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Structure and Function of HNK-1 Sulfotransferase

1999; Elsevier BV; Volume: 274; Issue: 36 Linguagem: Inglês

10.1074/jbc.274.36.25608

1083-351X

Edgar Ong, Jiunn‐Chern Yeh, Yili Ding, Ole Hindsgaul, Minoru Fukuda, Lars C. Pedersen, Masahiko Negishi,

Protein Kinase Regulation and GTPase Signaling

HNK-1 glycan, sulfo→3GlcAβ1→3Galβ1→4GlcNAc→R, is uniquely enriched in neural cells and natural killer cells and is thought to play important roles in cell-cell interaction. HNK-1 glycan synthesis is dependent on HNK-1 sulfotransferase (HNK-1ST), and cDNAs encoding human and rat HNK-1ST have been recently cloned. HNK-1ST belongs to the sulfotransferase gene family, which shares two homologous sequences in their catalytic domains. In the present study, we have individually mutated amino acid residues in these conserved sequences and determined how such mutations affect the binding to the donor substrate, adenosine 3′-phosphate 5′-phosphosulfate, and an acceptor. Mutations of Lys 128 , Arg 189 , Asp 190 , Pro 191 , and Ser 197 to Ala all abolished the enzymatic activity. When Lys 128 and Asp 190 were conservatively mutated to Arg and Glu, respectively, however, the mutated enzymes still maintained residual activity, and both mutant enzymes still bound to adenosine 3′,5′-diphosphate-agarose. K128R and D190E mutant enzymes, on the other hand, exhibited reduced affinity to the acceptor as demonstrated by kinetic studies. These findings, together with those on the crystal structure of estrogen sulfotransferase and heparan sulfate N -deacetylase/sulfotransferase, suggest that Lys 128 may be close to the 3-hydroxyl group of β-glucuronic acid in a HNK-1 acceptor. In contrast, the effect by mutation at Asp 190 may be due to conformational change because this amino acid and Pro 191 reside in a transition of the secondary structure of the enzyme. These results indicate that conserved amino acid residues in HNK-1ST play roles in maintaining a functional conformation and are directly involved in binding to donor and acceptor substrates.