2004年11月
Role of the carboxyl-terminal region in the activity of N-acetylglucosamine 6-O-sulfotransferase-1
JOURNAL OF BIOCHEMISTRY
- ,
- ,
- 巻
- 136
- 号
- 5
- 開始ページ
- 659
- 終了ページ
- 664
- 記述言語
- 英語
- 掲載種別
- 研究論文(学術雑誌)
- DOI
- 10.1093/jb/mvh162
- 出版者・発行元
- JAPANESE BIOCHEMICAL SOC
N-Acetylglucosamine 6-O-sulfotransferases (GlcNAc6STs) catalyze the transfer of sulfate from W-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 G1cNAcbeta1-6ManOMe 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)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.
- リンク情報
- ID情報
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- DOI : 10.1093/jb/mvh162
- ISSN : 0021-924X
- PubMed ID : 15632306
- Web of Science ID : WOS:000227092700014