Structure and function of HNK-1 sulfotransferase - Identification of donorand acceptor binding sites by site-directed mutagenesis

HNK-1 glycan, sulfo-->3GlcA beta 1-->3Gal beta 1-->4GlcNAc-->R, is uniquely enriched in neural cells and natural killer cells and is thought to play i mportant roles in cell-cell interaction. HNK-1 glycan synthesis is dependen t on HNK-1 sulfotransferase (HNK-1ST), and cDNAs encoding human and rat HNK -1ST have been recently cloned. HNK-1ST belongs to the sulfotransferase gen e 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 bind ing to the donor substrate, adenosine 3'-phosphate 5'-phosphosulfate, and a n acceptor. Mutations of Lys(128), Arg(189), Asp(190), Pro(191), and Ser(19 7) to Ala all abolished the enzymatic activity. When Lys(128) and Asp(190) were conservatively mutated to Arg and Glu, respectively, however, the muta ted enzymes still maintained residual activity, and both mutant enzymes sti ll bound to adenosine 3',5'-diphosphate-agarose. K128R and D190E mutant enz ymes, on the other hand, exhibited reduced affinity to the acceptor as demo nstrated by kinetic studies. These findings, together with those on the cry stal structure of estrogen sulfotransferase and heparan sulfate N deacetyla se/sulfotransferase, suggest that Lys(128) may be close to the 3-hydroxyl g roup of beta-glucuronic acid in a HNK-1 acceptor. In contrast, the effect b y mutation at Asp(190) may be due to conformational change because this ami no acid and Pro(191) reside in a transition of the secondary structure of t he enzyme. These results indicate that conserved amino acid residues in HNK -1ST play roles in maintaining a functional conformation and are directly i nvolved in binding to donor and acceptor substrates.