Molecular cloning and characterization of three carbohydrate sulfotransferases: Their possible relevance to the generation of L-selectin ligands

L-selectin, a lectin-like receptor, mediates rolling of lymphocytes on high endothelial venules (HEV) in secondary lymphoid organs by interacting with HEV-ligands. The HEV-ligands consist of mucin-like glycoproteins bearing O-linked carbohydrates. Candidate ligands are GlyCAM-1, CD34 and podocalyxin. Sialylation, fucosylation and sulfation of the ligands are required for optimal recognition by L-selectin. Structural analysis of the oligosaccharides of GlyCAM-1 revealed the presence of N-acetylglucosamine-6-sulfate and galactose-6-sulfate, in the context of sialyl Lewis x (sialyl 6-sulfo Lex and sialyl 6'-sulfo Lex, respectively). Sialyl 6-sulfo Lex is present on HEV in human lymphoid tissues. This thesis describes the molecular cloning and characterization of three carbohydrate sulfotransferases that may be relevant to the generation of L-selectin ligands in vivo.; Three distinct genes were identified as candidate sulfotransferases in the human expressed sequence tag databases by their homology with the cDNA sequence for a chicken galactose-6-O-sulfotransferase. The cDNAs corresponding to each of these genes were isolated by screening cDNA libraries utilizing conventional plaque-lifting techniques and PCR. Northern blot and in situ hybridization analysis indicated that two of these genes are broadly expressed, whereas the third is highly restricted to HEV.; The cDNAs encoding each of the genes were expressed in COS cells and extracts of the cells were tested for their ability to sulfate oligosaccharide acceptors in vitro. The sulfated acceptors were subjected to acid hydrolysis and the regiospecificity of sulfation was determined by high-pH anion-exchange chromatography. This analysis established that two of the cDNAs encode N-acetylglucosamine-6-O-sulfotransferases and one encodes a galactose-6-O-sulfotransferase. Coexpression of the sulfotransferase cDNAs with cDNAs encoding GlyCAM-1 or CD34 in COS cells showed that the sulfotransferases are capable of sulfating L-selectin ligands.; Flow cytometry analysis of CHO cells transfected with cDNAs encoding CD34, fucosyltransferase-VII and the sulfotransferase cDNAs indicated that (1) the two N-acetylglucosamine-6-O-sulfotramferases can confer expression of the sialyl 6-sulfo Lex epitope; (2) either of the three sulfotransferases can contribute to the generation of L-selectin ligand activity; (3) the galactose-6-sulfate and the N-acetylglucosamine-6-sulfate esters apparently synergize in enhancing L-selectin binding. Analysis in a parallel-plate flow chamber indicated that recombinant GlyCAM-1, when sulfated by either of the three enzymes, supports enhanced rolling of lymphocytes.