Undersulfated heparan sulfate in a Chinese hamster ovary cell mutant defective in heparan sulfate N-sulfotransferase.
Heparan sulfate N-sulfotransferase catalyzes the transfer of sulfate groups from adenosine 3'-phosphate, 5'-phosphosulfate to the free amino groups of glucosamine residues in heparan sulfate. We have identified a Chinese hamster ovary cell mutant, designated pgsE-606, which is 3-5-fold defective in N-sulfotransferase activity. The residual enzyme activity is indistinguishable from the wild-type enzyme with respect to Km values for adenosine 3'-phosphate,5'-phosphosulfate and N-desulfoheparin, pH dependence, Arrhenius activation energy, and thermal lability. The mutation is recessive, and mixing experiments indicate that the mutant does not produce soluble antagonists of N-sulfotransferase. Inspection of the heparan sulfate chains from the mutant showed that the extent of N-sulfation is reduced about 2-3-fold. The addition of sulfate to hydroxyl groups on the chain is reduced to a similar extent, suggesting that N-sulfation and O-sulfation are normally coupled. Nitrous acid fragmentation of the chains showed that N-sulfated glucosamine residues are spaced much less frequently than in heparan sulfate from wild-type cells. The close correlation of enzyme activity to the number and position of N-sulfate groups indicates that N-sulfotransferase plays a pivotal role in determining the extent of sulfation of heparan sulfate.
Bame
K J
KJ
Department of Biochemistry, School of Medicine, University of Alabama, Birmingham 35294.
Esko
J D
JD
D000818:Animals; D002460:Cell Line; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D005944:Glucosamine / Q000378:metabolism; D006025:Glycosaminoglycans / Q000378:metabolism*; D006497:Heparan Sulfate / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009154:Mutation; D010724:Phosphoadenosine Phosphosulfate / Q000378:metabolism; D013431:Sulfates / Q000378:metabolism*; D015238:Sulfotransferases; D013466:Sulfurtransferases / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics
D005944:Glucosamine; D006025:Glycosaminoglycans; D006497:Heparan Sulfate; D010724:Phosphoadenosine Phosphosulfate; D013431:Sulfates; D015238:Sulfotransferases; D013466:Sulfurtransferases; C021350:heparitin sulfotransferase