zymosan which induces phagocytosis. The release of elastase, myeloperoxidase and lactoferrin-containing granules was not at all or only slightly (less than 6%) induced either by fAhx-Leu-Phe-Ahx-Tyr-Leu or by zymosan particles. In contrast, type-I collagenase and gelatinase were secreted in significant amounts after treatment with these agents. The disruption of microfilaments by cytochalasin B and subsequent stimulation of PMNL with the formyl-peptide led to the secretion of elastase, myeloperoxidase and lactoferrin, and enhanced the release of gelatinase. Disruption of microtubules by incubation with colcemid resulted in inhibition of fAhx-Leu-Phe-Ahx-Thyr-Leu and fAhx-Leu-Phe-Ahx-Tyr-Leu/cytochalasin-B-induced granule release. Furthermore, we found different patterns of enzyme distribution after fractionation by centrifugation: most (greater than 90%) type-I collagenase and gelatinase was measured in the supernatant whereas 60-90% of elastase, myeloperoxidase and lactoferrin had partitioned into the cytoskeleton-containing pellet. Our results suggest that the two main types of secretory vesicles identified in PMNL (specific and azurophilic granules) consist of subpopulations. The differential association of the various types of granules with cytoskeletal elements may serve to control their sequential discharge.

Cell Fractionation

Effect of immunosuppressive drugs on the release of metalloproteinases from human polymorphonuclear leukocytes.

The concentration of the metalloproteinases type I collagenase and gelatinase was measured in isolated polymorphonuclear leukocytes (PMNLs) of renal transplant recipients treated either with cyclosporin A (CyA) and prednisolone (Pr) (n = 8) or azathioprine (Aza) and Pr (n = 8), and of healthy subjects (n = 12). PMNLs of CyA- and Aza-treated transplant patients displayed markedly higher gelatinase content (2427 +/- 489 and 3284 +/- 357 ng/10(7) cells) than PMNLs of controls (528 +/- 83 ng/10(7) cells). There was also a higher content of type I collagenase in PMNLs (3374 +/- 292 ng/10(7) cells) of Aza-treated patients and significantly elevated levels in PMNLs of patients receiving CyA (3625 +/- 229 ng/10(7) cells) compared with healthy subjects (2878 +/- 151 ng/10(7) cells). In contrast, neutrophil lactoferrin content was lower in transplant patients. Thus, immunosuppressive drugs may reduce the release of leukocyte proteinases, which are known for their deleterious role in proteolytic tissue and matrix breakdown. In vitro, the effects of different immunosuppressive drugs on the release of lactoferrin, collagenase and gelatinase were investigated on FMLPNTL-stimulated PMNLs isolated from healthy subjects. CyA but not Aza or Pr caused inhibition of gelatinase, collagenase and lactoferrin release.

Adult

Enzymatic semisynthesis of aprotinin homologues mutated in P' positions.

The replacement of amino acids in the P'1 and P'2 position of aprotinin, the bovine pancreatic trypsin inhibitor, is described. Using the "modified" inhibitor as starting material, with the hydrolyzed reactive-site peptide bond Lys15-Ala16, the residues P'1 (Ala16) and P'2 (Arg17) were split off by the action of aminopeptidase K. Incorporation of suitable dipeptides containing a basic residue (Lys or Arg) in the C-terminal position was carried out in a "one pot" reaction involving trypsin-catalyzed coupling. In this way, the native fragment Ala16-Arg17 was reintroduced and also replaced by Gly-Arg, Ala-Lys, and Leu-Arg yielding intact inhibitor molecules. The mechanism for incorporation of dipeptides was investigated by treating the aprotinin derivative with the Arg17-Ile18 peptide bond hydrolyzed with trypsin under proteosynthetic conditions. We established that only inhibitor molecules cleaved between Lys15 and Xaa16 are intermediates leading to the desired products. The inhibitory properties of the new aprotinin homologues were tested, and the significance of the P'1 residue for the inhibition of trypsin, kallikrein, and chymotrypsin was deduced.

Amino Acid Sequence

Chemical semisynthesis of aprotinin homologues and derivatives mutated in P' positions.

An extended concept for the replacement of amino acids in the P' region of aprotinin by chemical semisynthesis is presented. Either fragment condensation with dipeptides protected as tert-butyl ester or stepwise introduction of two single amino acid-tert-butyl esters into a partially esterified aprotinin derivative (with free Lys15-carboxyl group) lacking the amino acids Ala16 and Arg17 leads to aprotinin homologues and derivatives mutated in the P'1 and P'2 position. This method may complement the recently reported enzymatic synthesis by enabling access to aprotinin homologues and derivatives, which cannot be prepared enzymatically. The synthesis of [Ala17]BPTI and [seco-17/18]BPTI is described in detail.

Amino Acid Sequence

Leukodiapedesis, release of PMN leukocyte proteinases and activation of PMNL procollagenase.

Human polymorphonuclear leukocytes mainly protect the body against invading microorganisms. Therefore, cells must migrate to the site of infection, whereby they traverse the endothelial cell layer of the blood vessels, basement membranes and the connective tissue. We investigated the unknown mechanism of penetration of basement membranes by scanning electron microscopy. Our findings indicate a three-step mechanism--adhesion, locally limited proteolytic degradation of matrix components and mechanical loosening of the matrix. Finally, cells locomote in the stroma tissue to the site of infection. These processes are induced by formyl-peptides amongst others. In addition, an increased release of collagenase induced by formyl-peptides was observed. In contrast, incubation with prostaglandin F2 alpha led to significantly higher levels of secreted collagenase, while elastase failed to be detected in the supernatant of stimulated cells. Since it has been shown that collagenase is secreted in a latent form, we have focussed our attention on the activation mechanism of the proenzyme, which was investigated by determination of the N-terminal sequences of intermediate and final, activated forms.

Amino Acid Sequence

Enzymatic fragment substitution as a tool in protein design.

An easy and rapid enzymatic method is described which allows replacement of P'-residues in bovine pancreatic trypsin inhibitor. Insertion of Xaa-Arg or Xaa-Lys into a BPTI fragment lacking P1' = Ala16 and P2' = Arg17 was carried out in a "one pot" reaction catalysed by trypsin in the presence of 80% 1,4 butanediol.

Amino Acid Sequence