HNK-1 glycan, sulfo33GlcA133Gal134GlcNAc3 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. 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 -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.HNK-1 glycan is a unique carbohydrate expressed in a celltype specific manner (1, 2). In particular, HNK-1 glycan has been found in a number of neural cell adhesion molecules including the neural cell adhesion molecule, myelin-associated glycoprotein, L1, contactin, and P0 (3-7). Carbohydrate structural analysis of P0 showed that HNK-1 glycan is present in biantennary bisected N-glycan as sulfo3 GlcA133Gal-134GlcNAc132Man␣136[R3 Man␣133(GlcNAc134)]-Man134GlcNAc134GlcNAc3 Asn (8). The capping structure of this HNK-1 epitope is the same as those found in glycolipids such as sulfo33GlcA133Gal134GlcNAc133-Gal134Glc3ceramide (9, 10). The expression of HNK-1 glycan is spatially and temporally regulated and is found on migrating neural crest cells, cerebellum, and myelinating Schwann cells in motor neurons but not on those in the sensory neurons (11-13). Neural outgrowth of mouse motor neurons was facilitated much better by HNK-1 glycolipid substratum than sulfatide or GD1b ganglioside substratum (14). Complementary to this finding, the inhibition of neurite outgrowth by HNK-1 glycan was not observed once the sulfate group was removed (15). It has also been shown that the homophilic interaction of P0 depends on carbohydrate-protein interaction and anti-HNK-1 antibody inhibits this interaction (16,17).To elucidate the roles of HNK-1 glycan in molecular details, we and others have cloned a sulfotransferase HNK-1ST that forms HNK-...