New approaches for defining the molecular basis of anticoagulantly active heparan sulfate production

Mammalian cells synthesize heparan sulfate proteoglycans, which consist of core proteins with covalently linked glycosaminoglycans of 50-150 disaccharide units. The GAGs exhibit great structural diversity, which arises from differing arrangements of alternate disaccharide units. It has been hypothesized that HSPG may be involved in regulating the most basic aspects of cell biologic systems, such as adhesion, proliferation, and differentiation. However, considerable doubt exists about the specific nature of the above interactions because of a failure to isolate GAGs of unique monosaccharide sequence with appropriate biologic activities. We have demonstrated that mouse LTA cells synthesize cell surface heparan sulfate proteoglycans with regions of defined monosaccharide sequence that specifically interact with antithrombin (HSPGact). However, it remains unclear how HSPGact can be generated by a biosynthetic pathway with no simple template for directing the ordered assembly of monosaccharide units. To examine this issue, we treated LTA cells with ethylmethane sulfonate and then identified mutants that exhibit decreased antithrombin binding to heparan sulfate chains but possess no gross defects in glycosaminoglycan biosynthesis. After screening 40,000 colonies, we isolated seven stable mutants that synthesize 8-27% of the wild type HSPGact but produce normal amounts of other HSPG. These mutants are recessive in nature and fall into at least two different complementation groups. The delineation of the molecular basis of these defects should greatly improve our understanding of how cells synthesize HSPG with regions of defined monosaccharide sequence.