Characterization of a spleen sulphotransferase responsible for the 6-O-sulphation of the galactose residue in sialyl-N-acetyl-lactosamine sequences.

An enzyme which catalyses the transfer of sulphate from 3'-phosphoadenosine 5'-phosphosulphate (PAPS) to C-6 of galactose in the NeuAcalpha2-3Galbeta1-4GlcNAc (3'SLN) sequence has been found in rat spleen microsomes and its specificity indicates that it is well suited to participate in the assembly of 3'-sialyl-6'-sulpho-LacNAc [NeuAcalpha2-3Gal(6-SO4)beta1-4GlcNAc] and 3'-sialyl-6'-sulpho-LewisX [NeuAcalpha2-3Gal(6-SO4)beta1-4(Fucalpha1-3)GlcNAc] saccharide groups which have been implicated as selectin ligands. This sulphotransferase has a strict requirement for oligosaccharide acceptors which are capped by an alpha2-3-linked sialic acid residue, although GlcNAc in 3'SLN can be substituted by Glc, and Galbeta1-4GlcNAc can be replaced by Galbeta1-3GlcNAc without loss of activity. The finding that 3'-sialyl LewisX was inert as an acceptor suggested that fucosylation, in contrast with sialylation, follows the addition of the sulphate group. Since fetuin glycopeptides containing the NeuAcalpha2-3Galbeta1-4GlcNAc sequence had a similar affinity for the enzyme as the unattached 3'SLN, it would appear that the acceptor determinants reside primarily in the peripheral trisaccharide constellation. The position of the sulphate on C-6 of galactose was elucidated by Smith periodate oxidation, hydrazine/nitrous acid/NaBH4 treatment and elder (Sambucus nigra) bark lectin chromatography of the desialylated [35S]sulphate-labelled products 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 Km values determined for this enzyme were 4.7 microM 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-sulphotransferase is selectively distributed with maximal activity occurring in spleen which was substantially greater than thymus or lymph nodes. In contrast, other enzymes (i.e. PAPS:Gal-3-O-and GlcNAc-6-O-sulphotransferases) involved in the sulphation of sialyl-lactosamine and lactosamine sequences, which in the sulphated form are believed to also be selectin ligands, were more evenly distributed in lymphoid tissues. Relatively high activities for all three enzymes were found in brain.