Erik Martens

Zymography methods for visualizing hydrolytic enzymes

Zymography is a technique for studying hydrolytic enzymes on the basis of substrate degradation. It is a powerful, but often misinterpreted, tool yielding information on potential hydrolytic activities, enzyme forms and the locations of active enzymes. In this Review, zymography techniques are compared in terms of advantages, limitations and interpretations. With in gel zymography, enzyme forms are visualized according to their molecular weights. Proteolytic activities are localized in tissue sections with in situ zymography. In vivo zymography can pinpoint proteolytic activity to sites in an intact organism. Future development of novel substrate probes and improvement in detection and imaging methods will increase the applicability of zymography for (reverse) degradomics studies.

The gelatinase inhibitory activity of tetracyclines and chemically modified tetracycline analogues as measured by a novel microtiter assay for inhibitors

A quantitative nonisotopic solution assay for gelatinases and inhibitors was developed using biotinylated gelatin as enzyme substrate. In this assay, residual biotinylated substrate is sandwiched between avidin-coated plates and streptavidin-peroxidase and is quantified by the peroxidase reaction. This assay was useful for measuring gelatinase activities and defining the activities of gelatinase inhibitors. When 23 tetracycline analogues were compared, significant differences in gelatinase B inhibition were found between various compounds. 4-epioxytetracycline base, 4-epichlortetracycline, meclocyclinesulfosalicylate, and unmodified metacycline and minocycline proved to be the most potent gelatinase B (EC 3.4.24.35) inhibitors. The gelatinase B inhibitory activity of tetracyclines was clearly dissociated from their antimicrobial activity. The effect of high-molecular-weight inhibitors, such as monoclonal antibodies, was also demonstrable in the microtiter plate assay. In view of the pathophysiological function of gelatinases, the definition of gelatinase inhibitors with known efficacy, safety, and side effects is crucial for the treatment of diseases such as rheumatoid arthritis and multiple sclerosis. Particular tetracyclines fulfil these criteria and the described assay is useful for defining other gelatinase-inhibiting lead compounds.

CXCR3 determines strain susceptibility to murine cerebral malaria by mediating T lymphocyte migration toward IFN‐γ‐induced chemokines

Cerebral malaria (CM) results from the binding of infected erythrocytes and leukocytes to brain endothelia. The precise mechanisms underlying lymphocyte recruitment and activation in CM remain unclear. Therefore, the expression of various chemokines was quantified in brains of mice infected with Plasmodium berghei ANKA (PbA). Several chemokines attracting monocytes and activated T‐lymphocytes were expressed at high levels. Their expression was almost completely abrogated in IFN‐γ ligand and receptor KO mice, indicating that IFN‐γ is an essential chemokine inducer in vivo. Surprisingly, the expression levels of chemokines, IFN‐γ and also adhesion molecules in the brain were not lower in CM‐resistant Balb/c and DBA/2 mice compared to CM‐sensitive C57BL/6 and DBA/1 mice, although T lymphocyte sequestration in the brain was significantly less in CM‐resistant than in CM‐sensitive mice. This difference correlated with a higher up‐regulation of the CXC chemokine receptor (CXCR)‐3 on splenic T cells and a higher chemotactic response to IFN‐γ‐inducible protein‐10 (IP‐10) in C57BL/6 compared to Balb/c mice. In conclusion, parasite‐induced IFN‐γ in the brain results in high local expression levels of specific chemokines for monocytes and lymphocytes. The strain‐dependent susceptibility to develop CM is more related to the expression of CXCR3 in circulating leukocytes than to the chemokine expression levels in the brain.

Beta-hematin interaction with the hemopexin domain of gelatinase B/MMP-9 provokes autocatalytic processing of the propeptide, thereby priming activation by MMP-3.

Gelatinase B or matrix metalloproteinase-9 is involved in inflammation and in autoimmune and vascular diseases. In contrast to the constitutive and homeostatic matrix metalloproteinase-2, matrix metalloproteinase-9 is an inducible enzyme. Furthermore, it needs tight regulation, and a major control mechanism of its enzymatic activity is the activation of the latent enzyme by proteolysis of the 87 residue propeptide. Activated matrix metalloproteinase-9 is detected in many vascular or hematological disease states, including in an experimental model for cerebral malaria with Plasmodium berghei ANKA. However, insight into its activation mechanism is incomplete. In view of the association with hemorrhagic and hemolytic diseases, it was studied whether and how hemoglobin and its derivatives might activate pro-matrix metalloproteinase-9. Incubation of matrix metalloproteinase-9 with hemin or beta-hematin, the core constituent of hemozoin or malaria pigment, leads to differential autocatalysis of the propeptide, mediated by allosteric interaction with the hemopexin domain. The cleavage catalyzed by beta-hematin coincides with the first cleavage by stromelysin-1/matrix metalloproteinase-3, and preincubation of matrix metalloproteinase-9 with beta-hematin enhances the activation rate by matrix metalloproteinase-3 at least 6-fold. These findings suggest that reduction of hemorrhage and hemolysis might prevent matrix metalloproteinase-9-mediated inflammatory and vascular damages.

Analysis of gelatinases in complex biological fluids and tissue extracts.

G elatinase B (matrix metalloproteinase-9 [MMP-9]) and gelatinase A (MMP-2) are two closely related members of the MMP family that efficiently degrade denatured collagens or gelatins (Van den Steen et al, 2002). Specific MMPs play a major role in physiological processes, including angiogenesis, wound healing, bone remodeling, and cell migration. Moreover, MMP-9 and MMP-2 are key effector molecules in inflammation, autoimmunity (Opdenakker and Van Damme, 1994), and cancer (Sehgal et al, 1998). Analysis of both enzymes in complex biological samples, especially those with low gelatinase content, therefore is essential. We combined a miniaturized gelatin affinity chromatography with gelatin zymography and Western blot analysis. This strategy allows extremely sensitive and unambiguous detection of gelatinases. Gelatinases are often detected with specific antibodies or by substrate conversion assays. Gelatinase activity assays measure overall gelatinase activity often with the use of labeled gelatins (Paemen et al, 1996). Because these do not discriminate between gelatinase A and B and even other gelatin-degrading enzymes, specificity is low, especially in the analysis of complex biological samples. Introducing affinity prepurification with the use of monoclonal antibodies enhances the specificity (Hanemaaijer et al, 1998). Moreover, only activated enzymes are recognized. ELISA detects specific forms of MMP-9 or MMP-2. Unfortunately, this method does not necessarily differentiate between pro-enzyme and activated forms. Discrimination between different gelatin-degrading enzymes and their respective activation status may be achieved by zymography or Western blot analysis. Gelatin zymography detects picogram levels of MMP-9 (Masure et al, 1991). The sensitivity of Western blot analysis is usually lower and depends on the antibody affinity for MMP-9. In our hands, a combination of two monoclonal antibodies against mouse MMP-9 resulted in a detection limit of 100 pg of MMP-9. Nevertheless, such low detection limits are frequently not attained because the ratio of MMP-9 versus total protein is generally extremely low in crude samples, whereas the total protein load per lane is limited to 25 g so as not to distort the electrophoresis. After protein overloading, gelatinolysis may be eclipsed in zymography. Interference by gelatinolytic activity from other (abundant) enzymes constitutes an additional problem for zymography. For instance, in stomach extracts, pepsin is abundantly present and because this and other proteases also cleave gelatin, it will mask the gelatinases. To improve the ratio of MMP-9 versus total protein and to exclude interfering gelatinolytic activity, we developed a simple strategy for optimal preparation of complex samples, including tissue extracts. We used a miniaturized affinity chromatography purification step, taking advantage of gelatinases’ strong affinity for gelatin (Masure et al, 1991). For rapid and reproducible purification of the samples, we made use of mini-spin columns (Bio-Rad Laboratories, Hercules, California) and gelatin-Sepharose beads (Amersham Pharmacia Biotech, Uppsala, Sweden). Equilibration buffer was composed of 50 mM of Tris (pH 7.5), 0.5 M of NaCl, 10 mM of CaCl2, 0.01% Tween 20, and 5 mM of o-phenanthroline; washing buffer 1 had a similar composition except that the concentration of Tween 20 was increased to 0.05%. Washing buffer 2 was with omission of NaCl because high salt concentrations hinder the electrophoresis. The o-phenanthroline was added to the samples as a gelatinase inhibitor to prevent the gelatinolytic activity from breaking down the gelatin from the Sepharose beads, which are used for the affinity purification of the enzymes. The binding of gelatinases to gelatin-Sepharose is not disturbed by the presence of o-phenanthroline because this inhibitor acts by the chelation of the catalytic Zn , whereas binding to gelatin is mediated by the three fibronectin type II–like repeats. Elution buffer was at once the electrophoresis loading buffer and was composed of 100 mM of Tris/HCl (pH 6.8), 4% sodium dodecyl sulfate, 20% glycerol, and 200 g/ml of bromophenol blue as tracking dye. DOI: 10.1097/01.LAB.0000038556.54069.73

Gelatinase B is diabetogenic in acute and chronic pancreatitis by cleaving insulin

Genetic, endocrine, and environmental factors contribute to the development of diabetes. Much information has been gathered on the homeostasis mechanisms of glucose regulation by insulin‐producing pancreatic β cells. Here we demonstrate high expression levels of gelatinase B (matrix metalloproteinase‐9, MMP‐9) by neutrophils in acute pancreatitis and by ductular epithelial cells in chronic pancreatitis. Because gelatinase B processes cytokines and chemokines, we investigated whether and how gelatinase B cleaves insulin. Pure human neutrophil gelatinase B was found to destroy insulin by cleavage at 10 sites. Pancreatic islet and ductular cells are relatively spared in comparison with the complete destruction of acinar cells of the exocrine pancreas in chronic pancreatitis. High expression levels of gelatinase B are maintained in the immediate proximity of insulin‐secreting β cells. Consequently, diabetes may be worsened by enzymatic degradation of insulin by gelatinase B and by the consequent enhancement of the autoimmune process. Gelatinase B is diabetogenic in acute and chronic pancreatitis by cleaving insulin.

Gelatin degradation assay reveals MMP-9 inhibitors and function of O-glycosylated domain.

AIM To establish a novel, sensitive and high-throughput gelatinolytic assay to define new inhibitors and compare domain deletion mutants of gelatinase B/matrix metalloproteinase (MMP)-9. METHODS Fluorogenic Dye-quenched (DQ)™-gelatin was used as a substrate and biochemical parameters (substrate and enzyme concentrations, DMSO solvent concentrations) were optimized to establish a high-throughput assay system. Various small-sized libraries (ChemDiv, InterBioScreen and ChemBridge) of heterocyclic, drug-like substances were tested and compared with prototypic inhibitors. RESULTS First, we designed a test system with gelatin as a natural substrate. Second, the assay was validated by selecting a novel pyrimidine-2,4,6-trione (barbiturate) inhibitor. Third, and in line with present structural data on collagenolysis, it was found that deletion of the O-glycosylated region significantly decreased gelatinolytic activity (k(cat)/k(M) ± 40% less than full-length MMP-9). CONCLUSION The DQ™-gelatin assay is useful in high-throughput drug screening and exosite targeting. We demonstrate that flexibility between the catalytic and hemopexin domain is functionally critical for gelatinolysis.

Gelatinase B/matrix metalloproteinase-9 provokes cataract by cleaving lens βB1 crystallin

Cataract is a common cause of blindness and results from destruction of the microarchitecture of the lens. It is observed in many genetic syndromes, infections, inflammatory diseases and during aging. Fluctuations in lens density and light scattering by altered refraction index form the physical basis for this process, but the pathogenesis is poorly understood. Increased levels of gelatinase B/matrix metalloprotein‐ ase‐9 have been reported for cataract‐associated disor¬ders such as eye inflammation and diabetes. We dem¬onstrate that incubation of lenses with gelatinase B leads immediately to cataract. In complete eye extracts, βB1 crystallin was identified as the major gelatinase B substrate by combination of proteomics, mass spec¬trometry, and Edman degradation analysis. The cleav¬age of βB1 crystallin was also observed in vivo after endogenous gelatinase B‐induction by the chemokine granulocyte chemotactic protein‐2 in wild‐type mice but not in gelatinase B−/− mice.—Descamps, F. J., Mar¬tens, E., Proost, P., Starckx, S., Van den Steen, P. E., Van Damme, J., Opdenakker, G. Gelatinase B/matrix metalloproteinase‐9 provokes cataract by cleaving lens βB1 crystallin. FASEB J. 19, 29‐35 (2004)

In vivo activation of gelatinase B/MMP‐9 by trypsin in acute pancreatitis is a permissive factor in streptozotocin‐induced diabetes

Matrix metalloproteinases, in particular gelatinase B/MMP‐9, are key mediators in autoimmune diseases like multiple sclerosis and rheumatoid arthritis, but their pathogenic roles in diabetes are not well established. Gelatinase B has previously been shown to be upregulated in pancreas tissue from patients with acute and chronic pancreatitis and was suggested to exacerbate diabetes by cleaving insulin. In this study, the role of gelatinase B in diabetes was investigated using two streptozotocin‐induced animal models of type I diabetes. In both a hyperacute and a subacute model, gelatinase B upregulation was found to be associated with disease activity. However, gelatinase B deficiency did not significantly protect against diabetes development, and wild‐type and gelatinase B‐deficient animals behaved similarly in terms of β‐cell apoptosis or necrosis. The fact that gelatinase B was found almost exclusively as the inactive pro‐enzyme in most of the streptozotocin‐induced diabetic animals may explain the lack of a gelatinase B effect. On the contrary, gelatinase B was completely activated in a minority (15%) of wild‐type animals. This coincided with exocrine pancreatic inflammation, as revealed by the presence of active trypsin. The discovery of in vivo activation of progelatinase B by trypsin in acute pancreatitis is extended in a model of caerulein‐induced pancreatitis. In the latter model, trypsinogen activation is systematically achieved and gelatinase B is found in its active form. In conclusion, gelatinase B itself is not a causative factor but, when activated by endogenous trypsin, is a permissive factor for insulin degradation and diabetes. Copyright

Oligosaccharides of recombinant mouse gelatinase B variants

Gelatinase B (matrix metalloproteinase-9, MMP-9) contains three N-glycosylation sites and a Ser/Thr/Pro-rich type V collagen domain with repetitive attachment sites for O-linked sugars. Recombinant mouse gelatinase B was expressed in the yeast Pichia pastoris and the N-linked oligosaccharides of the truncated glycoprotein variants were analysed by in gel enzymatic release followed by mass spectrometry and normal phase HPLC. This technology, despite of the limiting amount of material, allowed the analysis of the formula of N- and O-linked sugars of the different glycoprotein variants. The 112/99- and 88-kDa gelatinase B forms each contained an oligomannose series (Man8GlcNAc2 to Man15GlcNAc2). Analysis of the hydrazine-released sugars showed that the O-linked oligosaccharides contained alpha1-2, alpha1-3 or alpha1-6 linked mannoses. These results were confirmed by lectin blot analysis of intact and glycosidase-treated enzyme variants.