Heparan sulfate biosynthesis enzymes EXT1 and EXT2 affect NDST1 expression and heparan sulfate sulfation.

Heparan sulfate (HS) proteoglycans influence embryonic development and adult physiology through interactions with protein ligands. The interactions depend on HS structure, which is determined largely during biosynthesis by Golgi enzymes. How biosynthesis is regulated is more or less unknown. During polymerization of the HS chain, carried out by a complex of the exostosin proteins EXT1 and EXT2, the first modification enzyme, glucosaminyl N-deacetylase/N-sulfotransferase (NDST), introduces N-sulfate groups into the growing polymer. Unexpectedly, we found that the level of expression of EXT1 and EXT2 affected the amount of NDST1 present in the cell, which, in turn, greatly influenced HS structure. Whereas overexpression of EXT2 in HEK 293 cells enhanced NDST1 expression, increased NDST1 N-glycosylation, and resulted in elevated HS sulfation, overexpression of EXT1 had opposite effects. Accordingly, heart tissue from transgenic mice overexpressing EXT2 showed increased NDST activity. Immunoprecipitaion experiments suggested an interaction between EXT2 and NDST1. We speculate that NDST1 competes with EXT1 for binding to EXT2. Increased NDST activity in fibroblasts with a gene trap mutation in EXT1 supports this notion. These results support a model in which the enzymes of HS biosynthesis form a complex, or a GAGosome.

Integrins are required for synchronous ommatidial rotation in the Drosophila eye linking planar cell polarity signalling to the extracellular matrix.

Integrins mediate the anchorage between cells and their environment, the extracellular matrix (ECM), and form transmembrane links between the ECM and the cytoskeleton, a conserved feature throughout development and morphogenesis of epithelial organs. Here, we demonstrate that integrins and components of the ECM are required during the planar cell polarity (PCP) signalling-regulated cell movement of ommatidial rotation in the Drosophila eye. The loss-of-function mutations of integrins or ECM components cause defects in rotation, with mutant clusters rotating asynchronously compared to wild-type clusters. Initially, mutant clusters tend to rotate faster, and at later stages they fail to be synchronous with their neighbours, leading to aberrant rotation angles and resulting in a disorganized ommatidial arrangement in adult eyes. We further demonstrate that integrin localization changes dynamically during the rotation process. Our data suggest that core Frizzled/PCP factors, acting through RhoA and Rho kinase, regulate the function/activity of integrins and that integrins thus contribute to the complex interaction network of PCP signalling, cell adhesion and cytoskeletal elements required for a precise and synchronous 90° rotation movement.

Cooperative effect of ribosomal protein s19 and Pim-1 kinase on murine c-Myc expression and myeloid/erythroid cellularity.

Diamond-Blackfan anemia is a bone marrow failure syndrome associated with heterozygous mutations in the ribosomal protein S19 (RPS19) gene in a subgroup of patients. One of the interacting partners with RPS19 is the oncoprotein PIM-1 kinase. We intercrossed Rps19 ( +/- ) and Pim-1 ( -/- ) mice strains to study the effect from the disruption of both genes. The double mutant (Rps19 ( +/- ) Pim-1 ( -/- )) mice display normal growth with increased peripheral white and red blood cell counts when compared to the w.t. mice (Rps19 ( +/+ ) Pim-1 ( +/+ )). Molecular analysis of bone marrow cells in Rps19 ( +/- ) Pim-1 ( -/- ) mice revealed up-regulated levels of c-Myc and the anti-apoptotic factors Bcl(2), Bcl(XL), and Mcl-1. This is associated with a reduction of the apoptotic factors Bak and Caspase 3 as well as the cell cycle regulator p21. Our findings suggest that combined Rps19 insufficiency and Pim-1 deficiency promote murine myeloid cell growth through a deregulation of c-Myc and a simultaneous up-regulation of anti-apoptotic Bcl proteins.

Enzymatically active N-deacetylase/N-sulfotransferase-2 is present in liver but does not contribute to heparan sulfate N-sulfation.

Heparan sulfate (HS) proteoglycans influence embryonic development through interactions with growth factors and morphogens. The interactions depend on HS structure, which is largely determined during biosynthesis by Golgi enzymes. NDST (glucosaminyl N-deacetylase/N-sulfotransferase), responsible for HS N-sulfation, is a key enzyme directing further modifications including O-sulfation. To elucidate the roles of the different NDST isoforms in HS biosynthesis, we took advantage of mice with targeted mutations in NDST1 and NDST2 and used liver as our model organ. Of the four NDST isoforms, only NDST1 and NDST2 transcripts were shown to be expressed in control liver. The absence of NDST1 or NDST2 in the knock-out mice did not affect transcript levels of other NDST isoforms or other HS modification enzymes. Although the sulfation level of HS synthesized in NDST1-/- mice was drastically lowered, liver HS from wild-type mice, from NDST1+/-, NDST2-/-, and NDST1+/- / NDST2-/- mice all had the same structure despite greatly reduced NDST enzyme activity (30% of control levels in NDST1+/- / NDST2-/- embryonic day 18.5 embryos). Enzymatically active NDST2 was shown to be present in similar amounts in wild-type, NDST1-/-, and NDST1+/- embryonic day 18.5 liver. Despite the substantial contribution of NDST2 to total NDST enzyme activity in embryonic day 18.5 liver (approximately 40%), its presence did not appear to affect HS structure as long as NDST1 was also present. In NDST1-/- embryonic day 18.5 liver, in contrast, NDST2 was responsible for N-sulfation of the low sulfated HS. A tentative model to explain these results is presented.

Intracellular coupling of the heavy chain of pre-alpha-inhibitor to chondroitin sulfate.

Pre-alpha-inhibitor is a serum protein consisting of two polypeptides, the heavy chain and bikunin, covalently linked through an ester bond between the chondroitin sulfate chain of bikunin and the alpha-carboxyl group of the carboxyl-terminal residue of the heavy chain. The heavy chain is synthesized with a carboxyl-terminal extension, which is cleaved off just before the link to bikunin is formed. Our earlier studies indicate that this extension mediates the cleavage, and we have now found that a short segment on the amino-terminal side of the cleavage site is also required for the reaction. Furthermore, we previously showed that coexpression of the heavy chain precursor and bikunin in COS-1 cells leads to linkage, and we have now used this system to identify a His residue in the carboxyl-terminal extension that is specifically required for the intracellular coupling of the two proteins. In addition, we have shown that another chondroitin sulfate-containing protein, decorin, will also form a complex with the heavy chain, as will free chondroitin sulfate chains. These results suggest that in vivo there might be other, as yet unknown, chondroitin sulfate-containing polypeptides linked to the heavy chain.

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.