RESUMEN
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.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Complejos Multienzimáticos/fisiología , Sulfato Adenililtransferasa/fisiología , Adenosina Trifosfato/química , Alelos , Animales , Tipificación del Cuerpo , Caenorhabditis elegans , Sulfatos de Condroitina/metabolismo , Clonación Molecular , ADN Complementario/metabolismo , Disacáridos/química , Electroforesis en Gel de Poliacrilamida , Escherichia coli/metabolismo , Eliminación de Gen , Genes Reporteros , Glicosaminoglicanos/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Microscopía Fluorescente , Modelos Genéticos , Músculos/metabolismo , Mutación , Neuronas/metabolismo , Fenotipo , Fosfoadenosina Fosfosulfato/química , Interferencia de ARN , Temperatura , TransgenesRESUMEN
The Galbeta1-->4(SO(3)(-)-->6)GlcNAc moiety is present in various N-linked and O-linked glycans including keratan sulfate and 6-sulfosialyl-Lewis X, an L-selectin ligand. We previously found beta1,4-galactosyltransferase (beta4GalT) activity in human colonic mucosa, which prefers GlcNAc 6-O-sulfate (6SGN) as an acceptor to non-substituted GlcNAc (Seko, A., Hara-Kuge, S., Nagata, K., Yonezawa, S., and Yamashita, K. (1998) FEBS Lett. 440, 307-310). To identify the gene for this enzyme, we purified the enzyme from porcine colonic mucosa. The purified enzyme had the characteristic requirement of basic lipids for catalytic activity. Analysis of the partial amino acid sequence of the enzyme revealed that the purified beta4GalT has a similar sequence to human beta4GalT-IV. To confirm this result, we prepared cDNA for each of the seven beta4GalTs cloned to date and examined substrate specificities using the membrane fractions derived from beta4GalT-transfected COS-7 cells. When using several N-linked and O-linked glycans with or without 6SGN residues as acceptor substrates, only beta4GalT-IV efficiently recognized 6SGN, keratan sulfate-related oligosaccharides, and Galbeta1-->3(SO(3)(-)-->6GlcNAcbeta1-->6) GalNAcalpha1-O-pNP, a precursor for 6-sulfosialyl-Lewis X. These results suggested that beta4GalT-IV is a 6SGN-specific beta4GalT and may be involved in the biosynthesis of various glycoproteins carrying a 6-O-sulfated N-acetyllactosamine moiety.