Harald Tschesche

Enhanced cleavage of type II collagen by collagenases in osteoarthritic articular cartilage.

We demonstrate the direct involvement of increased collagenase activity in the cleavage of type II collagen in osteoarthritic human femoral condylar cartilage by developing and using antibodies reactive to carboxy-terminal (COL2-3/4C(short)) and amino-terminal (COL2-1/4N1) neoepitopes generated by cleavage of native human type II collagen by collagenase matrix metalloproteinase (MMP)-1 (collagenase-1), MMP-8 (collagenase-2), and MMP-13 (collagenase-3). A secondary cleavage followed the initial cleavage produced by these recombinant collagenases. This generated neoepitope COL2-1/4N2. There was significantly more COL2-3/4C(short) neoepitope in osteoarthritis (OA) compared to adult nonarthritic cartilages as determined by immunoassay of cartilage extracts. A synthetic preferential inhibitor of MMP-13 significantly reduced the unstimulated release in culture of neoepitope COL2-3/4C(short) from human osteoarthritic cartilage explants. These data suggest that collagenase(s) produced by chondrocytes is (are) involved in the cleavage and denaturation of type II collagen in articular cartilage, that this is increased in OA, and that MMP-13 may play a significant role in this process.

Crystal structure of the complex formed by the membrane type 1-matrix metalloproteinase with the tissue inhibitor of metalloproteinases-2, the soluble progelatinase A receptor

The proteolytic activity of matrix metalloproteinases (MMPs) towards extracellular matrix components is held in check by the tissue inhibitors of metalloproteinases (TIMPs). The binary complex of TIMP‐2 and membrane‐type‐1 MMP (MT1‐MMP) forms a cell surface located ‘receptor’ involved in pro‐MMP‐2 activation. We have solved the 2.75 Å crystal structure of the complex between the catalytic domain of human MT1‐MMP (cdMT1‐MMP) and bovine TIMP‐2. In comparison with our previously determined MMP‐3–TIMP‐1 complex, both proteins are considerably tilted to one another and show new features. CdMT1‐MMP, apart from exhibiting the classical MMP fold, displays two large insertions remote from the active‐site cleft that might be important for interaction with macromolecular substrates. The TIMP‐2 polypeptide chain, as in TIMP‐1, folds into a continuous wedge; the A‐B edge loop is much more elongated and tilted, however, wrapping around the S‐loop and the β‐sheet rim of the MT1‐MMP. In addition, both C‐terminal edge loops make more interactions with the target enzyme. The C‐terminal acidic tail of TIMP‐2 is disordered but might adopt a defined structure upon binding to pro‐MMP‐2; the Ser2 side‐chain of TIMP‐2 extends into the voluminous S1′ specificity pocket of cdMT1‐MMP, with its Oγ pointing towards the carboxylate of the catalytic Glu240. The lower affinity of TIMP‐1 for MT1‐MMP compared with TIMP‐2 might be explained by a reduced number of favourable interactions.

The X-ray crystal structure of the catalytic domain of human neutrophil collagenase inhibited by a substrate analogue reveals the essentials for catalysis and specificity.

Matrix metalloproteinases are a family of zinc endopeptidases involved in tissue remodelling. They have been implicated in various disease processes including tumour invasion and joint destruction. These enzymes consist of several domains, which are responsible for latency, catalysis and substrate recognition. Human neutrophil collagenase (PMNL‐CL, MMP‐8) represents one of the two ‘interstitial’ collagenases that cleave triple helical collagens types I, II and III. Its 163 residue catalytic domain (Met80 to Gly242) has been expressed in Escherichia coli and crystallized as a non‐covalent complex with the inhibitor Pro‐Leu‐Gly‐hydroxylamine. The 2.0 A crystal structure reveals a spherical molecule with a shallow active‐site cleft separating a smaller C‐terminal subdomain from a bigger N‐terminal domain, composed of a five‐stranded beta‐sheet, two alpha‐helices, and bridging loops. The inhibitor mimics the unprimed (P1‐P3) residues of a substrate; primed (P1′‐P3′) peptide substrate residues should bind in an extended conformation, with the bulky P1′ side‐chain fitting into the deep hydrophobic S1′ subsite. Modelling experiments with collagen show that the scissile strand of triple‐helical collagen must be freed to fit the subsites. The catalytic zinc ion is situated at the bottom of the active‐site cleft and is penta‐coordinated by three histidines and by both hydroxamic acid oxygens of the inhibitor. In addition to the catalytic zinc, the catalytic domain harbours a second, non‐exchangeable zinc ion and two calcium ions, which are packed against the top of the beta‐sheet and presumably function to stabilize the catalytic domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural properties of matrix metalloproteinases

Abstract. Matrix metalloproteinases (MMPs) are involved in extracellular matrix degradation. Their proteolytic activity must be precisely regulated by their endogenous protein inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). Disruption of this balance results in serious diseases such as arthritis, tumour growth and metastasis. Knowledge of the tertiary structures of the proteins involved is crucial for understanding their functional properties and interference with associated dysfunctions. Within the last few years, several three-dimensional MMP and MMP-TIMP structures became available, showing the domain organization, polypeptide fold and main specificity determinants. Complexes of the catalytic MMP domains with various synthetic inhibitors enabled the structure-based design and improvement of high-affinity ligands, which might be elaborated into drugs. A multitude of reviews surveying work done on all aspects of MMPs have appeared in recent years, but none of them has focused on the three-dimensional structures. This review was written to close the gap.

Unusual solution properties of proline and its interaction with proteins

Proline in aqueous solution shows several properties which are unusual for low molecular weight substances. Investigations of solubility, density and viscosity revealed behaviour which is characteristic for hydrophilic colloids. 1H-NMR studies indicated a strong hydrogen bonding of water in proline solutions, especially at high concentrations of the solute. From these results it was concluded that proline forms aggregates by stepwise stacking and hydrophobic interaction of the pyrrolidine ring. Thus, the proposed multimer contans a hydrophobic backbone and hydrophilic groups on the surface, exposed to water. Proline solutions are able to increase the solubility of sparingly soluble proteins. The enhancement effect depends on the nature of the protein and on the proline concentration. It is postulated that by a hydrophobic interaction of proline with hydrophobic surface residues of proteins their hydrophilic area is increased. The presence of proline in solutions of the well soluble protein bovine albumin reduces the precipitation of this protein by ethanol and (NH4)2SO4, presumably by an increased water-binding capacity of the proline-protein solution.

A 25 kDa α2-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 23 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 α2‐microglobulin‐related protein from rats, a protein so far unknown in humans.

Activation of Type IV Procollagenases by Human Tumor-associated Trypsin-2

Increased production of proteinases, such as matrix metalloproteinases (MMPs), is a characteristic feature of malignant tumors. Some human cancers and cell lines derived from them also express trypsinogen, but the function of the extrapancreatic trypsin has remained unclear. In this study we cloned and sequenced trypsinogen-2 cDNA from human COLO 205 colon carcinoma cells and characterized the ability of the enzyme to activate latent human type IV procollagenases (proMMP-2 and proMMP-9). As shown by cloning and N-terminal amino acid sequencing, the amino acid sequence of tumor-associated trypsin-2 is identical to that of pancreatic trypsin-2. We found that both pancreatic trypsin-2 and tumor cell-derived trypsin-2 are efficient activators of proMMP-9 and are capable of activating proMMP-9 at a molar ratio of 1:1000, the lowest reported so far. Human trypsin-2 was a more efficient activator than widely used bovine trypsin and converted the 92-kDa proMMP-9 to a single 77-kDa product that was not fragmented further. The single peptide bond cleaved by trypsin-2 in proMMP-9 was Arg87-Phe88. The generation of the 77-kDa species coincided with the increase in specific activity of MMP-9. In contrast, trypsin-2 only partially activated proMMP-2. Trypsin-2 cleaved the Arg99-Lys100 peptide bond of proMMP-2 generating 62–65-kDa MMP-2 species. Trypsin-2-induced proMMP-2 and -9 conversions were inhibited by tumor-associated trypsin inhibitor added either prior to or during activation indicating that proMMPs were not activated autocatalytically. Trypsin-2 also activated proMMPs associated with tissue inhibitor of matrix metalloproteinases, the complexes of which are thought to be the major MMP forms in vivo. The ability of human tumor cell-derived trypsin-2 to activate latent MMPs suggests a role for trypsin-2 in initiating the proteinase cascade that mediates tumor invasion and metastasis formation.

Interleukin-8 synthesis and the onset of labor

Objective To examine the role of cytokines in cervical dilation. Methods In 55 patients undergoing cesarean delivery, we took samples from the lower uterine segment, the decidua, and the membranes. We determined the concentrations of interleukin (IL)-2, IL-8, tumor necrosis factor-α, matrix metalloproteinase (MMP)-8, and MMP-9 in the different tissues. Results Depending on the state of labor, we observed a significant increase (P < .001) in IL-8 concentrations in the lower uterine segment. The leukocyte enzymes MMP-8 and MMP-9 were highly significantly correlated with the IL-8 concentrations. Conclusion Interleukin-8 is critically involved in the process of parturition in humans. Interleukin-8 concentrations in the myometrium, decidua, and membranes correlated strongly with the observed MMP-8 and MMP-9 concentrations.

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.

Insights into MMP-TIMP interactions.

ABSTRACT: The proteolytic activity of the matrix metalloproteinases (MMPs) involved in extracellular matrix degradation must be precisely regulated by their endogenous protein inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). Disruption of this balance can result in serious diseases such as arthritis and tumor growth and metastasis. Knowledge of the tertiary structures of the proteins involved in such processes is crucial for understanding their functional properties and to interfere with associated dysfunctions. Within the last few years, several three‐dimensional structures have been determined showing the domain organization, the polypeptide fold, and the main specificity determinants of the MMPs. Complexes of the catalytic MMP domains with various synthetic inhibitors enabled the structure‐based design and improvement of high‐affinity ligands, which might be elaborated into drugs. Very recently, structural information also became available for some TIMP structures and MMP‐TIMP complexes, and these new data elucidated important structural features that govern the enzyme‐inhibitor interaction.