Chondroitin sulfate synthase-2. Molecular cloning and characterization of a novel human glycosyltransferase homologous to chondroitin sulfate glucuronyltransferase, which has dual enzymatic activities.

Chondroitin sulfate is found in a variety of tissues as proteoglycans and consists of repeating disaccharide units of N-acetylgalactosamine and glucuronic acid residues with sulfate residues at various places. We found a novel human gene (GenBank accession number AB086063) that possesses a sequence homologous with the human chondroitin sulfate glucuronyltransferase gene which we recently cloned and characterized. The full-length open reading frame encodes a typical type II membrane protein comprising 775 amino acids. The protein had a domain containing beta 3-glycosyltransferase motif but lacked a typical beta 4-glycosyltransferase motif, which is the same as chondroitin sulfate glucuronyltransferase, whereas chondroitin synthase had both domains. The putative catalytic domain was expressed in COS-7 cells as a soluble enzyme. Surprisingly, both glucuronyltransferase and N-acetylgalactosaminyltransferase activities were observed when chondroitin, chondroitin sulfate, and their oligosaccharides were used as the acceptor substrates. The reaction products were identified to have the linkage of GlcUA beta 1-3GalNAc and GalNAc beta 1-4GlcUA at the non-reducing terminus of chondroitin for glucuronyltransferase activity and N-acetylgalactosaminyltransferase activity, respectively. Quantitative real time PCR analysis revealed that the transcripts were ubiquitously expressed in various human tissues but highly expressed in the pancreas, ovary, placenta, small intestine, and stomach. These results indicate that this enzyme could synthesize chondroitin sulfate chains as a chondroitin sulfate synthase that has both glucuronyltransferase and N-acetylgalactosaminyltransferase activities. Sequence analysis based on three-dimensional structure revealed the presence of not typical but significant beta 4-glycosyltransferase architecture.

Evidence that rat hepatocytes co-express functional P2Y1 and P2Y2 receptors.

Previous studies have indicated the expression of multiple P2Y receptors by rat hepatocytes although they have not been identified. Here we show by reverse transcriptase-polymerase chain reaction (RT - PCR) that rat hepatocytes express mRNA encoding all of the four cloned rat P2Y receptors (P2Y(1), P2Y(2), P2Y(4) and P2Y(6)). The effects of UTP have been examined on single aequorin-injected rat hepatocytes. The [Ca(2+)](i) transients induced by UTP were indistinguishable from those induced by ATP in the same cell. The modulatory effects of elevated intracellular cyclic AMP concentration were the same on both UTP- and ATP-induced [Ca(2+)](i) transients. UDP, an agonist at the P2Y(6) receptor, failed to induce transients in hepatocytes, indicating that functional P2Y(6) receptors coupled to increased [Ca(2+)](i) are not expressed. The transients evoked by ADP were more sensitive to inhibition by suramin than those induced by either ATP or UTP. Within an individual cell, the transients induced by ATP and UTP were inhibited by the same concentration of suramin. This sensitivity of ATP and UTP responses to suramin suggests action through P2Y(2) rather than P2Y(4) receptors. Co-application of 30 microM pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) caused a decrease in frequency and amplitude of transients induced by ADP. ATP- and UTP-induced transients also displayed a decrease in amplitude in response to addition of PPADS, but this was accompanied by an increase in frequency of transients. In conclusion the data presented here are consistent with the co-expression of P2Y(1) and P2Y(2) receptors by rat hepatocytes.

Activation of pollen tube callose synthase by detergents. Evidence for different mechanisms of action.

In pollen tubes of Nicotiana alata, a membrane-bound, Ca(2+)-independent callose synthase (CalS) is responsible for the biosynthesis of the (1,3)-beta-glucan backbone of callose, the main cell wall component. Digitonin increases CalS activity 3- to 4-fold over a wide range of concentrations, increasing the maximum initial velocity without altering the Michaelis constant for UDP-glucose. The CalS activity that requires digitonin for assay (the latent CalS activity) is not inhibited by the membrane-impermeant, active site-directed reagent UDP-pyridoxal when the reaction is conducted in the absence of digitonin. This is consistent with digitonin increasing CalS activity by the permeabilization of membrane vesicles. A second group of detergents, including 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS), Zwittergent 3-16, and 1-alpha-lysolecithin, activate pollen tube CalS 10- to 15-fold, but only over a narrow range of concentrations just below their respective critical micellar concentrations. This activation could not be attributed to any particular chemical feature of these detergents. CHAPS increases maximum initial velocity and decreases the Michaelis constant for UDP-glucose and activates CalS even in the presence of permeabilizing concentrations of digitonin. Inhibition studies with UDP-pyridoxal indicate that activation by CHAPS occurs by recruitment of previously inactive CalS molecules to the pool of active enzyme. The activation of pollen tube CalS by these detergents therefore resembles activation of the enzyme by trypsin.

UTP induces vascular responses in the isolated and perfused canine epicardial coronary artery via UTP-preferring P2Y receptors.

1. Vasoconstrictor responses of the isolated and perfused canine epicardial coronary artery to uridine 5'-triphosphate (UTP) were analysed pharmacologically. 2. At basal perfusion pressure, UTP induced vasoconstriction in a dose-related manner and the vasoconstriction was sometimes followed by a slight vasodilatation at large doses (more than 10 nmol). The rank order of potency for vasoconstriction was UTP = UDP > ATP > TTP > or = ITP >> UMP. At raised perfusion pressure by 20 mM KCl, the vasoconstriction was not changed and a small vasodilatation was induced at large doses. The rank order of potency for vasodilatation was induced at large doses. The rank order of potency for vasodilatation was ATP >> ITP > or = UDP > UTP > or = TTP. The maximal vasodilator response to UTP was much less than that to ATP. UMP did not induce vasodilatation. 3. The P2X receptor agonist and desensitizing agent alpha, beta-methylene ATP (1 microM) and the P2 receptor antagonist suramin (100 microM) inhibited the vasoconstrictor responses to ATP but not those to UTP and UDP. The P2 receptor antagonist reactive blue 2 (30 microM) did not inhibit the vascular responses to UTP. 4. UTP (200 microM) desensitized the vasoconstrictor responses to UTP, but not either the vasodilator responses to UTP or the vasoconstrictor responses to ATP and UDP. UDP (200 microM) did not desensitize the vascular responses to UTP. 5. Preincubating the UDP stock solution and arterial preparation with hexokinase (10 and 1 uml-1, respectively) did not change the vasoconstrictor responses to UDP. 6. The Ca channel blocker diltiazem (1 microM) inhibited the vasoconstrictor responses to UTP but not those to ATP and UDP. Incubation in a Ca(2+)-free solution containing 1 mM EGTA inhibited the vascular responses to ATP, UTP and UDP. 7. Removal of the endothelium by an intraluminal injection of saponin (1 mg) inhibited the vasodilator responses to UTP. Indomethacin, a cyclo-oxygenase inhibitor (1 microM), inhibited the vasodilator responses to UTP, but NG-nitro-L-arginine, a nitric oxide synthase inhibitor (300 microM), did not have an inhibitory effect. 8. The results suggest that (1) UTP induces vasoconstriction via UTP-preferring P2Y receptors on the smooth muscle and vasodilatation via receptors different from those mediating the vasoconstriction induced by UTP and mediating the vasodilatation by ATP on the endothelium, through mainly the release of prostacyclin in the canine epicardial coronary artery; (2) UDP induces vasoconstriction via UDP-preferring P2Y receptors; and (3) L-type Ca ion channels are involved in the vasoconstriction induced by UTP, but not in that induced by UDP.

A novel G protein-coupled P2 purinoceptor (P2Y3) activated preferentially by nucleoside diphosphates.

A partial cDNA was isolated by hybridization screening of an embryonic chick brain library for P2Y purinoceptors. After extension to full length, it revealed an open reading frame that encoded a protein, P2Y3, of 328 amino acids that is nearest in sequence identity to the G protein-coupled P2 purinoceptors obtained by DNA cloning. Expression of P2Y3 in cRNA-injected Xenopus oocytes confirmed that this cDNA encodes a member of the metabotropic purinoceptor family, with a novel order for the relative activities of nucleotides. At 100 microM concentrations, ADP gave the highest activity, and UTP and UDP were also strongly active. When expressed in the human T cell line Jurkat, P2Y3 mediated transient increases in intracellular Ca2+ in response to various nucleotides. Again, an unusual agonist rank order was revealed, with uridine nucleotides being more potent than adenosine nucleotides and UDP being the most potent agonist tested (half-maximal concentration, 0.13 microM) and 10-fold more potent than UTP. 2-Methylthlo-ATP was of relatively low activity in both systems. The receptor transcript is expressed in brain, spinal cord, kidney, and lung and is highly abundant in the spleen but not in other peripheral tissues that we tested. The results indicated that P2Y3 is a previously unknown P2 purinoceptor subtype with a preference for nucleoside diphosphates.

Nucleotide receptor P2u partially mediates ATP-induced cell cycle progression of aortic smooth muscle cells.

mRNA of the P2u purinoceptor (or nucleotide receptor) is detected both by polymerase chain reaction or Northern blot analyses in cultured aortic smooth muscle cells. When added to the culture medium of these cells, UTP, a specific ligand of the P2u receptor, induces an increased expression of both immediate-early and delayed-early cell cycle-dependent genes. This induction demonstrates similar features (kinetics, concentration dependence) to those obtained after stimulation of aortic smooth cells by exogenous ATP, a common ligand for most P2 purinoceptors. In contrast, 2-methylthioATP, a preferential ligand for P2y purinoceptors, induces only a significant increase of immediate-early genes but not of delayed-early genes. Moreover, the 2-methylthioATP-induced responses (c-fos mRNA increase, free intracellular calcium transient) are lower than those induced by ATP or UTP and are complementary to those of UTP. These results demonstrate that functional P2u receptors are present on cultured aortic smooth muscle cells and suggest that the bulk of responses induced by extracellular ATP on cell cycle progression are mediated via P2u purinoceptors, a hypothesis confirmed by cytofluorometric studies. Since some ATP- or UTP-induced genes code for chemotactic proteins (monocyte chemoattractant protein-1 and osteopontin), this study suggests that these nucleotides may contribute to vascular or blood cell migration and proliferation and consequently to the genesis of arterial diseases.

Identification of a region of UDP-galactose:N-acetylglucosamine beta 4-galactosyltransferase involved in UDP-galactose binding by differential labeling.

The location of regions in the primary structure of UDP-galactose:N-acetylglucosamine beta 4-galactosyl-transferase (GT) that are involved in binding UDP-galactose has been investigated by differential chemical modification with two different reagents in the presence and absence of UDP-galactose. Treatment with periodate-cleaved UDP and NaCNBH3 resulted in a loss of 80% of GT activity, which was largely prevented by UDP-galactose. Stoichiometry of labeling and peptide maps of the modified enzyme samples indicated partial labeling at many sites. A major site of reaction in the absence of UDP-galactose that was essentially unmodified in its presence was found to correspond to Lys341 in the cDNA sequence of GT. As a second approach, the reactivities of the amino groups of GT were compared in the presence and absence of saturating levels of UDP-galactose by trace acetylation with [3H]acetic anhydride. UDP-galactose binding was found to perturb the reactivities of a number of lysines in the C-terminal region of GT, the most pronounced effect being a reduction in the reactivity of Lys351. The two procedures thus identified a region between residues 341 and 351 as being associated with UDP-galactose binding. This region overlaps a small section in the sequence of GT that was previously noted to be similar to part of bovine alpha-1,3-galactosyltransferase (Joziasse, D. H., Shaper, J. H., Van den Eijnden, D. H., Van Tunen, A. J., and Shaper, N. L. (1989) J. Biol. Chem. 264, 14290-14297). Sequence comparisons indicate that extended regions at the C terminus of each enzyme encompassing this area may represent homologous UDP-galactose-binding domains.