Molecular Cloning and Expression of anN-Acetylgalactosamine-4-O-sulfotransferase That Transfers Sulfate to Terminal and Non-terminal β1,4-LinkedN-Acetylgalactosamine

Abstract

-7), and triggering preferential neurite outgrowth (8). HNK-1, an example of such a sulfated glycan, has the structure SO 4 -3-GlcUA␤1,3Gal␤1,4GlcNAc-R and is found at the nonreducing termini of glycoprotein and glycolipid oligosaccharides (9, 10). The structure was first identified with monoclonal antibodies as an epitope on human natural killer cells (11). The HNK-1 epitope is a hallmark of many neural recognition molecules and displays phylogenetic conservation (12), highlighting its functional importance. It is expressed in the central and peripheral nervous systems during development and regeneration (13,14) and has recently been recognized to be a crucial player in synaptic plasticity involving inhibitory interneurons in the hippocampus (15). HNK-1 is also a predominant autoantigen in demyelinating diseases of the peripheral nervous system (16).Expression cloning of the rat and human sulfotransferases responsible for synthesis of the HNK-1 epitope revealed the presence of two sequence motifs that are associated with 3Ј-phosphoadenosyl-5Ј-phosphosulfonate (PAPS) binding (17,18) and are common among all sulfotransferases cloned to date (19 -21). Despite the presence of these motifs, HNK-1 sulfotransferase shows only limited similarity to other sulfotransferases outside of the PAPS-binding regions. Nonetheless, three additional sulfotransferases, chondroitin 4-O-sulfotransferase 1 (C4ST-1) (22, 23), chondroitin 4-O-sulfotransferase 2 (C4ST-2) (23), and N-acetylgalactosamine-4-O-sulfotransferase 1 (GalNAc-4-ST1) (24, 25), were identified based on their homology to the catalytic C-terminal domain of HNK-1 ST. C4ST-1 and C4ST-2 (Fig. 2) show 25 and 22% identity to HNK-1 ST, respectively, whereas GalNAc-4-ST1 is 23% identical to HNK-1 ST. In addition to the motifs associated with binding PAPS, HNK-1 ST, C4ST-1, C4ST-2, and GalNAc-4-ST1 have three additional motifs of unknown function that are located C-terminal to the PAPS-binding motifs. While homolog-

Molecular Cloning and Characterization of a Dermatan-specific N-Acetylgalactosamine 4-O-Sulfotransferase

“…Although D4ST-1 has a hydrophobic sequence near its amino terminus that is predicted to act as a transmembrane domain, only 3% of the activity is retained by the transfected cells as compared with that released into the culture medium. The D4ST-1 released into the medium may reflect a proteolytic clip, as has been seen for a number of transferases including GalNAc-4-ST1 and GalNAc-4-ST2 (15,16,34,35); however, this possibility has not been formally addressed for D4ST-1. While D4ST-1 is able to transfer sulfate to both dermatan and chondroitin, the rate of transfer is nearly 10-fold greater to dermatan than to chondroitin when the same substrate concentrations are used (Table I).…”

“…The additional exons in GalNAc-4-ST1 and GalNAc-4-ST2 encode the cytosolic domain, transmembrane domain, and much of the stem region. The stem region appears to confer the specificity for terminal rather than internal GalNAc on GalNAc-4-ST1 and GalNAc-4-ST2 (15,16). While 5Ј-rapid amplification of cDNA ends and attempts to amplify predicted sequences did not yield the entire 5Ј-UTR for the D4ST-1 cDNA, there are a number of indications that the entire coding sequence for the ORF is contained in the cDNA sequence we have obtained.…”

“…C4ST-2 is, however, reported to be a GalNAc-4-sulfotransferase that transfers sulfate to internal UA-GalNAc sequences (12). In contrast to D4ST-1, C4ST-1, and C4ST-2, GalNAc-4-ST1 and GalNAc-4-ST2 both transfer sulfate to GalNAc␤1,4GlcNAc␤-sequences found at the termini of N-and O-linked oligosaccharides (15,16). Since deletion of the stem region from both GalNAc-4-ST1 and GalNAc-4-ST2 converts these terminal sulfotransferases to forms that will transfer sulfate to internal GlcUA-GalNAc sequences as well as to terminal GalNA c␤1,4GlcNAc␤-sequences, the high percentage of identical amino acids, 24.7 and 21.0% respectively, is not surprising.…”

“…Two additional GalNAc-4-O-sulfotransferases, GalNAc-4-ST1 and GalNAc-4-ST2, that add sulfate to terminal ␤1,4-linked GalNAc in the sequence GalNAc␤1,4GlcNAc-R on Nlinked oligosaccharides such as are present on the glycoprotein hormones lutropin and thyrotropin were cloned and also found to be homologous to HNK-1 ST. The intracellular forms of GalNAc-4-ST1 and GalNAc-4-ST2 that are located in the Golgi do not add sulfate to the nonterminal GalNAc residues in either chondroitin or dermatan (15,16).…”

Specificities of Three Distinct Human Chondroitin/Dermatan N-Acetylgalactosamine 4-O-Sulfotransferases Demonstrated Using Partially Desulfated Dermatan Sulfate as an Acceptor

“…However, the detailed properties of C4STs and the precise spatial arrangement of IdoUA residues and 4-O-sulfation sites have not been investigated. In addition to C4ST-1 and C4ST-2, several other sulfotransferases that belong to the same HNK-1 sulfotransferase (HNK-1ST) family have also been cloned, although they catalyze different regioselective sulfations of other types of glycan chains: HNK-1ST, which facilitates 3-O-sulfation of a GlcUA residue in HNK-1 antigen precursor oligosaccharide chains of glycoproteins and/or glycolipids (33,34), and N-acetylgalactosamine 4-O-sulfotransferases-1 and -2 (GalNAc4ST-1 and GalNAc4ST-2), which catalyze 4-O-sulfation at the nonreducing terminal GalNAc residues in the GalNAc␤1-4GlcNAc␤1-containing sequences in both N-and O-glycans (35)(36)(37)(38).…”