Biomedical subjects

Gunnar Pejler

Publications and source records attributed to Gunnar Pejler.

26 records · Page 2Linked to original sources

PubMed2003

Human mast cell beta-tryptase is a gelatinase.

Remodeling of extracellular matrix is an important component in a variety of inflammatory disorders as well as in normal physiological processes such as wound healing and angiogenesis. Previous investigations have identified the various matrix metalloproteases, e.g., gelatinases A and B, as key players in the degradation of extracellular matrix under such conditions. Here we show that an additional enzyme, human mast cell beta-tryptase, has potent gelatin-degrading properties, indicating a potential contribution of this protease to matrix degradation. Human beta-tryptase was shown to degrade gelatin both in solution and during gelatin zymographic analysis. Further, beta-tryptase was shown to degrade partially denatured collagen type I. beta-Tryptase bound strongly to gelatin, forming high molecular weight complexes that were stable during SDS-PAGE. Mast cells store large amounts of preformed, active tryptase in their secretory granules. Considering the location of mast cells in connective tissues and the recently recognized role of mast cells in disorders in which connective tissue degradation is a key event, e.g., rheumatoid arthritis, it is thus likely that tryptase may contribute to extracellular matrix-degrading processes in vivo.

Animals↗

PubMed2003

Polycationic peptides as inhibitors of mast cell serine proteases.

When mast cells are activated, e.g. during allergic responses, they secrete the serine proteases chymase and tryptase, which both are complex-bound to heparin proteoglycan in vivo. Previous reports have demonstrated potent pro-inflammatory effects of both tryptase and chymase in different animal models, suggesting that these serine proteases may be relevant targets for therapeutic intervention. Recent investigations have shown that heparin-binding compounds can cause tryptase inhibition and it has been suggested that the inhibitory activity of such compounds is due to interference with the binding of heparin to tryptase. Here we tested various polycationic peptides for their ability to inhibit heparin-free human recombinant betaI-tryptase. We demonstrate powerful direct inhibition of tryptase (IC(50) values approximately 1-100 nM) by poly-Arg and poly-Lys of different molecular weights. Poly-Arg and poly-Lys showed predominantly competitive inhibition kinetics, although decreases in the k(cat) values for the chromogenic substrate S-2288 were also observed. Peptides built up from heparin-binding motifs were also inhibitors of tryptase, albeit of lower efficiency than poly-Arg/Lys. Tryptase inhibition was strongly dependent on the size of the polycationic peptides. The various polycationic peptides were also inhibitory for heparin-dependent activities of chymase. The tryptase inhibition caused by the polycationic peptides could be reversed by adding heparin. After heparin-induced rescue of tryptase activity, the major part of the tryptase activity was sensitive to inhibition by bovine pancreatic trypsin inhibitor, whereas tryptase before addition of polycationic peptide was completely resistant. Taken together, our findings indicate that polycationic peptides can be used as powerful agents for combined inhibition of mast cell tryptase and chymase.

Animals↗

PubMed2003

Formation of active monomers from tetrameric human beta-tryptase.

Tryptase is a serine protease that is stored at low pH in the mast cell secretory granules in complex with heparin proteoglycan. When mast cells are activated, e.g. during allergic responses, the tryptase/heparin complexes are released together with a variety of other preformed inflammatory mediators. Previous crystallization of human beta-tryptase revealed a unique tetrameric structure with all of the active sites facing a central pore that has a limited accessibility both for potential substrates as well as for protease inhibitors. In this study we examined whether human beta-tryptase, in addition, could form active monomers. Incubation of recombinant tetrameric human beta-tryptase at neutral pH and 37 degrees C, followed by gel-filtration analysis using a running buffer containing pig mucosal heparin, led to the formation of enzymically active compounds that were of a size compatible with tryptase monomers in complex with heparin. The monomers were, in contrast to tryptase in the tetrameric form, inhibited by bovine pancreatic trypsin inhibitor. Further, the monomers, but not the tetramers, degraded fibronectin. Formation of active monomers was more pronounced at pH 7.5 than at pH 6.0 and was not detected at room temperature or at high heparin/tryptase ratios. The present findings thus introduce the possibility that human beta-tryptase, after mast cell degranulation and exposure to neutral pH in the tissue, may dissociate into active monomers with properties that are distinct from the tetrameric counterpart. Possibly, some of the biological activities of human tryptase may be attributable to active tryptase in its monomeric rather than tetrameric form.

Aprotinin↗

PubMed2003

Secretion of macrophage urokinase plasminogen activator is dependent on proteoglycans.

The importance of proteoglycans for secretion of proteolytic enzymes was studied in the murine macrophage cell line J774. Untreated or 4beta-phorbol 12-myristate 13-acetate (PMA)-stimulated macrophages were treated with hexyl-beta-d-thioxyloside to interfere with the attachment of glycosaminoglycan chains to their respective protein cores. Activation of the J774 macrophages with PMA resulted in increased secretion of trypsin-like serine proteinase activity. This activity was completely inhibited by plasminogen activator inhibitor 1 and by amiloride, identifying the activity as urokinase plasminogen activator (uPA). Treatment of both the unstimulated or PMA-stimulated macrophages with xyloside resulted in decreased uPA activity and Western blotting analysis revealed an almost complete absence of secreted uPA protein after xyloside treatment of either control- or PMA-treated cells. Zymography analyses with gels containing both gelatin and plasminogen confirmed these findings. The xyloside treatment did not reduce the mRNA levels for uPA, indicating that the effect was at the post-translational level. Treatment of the macrophages with xylosides did also reduce the levels of secreted matrix metalloproteinase 9. Taken together, these findings indicate a role for proteoglycans in the secretion of uPA and MMP-9.

Amiloride↗

PubMed2002

Rat mast cell protease 4 is a beta-chymase with unusually stringent substrate recognition profile.

Activated mast cells release a variety of potent inflammatory mediators including histamine, cytokines, proteoglycans, and serine proteases. The serine proteases belong to either the chymase (chymotrypsin-like substrate specificity) or tryptase (trypsin-like specificity) family. In this report we have investigated the substrate specificity of a recently identified mast cell protease, rat mast cell protease-4 (rMCP-4). Based on structural homology, rMCP-4 is predicted to belong to the chymase family, although rMCP-4 has previously not been characterized at the protein level. rMCP-4 was expressed with an N-terminal His tag followed by an enterokinase site substituting for the native activation peptide. The enterokinase-cleaved fusion protein was labeled by diisopropyl fluorophosphate, demonstrating that it is an active serine protease. Moreover, rMCP-4 hydrolyzed MeO-Suc-Arg-Ala-Tyr-pNA, thus verifying that this protease belongs to the chymase family. rMCP-4 bound to heparin, and the enzymatic activity toward MeO-Suc-Arg-Ala-Tyr-pNA was strongly enhanced in the presence of heparin. Detailed analysis of the substrate specificity was performed using peptide phage display technique. After six rounds of amplification a consensus sequence, Leu-Val-Trp-Phe-Arg-Gly, was obtained. The corresponding peptide was synthesized, and rMCP-4 was shown to cleave only the Phe-Arg bond in this peptide. This demonstrates that rMCP-4 displays a striking preference for bulky/aromatic amino acid residues in both the P1 and P2 positions.

Amino Acid Sequence↗

PubMed2002

Altered storage of proteases in mast cells from mice lacking heparin: a possible role for heparin in carboxypeptidase A processing.

Heparin-deficient mice, generated by gene targeting of N-deacetylase/N-sulfotransferase-2 (NDST-2), display severe mast cell defects, including an absence of stored mast cell proteases. However, the mechanism behind these observations is not clear. Here we show that NDST-2+/+ bone marrow-derived mast cells cultured in the presence of IL-3 synthesise, in addition to highly sulphated chondroitin sulphate (CS), small amounts of equally highly sulphated heparin-like polysaccharide. The corresponding NDST-2-/- cells produced highly sulphated CS only. Carboxypeptidase A (CPA) activity was detected in NDST+/+ cells but was almost absent in the NDST-/- cells, whereas tryptase (mouse mast cell protease 6; mMCP-6) activity and antigen was detected in both cell types. Antigen for the chymase mMCP-5 was detected in NDST-2+/+ cells but not in the heparin-deficient cells. Northern blot analysis revealed mRNA expression of CPA, mMCP-5 and mMCP-6 in both wild-type and NDST-2-/- cells. A approximately 36 kDa CPA band, corresponding to proteolytically processed active CPA, as well as a approximately 50 kDa pro-CPA band was present in NDST-2+/+ cells. The NDST-2-/- mast cells contained similar levels of pro-CPA as the wild-type mast cells, but the approximately 36 kDa band was totally absent. This indicates that the processing of pro-CPA to its active form may require the presence of heparin and provides the first insight into a mechanism by which the absence of heparin may cause disturbed secretory granule organisation in mast cells.

Actins↗

PubMedDate not supplied

Mast cell cathepsins C and S control levels of carboxypeptidase A and the chymase, mouse mast cell protease 5.

Carboxypeptidase A (CPA) is a metalloprotease, residing in the mast cell secretory granules together with chymases and tryptases. Little information is available with respect to the mechanisms that maintain or regulate the levels of stored proteases in the mast cell secretory granules. In this study we examined whether cathepsins C and S may be involved in the control of the levels of mast cell proteases. Mast cells cultured from bone marrow of cathepsin C- or S-null mice expressed higher levels of CPA protein and activity than cells from wild-type mice. Similar increases in protein were observed for the mouse chymase, mast cell protease-5 (mMCP-5), but not for the tryptase, mMCP-6. Steady-state levels of CPA and mMCP-5 mRNA were similar in wild-type and cathepsin C-null mast cells, indicating that post-transcriptional mechanisms explain the observed cathepsin C-dependence of CPA and mMCP-5 expression. The present study thus indicates novel roles for cathepsins C and S in regulating the levels of stored proteases in the mast cell secretory granules.

Actins↗

PubMedDate not supplied

Mast cell-dependent activation of pro matrix metalloprotease 2: A role for serglycin proteoglycan-dependent mast cell proteases.

The formation of active matrix metalloprotease-2 (MMP-2) requires the proteolytic processing of proMMP-2, a process that can occur through the formation of a ternary complex between proMMP-2, the tissue inhibitor of metalloprotease-2 and membrane type 1-MMP. However, other activation mechanisms have been suggested, and in this study we investigated whether mast cells (MCs) may play a role in the activation of proMMP-2. Murine peritoneal cells, a mixture of macrophages, lymphocytes and MCs, were cultured ex vivo. Addition of proMMP-2 to resting peritoneal cell cultures resulted in only slow conversion of proMMP-2 into the active enzyme. However, when MC degranulation was provoked using a calcium ionophore, proMMP-2 processing was markedly enhanced. When the peritoneal cell populations were depleted in MCs, proMMP-2 processing was abrogated, but was reconstituted when purified MCs were added to the depleted cultures. ProMMP-2 processing was sensitive to serine protease inhibitors, but not to inhibitors of other classes of proteases. Furthermore, proMMP-2 processing was completely abrogated in cells lacking serglycin, a proteoglycan that has previously been shown to mediate storage of a variety of MC serine proteases. Taken together, these results suggest a novel mode of proMMP-2 activation mediated by serglycin-dependent MC serine proteases.

Animals↗