Purification of heparan sulfate D-glucosaminyl 3-O-sulfotransferase

Abstract

The cellular generation of proteoglycans with anticoagulant heparan sulfate (HSPG(act)) is determined by microspinal '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 comigrated 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 microspinal 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 RS(act) precursor into HS(act). The kinetic parameters of purified 3-0- 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.