Altered processing of fibronectin in mice lacking heparin. a role for heparin-dependent mast cell chymase in fibronectin degradation

We have previously generated a mouse strain with a defect in its heparin biosynthesis by targeting the gene for N-deacetylase/N-sulfotransferase-2 (NDST-2). The NDST-2(-/-) mice show reduced levels of various mast cell mediators such as histamine and various heparin-binding mast cell proteases, including chymases, tryptases, and carboxypeptidase A. In this work we have addressed the possible functional consequences of the lack of sulfated heparin. Peritoneal cells were harvested from normal and NDST-2(-/-) mice. After culturing the cells, conditioned media were collected and were subjected to SDS-polyacrylamide gel electrophoresis under reducing conditions. Several differences in the protein patterns were observed, including the presence of large amounts of a approximately 250-kDa protein in medium from NDST-2(-/-) mice that was absent in normal controls. Peptide microsequencing revealed identity of this protein with fibronectin. Western blot analysis showed the presence of fibronectin degradation products in cell cultures from normal mice, which were absent in cultures from NDST-2(-/-) animals. Further experiments showed that the degradation of fibronectin observed in cell cultures from NDST-2(+/+) mice was catalyzed by mast cell chymase in a strongly heparin-dependent manner. This report thus indicates a biological function for chymase/heparin proteoglycan complexes in fibronectin turnover.     

Serglycin: a structural and functional chameleon with wide impact on immune cells

Among the different proteoglycans expressed by mammals, serglycin is in most immune cells the dominating species. A unique property of serglycin is its ability to adopt highly divergent structures, because of glycosylation with variable types of glycosaminoglycans when expressed by different cell types. Recent studies of serglycin-deficient animals have revealed crucial functions for serglycin in a diverse array of immunological processes. However, its exact function varies to a large extent depending on the cellular context of serglycin expression. Based on these findings, serglycin is emerging as a structural and functional chameleon, with radically different properties depending on its exact cellular and immunological context.     

Histidines are critical for heparin-dependent activation of mast cell tryptase

Mast cell tryptase is a tetrameric serine protease that is stored in complex with negatively charged heparin proteoglycans in the secretory granule. Tryptase has potent proinflammatory properties and has been implicated in diverse pathological conditions such as asthma and fibrosis. Previous studies have shown that tryptase binds tightly to heparin, and that heparin is required in the assembly of the tryptase tetramer as well as for stabilization of the active tetramer. Because the interaction of tryptase with heparin is optimal at acidic pH, we investigated in this study whether His residues are of importance for the heparin binding, tetramerization, and activation of the tryptase mouse mast cell protease 6. Molecular modeling of mouse mast cell protease 6 identified four His residues, H35, H106, H108, and H238, that are conserved among pH-dependent tryptases and are exposed on the molecular surface, and these four His residues were mutated to Ala. In addition, combinations of different mutations were prepared. Generally, the single His-Ala mutations did not cause any major defects in heparin binding, activation, or tetramerization, although some effect of the H106A mutation was observed. However, when several mutations were combined, large defects in all of these parameters were observed. Of the mutants, the triple mutant H106A/H108A/H238A was the most affected with an almost complete inability to bind to heparin and to form active tryptase tetramers. Taken together, this study shows that surface-exposed histidines mediate the interaction of mast cell tryptase with heparin and are of critical importance in the formation of active tryptase tetramers.     

Occurrence of limnic micro-crustaceans in relation to temperature and oxygen

Information on the distribution of 56 micro-crustaceans from different types of waters in south and central Sweden was analyzed to reveal their relationships to temperature and oxygen content. A very wide tolerance range concerning these factors was found for most species.     

On the ecology of Lecane (Rotifera)

A material consisting of 49 taxa of Lecane (Rotifera), from diverse waters in south and Central Sweden, was analyzed to reveal their relationships to substrate and habitat. Most species did not show any pronounced preference for any substrate, occurring in several diverse substrates, and especially in periphytic environments. Only a few species seemed to specialize in bog substrates. Some taxa were found, sometimes at a high frequency, on an artificial substrate, white cotton, indicating a high degree of mobility.     

A prothrombinase complex of mouse peritoneal macrophages

Addition of prothrombin to mouse peritoneal macrophages in vitro resulted in the formation of a thrombin-like enzyme, as demonstrated by use of the luminogenic peptide substrate S-2621. The prothrombinase activity was sedimented by high-speed centrifugation following homogenization of the cells and was abolished by treatment of the cells with the nonionic detergent Triton X-100 at 0.02% concentration. Moreover, the activity was drastically reduced by maintaining cultures in the presence of warfarin and, presumably due to competitive substrate inhibition, by adding S-2222, a chromogenic peptide substrate for Factor Xa. These findings suggest that prothrombin cleavage is catalyzed by Factor Xa at the macrophage surface. The generated thrombin was inhibited by antithrombin, and this reaction was accelerated by heparin with high affinity for antithrombin but not by the corresponding oligosaccharides composed of 8-14 monosaccharide units. Such oligosaccharides which are capable of accelerating the inactivation of Factor Xa by antithrombin, inhibited thrombin formation from prothrombin in the macrophage cultures, presumably by promoting inactivation by antithrombin of Factor Xa in a prothrombinase complex. Activation of the macrophage coagulation system, as proposed to occur in certain inflammatory conditions, thus may be modulated at various levels by heparin, or heparin oligosaccharides, released from mast cells.     

Structure and antithrombin-binding properties of heparin isolated from the clams Anomalocardia brasiliana and Tivela mactroides

Heparin with high anticoagulant activity was isolated from the two marine clam species Anomalocardia brasiliana and Tivela mactroides. A large portion of the polysaccharide chains of both preparations bound with high affinity to immobilized antithrombin. Titrations monitored by tryptophan fluorescence showed that clam polysaccharide chains with Mr approximately 22,500 contained up to three binding sites for antithrombin and that the binding constants for the interaction of these chains with antithrombin were higher than those reported for mammalian heparin of comparable size. Structural analysis of clam heparin fractions and subfractions of clam heparin with differing affinity for immobilized antithrombin revealed the presence of large amounts (up to 25-30% of the total disaccharide units) of the 3-O-sulfated saccharide sequences (-GlcNSO3)-GlcA-GlcNSO3(3-OSO3)- and (-GlcNSO3)-GlcA-GlcNSO3(3,6-di-OSO3)-, previously identified as unique markers for the antithrombin-binding region of heparin. The content of these saccharide sequences was found to increase with increasing affinity of the parent polysaccharide for antithrombin. Structural analysis of the clam heparins also demonstrated the occurrence of a novel saccharide sequence, tentatively identified as (-GlcNSO3)-IdA-GlcNSO3(3,6-di-OSO3)-, that has not previously been found in heparin or related polysaccharides. The contents of this latter sequence, at most 3-4% of the total disaccharide units, showed no correlation with the affinity for antithrombin.     

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.     

Proteolytic Histone Modification by Mast Cell Tryptase,a Serglycin Proteoglycan-dependent Secretory Granule Protease

A hallmark feature of mast cells is their high content of cytoplasmic secretory granules filled with various preformed compounds, including proteases of tryptase-, chymase-, and carboxypeptidase A3 type that are electrostatically bound to serglycin proteoglycan. Apart from participating in extracellular processes, serglycin proteoglycan and one of its associated proteases, tryptase, are known to regulate cell death by promoting apoptosis over necrosis. Here we sought to outline the underlying mechanism and identify core histones as primary proteolytic targets for the serglycin-tryptase axis. During the cell death process, tryptase was found to relocalize from granules into the cytosol and nucleus, and it was found that the absence of tryptase was associated with a pronounced accumulation of core histones both in the cytosol and in the nucleus. Intriguingly, tryptase deficiency resulted in defective proteolytic modification of core histones even at baseline conditions, i.e. in the absence of cytotoxic agent, suggesting that tryptase has a homeostatic impact on nuclear events. Indeed, tryptase was found in the nucleus of viable cells and was shown to cleave core histones in their N-terminal tail. Moreover, it was shown that the absence of the serglycin-tryptase axis resulted in altered chromatin composition. Together, these findings implicate histone proteolysis through a secretory granule-derived serglycin-tryptase axis as a novel principle for histone modification, during both cell homeostasis and cell death.     

Serglycin is a major proteoglycan in polarized human endothelial cells and is implicated in the secretion of the chemokine GROalpha/CXCL1

Proteoglycan (PG) expression was studied in primary human umbilical vein endothelial cells (HUVEC). RT-PCR analyses showed that the expression of the PG serglycin core protein was much higher than that of the extracellular matrix PG decorin and the cell surface PG syndecan-1. PG biosynthesis was further studied by biosynthetic [(35)S]sulfate labeling of polarized HUVEC. Interestingly, a major part of (35)S-PGs was secreted to the apical medium. A large portion of these PGs was trypsin-resistant, a typical feature of serglycin. The trypsin-resistant PGs were mainly of the chondroitin/dermatan sulfate type but also contained a minor heparan sulfate component. Secreted serglycin was identified by immunoprecipitation as a PG with a core protein of ～30 kDa. Serglycin was furthermore shown to be present in perinuclear regions and in two distinct types of vesicles throughout the cytoplasm using immunocytochemistry. To search for possible serglycin partner molecules, HUVEC were stained for the chemokine growth-related oncogene α (GROα/CXCL1). Co-localization with serglycin could be demonstrated, although not in all vesicles. Serglycin did not show overt co-localization with tissue-type plasminogen activator-positive vesicles. When PG biosynthesis was abrogated using benzyl-β-D-xyloside, serglycin secretion was decreased, and the number of vesicles with co-localized serglycin and GROα was reduced. The level of GROα in the apical medium was also reduced after xyloside treatment. Together, these findings indicate that serglycin is a major PG in human endothelial cells, mainly secreted to the apical medium and implicated in chemokine secretion.