Asparagine 405 of heparin lyase II prevents the cleavage of glycosidic linkages proximate to a 3-O-sulfoglucosamine residue

► Heparin and heparan sulfate contain a rare 3-O-sulfoglucosamine residue. ► 3-O-Sulfoglucosamine is critical for anticoagulation and virus recognition. ► The glycosidic linkage proximate to this 3-O-sulfoglucosamine is resistant to heparin lyase cleavage. ► Protein engineering of heparin lyase II broadened its specificity towards 3-O-sulfoglucosamine containing substrates.