Abstract
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.
Highlights
Tant role [2, 3]
We showed that pps-1 is involved in controlling several aspects of both embryonic and postembryonic development, including molting, changes in cell shape, and patterning of epithelial and muscle cells
PAPSS1 is localized in nuclei, whereas PAPSS2 is in cytoplasm [50]
Summary
Strains—Most of the nematode and Escherichia coli strains used were from the Caenorhabditis Genetic Center. 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. Adenosine 5Ј-phospho[35S]sulfate (APS) was prepared from 2.5 MBq of [35S]PAPS using 50 l of alkaline phosphatase beads (derived from bovine intestinal mucosa; 1280 units/g acrylic beads; Sigma) in 200 l of 50 mM Tris-HCl (pH 8.0) at 37 °C for 15 min, followed by paper electrophoresis. To determine APS kinase activities, mix staged pps-1 RNAi-untreated and -treated nematodes were harvested by washing and sucrose flotation as described [38]. Todes 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 harvested by washes. Lethality of pps-1 mutants was scored as described under “Experimental Procedures.”
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