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 Ndeacetylase/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.
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.
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 (≈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.
Pre-␣-inhibitor is a plasma protein whose physiological function is still unknown, but in vitro studies suggest that it might be involved in inflammatory reactions. Pre-␣-inhibitor consists of a 25-and a 75-kDa polypeptide: bikunin and heavy chain 3 (H3), respectively. H3 is synthesized with a 30-kDa C-terminal extension, which is released in the Golgi complex through cleavage between an Asp and a Pro residue. We now provide evidence that this cleavage is triggered by the low pH in the late Golgi and occurs through an intramolecular process. First, incubation in vitro of the H3 precursor (proH3) at pH 6.0 or lower results in rapid cleavage of the protein. Second, the rate of the cleavage reaction does not depend on the concentration of proH3 and is not affected by the presence of various protease inhibitors. Third, raising the pH in organelles of cells producing proH3 abolishes cleavage during secretion. The amino acid residues near the cleavage site of proH3 differ from those of previously described self-cleaving proteins, indicating that the mechanisms of scission are different.
Pre-alpha-inhibitor is a serum protein consisting of two polypeptides: bikunin of 16 kDa, which carries an 8 kDa chondroitin sulphate chain, and heavy chain 3 (H3) of 74 kDa. The two polypeptides are linked through an ester bond between an internal N-acetylgalactosamine residue of the chondroitin sulphate chain and the C-terminal aspartic acid residue of H3. Both bikunin and H3 are synthesized by hepatocytes and become linked as they pass through the Golgi complex. H3 is synthesized with both N- and C-terminal extensions which are released during intracellular transport. To be able to analyse the assembly of pre-alpha-inhibitor in detail, we have cloned and sequenced the cDNA of rat H3. Upon expression of the protein in COS-1 cells, both propeptides were found to be released. Furthermore, co-expression of H3 and bikunin resulted in the two polypeptides becoming coupled, indicating that cells other than hepatocytes may have the capacity to form chondroitin sulphate-containing links.
Pre-␣-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 ␣-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.
Pre-␣-inhibitor is a serum protein consisting of two polypeptides named bikunin and heavy chain 3 (H3). Both polypeptides are synthesized in hepatocytes and while passing through the Golgi complex, bikunin, which carries a chondroitin sulfate chain, becomes covalently linked to the COOH-terminal amino acid residue of H3 via its polysaccharide. Immediately prior to this reaction, a COOH-terminal propeptide of 33 kDa is cleaved off from the heavy chain. Using COS-1 cells transfected with rat H3, we found that in the absence of bikunin, the cleaved propeptide remained bound to the heavy chain and that H3 lacking the propeptide sequence did not become linked to coexpressed bikunin. Sequencing of H3 secreted from COS-1 cells showed that part of the molecules had a 12-amino acid residue long NH 2 -terminal propeptide. Cleavage of this propeptide, which occurred in the endoplasmic reticulum, was found to require basic amino acid residues at P 1 , P 2 , and P 6 suggesting that it is mediated by a Golgi enzyme in transit. Deletion of the NH 2 -terminal propeptide or blocking of its release affected neither transport nor coupling of the heavy chain to bikunin.
Diamond Blackfan anemia (DBA) 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. We have previously generated a mouse model with targeted disruption of Rps19 25 . TheRps19 null mouse is lethal prior to implantation whereas the Rps19 heterozygous mice show a normal phenotype 25 . To clarify the significance of the previously reported RPS19 and PIM-1 interaction we generated mice with targeted disruptions of Rps19 and/or Pim-1. We present herein the results from the morphological and molecular analysis of hematopoietic organs in these mice strains. Our findings support a combined effect of Rps19 and Pim-1 mutations on myeloid proliferation. Materials and methods Generation of mice carrying Rps19 and Pim-1 disruptions Rps19+/-mice 25 were bred with Pim-1 -/-mice 18 for several generations on a C57/Bl6 background to produce offspring with the genotypes Rps19
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