Role of the carboxyl-terminal region in the activity of N-acetylglucosamine 6-o-sulfotransferase-1.
N-Acetylglucosamine 6-O-sulfotransferases (GlcNAc6STs) catalyze the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to the C-6 position of non-reducing N-acetylglucosamine. N-acetylglucosamine 6-O-sulfotransferase-1 (GlcNAc6ST-1) is the first cloned GlcNAc6ST and is involved in the synthesis of the L-selectin ligand. We noticed conserved C-terminal segments among GlcNAc6STs and produced mutant enzymes to reveal the functional significance. Mutant enzymes were transiently expressed as fusion proteins with protein A in COS-7 cells, and some of them were purified to homogeneity by IgG Sepharose column chromatography. Deletion of a C-terminal segment (amino acid numbers 479-483) resulted in a complete loss of the activity, when assayed using GlcNAcbeta1-6ManOMe as a substrate. Upon site-directed mutagenesis of the C-terminal region, three mutants, L477A, L478A and L483A, exhibited reduced activity. The K(M )values for GlcNAcbeta1-6ManOMe of L477A and L478A were 4 times higher than the K(M) of the wild-type enzyme, while that of L483A was unchanged. On the other hand the K(M )for PAPS of L483A was 3 times higher than that of the wild-type enzyme, while the values of L477A and L478A were unchanged. Furthermore, the L477A mutant acted on a core 3 structure (GlcNAcbeta1-3GalNAc-pNP), while the wild-type enzyme does not. These results demonstrate a role for leucine residues in the C-terminal region in the enzymatic activity.
Chen
Lan
L
Department of Biochemistry, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan.
Ichihara-Tanaka
Keiko
K
Muramatsu
Takashi
T
D000818:Animals; D019556:COS Cells; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D015238:Sulfotransferases / Q000378:metabolism* / Q000502:physiology*; D000097811:Carbohydrate Sulfotransferases
D015238:Sulfotransferases; D000097811:Carbohydrate Sulfotransferases