Molecular cloning and identification of 3'-phosphoadenosine 5'-phosphosulfate transporter

Nucleotide sulfate, namely 3'-phosphoadenosine 5'-phosphosulfate (PAPS), is a universal sulfuryl donor for sulfation. Although a specific PAPS transporter is present in Golgi membrane, no study has reported the corresponding gene. We have identified a novel human gene encoding a PAPS transporter, which we have named PAPST1, and the Drosophila melanogaster ortholog, slalom (sll). The amino acid sequence of PAPST1 (432 amino acids) exhibited 48.1% identity with SLL (465 amino acids), and hydropathy analysis predicted the two to be type III transmembrane proteins. The transient expression of PAPST1 in SW480 cells showed a subcellular localization in Golgi membrane. The expression of PAPST1 and SLL in yeast Saccharomyces cerevisiae significantly increased the transport of PAPS into the Golgi membrane fraction. In human tissues, PAPST1 is highly expressed in the placenta and pancreas and present at lower levels in the colon and heart. An RNA interference fly of sll produced with a GAL4-UAS system revealed that the PAPS transporter is essential for viability. It is well known that mutations of some genes related to PAPS synthesis are responsible for human inherited disorders. Our findings provide insights into the significance of PAPS transport and post-translational sulfation.     

Purification of the testicular galactolipid: 3'-phosphoadenosine 5'-phosphosulfate sulfotransferase.

The rat testicular galactolipid sulfotransferase has been purified by affinity chromatography using 3'5'-adenosine diphosphate-agarose affinity chromatography. Both galactosyl glycerolipid and galactosyl ceramide were effective substrates with Km values of 4.8 and 1.1 microM respectively. A single protein of molecular mass 56 kDa was present in the purified sulfotransferase preparation as monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. Specific photoaffinity substrate labeling, using an azido derivative of galactosyl ceramide, was used to identify this protein, both in crude extracts and when purified. The protein was also selectively phosphorylated in the presence of the rat testicular galactolipid sulfotransferase stimulatory protein kinase.     

Sulfohydrolytic degradation of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) and adenosine 5'-phosphosulfate (APS) by enzymes of a nucleotide pyrophosphatase nature

The sulfohydrolytic activity to degrade active sulfate (3'-phosphoadenosine 5'-phosphosulfate, PAPS) and its precursor, APS (adenosine 5'-phosphosulfate), with a pH optimum at 9.5 was found to be widely distributed in various tissues of rats. In the liver, the activity was located in plasma membranes and endoplasmic reticula. Triton X-100 solubilized rough and smooth endoplasmic reticula gave two peaks of the activity on gel filtration, both of which had nucleotide pyrophosphatase activities, hydrolyzing the pyrophosphate linkages of ATP, NAD, and UDP-Glc, and the phosphodiester linkage of PNTP (p-nitrophenyl-thymidine 5'-monophosphate) besides PAPS and APS.     

Molecular cloning and characterization of a novel human protein phosphatase,LMW-DSP3

Reversible phosphorylation is recognized to be a major mechanism for the control of intracellular events in eukaryotic cells. From a human fetal brain cDNA library, we isolated a cDNA clone encoding a novel dual specificity protein phosphatase, which showed 88% identity with previously reported mouse LMW-DSP3 at the amino acid level. The deduced protein had a single dual-specificity phosphatase catalytic domain, and lacked a cdc25 homology domain. LMW-DSP3 was expressed in the heart, lung, liver, and pancreas, and the expression level in the pancreas was highest. The LMW-DSP3 gene was located in human chromosome 2q32, and consisted of five exons spanning 21kb of human genomic DNA. LMW-DSP3 fused to GST showed phosphatase activity towards p-nitrophenyl phosphate which was optimal at pH 7.0 and 40 degrees C, and the activity was enhanced by Ca(2+) and Mn(2+). The phosphatase activity of LMW-DSP3 was inhibited by orthovanate. LMW-DSP3 showed phosphatase activity toward oligopeptides containing pSer/Thr and pTyr, indicating that LMW-DSP3 is a protein phosphatase with dual substrate specificity.     

Molecular cloning and characterization of a novel dual specificity phosphatase, MKP-5.

A group of dual specificity protein phosphatases negatively regulates members of the mitogen-activated protein kinase (MAPK) superfamily, which consists of three major subfamilies, MAPK/extracellular signal-regulated kinase (ERK), stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), and p38. Nine members of this group of dual specificity phosphatases have previously been cloned. They show distinct substrate specificities for MAPKs, different tissue distribution and subcellular localization, and different modes of inducibility of their expression by extracellular stimuli. Here we have cloned and characterized a novel dual specificity phosphatase, which we have designated MKP-5. MKP-5 is a protein of 482 amino acids with a calculated molecular mass of 52.6 kDa and consists of 150 N-terminal amino acids of unknown function, two Cdc25 homology 2 regions in the middle, and a C-terminal catalytic domain. MKP-5 binds to p38 and SAPK/JNK, but not to MAPK/ERK, and inactivates p38 and SAPK/JNK, but not MAPK/ERK. p38 is a preferred substrate. The subcellular localization of MKP-5 is unique; it is present evenly in both the cytoplasm and the nucleus. MKP-5 mRNA is widely expressed in various tissues and organs, and its expression in cultured cells is elevated by stress stimuli. These results suggest that MKP-5 is a novel type of dual specificity phosphatase specific for p38 and SAPK/JNK.     

Molecular cloning and functional characterization of human vesicular glutamate transporter 3

Glutamate is the major excitatory neurotransmitter in the mammalian CNS. It is loaded into synaptic vesicles by a proton gradient-dependent uptake system and is released by exocytosis upon stimulation. Recently, two mammalian isoforms of a vesicular glutamate transporter, VGLUT1 and VGLUT2, have been identified, the expression of which enables quantal release of glutamate from glutamatergic neurons. Here, we report a novel isoform of a human vesicular glutamate transporter (hVGLUT3). The predicted amino acid sequence of hVGLUT3 shows 72% identity to both hVGLUT1 and hVGLUT2. hVGLUT3 functions as a vesicular glutamate transporter with similar properties to the other isoforms when it is heterologously expressed in a neuroendocrine cell line. Although mammalian VGLUT1 and VGLUT2 exhibit a complementary expression pattern covering all glutamatergic pathways in the CNS, expression of hVGLUT3 overlaps with them in some brain areas, suggesting molecular diversity that may account for physiological heterogeneity in glutamatergic synapses.     

Human 3'-phosphoadenosine 5'-phosphosulfate synthetase (isoform 1, brain): kinetic properties of the adenosine triphosphate sulfurylase and adenosine 5'-phosphosulfate kinase domains

Recombinant human 3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthetase, isoform 1 (brain), was purified to near-homogeneity from an Escherichia coli expression system and kinetically characterized. The native enzyme, a dimer with each 71 kDa subunit containing an adenosine triphosphate (ATP) sulfurylase and an adenosine 5'-phosphosulfate (APS) kinase domain, catalyzes the overall formation of PAPS from ATP and inorganic sulfate. The protein is active as isolated, but activity is enhanced by treatment with dithiothreitol. APS kinase activity displayed the characteristic substrate inhibition by APS (K(I) of 47.9 microM at saturating MgATP). The maximum attainable activity of 0.12 micromol min(-1) (mg of protein)(-1) was observed at an APS concentration ([APS](opt)) of 15 microM. The theoretical K(m) for APS (at saturating MgATP) and the K(m) for MgATP (at [APS](opt)) were 4.2 microM and 0.14 mM, respectively. At likely cellular levels of MgATP (2.5 mM) and sulfate (0.4 mM), the overall endogenous rate of PAPS formation under optimum assay conditions was 0.09 micromol min(-1) (mg of protein)(-1). Upon addition of pure Penicillium chrysogenum APS kinase in excess, the overall rate increased to 0.47 micromol min(-1) (mg of protein)(-1). The kinetic constants of the ATP sulfurylase domain were as follows: V(max,f) = 0.77 micromol min(-1) (mg of protein)(-1), K(mA(MgATP)) = 0.15 mM, K(ia(MgATP)) = 1 mM, K(mB(sulfate)) = 0.16 mM, V(max,r) = 18.7 micromol min(-1) (mg of protein)(-1), K(mQ(APS)) = 4.8 microM, K(iq(APS)) = 18 nM, and K(mP(PPi)) = 34.6 microM. The (a) imbalance between ATP sulfurylase and APS kinase activities, (b) accumulation of APS in solution during the overall reaction, (c) rate acceleration provided by exogenous APS kinase, and (d) availability of both active sites to exogenous APS all argue against APS channeling. Molybdate, selenate, chromate ("chromium VI"), arsenate, tungstate, chlorate, and perchlorate bind to the ATP sulfurylase domain, with the first five serving as alternative substrates that promote the decomposition of ATP to AMP and PP(i). Selenate, chromate, and arsenate produce transient APX intermediates that are sufficiently long-lived to be captured and 3'-phosphorylated by APS kinase. (The putative PAPX products decompose to adenosine 3',5'-diphosphate and the original oxyanion.) Chlorate and perchlorate form dead-end E.MgATP.oxyanion complexes. Phenylalanine, reported to be an inhibitor of brain ATP sulfurylase, was without effect on PAPS synthetase isoform 1.     

Protamine increases the affinity of 3'-phosphoadenosine 5'-phosphosulfate toward a sulfotransferase from chicken embryo epiphyseal cartilage

A 3' -phosphoadenosine 5' -phosphosulfate (PAPS):chondroitin sulfate sulfotransferase from chicken embryo epiphyseal cartilage, which was partially purified, exhibited a molecular mass of 150 kDa. The enzymatic sulfation of totally desulfated chondroitin was activated up to 12-fold by protamine while the sulfation of partially sulfated chondroitin was activated only 3-fold. Protamine increased the affinity of the enzyme for PAPS about 4-fold when partially desulfated chondroitin was used as sulfate acceptor. The S 0.5 for the totally desulfated chondroitin was not affected by protamine, while high PAPS concentration slightly increased the affinity of the enzyme for the same sulfate acceptor. The possible role of these substances in the regulation of the sulfation of chondroitin sulfate is discussed.     

Molecular cloning and characterization of Crmdr1, a novel MDR-type ABC transporter gene from Catharanthus roseus.

A novel gene encoding a MDR-like ABC transporter protein was cloned from Catharanthus roseus, a medicinal plant with more than 120 kinds of secondary metabolites, through rapid amplification of cDNA ends (RACE). This gene (named as Crmdr1; GenBank accession no.: DQ660356) had a total length of 4395 bp with an open reading frame of 3801 bp, and encoded a predicted polypeptide of 1266 amino acids with a molecular weight of 137.1 kDa. The CrMDR1 protein shared 59.8, 62.5, 60.0 and 58.2% identity with other MDR proteins isolated from Arabidopsis thaliana (AAD31576), Coptis japonica (CjMDR), Gossypium hirsutum (GhMDR) and Triticum aestivum (TaMDR) at amino acid level, respectively. Southern blot analysis showed that Crmdr1 was a low-copy gene. Expression pattern analysis revealed that Crmdr1 constitutively expressed in the root, stem and leaf, but with lower expression in leaf. The domains analysis showed that CrMDR1 protein possessed two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs) arranging in "TMD1-NBD1-TMD2-NBD2" direction, which is consistent with other MDR transporters. Within NBDs three characteristic motifs common to all ABC transporters, "Walker A", "Walker B" and C motif, were found. These results indicate that CrMDR1 is a MDR-like ABC transporter protein that may be involved in the transport and accumulation of secondary metabolites.     

Studies on bovine adrenal estrogen sulfotransferase. III. Facile synthesis of 3'-phospho- and 2'-phosphoadenosine 5'-phosphosulfate.

A practical synthesis of 3'-phosphoadenosine 5'-phosphosulfate (IV) in yields of 68-72% from adenosine 2',3'-cyclic phosphate 5'-phosphate (II) is described. Reaction of II with triethylamine-N-sulfonic acid affords adenosine 2',3'-cyclic phosphate 5'-phosphosulfate (III) which, on treatment with ribonuclease-T2, provides IV. Spleen phosphodiesterase, on the other hand, converts III to 2'-phosphoadenosine 5'-phosphosulfate (V). The biological activity of IV, measured by sulfate transfer to [6,7-3H2]estrone as mediated by bovine adrenal estrone sulfotransferase (3'-phosphoadenylyl-sulfate:estrone 3-sulfotransferase, EC 2.8.2.4), is identical with that obtained with a sample of IV prepared by an established biochemical procedure. By contrast, V exhibits approximately one-third the activity of the natural isomer.     

Molecular cloning and characterization of AAAS-V2, a novel splice variant of human AAAS

Triple-A syndrome (MIM 231550; also known as Allgrove syndrome) is an autosomal recessive disorder characterized by adrenocorticotropin hormone (ACTH)-resistant adrenal insufficiency, achalasia of the oesophageal cardia and alacrima. Much initial molecular analysis supported that Triple-A syndrome was caused by mutations in AAAS, a WD-repeat protein gene. Here we report cloning and characterization of a novel splice variant of human AAAS, which we named AAAS-v2, which is located on the human chromosome 12p13. The cDNA is 1703bp, encoding a 513-amino acid polypeptide, which contains three WD40 domains, one less than the original which we called AAAS-v1 (Gen Bank: NM_015665.3). RT-PCR analysis in our work revealed that AAAS-v2 and AAAS-v1 were ubiquitously detected in human multiple tissue cDNA (MTC) panels (CLONTECH).The nucleotide sequence reported in this paper has been submitted to GenBank under accession number AY237818.Xin Li: These two authors contributed equally to this paper.Chaoneng Ji: These two authors contributed equally to this paper.