Purification of Heparan Sulfate D-Glucosaminyl 3-O-Sulfotransferase*

The cellular generation of proteoglycans with anticoagulant heparan sulfate (HSPG^act) is determined by microsomal “HS^act conversion activity” that functions in concert with the sulfate donor 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to convert nonanticoagulant heparan sulfate (HS^inact) to anticoagulant heparan sulfate (HS^act) (Shworak, N. W., Fritze, L. M. S., Liu, J., Butler, L. D., and Rosenberg, R. D. (1996) J. Biol. Chem. 271, 27063-27071). Suspension cultures of L-33⁺ cells in serum-free medium produce HSPG^act and secrete HS^act conversion activity. The secreted protein exhibiting HS^act conversion activity was isolated by subjecting large volumes of conditioned suspension culture medium to heparin-AF Toyopearl affinity chromatography, Mono Q-FPLC, TSK SW3000-HPLC, and 3′,5′-ADP-agarose affinity chromatography. The final product was purified ∼700,000-fold relative to cellular material with a 5% overall recovery of HS^act conversion activity. The isolated protein migrated on SDS-polyacrylamide gel electrophoresis as a broad band of M_r = 46,000 and co-migrated on nondenaturing acidic pH gel electrophoresis with HS^act conversion activity. The purified component was identified as heparan sulfate D-glucosaminyl 3-O-sulfotransferase because it transferred sulfate from [³⁵S]PAPS to the 3-O-position of D-glucosamine and D-glucosamine 6-O-sulfate of HS^act precursor and HS^inact precursor to produce nearly equivalent amounts of labeled HS^act and HS^inact. The exhaustive modification of wild-type LTA cell [³⁵S]HS with either microsomal HS^act conversion activity or purified enzyme increased HS^act content from 9 to ∼36%, which indicates that microsomal HS^act conversion activity predominantly reflects the level of a 3-O-sulfotransferase that converts HS^act precursor into HS^act. The kinetic parameters of purified 3-O-sulfotransferase were determined for modification of HS^act precursor and HS^inact precursor. The apparent K_M* and V_max* with respect to PAPS concentration for sulfation of HS^act precursor and HS^inact precursor were 2.4 μM and 23 fmol of sulfate/min/ng of enzyme and 2.1 μM and 38 fmol of sulfate/min/ng of enzyme, respectively. There was substrate inhibition of the sulfation reaction at elevated HS concentration. The apparent K_M* and V_max* with respect to GAG concentration for sulfation of HS^act precursor and HS^inact precursor were 16 nM and 120 fmol of sulfate/min/ng of enzyme and 17 nM and 240 fmol of sulfate/min/ng of enzyme, respectively. The observation that purified 3-O-sulfotransferase catalyzes sulfation of HS^act precursor and HS^inact precursor in conjunction with a documented discordant regulation of 3-O-sulfate content in HS^inact and HS^act suggests that two discrete forms of the enzyme may exist.