Substrate specificity of the heparan sulfate hexuronic acid 2-O-sulfotransferase

The interaction of heparan sulfate with different ligand proteins depends o n the precise location of O-sulfate groups in the polysaccharide chain. We have previously shown that overexpression in human kidney 293 cells of a mo use mastocytoma 2-O-sulfotransferase (2-OST), previously thought to catalyz e the transfer of sulfate from 3 ' -phosphoadenosine 5 ' -phosphosulfate to C2 of L-iduronyl residues, preferentially increases the level of 2-O-sulfa tion of D-glucuronyl units [Rong, J., Habuchi, H., Kimata, K., Lindahl, U., and Kusche-Gullberg, M. (2000) Biochem. J. 346, 463-468]. In the study pre sented here, we further investigated the substrate specificity of the mouse mastocytoma 2-OST. Different polysaccharide acceptor substrates were incub ated with cell extracts from 2-OST-transfected 293 cells together with the sulfate donor 3 ' -phosphoadenosine 5 ' -phospho[S-35]sulfate. Incubations with O-desulfated heparin, predominantly composed of [(4)alpha IdoA(1)-(4)a lpha GlcNSO(3)(1)-](n), resulted in 2-O-sulfation of iduronic acid. When, o n the other hand, an N-sulfated capsular polysaccharide from Escherichia co li K5, with the structure [(4)beta GlcA(1)-(4)alpha GlcNSO(3)(1)-](n), was used as an acceptor, sulfate was transferred almost exclusively to C2 of gl ucuronic acid. Substrates containing both iduronic and glucuronic acid resi dues in about equal proportions strongly favored sulfation of iduronic acid . In agreement with these results, the 2-OST was found to have a similar to 5-fold higher affinity for iduronic acid-containing substrate disaccharide units (K-m similar to 3.7 muM) than for glucuronic acid-containing substrat e disaccharide units (K-m similar to 19.3 muM).