CHARACTERIZATION OF A SPLEEN SULFOTRANSFERASE RESPONSIBLE FOR THE 6-O-SULFATION OF THE GALACTOSE RESIDUE IN SIALYL-N-ACETYL-LACTOSAMINE SEQUENCES

An enzyme which catalyses the transfer of sulphate from 3'-phosphoaden osine 5'-phosphosulphate (PAPS) to C-6 of galactose in the NeuAc alpha 2-3Gal beta 1-4GlcNAc (3'SLN) sequence has been found in rat spleen m icrosomes and its specificity indicates that it is well suited to part icipate in the assembly of 3'-sialyl-6'-sulpho-LacNAc [NeuAc alpha 2-3 Gal(6-SO4)beta 1-4GlcNAc] and 3'-sialyl-6'-sulpho-Lewis(x) [NeuAc alph a 2-3Gal(6-SO4)beta 1-4(Fuc alpha 1-3)GlcNAc] saccharide groups which have been implicated as selectin ligands, This sulphotransferase has a strict requirement for oligosaccharide accepters which are capped by an alpha 2-3-linked sialic acid residue, although GlcNAc in 3'SLN can be substituted by Glc, and Gal beta 1-4GlcNAc can be replaced by Gal b eta 1-3GlcNAc without loss of activity. The finding that 3'-sialyl Lew is(x) was inert as an acceptor suggested that fucosylation, in contras t with sialylation, follows the addition of the sulphate group, Since fetuin glycopeptides containing the NeuAc alpha 2-3Gal beta 1-4GlcNAc sequence had a similar affinity for the enzyme as the unattached 3'SLN , it would appear that the acceptor determinants reside primarily in t he peripheral trisaccharide constellation. The position of the sulphat e on C-6 of galactose was elucidated by Smith periodate oxidation, hyd razine/nitrous acid/NaBH4 treatment and elder (Sambucus nigra) bark le ctin chromatography of the desialylated [S-35]sulphate-labelled produc ts of the enzyme. Assays carried out with 3'SLN as acceptor indicated that the sulphotransferase had a pH optimum between 6.5 and 7.0 and a dependence on a bivalent cation best met by Mn2+ (12-25 mM); Triton X- 100 (0.02 to 0.35 %) brought about maximal stimulation. Tentative K-m values determined for this enzyme were 4.7 mu M for PAPS, and 0.72 mM and 1.16 mM for 3'SLN and fetuin glycopeptides respectively, A survey of several rat organs indicated that the PAPS: 3'SLN-6-O-sulphotransfe rase is selectively distributed with maximal activity occurring in spl een which was substantially greater than thymus or lymph nodes, In con trast, other enzymes (i.e. PAPS:Gal-3-O- and GlcNAc-6-O-sulphotransfer ases) involved in the sulphation of sialyl-lactosamine and lactosamine sequences, which in the sulphated form are believed to also be select in ligands, were more evenly distributed in lymphoid tissues, Relative ly high activities for all three enzymes were found in brain.