Characterization and mutagenesis of Gal/GlcNAc-6-O-sulfotransferases.
The installation of sulfate groups on the carbohydrate residues of glycoproteins, glycolipids, and glycosaminoglycans is a critical posttranslational modification that occurs in all higher eukaryotes. The Gal/GalNAc/GlcNAc-6-O-sulfotransferases (GSTs) are a recently discovered family of carbohydrate sulfotransferases that share significant sequence homology at the amino acid level and mediate a number of different biological processes such as leukocyte adhesion at sites of chronic inflammation. Structural and mechanistic studies of this family of sulfotransferases have been hindered by the lack of a productive recombinant protein expression system. We developed a baculovirus expression system for five of the seven cloned GSTs and determined their kinetic parameters using both thin-layer chromatography and a recently developed polymer dot-blot assay. We used these tools to perform the first site-directed mutagenesis study of a member of this sulfotransferase family, GST2. Using sequence alignments with other carbohydrate and cytosolic sulfotransferases, we selected residues within the putative binding regions for 3'-phosphoadenosine 5'-phosphosulfate (PAPS) and the carbohydrate substrate for mutagenesis. Kinetic analysis of the mutants identified residues that are essential for catalytic activity. These results should facilitate mechanistic studies and the development of small molecule inhibitors of this enzyme family to ameliorate chronic inflammatory diseases.
Grunwell
Jocelyn R
JR
Department of Chemistry, Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, USA.
Rath
Virginia L
VL
Rasmussen
Jytte
J
Cabrilo
Zeljka
Z
Bertozzi
Carolyn R
CR
D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002241:Carbohydrates / Q000737:chemistry / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D005822:Genetic Vectors / Q000138:chemical synthesis; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000737:chemistry; D010724:Phosphoadenosine Phosphosulfate / Q000737:chemistry; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity / Q000235:genetics; D015238:Sulfotransferases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D000097811:Carbohydrate Sulfotransferases
D000581:Amidohydrolases; D002241:Carbohydrates; D010710:Phosphates; D010724:Phosphoadenosine Phosphosulfate; D011994:Recombinant Proteins; D015238:Sulfotransferases; D000097811:Carbohydrate Sulfotransferases; C499549:CHST10 protein, human; C498300:CHST9 protein, human; C499545:Chst10 protein, mouse; C499547:Chst10 protein, rat; C109318:HNK-1 sulfotransferase; C416670:N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase; C092527:N-acetylgalactosamine-4-sulfotransferase; C021350:heparitin sulfotransferase