Purification and characterization of a lymph node sulfotransferase responsible for 6-O-sulfation of the galactose residues in 2 '-fucosyllactose and other sialyl Lewis(X)-related sugars

A microsomal galactose 6-O-sulfotransferase (Gal-6-O-Stase) from porcine ly mph nodes, able to transfer the sulfate group from adenosine 3'-phosphate 5 '-phosphosulphate (PAPS) onto 2'-fucosyllactose (2'-FL) and other sialyl Le wis(x) (sLe(x))-related sugars, has been purified and characterized. The en zyme was purified to about 35,000-fold by a combination of conventional and affinity chromatographic steps. The purified enzyme preparation exhibited two protein bands at around 80-90 and 170 kDa on 7.5% SDS-PAGE under reduci ng conditions. Both of these protein bands always comigrated in the gel whe n peak fractions containing Gal-6-O-Stase activity from the 3',5'-ADP-agaro se column were subjected to 6% SDS-PAGE under reducing conditions. These pr otein bands also showed similar binding patterns to WGA (wheat germ aggluti nin), Con A (concanvalin A), and EBA (elderberry agglutinin). Similarly whe n the enzyme preparation after the hydroxylapatite step was photolabeled wi th 8-azido-[P-32]-PAPS, both 80-90 and 170 kDa protein bands were labeled i n a specific manner. These results suggest a possible association of these two protein bands with the enzyme activity. The carbohydrate substrate spec ificity of this enzyme suggests that it is well suited to catalyze the sulp honation at the C-6 position of the galactose residues of oligosaccharides that are structurally similar to sLe(x). Furthermore, a survey of several p orcine organs revealed that this enzyme was selectively expressed in lympho id tissues such as lymph nodes (peripheral and mesenteric) and spleen. Thes e findings suggest that this enzyme may be involved in the assembly of 3'-s ialyl-6'-sulfo Lewis(x), the major capping group of HEV-ligands for L-selec tin. (C) 1999 Academic Press.