Cell-free synthesis of anticoagulant heparan sulfate reveals a limiting converting activity that modifies an excess precursor pool

Abstract

LTA cells synthesize a minor population of heparan sulfate proteoglycans (HSPG(act)) bearing anticoagulant heparan sulfate (HS(act)) with a specific monosaccharide sequence that accelerates the action of antithrombin (AT). LTA cells also synthesize a major population of heparan sulfate proteoglycans endowed with nonanticoagulant heparan sulfate (HS(inact)) lacking the AT- binding site. To investigate the pathway-specific features of HSPG(act) generation, we established a novel detergent-containing cell-free system with unlabeled and labeled microsomes from wild-type and variant LTA cells, respectively. The unlabeled microsomes provide 'HS(act) conversion activity' that requires 3'-phosphoadenosine 5'-phosphosulfate to convert [35S]HSPG(inact) into [35S] HSPG(act), presumably by sulfation. The reaction kinetics demonstrate that the rate of HS(act) synthesis is constant over the first 4 h of incubation. During this time, the rate of HS(act) production is linearly dependent on the amount of unlabeled LTA microsomal protein over a range of 10 to 50 μg as well as on the level of [35S]HS substrate over a range of 0.4 to 4.0 μg, microsomal protein. Compared with labeled microsomes, equivalent or slightly greater levels of HS(act) were generated from 35S-labeled HSPG, microsomal HS, or cell surface HS, which demonstrates that HS(inact) is the minimal substrate and that large amounts of HS(act) precursor exit the Golgi apparatus. Indeed, extensive modification of wild-type LTA cell surface [35S]HS elevated HS(act) content from 9 to 35%. The hypothesis that microsomal HS(act) conversion activity predicts the cellular rate of HS(act) generation was tested with wild-type or variant LTA cells in which production of HS(act) has been significantly altered by mutagenesis or overexpression of core protein or growth conditions. The data demonstrate that microsomal HS(act) conversion activity accurately reflects the cellular rate of HS(act) synthesis over a very wide range of conditions. The possibility that the reduced HS(act) generation is due to an inhibitor was excluded by mixing experiments. The possibility that reduced HS(act) generation is caused by decreased levels of HS(act) precursor was excluded as equivalent levels of HS(act) were formed from wild-type and variant [35S]HS. Based upon the above data, the LTA cell microsomal HS(act) conversion activity contains one or more limiting components that kinetically regulate the rate of cellular HS(act) generation and the levels of HS(act) precursor in HS greatly exceed HS(act) production.