Essential Roles of 3′-Phosphoadenosine 5′-Phoshosulfate Synthase in Embryonic and Larval Development of the Nematode Caenorhabditis elegans
2006; Elsevier BV; Volume: 281; Issue: 16 Linguagem: Inglês
10.1074/jbc.m601509200
ISSN1083-351X
AutoresKatsufumi Dejima, Akira Seko, Katsuko Yamashita, Keiko Gengyo‐Ando, Shohei Mitani, Tomomi Izumikawa, Hiroshi Kitagawa, Kazuyuki Sugahara, Souhei Mizuguchi, Kazuya Nomura,
Tópico(s)Parasites and Host Interactions
ResumoSulfation of biomolecules, which is widely observed from bacteria to humans, plays critical roles in many biological processes. All sulfation reactions in all organisms require activated sulfate, 3′-phosphoadenosine 5′-phosphosulfate (PAPS), as a universal donor. In animals, PAPS is synthesized from ATP and inorganic sulfate by the bifunctional enzyme, PAPS synthase. In mammals, genetic defects in PAPS synthase 2, one of two PAPS synthase isozymes, cause dwarfism disorder, but little is known about the consequences of the complete loss of PAPS synthesis. To define the developmental role of sulfation, we cloned a Caenorhabditis elegans PAPS synthase-homologous gene, pps-1, and depleted expression of its product by isolating the deletion mutant and by RNA-mediated interference. PPS-1 protein exhibits specific activity to form PAPS in vitro, and disruption of the pps-1 gene by RNAi causes pleiotropic developmental defects in muscle patterning and epithelial cell shape changes with a decrease in glycosaminoglycan sulfation. Additionally, the pps-1 null mutant exhibits larval lethality. These data suggest that sulfation is essential for normal growth and integrity of epidermis in C. elegans. Furthermore, reporter analysis showed that pps-1 is expressed in the epidermis and several gland cells but not in neurons and muscles, indicating that PAPS in the neurons and muscles is provided by other cells. Sulfation of biomolecules, which is widely observed from bacteria to humans, plays critical roles in many biological processes. All sulfation reactions in all organisms require activated sulfate, 3′-phosphoadenosine 5′-phosphosulfate (PAPS), as a universal donor. In animals, PAPS is synthesized from ATP and inorganic sulfate by the bifunctional enzyme, PAPS synthase. In mammals, genetic defects in PAPS synthase 2, one of two PAPS synthase isozymes, cause dwarfism disorder, but little is known about the consequences of the complete loss of PAPS synthesis. To define the developmental role of sulfation, we cloned a Caenorhabditis elegans PAPS synthase-homologous gene, pps-1, and depleted expression of its product by isolating the deletion mutant and by RNA-mediated interference. PPS-1 protein exhibits specific activity to form PAPS in vitro, and disruption of the pps-1 gene by RNAi causes pleiotropic developmental defects in muscle patterning and epithelial cell shape changes with a decrease in glycosaminoglycan sulfation. Additionally, the pps-1 null mutant exhibits larval lethality. These data suggest that sulfation is essential for normal growth and integrity of epidermis in C. elegans. Furthermore, reporter analysis showed that pps-1 is expressed in the epidermis and several gland cells but not in neurons and muscles, indicating that PAPS in the neurons and muscles is provided by other cells. Body and tissue morphologies are generated by orchestrated events of cell movements and cell shape changes at an appropriate time and place. In the nematode Caenorhabditis elegans, which is an ideal model organism for the study of morphogenesis, embryos undergo a 4-fold increase in length and a 3-fold decrease in circumference without cell division after the cell proliferation phase (1Priess J.R. Hirsh D.I. Dev. Biol. 1986; 117: 156-173Crossref PubMed Scopus (309) Google Scholar). The elongation process requires proper patterning and shape change of the epidermis (hypoderms) and body wall muscle cells adjacent to the hypodermis in C. elegans (2Simske J.S. Hardin J. BioEssays. 2001; 23: 12-23Crossref PubMed Google Scholar, 3Ding M. Woo W.M. Chisholm A.D. Exp. Cell Res. 2004; 301: 84-90Crossref PubMed Scopus (31) Google Scholar). In the patterning and shape change of the hypodermis and the body wall muscle, both the basement membrane, which is positioned between them, and dynamic cytoskeletal changes play an important role (2Simske J.S. Hardin J. BioEssays. 2001; 23: 12-23Crossref PubMed Google Scholar, 3Ding M. Woo W.M. Chisholm A.D. Exp. Cell Res. 2004; 301: 84-90Crossref PubMed Scopus (31) Google Scholar). On the other hand, the extracellular cuticle that surrounds the hypodermis contributes to the maintenance of the final shape of the worms after the elongation (1Priess J.R. Hirsh D.I. Dev. Biol. 1986; 117: 156-173Crossref PubMed Scopus (309) Google Scholar). Sulfation, a common chemical modification of biomolecules, is critical for many biological processes. For example, sulfation of lipooligo-saccharide signals determines the symbiosis between the bacteria Rhizobium meliloti and alfalfa (4Roche P. Debelle F. Maillet F. Lerouge P. Faucher C. Truchet G. Denarie J. Prome J.C. Cell. 1991; 67: 1131-1143Abstract Full Text PDF PubMed Scopus (320) Google Scholar). In vertebrates, tyrosine sulfation of chemokine receptor CCR5 facilitates HIV entry (5Farzan M. Mirzabekov T. Kolchinsky P. Wyatt R. Cayabyab M. Gerard N.P. Gerard C. Sodroski J. Choe H. Cell. 1999; 96: 667-676Abstract Full Text Full Text PDF PubMed Scopus (601) Google Scholar), and terminal SO4-4GalNAc of the pituitary hormone lutropin, which plays a critical role in the expression of hormone activity, modulates the circulatory half-life of the hormone (6Baenziger J.U. Kumar S. Brodbeck R.M. Smith P.L. Beranek M.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 334-338Crossref PubMed Scopus (159) Google Scholar). Proper sulfation of glycosaminoglycans (GAGs) 2The abbreviations used are: GAG, glycosaminoglycan; PAPS, 3′-phosphoadenosine 5′-phosphosulfate; APS, adenosine 5′-phosphosulfate; HS, heparan sulfate; dsRNA, double-stranded RNA; RNAi, RNA interference; DIC, differential interference contrast; GFP, green fluorescent protein; EGFP, enhanced green fluorescent protein. 2The abbreviations used are: GAG, glycosaminoglycan; PAPS, 3′-phosphoadenosine 5′-phosphosulfate; APS, adenosine 5′-phosphosulfate; HS, heparan sulfate; dsRNA, double-stranded RNA; RNAi, RNA interference; DIC, differential interference contrast; GFP, green fluorescent protein; EGFP, enhanced green fluorescent protein. is required for interactions with several extracellular signaling molecules; therefore, disrupting the genes that encode enzymes mediating sulfation reactions causes dorso/ventral or segment polarity defects in Drosophila (7Sen J. Goltz J.S. Stevens L. Stein D. Cell. 1998; 95: 471-481Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 8Lin X. Perrimon N. Nature. 1999; 400: 281-284Crossref PubMed Scopus (414) Google Scholar) and induces abnormal mast cells (9Humphries D.E. Wong G.W. Friend D.S. Gurish M.F. Qiu W.T. Huang C. Sharpe A.H. Stevens R.L. Nature. 1999; 400: 769-772Crossref PubMed Scopus (360) Google Scholar, 10Forsberg E. Pejler G. Ringvall M. Lunderius C. Tomasini-Johansson B. Kusche-Gullberg M. Eriksson I. Ledin J. Hellman L. Kjellen L. Nature. 1999; 400: 773-776Crossref PubMed Scopus (404) Google Scholar) or renal agenesis in mice (11Bullock S.L. Fletcher J.M. Beddington R.S. Wilson V.A. Genes Dev. 1998; 12: 1894-1906Crossref PubMed Scopus (398) Google Scholar). Furthermore, sulfation of several carbohydrates also plays important roles in nervous system development, particularly in axon guidance (12Lee J.S. Chien C.B. Nat. Rev. Genet. 2004; 5: 923-935Crossref PubMed Scopus (102) Google Scholar). All sulfation reactions in all organisms require 3′-phosphoadenosine 5′-phosphosulfate (PAPS) as a donor. The formation of PAPS involves two catalytic reactions (13Robbins P.W. Lipmann F. J. Biol. Chem. 1958; 233: 681-685Abstract Full Text PDF PubMed Google Scholar). The first reaction is carried out by ATP-sulfurylase (EC 2.7.7.4), resulting in the formation of adenosine 5′-phosphosulfate (APS). The second reaction is carried out by APS kinase (EC 2.7.1.25), resulting in the formation of PAPS. Synthesized PAPS is translocated into the Golgi apparatus by the PAPS transporter (14Kamiyama S. Suda T. Ueda R. Suzuki M. Okubo R. Kikuchi N. Chiba Y. Goto S. Toyoda H. Saigo K. Watanabe M. Narimatsu H. Jigami Y. Nishihara S. J. Biol. Chem. 2003; 278: 25958-25963Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 15Luders F. Segawa H. Stein D. Selva E.M. Perrimon N. Turco S.J. Hacker U. EMBO J. 2003; 22: 3635-3644Crossref PubMed Scopus (66) Google Scholar); then several sulfotransferases transfer sulfate onto proteins and carbohydrates from PAPS. In animals, ATP-sulfurylase and APS kinase activity are performed by the bifunctional enzyme, PAPS synthase (16Strott C.A. Endocr. Rev. 2002; 23: 703-732Crossref PubMed Scopus (378) Google Scholar, 17Venkatachalam K.V. IUBMB Life. 2003; 55: 1-11Crossref PubMed Scopus (78) Google Scholar), although in plants and in simple organisms, including bacteria, fungi, and yeast, the two enzymes are present on separate peptides. In higher organisms, including humans, mice, guinea pigs, and chickens, PAPS synthase exists as two isozymes, PAPSS1 and PAPSS2. However, there is only a single PAPS synthase in lower animals. Mutations in the PAPSS2 gene cause developmental dwarfism disorders: autosomal recessive spondyloepimetaphyseal dysplasia in humans and brachymorphism in mice (18ul Haque M.F. King L.M. Krakow D. Cantor R.M. Rusiniak M.E. Swank R.T. Superti-Furga A. Haque S. Abbas H. Ahmad W. Ahmad M. Cohn D.H. Nat. Genet. 1998; 20: 157-162Crossref PubMed Scopus (176) Google Scholar, 19Kurima K. Warman M.L. Krishnan S. Domowicz M. Krueger R Jr C. Deyrup A. Schwartz N.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8681-8685Crossref PubMed Scopus (129) Google Scholar). The disorder in brachymorphic (bm) mice is due to a glycine 79 to arginine (G79R) mutation in the APS kinase domain of PAPSS2, which fails to synthesize PAPS (19Kurima K. Warman M.L. Krishnan S. Domowicz M. Krueger R Jr C. Deyrup A. Schwartz N.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8681-8685Crossref PubMed Scopus (129) Google Scholar, 20Sugahara K. Schwartz N.B. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 6615-6618Crossref PubMed Scopus (106) Google Scholar). A decrease in the formation of PAPS in cartilage in bm mice induces undersulfation of chondroitin sulfate, a major component of extracellular matrix in cartilage; chondroitin sulfate interacts with many extracellular matrix molecules, such as type II collagen. Subsequently, bm mice exhibit abnormalities in limb and axial skeletal length (21Orkin R.W. Pratt R.M. Martin G.R. Dev. Biol. 1976; 50: 82-94Crossref PubMed Scopus (101) Google Scholar, 22Orkin R.W. Williams B.R. Cranley R.E. Poppke D.C. Brown K.S. J. Cell Biol. 1977; 73: 287-299Crossref PubMed Scopus (38) Google Scholar) as well as in blood clotting and bleeding time (23Rusiniak M.E. O'Brien E.P. Novak E.K. Barone S.M. McGarry M.P. Reddington M. Swank R.T. Mamm. Genome. 1996; 7: 98-102Crossref PubMed Scopus (15) Google Scholar, 24Schwartz N.B. Kelley R.O. Goetinck P.F. MacCabe J.A. Limb Development and Regeneration. Part B. Liss, New York1983: 97-103Google Scholar). Mutation in PAPSS2 does not cause lethality in mice, although PAPSS2 is inactive for PAPS synthesis in bm mice; in addition, expression of PAPSS1 is more ubiquitous than that of PAPPS2 (25Girard J.P. Baekkevold E.S. Amalric F. FASEB J. 1998; 12: 603-612Crossref PubMed Scopus (51) Google Scholar, 26Xu Z.H. Otterness D.M. Freimuth R.R. Carlini E.J. Wood T.C. Mitchell S. Moon E. Kim U.J. Xu J.P. Siciliano M.J. Weinshilboum R.M. Biochem. Biophys. Res. Commun. 2000; 268: 437-444Crossref PubMed Scopus (55) Google Scholar, 27Fuda H. Shimizu C. Lee Y.C. Akita H. Strott C.A. Biochem. J. 2002; 365: 497-504Crossref PubMed Scopus (51) Google Scholar). These facts indicate that PAPSS1 is the major PAPS synthase in mice; however, to date there have been no reports of genetic deficiencies in the PAPSS1 gene or in PAPSS1 and PAPSS2 knock-out mice. Also unknown are the exact cell types and molecular mechanisms of deficiencies resulted from PAPS synthase disruption. Gene knock-out studies of PAPS synthases will provide valuable information about the physiological roles of sulfation. However, as observed in bm and spondyloepimetaphyseal dysplasia, inactivation of PAPS synthesis would mainly cause defects in cartilage-containing chondroitin sulfate, one of the most abundant sulfated molecules, making it difficult to elucidate the physiological roles of other sulfated molecules in vertebrates (18ul Haque M.F. King L.M. Krakow D. Cantor R.M. Rusiniak M.E. Swank R.T. Superti-Furga A. Haque S. Abbas H. Ahmad W. Ahmad M. Cohn D.H. Nat. Genet. 1998; 20: 157-162Crossref PubMed Scopus (176) Google Scholar, 19Kurima K. Warman M.L. Krishnan S. Domowicz M. Krueger R Jr C. Deyrup A. Schwartz N.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8681-8685Crossref PubMed Scopus (129) Google Scholar). Importantly, although chondroitin proteoglycans are the primary extracellular matrix components in C. elegans, as in higher organisms (28Mizuguchi S. Uyama T. Kitagawa H. Nomura K.H. Dejima K. Gengyo-Ando K. Mitani S. Sugahara K. Nomura K. Nature. 2003; 423: 443-448Crossref PubMed Scopus (218) Google Scholar), sulfated forms of chondroitin GAGs have not been detected by biochemical analysis (29Yamada S. Van Die I. Van den Eijnden D.H. Yokota A. Kitagawa H. Sugahara K. FEBS Lett. 1999; 459: 327-331Crossref PubMed Scopus (96) Google Scholar, 30Toyoda H. Kinoshita-Toyoda A. Selleck S.B. J. Biol. Chem. 2000; 275: 2269-2275Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). On the other hand, as in vertebrates, heparan sulfate (HS) proteoglycans are present in C. elegans, although they are not the major proteoglycans in the nematode (29Yamada S. Van Die I. Van den Eijnden D.H. Yokota A. Kitagawa H. Sugahara K. FEBS Lett. 1999; 459: 327-331Crossref PubMed Scopus (96) Google Scholar, 30Toyoda H. Kinoshita-Toyoda A. Selleck S.B. J. Biol. Chem. 2000; 275: 2269-2275Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). The C. elegans genome contains a set of sulfotransferases (hst-1, hst-2, hst-3, and hst-6) involved in sulfation of HS in contrast to that of chondroitin sulfate. Although several reports have suggested that sulfation of HS plays a role in cell migration and axon guidance in C. elegans (31Bulow H.E. Hobert O. Neuron. 2004; 41: 723-736Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 32Kinnunen T. Huang Z. Townsend J. Gatdula M.M. Brown J.R. Esko J.D. Turnbull J.E. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 1507-1512Crossref PubMed Scopus (64) Google Scholar), little is known about how PAPS synthesis affects developmental processes. In this study, we show that a single C. elegans ortholog of the PAPS synthase gene pps-1 is present in the nematode genome and is expressed in the epidermis and several gland cells but not in neurons and muscles. Disruption of the pps-1 gene caused pleiotropic developmental defects and abnormality in patterning of muscles and shape change of epidermis. Our results showed for the first time that PAPS synthase is indispensable for normal growth and development and is involved in pattern formation and cell shape change of epidermis in C. elegans. Strains—Most of the nematode and Escherichia coli strains used were from the Caenorhabditis Genetic Center. The deletion mutant strain tm1109 was isolated from pools of worms mutagenized by the UV/TMP method (33Gengyo-Ando K. Mitani S. Biochem. Biophys. Res. Commun. 2000; 269: 64-69Crossref PubMed Scopus (167) Google Scholar). Primers used for PCR screening and genotyping of the deletion allele were as follows: T14G10#F1 (5′-CCGCCTACCACTTCTTAGTA-3′), T14G10#R1 (5′-CCGTGCCAATCCTCGCATCT-3′), T14G10#F2 (5′-TGCCCACGTATCATATGCTG-3′), and T14G10#R2 (5′-AGCAGGATCACGTCCCACTA-3′). RNAi by Feeding—The protocol for RNAi by feeding was based on described methods (34Kamath R.S. Martinez-Campos M. Zipperlen P. Fraser A.G. Ahringer J. Genome Biol. 2001; 2 (RESEARCH0002)PubMed Google Scholar, 35Timmons L. Court D.L. Fire A. Gene (Amst.). 2001; 263: 103-112Crossref PubMed Scopus (1336) Google Scholar). The cDNA clone for T14G10.1 (pps-1) was amplified by PCR from total cDNA of N2 worms. Primers for the T14G10.1 cDNA were 5′-TGCTCACTCCACGGGATGAA-3′ and 5′-AGTTTGAGTTTTGTAGTGATTTGTAG-3′. A fragment of cDNA was cloned into the L4440 (pPD129.36) vector, and the cloned plasmids were transformed into E. coli HT115 (DE3). A single colony of HT115 (DE3) containing the plasmid was grown in LB culture medium for 8 h and seeded onto NGM agar plates (80 μl/plate), which were incubated at 37 °C overnight. Following the addition of 2 mm isopropyl-β-d-thiogalactoside (100 μl/plate), the cells were cultivated for 4 h to induce the expression of double-stranded RNA (dsRNA). HT115 harboring the plasmid pPD129.36 without any insert was used as control. For the assay of PAPS synthase activity, mixed stage hermaphrodites were used, and for phenotypic characterization and the assay of lethality, L4 or L3 hermaphrodites were transferred on the plates, and dsRNA was introduced into the nematode by feeding. cDNA Cloning of C. elegans PAPS Synthase—The cDNA encoding the full open reading frame of C. elegans PAPS synthase (accession number NM_069456) was amplified by PCR from C. elegans cDNAs, which were prepared from large scale mixed stage culture of N2 worms (36Stiernagle T. C. elegans: A Practical Approach.in: Hope I.A. Oxford University Press, Oxford1999: 51-68Google Scholar). The oligonucleotide primers used were 5′-tttgtcgacATGCTCACTCCACGGGAT-3′ (forward primer) and 5′-tttaagcttAGTTTGAGTTTTGTAGTG-3′ (reverse primer). The sequences shown by lowercase letters indicate appropriate restriction sites. The amplified cDNAs were digested with SalI and HindIII and cloned into pBluescript II SK(+). The plasmids were sequenced using an Applied Biosystems Prism 310 Genetic Analyzer (PE Biosystems). The cDNA with the precise sequence was recloned into pQE-9-EK, which was prepared from pQE-9 (Qiagen) by insertion of an enterokinase cleavage site (DDDDK) using the QuikChange site-directed mutagenesis kit (Stratagene) and two oligonucleotides, 5′-CCATCACCATCACGATGACGATGACAAAGGATCCGTCGACC-3′ and its complement. The resulting pQE-9-EK-PAPS was transformed using the E. coli M15 strain (Qiagen). LB medium (80 ml) with 100 μg/ml ampicillin and 25 μg/ml kanamycin was inoculated with 0.8 ml of overnight culture of the transformants and then shaken at 30 °C until the absorbance at 600 nm reached 1.0. After the addition of isopropyl-β-d-thiogalactoside to a final concentration of 0.1 mm, the bacteria were cultured at 20 °C for 16 h and harvested by centrifugation. The pellet was suspended in 10 ml of 20 mm sodium phosphate, 0.3 m NaCl, and 10 mm imidazole (pH 8.0) (buffer A) and sonicated. A milliliter of 5% (v/v) Triton X-100 was added, and the mixture was kept on ice for 30 min. After centrifugation, the supernatant was applied to an Ni2+-nitrilotriacetic acid-agarose (Qiagen) (0.7 × 1.3 cm; equilibrated with buffer A containing 0.5% (v/v) Triton X-100). After washing with buffer A, the recombinant protein was eluted with 0.25 m imidazole, 20 mm sodium phosphate, and 0.3 m NaCl (pH 8.0). Finally, 1.7 mg of the protein was obtained. Protein concentration was determined by a Bio-Rad protein assay dye reagent using bovine serum albumin as a standard. Assay of PAPS Synthase Activity—Free [35S]sulfate was prepared from 2.5 MBq of adenosine 3′-phosphate 5′-phospho[35S]sulfate (PAPS, 71.2 GBq/mmol; PerkinElmer Life Sciences) by mild acid hydrolysis (0.5 ml of 0.02 n HCl, 100 °C, 15 min), followed by paper electrophoresis (pyridine/acetic acid/water = 3:1:387, pH 5.4). Adenosine 5′-phospho[35S]sulfate (APS) was prepared from 2.5 MBq of [35S]PAPS using 50 μl of alkaline phosphatase beads (derived from bovine intestinal mucosa; 1280 units/g acrylic beads; Sigma) in 200 μl of 50 mm Tris-HCl (pH 8.0) at 37 °C for 15 min, followed by paper electrophoresis. APS kinase activity was assayed according to the methods of Geller et al. (37Geller D.H. Henry J.G. Belch J. Schwartz N.B. J. Biol. Chem. 1987; 262: 7374-7382Abstract Full Text PDF PubMed Google Scholar). Briefly, 10 μl of the solution containing 16 kBq of [35S]APS, 0.1 m HEPES-NaOH (pH 7.2), 40 mm MgCl2, and 20 mm disodium ATP (Grade II; Sigma) was mixed with 10 μl of enzyme suspension (lyophilized powders were suspended in 20 mm HEPES-NaOH (pH 7.2), 0.15 m NaCl) and incubated at 37 °C for 10 min. The reaction was stopped by the addition of 20 μl of ice-cold ethanol, and the mixture was immediately applied to paper electrophoresis with the same conditions as described above. The paper was scanned using a radiochromatogram scanner RITA Star (Straubenhardt, Germany). [35S]PAPS fractions were cut off and counted for the radioactivity. To determine APS kinase activities, mix staged pps-1 RNAi-untreated and -treated nematodes were harvested by washing and sucrose flotation as described (38Johnstone I.L. C. elegans: A Practical Approach.in: Hope I.A. Oxford University Press, Oxford1999: 201-225Google Scholar). Three hundred L4-staged tm1109 heterozygous and wild-type nematodes were placed on NGM agar plates with a lawn of OP50 E. coli cells by picking. They were cultured at 20 °C for 24 h and then harvested by washes. Because of the small amount, we omitted the sucrose flotation step that could cause loss of collected nematodes. Total PAPS synthase activity was assayed with the same conditions for APS kinase described above, except that 8.0 kBq of [35S]sulfate was used as a substrate instead of [35S]APS. Analysis of GAGs—GAGs were prepared from dried homogenates of wild-type or RNAi-treated nematode (dry weight 55 or 119 mg, respectively). GAG chains were released from the proteoglycan core proteins by the sodium borohydride treatment. It should be noted that the amount of HS in C. elegans was so small (28Mizuguchi S. Uyama T. Kitagawa H. Nomura K.H. Dejima K. Gengyo-Ando K. Mitani S. Sugahara K. Nomura K. Nature. 2003; 423: 443-448Crossref PubMed Scopus (218) Google Scholar) that 100 μg of shark cartilage chondroitin 6-O-sulfate (Seikagaku Corp.), which contained a negligible proportion of nonsulfated disaccharides, was added as a carrier after the borohydride treatment but before the purification steps. The unsaturated disaccharides were produced by digestion with chondroitinase ABC or a mixture of heparitinases I and II, and then the digests were derivatized with 2-aminobenzamide and analyzed by high performance liquid chromatography as described previously (28Mizuguchi S. Uyama T. Kitagawa H. Nomura K.H. Dejima K. Gengyo-Ando K. Mitani S. Sugahara K. Nomura K. Nature. 2003; 423: 443-448Crossref PubMed Scopus (218) Google Scholar, 29Yamada S. Van Die I. Van den Eijnden D.H. Yokota A. Kitagawa H. Sugahara K. FEBS Lett. 1999; 459: 327-331Crossref PubMed Scopus (96) Google Scholar). Phenotypic Analysis—We determined the extent of lethality of the pps-1 mutant by picking late L4 heterozygous strains to separate plates, allowing them to lay eggs for 24 h, and transferring them to a new plate daily for the next 3 days. Eggs unhatched after 20, 24, and 40 h were scored as embryonic lethal at 25, 20, and 15 °C, respectively. Larvae that failed to develop into L4 or adult stage after 40, 46, and 96 h were scored as larval arrest/lethal at 25, 20, and 15 °C, respectively. To determine the embryonic phenotype of pps-1 RNAi, embryos were dissected from the adult animals that had had dsRNA introduced for the indicated time. The embryos were cultured for an additional 18 h in M9, and various phenotypes were scored. To determine the larval phenotype of pps-1 RNAi-treated animals, embryos were dissected from the adult animals that were fed with pps-1 dsRNA for 40 h. The dissected embryos were cultured for an additional 18 h in M9 and placed onto the dsRNA-expressing bacteria, and developmental stages were scored after 40 h. Phalloidin staining was performed as described previously (39Costa M. Draper B.W. Priess J.R. Dev. Biol. 1997; 184: 373-384Crossref PubMed Scopus (102) Google Scholar). Reporter Constructs and Transgenic Lines—The pps-1p::egfp and myo-3p::egfp translational reporter constructs were generated by PCR amplification of a 3249-bp upstream sequence of the pps-1 ATG using the primers 5′-TTTTTATCCCATTTGCCATT-3′ and 5′-CATGGTTATATAGGTAGTCCCACG-3′ and a 2453-bp upstream sequence of the myo-3 ATG using the primers 5′-GGCTGCAACAAAGATCAGGT-3′ and 5′-CATTTCTAGATGGATCTAGTG-3′, respectively; ligation into the XcmI-digested vector pFX_EGFPT (40Izumikawa T. Kitagawa H. Mizuguchi S. Nomura K.H. Nomura K. Tamura J. Gengyo-Ando K. Mitani S. Sugahara K. J. Biol. Chem. 2004; 279: 53755-53761Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) using the TA-cloning strategy to fuse the coding sequence region of EGFP then followed. The translational pps-1(FL)::egfp reporter construct was generated by PCR amplification of a 5495-bp genomic fragment, including the 3 kb of the potential promoter region. The fragment was amplified by PCR from C. elegans genomic DNA as a template using a 5′-primer (5′-TTTTTATCCCATTTGCCATT-3′) and a 3′-primer (5′-GTTTGAGTTTTGTAGTGATTTGTAGTAG-3′) located just before the stop codon and ligated into the vector pFX_EGFPT as described above. The translational dpy-7p::dsred reporter was generated by PCR amplification of 428 bp upstream of the dpy-7 ATG using the primers 5′-TCGAAAGTCTCTCCGGTAGC-3′ and 5′-CATTTATCTGGAACAAAATGTAAG-3′, followed by ligation into XcmI-digested vector pFX_DsRedXT (40Izumikawa T. Kitagawa H. Mizuguchi S. Nomura K.H. Nomura K. Tamura J. Gengyo-Ando K. Mitani S. Sugahara K. J. Biol. Chem. 2004; 279: 53755-53761Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) using the TA-cloning strategy to fuse the coding sequence region of DsRed. PCR was carried out with Platinum TaqDNA polymerase High Fidelity (Invitrogen). Germ line transformation was done as described by Mello et al. (41Mello C.C. Kramer J.M. Stinchcomb D. Ambros V. EMBO J. 1991; 10: 3959-3970Crossref PubMed Scopus (2422) Google Scholar). The transgenic line was generated by injecting the experimental DNA at a concentration of 10-100 μg/ml into the distal gonads. Transgenic lines containing reporter constructs were isolated. Live transgenic worms were anesthetized with a 10 mm sodium azide solution, placed on an 8-well printed microscope slide glass (Matsunami Glass), and examined using four-dimensional microscopy (a DMRXA full automatic microscope with differential interference contrast (DIC) and fluorescent optics; Leica) as described (28Mizuguchi S. Uyama T. Kitagawa H. Nomura K.H. Dejima K. Gengyo-Ando K. Mitani S. Sugahara K. Nomura K. Nature. 2003; 423: 443-448Crossref PubMed Scopus (218) Google Scholar). The images were processed using MetaMorph software (version 6.1r5; Universal Imaging). Identification and PAPS Synthase Activity of C. elegans PPS-1—A data base search of the complete C. elegans genome sequence revealed the presence of only one PAPS synthase homolog pps-1 (T14G10.1) on chromosome IV. The predicted pps-1 gene product consists of 652 amino acid residues and shows 57% identity both with the human PAPSS1 and PAPSS2a at the amino acid level. PPS-1 has a putative nuclear localization signal sequence at the N terminus. The APS kinase domain (Pfam: PF01583) and ATP sulfurylase domain (Pfam: PF01747) occupy one-third of the N terminus and two-thirds of the C terminus of the sequence, respectively. The APS kinase domain contains a nucleotide-binding P-loop motif (GXXGXGK(S/T)) that is a critical site in APS kinase activity of mammalian PAPS synthases. On the other hand, the ATP sulfurylase domain has another type of nucleotide binding motif, HXGH, required for ATP sulfurylase activity of mammalian PAPS synthases (Fig. 1A) To assess biochemically whether the protein encoded in the PAPS synthase gene could synthesize PAPS from free sulfate and ATP, we isolated the cDNA and expressed the protein in E. coli M15 cells. SDS-PAGE analysis of the purified recombinant protein showed one band with a molecular mass of 73 kDa (Fig. 1B). Using [35S]sulfate and [35S]APS, we assayed total PAPS synthase and APS kinase activities, respectively. [35S] PAPS was produced in both assays (Fig. 1, C and D). Specific activities of the protein for total PAPS synthase and APS kinase were 0.80 nmol/min/mg protein and 63 nmol/min/mg protein, respectively. It should be noted that in the total PAPS synthase assay, an intermediate product, [35S]APS, was scarcely detected, suggesting that APS produced from sulfate and ATP was quickly converted to PAPS. pps-1 Reporter Constructs Are Widely Expressed in Epidermal Cells and Gland Cells, but Not in Muscle Cells and Neurons, and Are Predominantly Localized to the Nucleus—To determine the expression pattern of pps-1, two different types of pps-1 genomic fragments were cloned into a reporter gene vector. Because the PCR product that contained the 3-kb sequence from the predicted ATG was sufficient to rescue the lethality of the pps-1 mutant (see below; Table 1), the same 3-kb region was cloned into the reporter vector as the promoter-reporter construct pps-1p::EGFP. On the other hand, the 5-kb sequence upstream from the predicted stop codon that contains both the promoter and open reading frame sequence was cloned into the reporter vector as a translational fusion construct, pps-1(FL)::EGFP (Fig. 1E). The pps-1p::EGFP reporter is widely but tissue-specifically expressed in somatic cells. pps-1p::EGFP is strongly expressed in seam cells (Fig. 2, A, H, and H′), gland cells (Fig. 2B), and neuronal support cells (amphid sheath cells; Fig. 2, C-C″) throughout development. Relatively weak expression was also detected in the hypodermis (Fig. 2D) and the phasmid support cells during larval development. Additionally, weak expression was observed in the intestine at the adult stage (Fig. 2, E and E′). Unexpectedly, no signal was found in the neurons and muscles.TABLE 1The pps-1 null phenotype is a lar
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