Enzymatic access to the rare ΔUA (1→4) Glc 3, 6, N-sulfated heparin disaccharide, implications for heparin quality control

The sulfated glycosaminoglycan heparin is the most commonly used pharmaceutical anticoagulant worldwide. Heparin, which is extracted primarily from porcine sources, has a complex heterogeneous structure, resulting in a highly variable pharmaceutical product susceptible to contamination. As a by-product of the food industry, heparin is also limited by production capacity, giving rise to concerns that demand will outstrip supply. The anticoagulant activity of heparin derives principally from the AGA*IA pentasaccharide sequence, containing a rare 3-O-sulfated glucosamine, which binds and activates antithrombin. Analytical heparin digestion by the widely usedPedobacter heparinuslyases has limited activity in regions of 3-O-sulfation, rendering these enzymes poorly suited to study anticoagulant sequences. Here, we provide structural and functional characterization of aBacteroides eggerthiilyase that exhibits highly efficient heparin depolymerization, with specificity distinct toP. heparinus. Using a panel of biophysical and structural techniques, we demonstrate thatB. eggerthiilyase effectively liberates the rare GA* disaccharide, a key indicator of anticoagulant potential, from the defined heparin pentasaccharide fondaparinux. We envision superior cleavage byB. eggerthiilyases will enable the future quantitative, direct detection of anticoagulant relevant 3-O-sulfated sequences, delivering complementary structural information to existing analytical methods, with clear utility for pharmaceutical quality control workflows.