Maria Thuveson

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.

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 (≈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.

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.

The low pH in trans-Golgi triggers autocatalytic cleavage of pre-alpha -inhibitor heavy chain precursor.

Pre-α-inhibitor is a plasma protein whose physiological function is still unknown, but in vitrostudies suggest that it might be involved in inflammatory reactions. Pre-α-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.

INTRACELLULAR PROTEOLYTIC PROCESSING OF THE HEAVY CHAIN OF RAT PRE-ALPHA -INHIBITOR : THE COOH-TERMINAL PROPEPTIDE IS REQUIRED FOR COUPLING TO BIKUNIN

Pre-α-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 NH2-terminal propeptide. Cleavage of this propeptide, which occurred in the endoplasmic reticulum, was found to require basic amino acid residues at P1, P2, and P6 suggesting that it is mediated by a Golgi enzyme in transit. Deletion of the NH2-terminal propeptide or blocking of its release affected neither transport nor coupling of the heavy chain to bikunin.

Binding of Zn2+ to the plasma protein inter-α-inhibitor

Abstract Inter-α-inhibitor (IαI) is a serum protein consisting of a chondroitin-sulfate-containing protein of 25 kDa (bikunin) and two other polypeptides of 75–80 kDa (heavy chains 1 and 2). The physiological function of IαI is unclear but recent results suggest that it is required for the formation of the extracellular matrix of certain cell types and that it has anti-inflammatory activity. It was previously reported that IαI isolated from serum contains bound Zn2+, but details of this binding are lacking. Using equilibrium dialysis, we have found that when the free Zn2+ concentration is raised from 0.3 to 50 μmol/L, the number of bound ions increases from 0.1 to 7. The concentration of free Zn2+ in plasma is in the nanomolar range; our results therefore suggest that inter-α-inhibitor does not contain stoichiometric amounts of zinc ions under normal in vivo conditions.